CA2935570A1 - The use of transgenic plant for recovery of non-transgenic hybrids - Google Patents

Abstract

A method is disclosed of producing a hybrid plant system for breeding purposes using a transgenic plant as a intermediate to create a non-transgenic plant.

Description

THE USE OF TRANSGENIC PLANT FOR RECOVERY OF NON-TRANSGENIC
HYBRIDS
PRIORITY
The present invention claims priority to U.S. Provisional Patent Application Ser. No.
61/922,454 filed December 31, 2013, the disclosure of which is hereby incorporated by reference in its entirety.
GOVERNMENT SUPPORT
The present invention was developed, in part, by support from the United States Government by the Department of Energy under Contract Grant No. DE-FG36-08G088070.
The United States Government has certain rights to the present invention.
FIELD OF THE INVENTION
The present invention relates to plant genome modification methods that result in the recovery of progeny from wide inter- and intra- varietal specific and generic hybrid plants.
This invention relates generally to the field of plant genetics. More particularly, it concerns production of non-genetically modified hybrid plants using transgenic hybrids and methods for production and use thereof.
BACKGROUND
As population growth increases worldwide, now exceeding 7 billion with projected increases to over 9 billion in the next three decades, the need to improve agricultural production has been considered by some to be a moral imperative. In addition, concomitant with population growth, the need for sustainable energy resources becomes more apparent as reliance on fossil fuels strains the global environment, economy and international security as struggles for remaining assets swell and availability of arable land and water are increasingly challenged. The utilization of dedicated crops as a source of bioenergy from renewable resources is a goal with great relevance to current ecological and world economic issues and presents compelling challenges to world agriculture.
While many alternative energy sources, including wind, wave, tidal, solar, and nuclear provide means to generate electricity, renewable bioenergy crops present a solution for liquid transportation fuel production. Many countries, such as the USA and China, have issued increasingly aggressive targets for renewable energy over time; these will certainly require new dedicated bioenergy crops and fuel platforms. Domesticated crops, such as maize, canola, oil palm, sunflower, sugarcane, and soybean are currently used for large scale commercial biofuels production. One concern is whether sufficient amounts of renewable materials can be supplied without impacting the cost of arable agricultural land, competing with food production, and harming the environment. In 2000, the U.S.
Department of Energy's (DOE's) Bioenergy Feedstock Development Program (BFDP) at Oak Ridge National Laboratory (ORNL) issued the Bioenergy Feedstock Development Program Status Report, which reported on a survey of potential bioenergy plants including over 100 woody trees and 35 herbaceous species. For a variety of reasons, production of liquid fuels from dedicated nonfood crops as cellulosic sources, such as switchgrass, sorghum, Miscanthus, Energy Cane willow, and poplar, is widely understood as a necessary development.
The development of switchgrass (Panicum virgatum L.) as a bioenergy resource, serves as an important example for the purpose of the current review.
Switchgrass (Panicum virgatum L.) and related species have become dominant candidates as feedstocks for cellulosic biomass that will be used for the production of various types of biofuels.
Switchgrass is native and widespread throughout most of North America and, importantly, grows on marginal lands that are not competitive with food production resources. The DOE
survey chose switchgrass as one of the most promising cellulosic feedstocks based on its high net energy balance ratio (NEB), stand longevity, perennial growth habit, ability to grow on marginal lands (Conservation Reserve Program, CRP, land has been cited as a possible resource for production), low inputs, natural pest and disease resistance, and high biomass yield.
Currently, switchgrass cultivars are estimated to produce a net energy yield (NEY) of 60 Gigajoules per hectare per year providing potentially 540% more renewable energy available than non-renewable energy consumed and reducing greenhouse gas emission by 94% in comparison to gasoline. Meeting the goals of the US DOE billion-ton annual supply of biomass translates into 5% of the nation's power, 20% of the nation's transportation fuels, and 25% of the nation's chemicals by 2030. This goal is equivalent to 30% of current petroleum consumption. USDA/DOE projects that 42 million acres of cropland will be competitive producing an average of 4.2 dry tons per acre of perennial grasses at $40/dry ton.
Yields from the best clones of perennial grasses were generally 8 tons per acre or higher, and the highest yields of existing clones is 15.5 dry tons per acre.
Limitations in the current availability of bioenergy feedstocks are considered a major problem for large commercial applications particularly for production of next-generation non-ethanol liquid biofuels, such as green diesel and gasoline, synfuels, biobutanol, aviation fuels and other hydrocarbon biofuels, essential for large-scale transportation energy demand. To

2 fully realize the potential of biofuels, the power of advanced genetic and biotechnology tools need to be brought to bear on the improvement of bioenergy crops, most of which are largely considered under domesticated. Technologies such as hybrid plant systems, genomics, association genetics, marker assisted breeding, bioinformatics, advanced tissue culture, and transgenics, are just a few of the technologies that promise increased yield, processability, and regional adaptation of biofuel crops. Assuming an intensive genetic and research program, the feasibility of obtaining much higher yields and NEB ratios over millions of acres of bioenergy crops is supported by modeling.
Conventional plant breeding is the science that utilizes intentional crosses between individuals with different genetic constitutions with intentional trait selection followed by subsequent crosses. The conventional breeding process produces new hybrids varieties and indeed new species with desirable traits. These new plant types are the result of sexual recombination of genes mostly accomplished during meiosis. However additions, duplications, deletions, insertions and rearrangements of chromosomal sets, fragments and individual genes as well as noncoding DNAs also can play a significant role.
These results can occur between different lines and varieties, species, genera, families or even more distant relatives and recent new breeding technologies will play significant role in future cultivar development.
New breeding and genetic technologies for the improvement of perennial non-food plants specifically as feedstocks with biofuels related traits are important for perennial grasses, like switchgrass, Miscanthus, Energy Cane, and sweet sorghum but also short-rotation trees, such as Eucalyptus, Salix, Paulownia, and Populus.
Conventional hybrid breeding techniques have been applied to certain bioenergy crops. Given the under domesticated status of many bioenergy plant species, crop improvement can be greatly facilitated by the creation of hybrid plants, trait selection, genomics, association genetics and exploitation of heterosis. Hybrid plant performance is mainly determined by the degree of heterosis defined by superiority of the heterozygous F 1 hybrid compared with parental types.
Heterosis can cause dramatic improvements in various aspects of perennial plant performance such as seed yield and size, floral number and size, first year biomass yield, second year biomass yield and other agronomic traits by recombination of genetic variation through intraspecific or interspecific hybrid production. Panmictic-midparent heterosis describes the improvements in F 1 progeny of two random mating populations. In allogamous grass species hybrid performance is difficult to measure because of obfuscating ploidy effects and high degrees of population heterozygosity. However, breeding schemes have been shown that are

3 aimed to more efficiently exploit heterosis in bioenergy grasses based on fact that they are (1) cross pollinating (2) wind pollinated, (3) produce a large amount of seed per plant and, (4) exhibit a strong self-incompatibility which can be exploited for hybrid seed production.
Certain work has provides a large background to heterosis and breeding in switchgrass. Current commercial switchgrass varieties are improved populations or synthetic cultivars developed using breeding methods from additive genetic variation. To produce hybrid cultivars, switchgrass breeders have not relied on non-additive genetic variance except where the hybrids can be vegetatively propagated. Heterosis, also known as hybrid vigor, must be addressed on a trait by trait basis, and is defined by some as "the positive difference between the hybrid and the mean of the two parents."
The phenomenon of hybrid vigor is best known as observed in maize breeding and the term heterosis was first coined in 1914 regarding hybrid maize. Heterosis and identification of heterotic groups has played an essential role in maize becoming the highest tonnage crop worldwide in 2001. As further breeding programs are established with emphasis on biofuel -specific traits, bioenergy cultivars could benefit from exploitation of hybrid vigor for biomass production and identification of heterotic groups in ways that have driven maize, rice and wheat to the forefront of worldwide food production. The major problems addressed in the present review are that the production, analysis, and commercialization of hybrids by the current conventional methods is cumbersome, time-consuming, cost and time prohibitive, and not broadly applicable. The present invention is intended to present solutions that seek to overcome these obstacles.
Hybrids can be selected for desirable phenotypes contributed by either parent;

including bioenergy traits, such as carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus). Genomic and marker assisted breeding can be deployed to characterize parental genomic contribution and to follow traits in subsequent downstream

4 breeding for varietal development. Newly created hybrids can be sexually crossed and/or vegtetatively propagated, depending on the crop species and utility.
The application of genetic modification (GM) will provide additional powerful approaches for improvement of traits important to the development of energy crops. The regulatory parameters for row crops such as maize, soybean, cotton, and canola are already well established and many gene flow studies on these plants have been conducted. The genetic improvement of food row crops has been greatly accelerated through advanced applications of tools of biotechnology and advanced breeding and undoubtedly this same model will be useful for improving perennial bioenergy feedstocks. Many traits are currently being tested for specific biofuel crop applications. Some of these traits have been already developed for row crops including drought and pest resistance, increased yield, and decreased inputs while other transgenic traits are being designed to specifically enhance biofuel production; e.g., biofuel-specific traits such as production of glycosyl hydrolases, biopolymers and other co-products, altered sugar profiles, low starch or low lignocellulose fibers, cell wall biosynthesis proteins for increased cellulose, decreased lignin, improved biomass yields per acre, decreased inputs are all traits that can be engineered to increase fuel production per acre. In fact, several commercial companies have based their business models on applications for improvement of biofuels crops and microbes using genetic modification.
Commercial-scale production of some transgenic plants have been suggested by some to lead to undesirable environmental and agricultural consequences including transgene escape to wild and non-transgenic relatives. One obstacle then that arises regarding transgenic improvement of perennials used for bioenergy is the propensity of these plants to be open pollinated with the undesirable capacity of outcrossing to non-transgenic and wild relative species. Current information strongly indicates the potential for gene flow in open pollinated GM grasses. Moreover the release of open pollinated round-up resistant creeping bentgrass resulted in landscape wide gene flow. Gene confinement has been considered as a technical barrier to the development and release of transgenic perennial plants. Thus, to realize the full potential of agricultural biotechnology for dedicated energy crops enhancement, the commercial and environmental impacts of gene flow must be addressed.
Several questions however, arise when considering current research on gene confinement and the degree of stringency required for release of a GM
perennial plant into the environment for commercial applications, such as is anticipated for the production of switchgrass and other perennials as a biofuels feedstocks. Will the control of pollen flow alone be adequate, or will a control for seed scatter, or vegetative tillering also be required?
Can a system be developed that will be flexible enough to allow additional gene stacking without the cost and time involved with the production of new transgenic lines? Given current regulatory concerns, transgenic GM traits might never be deregulated for commercial release without a robust gene confinement strategy. GM technologies with significant potential for bioenergy crop improvement may be difficult or impossible to commercialize without an adequate gene confinement strategy that will allow them to progress through the deregulation process.
Progress on transgenic improvement of perennial plants used for biofuels will likely not happen without an operating platform technology to impede transgene escape of the traits they have engineered. The USDA-APHIS-BRS regulates the environmental release of transgenic plants. Permits are required for all non-deregulated transgenic plants to be grown outside of containment greenhouses. The value of BRS' to both biosafety and innovation in transgenic field testing is apparent inasmuch that transgenic releases in the US do not require costly permitting or undue paperwork. However, permits often are accompanied by additional requirements. For example, in the field testing of transgenic switchgrass, researchers are required to prevent flowering and set seed; i.e., by the mechanical removal of flowers prior to anthesis. BRS currently considers the planting of transgenic switchgrass to be a case that requires the imposition of a stringent set of precautions to avoid gene flow when the first field tests were performed, even though the transgenics contain only non-herbicide selectable and scoreable marker genes. The current review addresses the evaluation of confinement and sets the stage for monitoring methodologies for field trials of genetically modified organisms related to biofuels crops.
The process of US deregulation includes lengthy reviews and data collection spanning different environments over several years with consideration of several factors including biology, geography and ecology of the plant, the genes and traits of interest, the possibility of gene flow to wild and non-transgenic relatives, the possibility of weediness or invasiveness, and unintended consequences to other organisms. The process has been considered by some to be overly cumbersome, excessively expensive and progress prohibitive, yet cautious, protective of the environment and the public interest. It is important therefore, to assess individual bioenergy feedstock species independently and to evaluate the introduced traits or characteristics to determine if they could enhance the vigor or invasiveness of wild or weedy relatives or have other detrimental effects. While some traits may pose relatively few ecological risks (e.g., herbicide tolerance), other traits have the potential for unintended consequences and invasiveness (e.g., drought and pest tolerance). Without an adequate gene confinement strategy in place vast amounts of research and development on these improvements will be wasted.
Most of the next-generation dedicated energy crops will be perennial trees and grasses. Many species that are being seriously considered to play a major role in the developing biofuels industry have wild relatives in the regions where they will be grown. In addition, for some prominent feedstocks, such as switchgrass, there is an absence of data on gene flow. The regulatory data requirements or constraints for gene flow are still unclear.
While one may assume that transgene containment is the goal, acceptable levels of transgene escape need to be practically defined. Considering the cost of deregulation and the subsequently imposed market restrictions, the risks and benefits of some regulatory requirements may need to be reconsidered i.e., modified without unduly compromising safety. The development of effective genetic containment strategies and evaluation of the efficacy of genetic techniques, to prevent gene transfer or outcrossing is a major priority for genetic improvement of perennial plants used for biofuels.
Various transgenic gene confinement strategies have been devised based on hybrid plant systems. The methods for transgenic hybrid plant gene confinement that have been developed so far include seed-based gene confinement, the Gene Deletor system, and various total sterility concepts. One of the best known is the GeneSafe Technology, known more commonly by the nomme fatal Terminator uses an inducible site specific recombinase system (Cre/lox) to produce seed that will germinate but never again set fertile progeny. Despite its poor press and public perception issues this type of technology would be well suited to gene confinement for improved bioenergy crops. Therefore, while systems based on recombinase excision technologies have proven practical for removal of specific DNA
insertions and then propagated as specific lines, and even produced commercially as in the case of high lysine corn produced by Monsanto the inability to use these systems for gene confinement and hybrid plant systems is evident by the lack of reports in peer reviewed and patent literature.
While reviewed on a case-by-case, species-by-species, and trait-by-trait basis by USDA APHIS-BRS, bioconfinement of engineered genes in perennial plants used for cellulosic biofuels will accordingly likely be a prerequisite for deregulation and commercial production of these plants. Bioconfinement of transgenes is thus an obvious regulatory, economic, environmental and biosafety objective for the release and commercialization of transgenic bioenergy feedstocks. One desirable objective then is to create a flexible and simple hybrid system useful for the breeding of GM and non-GM bioenergy crops while providing for gene confinement in perennial plants practical for biofuels. In addition, these systems will provide the biological materials to evaluate ecological gene flow in transgenic perennial plants.
In particular then, this review for this patent aims to present the background to (1) evaluate the current population and synthetic breeding schemes relevant to bioenergy crops, with an emphasis on grasses, and especially switchgrass, (2) present novel GM
and Non-GM
approaches to hybrid plant development, and (3) address gene confinement strategies pertinent to release of GM improved bioenergy crops.
Conventional population and synthetic breeding schemes in perennial ryegrass (Lolium perenne L.) and switchgrass (Panicum virgatum L.) are relevant to for hybrid plant development in bioenergy crops generally. Both of those species are characterized by highly effective self-incompatibility systems which promote high levels of cross-pollination, outcrossing and hence heterozygosity. Recurrent selection in population breeding schemes results in continuous cultivar improvement. Whereas the use of crosses among a limited number of selected parents which is followed by multiplication trough repeated open pollinations characterize synthetic breeding schemes. Since both of these schemes are based on open pollination which ensures random mating result in panmictic populations and continuous improvement. However, only relatively low degrees of improvement have been reported presumably due to negative correlations between diverse target traits, such as biomass and seed yields and the long breeding cycles in allogamous grass species.
Additionally these schemes may result in limited degrees of success due to incomplete exploitation of heterosis.
Over recent years large amounts of morphological and genetic information have accumulated about switchgrass cultivars and populations. Two main ecotypic classifications have been characterized; (U) upland types, indigenous to low-flood risk upland areas in North America, and; lowland types (L), common to the flood plain regions of North America.
These two ecotypes show distinct morphological and physiological characteristicsand the genetic distinctiveness of their nuclear genomes has been established on the basis of cluster analysis of RAPD markers, identification of RFLP markers, and cluster analysis of EST-SSRs markers. Hence there are significant barriers to the inter-breeding of these ecotypes.
Ploidy levels vary within switchgrass ecotypes, ranging from diploid (2n=2x=18) to duodecaploid (2n=12x=108), with all lowland ecotypes identified as tetraploids (2n=4x=36) while upland types can be tetraploids or octaploids (2n=8x=72). Mixed ploidy levels among accessions and within cultivars has also been observed. Further ploidy level analysis has been performed through mitotic chromosome counts and flow cytometry. Finally, because of the breeding restrictions between commercial varieties, particularly upland and lowland ecotypes, it would be extremely useful to develop methodologies for the recovery of wide intra-and interspecific hybrids.
Switchgrass, is a highly heterozygous, anemophilous obligate outcrosser with both pre-fertilization and post-fertilization self incompatibility systems present.
Self-incompatibility prevents self-fertilization thus maintaining high population degress of heterozygosity. Gametophytic self-incompatibility in grasses is controlled by two loci, S and Z. None of the self-compatibility genes have been cloned. Self incompatibility demonstrated by some in switchgrass show that in controlled crosses between octoploid x octoploid, octoploid x tetraploid, tetraploid x octoploid post-fertilization abortion occurs in many cases 20-40 DAP and maturing caryopses are easily isolated. These observations provide a sound background for the isolation of hybrid precursors. With ample phenotypic and genetic diversity characterized within and among switchigrass cultivars and populations, data on heterosis and hybrid plant development is limited then by the time-consuming and laborious process to recover hybrid plants and fertile alloploids. Controlled hybridization techniques, based on floral emasculation and mutual pollination by bagging inflorescences, have been used in recovering both population hybrids and specific hybrids of switchgrass. Through these techniques intraspecific crosses between upland and lowland ecotypes, and between spatially separated populations have yielded viable hybrid plants, which do display heterosis.
Although these methods are accurate and promising, they are tedious, time consuming and produce low numbers of candidate progeny. Additionally, analysis and verification of hybrid plants requires extensive phenotypic observation and measurements based on morphological characteristics before molecular analysis can verify the hybrid genotype.
As has been demonstrated, a set of subpopulations, ectotypes or heterotic groups can be used in crosses for the development of population hybrids. The resulting population hybrids will be a blend of inter- and intra-population crosses. Certain work shows population hybrid breeding schemes which exploit self-incompatibility with the goal to maximize the amount of hybrid seed derived from interpopulation crosses. Populations can be grown side by side but in different ratios whereby one population will contribute more pollen to the pollen cloud compared with the other, forcing the hybrid cross for production purposes.
Alternatively, one parent (A) could be arranged to surround another parental line (B) thus creating a pollen cloud sufficient to recover a successful population cross (shown in Figure 1 at I). In this strategy the parental line (A) is spatially separated with respect to parent (B), each, so that the harvested seed from the pollen recipient will comprise at least 75% hybrid seed. In another scheme populations used as pollen donors (A) could be selected specifically for the trait of high pollen production and then grown side by each to force a hybrid cross (shown in Figure 1 at II). This latter strategy could also be accomplished by the development of transgenic nuclear male and female sterile lines specifically for this purpose (shown in Figure 1 at III) where the pollen donor line (A) surround the male sterile line (B).
Similarly the creation of transgenic nuclear stable male and female sterile lines with dual herbicide resistance markers would be useful for commercial hybrid production(shown in Figure 1 at IV). In this scheme the female sterile line (A) would be resistant to one herbicide, bialaphos, for example, while the male sterile line (B) would be resistant to a second herbicide, such a gylphosate. The hybrid cross therefore could be selected for with both herbicides and expected to be totally sterile. Lines A and B would be useful for the generation of recurrent inbred lines. This scheme would also find application for the prevention of transgene escape and gene confinement. These latter strategies (III and IV) will be discussed below.
Synthetic breeding schemes rely on the use of a limited number of selected parents, where the parents of a single cross hybrid are two inbred lines. In perennial grasses such as switchgrass, inbred line development is impaired by self-incompatibility and inbreeding depression. Thus single cross hybrids from heterozygous parents resulting in segregating F 1 populations are more likely for perennial grasses and comparable to di-hybrid crosses in maize.
There remains a need, therefore, for an improved extension of breeding applications via hybridization of distinct heterotic groups (ectotypes) and near relatives.
SUMMARY
The need for the world to increase its efforts on sustainable food and energy production and the role that advanced genetics will play are well understood and widely known. Rapid genetic improvement of the most domesticated crop plants is desirable for current and projected global agricultural needs. Addressing these needs is anticipated by current genomics, bioinformatics, association genetics, marker assisted breeding, conventional genetics and other non-GMO approaches. While transgenics offer access to traits outside the conventional breeding pool they are time consuming, costly, and involve unresolved issues regarding public acceptance, governmental deregulation and commercial release. This invention involves the novel use of transgenic herbicide resistance from crosses for recovery of new hybrids. A new hybrid in this context refers to a genetic conduit for incorporation of new genes and conferred traits into new hybrids or varieties.
The hybrid intermediate provides a mechanism for importing many new genes and large amounts of genetic material that cannot be otherwise moved through common conventional breeding program materials. In addition, the creation of these intermediates provides new de novo genetic material that arises from these wide varietal, species or genera crosses which would not be possible using traditional plant breeding techniques.
This technology will be useful in several way: (1) to rapidly produce new hybrids afor transfer of useful traits such as biotic (including pest resistance, drought and heat tolerance), abiotic stress (including herbicide resistance), yield and heterosis; (2) to combine near and distantly related germplasm to create new hybrids, and (3) to use genome editing functions and subsequently remove the transgen; and (4) to use transgenics as 'bridge intermediates' to create new hybrids otherwise difficult or imposssible to create.
These wide cross hybrids are then in backcrosses or outcrosses to remove the transgene facilitating recovery of non-transgenic hybrids that can be rapidly introduced to the agricultural market without costly R&D and deregulation of GM development.
Using this novel system the problem of new varietal development can be addressed by the creation of new hybrid plants which include novel traits, allows for advanced trait selection using genomic assisted breeding technologies, and the exploitation of heterosis.
Hybrid plants incorporate new genetic material in a breeding program that can result in dramatic improvements in various aspects of plant performance such as yield, including, but not limited to: fruit, biomass, grain, root or tuber and seed yield; plant size, color, or texture;
plant growth rate; floral timing; floral numbers and size; secondary metabolite production and yields; first year and, second year biomass yield in bioenergy crops; root mass; water use efficiency; insect and pest tolerance, avoidance, or protection; drought, cold, and salt-tolerance; more efficient use of nutrients and, many other important agronomic traits. New traits are introduced by recombination of genetic variation through intra- or inter- varietal, specific or generic hybrid bridge intermediates and subsequent production through conventional breeding. The specific embodiments of this invention involve the use of methods for high scale production of hybrid plants through wide crosses, and recovery of hybrids in crop species.
The demonstration of four specific embodiments of this invention teach the methods for high scale production of hybrid plants through wide crosses, recovery and use of appropriate hybrids in switchgrass (Panicum viratum L.) and extension of these techniques to other grasses as well as cereal crops and by extension all plants. During the practice of this invention new transgenic lines are generated for aiding in the improved efficiency for creating hybrids according to various plant species and hybrid recovery by using transgenic herbicide or antibiotic resistance which may be linked with male or female sterility and advanced tissue culture approaches. Through these efforts one practiced in the art expects to create new hybrid plants as demonstrated herein. Similarly, one practiced in the art can use these techniques on any other flowering plant (angiosperms), expeting to segregate the trans genes in the F 1 BC1 population resulting in a non transgenic population with hybrid traits or other effected mutations (as such for genome editing). Hence, the transgenic traits can be selected against (crossed out) in the F2 population to recover hybrids that are essentially non-transgenic. In addition, crosses that result in sterile hybrids can be used as vegetatively propagated transgenic crops with 100% gene confinement. The anticipated results for the extension of this method to other angiosperms has direct and short and long term commercial application. The invention creates new varieties that will be superior in the marketplace, virtually replacing existing varieties.
Switchgrass and its related species are well known as bioenergy crops. There are global economic, political, US national security and environmental pressures to increase renewable biofuel production and utilization, to offset gasoline and diesel fuel use, especially in the liquid fuel transportation sector.
In accordance with certain embodiments, the present invention involves the recovery of progeny from wide crosses in flowering plants using transgenic plants to recover hybrids for the production of non-genetically modified hybrids via backcrossing or outcrossing and conventional breeding. The fact that wide crosses occur in nature is visible in many extant species of plants and animals. However, the frequency of fertile progeny from wide crosses in nature is low but exploitation of such events would be very useful for crop breeding purposes. The introgression of genes from wide crosses will increase genetic diversity and allow trait introduction that does not include transgenes, which will shorten the breeding and commercialization processes. This patent teaches methods for the establishment of an efficient breeding platform for agricultural improvement of members of the Monocotyledonea.
In one embodiment, one of the monocot parental types are transgenic members of the Poacea, such as switchgrass (Panicum virgatum L. cv Alamo). The second parental type is also a member of the Poacea, such as but not limited to, Andropogon sp., Panicum, sp., Pennisetum sp., Zea sp., Saccharum sp., Miscanthus sp., a Saccharum sp. x Miscanthus sp.

hybrids, Erianthus sp., Tripsicum sp., or Zea X Tripiscum sp. hybrids, In general the procedure begins with a transgenic monocot parent with a transgenic selectable marker (typically, but not limited to a selectable marker conferring resistance to an antibiotic or herbicide that can be used for recovery of primary transgenics) as a maternal or paternal parent. In some crosses each parent may be independent transgenic events, containing the same or different selectable markers. The transgenic parent is used in wide crosses, defined as inter- and intra-varietial, inter- and intra-specific as well as inter-generic crosses. Recovery of putative wide crosses is accomplished and progeny are screened for the presence of the transgenic selectable marker. In one embodiment, for example, transgenic herbicide resistant Panicum virgatum L. cv Alamo may be used in an inter-specific cross with non-transgenic Atlantic Coastal Panicgrass (Panicum amarum, Ell. var. amarulum). Note that directionality (maternal X paternal) does not matter to the practice of hybrid plant recovery. The F 1 progeny from the wide crosses may be fertile, producing viable seeds which germinate to produce healthy fertile plants that can be used in backcrosses to wild type non-transgenic Panicum virgatum L. cv Alamo. The subsequent F 1BC1 population is then germinated from the resultant seed. The F 1 BC1 seedlings are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F 1 BC1 hybrid population is then used in downstream varietal and breeding applications. Using techniques such as Genotyping-by-Sequencing (GBS), hybrids can be selected for desirable phenotypes contributed by either parent; including bioenergy traits, such as carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus). Genomic and marker assisted breeding is deployed characterize parental genomic contribution and to follow traits in subsequent downstream breeding for varietal development. Hybrids can be sexually crossed and/or vegtetatively propagated. This technique is generally applicable to all sexually repro cucing plants.

In a second embodiment, transgenic herbicide resistant Panicum virgatum L. cv Alamo may used in difficult to recover interspecific and/or intervarietal crosses to identify and define progeny useful for production of fertile non-transgenic hybrids. In this example, using a self-compatible intervarietial cross between transgenic herbicide resistant Panicum virgatum L. cv Alamo (4x) and non-transgenic Panicum virgatum L. cv Kanlow (4x); where (x) is the basal number of chromosomes, and in Panicum sp. x=9, therefore, 4x refers to a tetraploid where 2n=4x=36 chromosomes, and 8x refers to an octaploid where 2n=
8x = 72 chromosomes. Note that in this example directionality (maternal X paternal) also does not matter to the practice of hybrid plant recovery. The Fl progeny from the wide crosses may be fertile, producing viable seeds which germinate to produce healthy fertile plants that can be used in backcrosses to wild type non-transgenic Panicum virgatum L. cv Alamo. The subsequent F2 population is then germinated from the resultant seed. The F2 seedlings are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment. This type of cross will be useful for the combination of trits in many crop species.
In a third embodiment, transgenic herbicide resistant Panicum virgatum L. cv Alamo may used in to recover rare intra- or inter-specific crosses between self-incompatible parents to identify and define progeny useful for production of fertile hybrids. In this example Genotyping-by-Sequencing (GBS) is used to analyze varietal and breeding applications in the F 1 BC1 population. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment. GBS data and other molecular and phenotypic information indicates the absence of all transgene sequences, Ti plasmid backbone and any Agrobacterium sequences demonstrating that all of the F 1BC1 progeny are non-transgenic. This technique therefore can be applied to genome editing procedures in plants resulting in non-GMO site directed mutagenesis populations.
In yet a fourth embodiment, this invention will generate a series of intra-and inter-specific wide crosses in switchgrass and related species. The breeding platform will utilize transgenic male and female sterile lines from a switchgrass variety, Panicum virgatum L. cv.
Alamo and herbicide selection for recovery of wide intra- and inter-specific Fl crosses. The male and female sterile lines are used to increase the efficacy to recover wide crosses as relatively rare events and force otherwise unlikely crosses. In some cases this can also be facilitated by embryo rescue. Fl hybrids can be backcrossed to the reference Alamo cultivar to segregate away the transgene to generate a non-transgenic BC1 population.
Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment. Phenotypic analysis is conducted on the non-transgenic population in regionally selected field plots and phenotypic data is statistically correlated to genetic variation.
Variation is assessed using genome-resequencing technologies and this data, along with phenotypic information is used to establish a computational and statistical pipeline to identify, map and introgress variation associated with biomass and other bioenergy traits.
This fundamental approach will take advantage of current transgenic, genetic and genomic work already in place for the development of regionally selected cultivars. The long-term goal of this program is to develop new publicly available switchgrass cultivars with bioenergy-relevant characteristics, including; biomass yield, quantity, and quality; pest resistance, drought tolerance, and, environmental adaptation.
BRIEF DESCRIPTION OF THE DRAWINGS
The following description will be further understood with reference to the accompanying drawings in which:
Figure 1 shows an illustrative diagrammatic view of breeding schemes to produce population hybrids;
Figure 2 shows an illustrative diagrammatic view of a switchgrass transmformation sequence;
Figure 3 shows an illustrative diagrammatic view of correlation between PCR
and herbicide resistance and the introduced bar gene construct;
Figures 4A and 4B show illustrative diagrammatic views of recovery schemes of interspecific (Figure 4A) and intervarietal (Figure 4B) crosses using herbicide selection as a marker;
Figures 5A ¨ 5H show illustrative views of molecular characteristics of the wild-type Panicum virgatum and amarum;
Figure 6A ¨ 6D show illustrative views of phynotypic characteristics of the wild-type Panicum virgatum and amarum;
Figure 7 shows an illustrative diagrammatic view of the development and characterization of non-trangenic FlBC 1 populations derived from a transgenic hybrid;
Figures 8 shows the ACP fraction in F1BC1 offspring17;
Figures 9A and 9B show illustrative graphical representations of distributions of ACP
alleles at polymorphic sites;

Figure 10 shows an illustrative diagrammatic view of distribution variant sites and synteny alignment with the Setaria italic genome;
Figure 11 shows an illustrative diagrammatic view of ACP and Alamo varients in each of the 83 F1BC1 lines individually as a heatmap for the total variant calls;
Figure 12 shows an illustrative diagrammatic view of contribution of ACP
alleles across the Switchgrass genome;
Figure 13 shows an illustrative diagrammatic view of a general strategy for commercial hybrid production for a tomato;
Figures 14A and 14B show an illustrative diagrammatic views of a specific strategy for commercial hybrid production of a tomato;
Figures 15A and 15B show an illustrative diagrammatic views of strategies for the creation of intergenic hybrids as crosses between tomatoes and eggplants;
Figure 16 shows an illustrative diagrammatic views of tomato varieties in accordance with embodiments of the invention;
Figure 17 shows an illustrative diagrammatic view of male sterility lines generated through the introduction of specific promotors; and Figure 18 shows an illustrative diagrammatic view of physical linkage of herbicide resistance with male and femail sterility transgenes that may be used for the creation of hybrid-hybrid breeding populations.
The drawings are shown for illustrative purposes only.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Novel Non-Transgenic and Transgenic Approaches to Hybrid Plant Development As has been pointed out, in polyploid switchgrass, preferential pairing increases the probability to capture heterosis by reduction of the number of possible gametes if genotypes with different allelic frequencies are crossed. They recognize that the challenge is to select true hybrid progeny in a semihybrid design. Therefore novel designs have been generated to direct the selection of true hybrid progeny through wide cross selection and the generation of bridge intermediates. These designs include the use of embryo rescue, transgenic herbicide resistance, and transgenic male and female sterility to select hybrid progeny in a semihybrid.
Transgenes can then be selected against in subsequent backcrosses, when fertile progeny are recovered, to derive non-transgenic hybrids. Alternatively, the generation of infertile outcomes present promising sterility mechanisms for gene confinement of GM
lines.

The use of herbicide resistance has been suggested as a possible mechanism to select hybrid progeny in forage grass cultivar development. In that strategy, the two populations must differ in their ability for resistance to two separate herbicides. The source of the herbicide resisatnce could be naturally occurring through selection, of transgenically introduced and the hybrid cross recovered by treatment with both herbicides.
It may however, be difficult to restrict herbicide resistance to particular parental populations and that fixation of the herbicide resisatnce genes may be challenging in a polyploid species. These obstacles may be overcome however by genomic assisted breeding of hybrid progeny populations.
Switchgrass has been genetically modified via particle bombardment or Agrobacterium-mediated transformation using callus cultures induced from mature caryopses or inflorescence and nodal explants and employed either hygromycin or bialaphos selection.
Agrobacterium-mediated transformation has been successfully applied to switchgrass with generally low numbers of T-DNA insertions and transmission to progeny as Mendelian loci without rearrangements. The Kausch lab has demonstrated a high level of proficiency for switchgrass transformation and significant improvements to previously published procedures have been developed, and therefore, high-throughput genetic transformation of switchgrass is now routine. Passage of transgenes to progeny is critically important to immortalization of transgenic lines and incorporation of the transgenic material into useful breeding programs.
The first genetically-modified, asexually-cloned plants generated in tissue culture during a genetic transformation experiment are known as To "events." These plants can be molecularly "characterized" by the nature of the T-DNA insertion into the plant genome. Ti plants are derived from selfs or crosses using To plants to generate progeny designated as Ti plants, and the subsequent generation is referred to as T2. However, Ti, and T2 generation designations are often also referred to as Fi and F2, respectively. Certain studies have greatly increased our knowledge of transformation efficiency, extended the genotype range of transformation to various switchgrass cultivars and Atlantic Coastal Panicgrass (Panicum amarum, Ell. var. amarulum.) and shows that transgenic (Ti) progeny can be readily obtained from crosses with wild-type plants.
Figure 1, for example shows breeding schemes to produce population hybrids. In Figure 1, at I) a Parent A produces a pollen cloud sufficient to swamp out pollen from B to force the hybrid production. In Figure 1, a II) a Parent A is selected to as a genotype that out-produces that of parent B. In Figure 1, at III) the use is shown of male sterility, CMS, nuclear, or transgenic as parent B surrounded by a pollen donor A parent. In Figure 1, at IV) the use is shown of transgenic female sterile herbicide resistant parent A in alternating rows with male sterile parent B resistant to a second herbicide for totally sterile hybrid production.
TransgenicIntermediates as Breeding Tools The derivation of wide crosses can result in important breeding stocks.
Controlled reproduction in plants has been proscribed by plant breeding using a variety of techniques.
Plant breeding techniques such as trait selection from and crossing with wild relatives, domestication, involving selection of agriculturally important traits over time, such as nonshattering in rice, applications of genetics, since Mendel, and later the ability to induce mutations using chemical and radioactive mutagens, wide crosses, by forcing otherwise rare crosses, and eventually the use of gene transfer and genetic modification to include genes from outside the usual breeding pools even across Kingdom barriers. The ability of combine genetics from various gene pools from closely or distantly related plants is therefore well understood in its importance to plant breeding and agriculture generally, regardless of the methods used.
One example includes maize, which is usually a sexually reproducing monecious plant, where techniques for hybridization include using controlled pollination are frequently employed. Controlling breeding crosses in maize involves intentionally repeating two basic steps: (1) evaluation and trait selection from a series of genotypes, and (2) self-pollinating to produce inbred lines or crossing among the most superior plants to obtain the next generation of genotypes or progeny. Controlled pollinations in maize are efficiently possible because of the monecious nature of the plant. The male (tassel) is located as the terminal inflorescence, whereas the female (ear) is borne laterally on the stem. This allows two procedures, detasseling and hand pollination which facilitate controlled crosses.
Using transgenes to control male and female sterility is one option (See SEQ
ID No.s 1-4). However, the use of bridge intermediates is an additonal approach where complenarty species can be crossed and then backcrossed to confer usefull traits.
Some plants can hybridize with their wild or closely related offspring. Zea diploperennis (also referred to as Diploperennis), is a diploid perennial teosinte grass and a wild relative of maize. Diploperennis has the same chromosome number as maize (2n=20), and hybridize naturally with it to produce interspecific hybrid progeny. They are however, in the same genus. The use of transgenics to select such wide crosses would therefore be useful.

Tripsacum is a rhizomatous perennial grass, different genus and a distant relative of maize. Tripsacum is polyploid with a different chromosome number from either maize (x=18, 2n=36 or 2n=72) or Diploperennis. Tripsacum x maize hybrids are not known to naturally form fertile hybrids with maize or the wild Zeas. The progeny of (maize x Tripsacum or reciprocal crosses) have been obtained by artificial methods and have ten maize chromosome, plus an additional either 18 or 36 Tripsacum chromosomes. These hybrids are usually male sterile. Ity has been shown that female fertility can be partially restored using techniques that eliminate most of the Tripsacum chromosomes. Tripsacum x maize (Zea mays L.) crosses can be obtained by employing Tripsacum as the pollen (male) donor which have unreduced gametes with a complete set of Zea chromosomes and a complete set of Tripsacum chromosomes. A maize x Tripsacum reciprocal cross (maize as the pollen donor) was reported that required conventional surgical embryo rescue culture techniques to bring the embryo to maturity. The resulting plants were sterile. When crossed with teosinte, Maize/Tripsacum hybrids have been shown to produce a trigenomic hybrid that has a total of 38 chromosomes. This combination of intergeneric crosses contains 10 chromosomes from maize, 18 from Tripsacum, and 10 from teosinte. This trigenomic hybrid combination produces plants which are all male sterile with a high degree of female infertility.
Others showed that crosses between Diploperennis and Tripsacum resulted in viable, fully fertile plants with chromosome numbers of 2n=20. the success of the crosses between Based on known crossability relationships between Then crosses between Zea and Tripsacum and the results in the reduction in chromosome number in the interspecific crosses. The fertility of plants resulting from the cross made reciprocally with Tripsacum as pollen donor and pollen recipient was unexpected. These results provide an example for the utility of a transgenic bridge intermediate that can be widely useful in the breeding process.
The total chromosome lengths of Tripsacum, and Diploperennis are almost equal although their chromosomal base numbers are different (x=10 in Zea and x=18 in Tripsacum) as previously mentioned. It is not easy to obtain a hybrid plant when crossing Tripsacum and Diploperennis are rare and hybrid plants have not proven easy to obtain experimentally. For example, to recover mature or viable seed, thousands of meticulous pollinations may be required. When recovered, seed from these wide crosses often produce plantlets that die within days of germination as they form weak root and shoot systems. However, occasionally the rare cross is occasionally successfully recovered and has been attributed to the rare occurrence when precise alignments occur between homologous regions of the similar lengths and syntenic chromosomes of Tripsacum and Diploperennis.

These rare fertile Tripsacum/ Diploperennis hybrid crosses provide the opportunity for directly crossing the recombined intergeneric germplasm with maize.
Therefore, Tripsacum/ Diploperennis hybrid crosses demonstrate the utility of a genetic bridge intermediate that can be widely useful in the breeding process. As such the bridge intermediate can be understood as a non-transgenic genomic delivery method for the purpose of recombining useful characteristic across normal breeding barriers. By providing a genetic intermediate bridge for incorporating Tripsacum genes into maize, Tripsacum x Diploperennis hybrids provide an example for the utility of wide crosses for delivery of new genetic materials that may be derived from wide varietal, species, genera and more distant relatives that can then be moved though the use of traditional conventional breeding plant breeding programs.
The Importance of Genomic Assisted Breeding and Wide Crosses for New Hybrid Plant Development The use of intermediate transgenic genetic materials used in wide cross hybrid generation can be used to selecte for new and useful traits. These can then be moved into useful breeding programs by advanced genomics, marker assisted breeding (MAB), Genotype-By-Sequencing (GBS) and genomic assisted breeding (GAB) technologies.

Hybrids can be selected for desirable phenotypes contributed by either parent;
including biotic and biotic stress tolerances, yield, and broad agricultural applications traits, such as;
perennialism, carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, response to high levels of atmospheric CO2 are in process, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus). Genomic and marker assisted breeding is deployed characterize parental genomic contribution and to follow traits in subsequent downstream breeding for varietal development. Hybrids can be sexually crossed, used as intermediates and/or vegtetatively propagated. The transgene(s) can be segrated resulting in non-transgenic hybrids with conferred characteristics.
The importance of using GAB for a successful breeding program in this scheme is indispensible. The use of genetically modified plants for the recovery of non-genetically modified hybrids from wide crosses is possible but only useful if coupled with a significant GAB program to follow the outcomes. Fl hybrids could be backcrossed to the reference cultivars (such as cv Alamo for switchgrass), and if transgenic were used to create bridge intermediates, to segregate away the transgene to generate a non-transgenic F

population. Phenotypic analysis could be conducted on the non-transgenic population in regionally selected field plots and phenotypic data could then be statistically correlated to genetic variation. Variation could be assessed using genome-resequencing technologies and this data, along with phenotypic information used to establish a computational and statistical pipeline to identify, map and introgress variation associated with biomass and other bioenergy traits described in the previous paragraph. To further develop appropriate population breeding blocks, BC1 individuals, selected by their inter-specific genome composition, could be further developed by sib-mating to develop a series "Recombinant Admixture Lines" (RALs) as a public genetic resource.
This fundamental approach will take advantage current transgenic, genetic and genomic work already in place for the development of regionally selected cultivars. Thus, wide crosses have been used as a method in plant breeding for decades and proven to be a useful method for transferring novel genetic materials and traits for new cultivar development. The use of wide crosses may be exploited to generate valuable bridge intermediates for hybrid plant development as exemplified in switchgrass. New traits will be identified and linked to genomic specific sequences. These new hybrids will only be successfully implemented into breeding programs when facilitated by GAB.
Various embodiements have been designed for simple recovery of wide crosses resulting in inter-varietial, intraspecific and intergeneric hybrid plants by combining novel applications of transgenics, selection for herbicide resistance and classical breeding techniques. At first glance this seems contradictory to previous statements concerning the time consuming and costly application of using transgenics, however this technology aims to use transgenic herbicide resistance as a selectable marker in switchgrass and related species for recovery ofhybrids and crossing out the transgene in the subsequent backcrossed generation.
Therefore, in its simplest application, a line of transgenic switchgrass with a dominant herbicide-resistance selectable marker gene can serve as the paternal parent in the proposed intra-specific and inter-specific crosses. Transgenic herbicide resistant switchgrass (cv Alamo) plants have been developed in the Kausch laboratory and were used to pollinate wild-type individuals of alternate switchgrass varieties or Panicum species.
By isolating entire flowering switchgrass plants in controlled crosses within individual pollen cages, as opposed to bagging inflorescences, the chances of recovering hybrid plantlets is increased dramatically. A simple herbicide treatment of seedlings from the maternal wild-type plant verifies the hybrid nature of the offspring. The transgenic traits can then be selected against in the F2 population to recover herbicide sensitive hybrids that are essentially non-transgenic. These hybrids can be verified as non-transgenic using our genomics and sequencing approaches and thus can be rapidly introduced to the commercial market without the costly and time consuming process of deregulation.
The opportunity addressed then by these schemes is to provide the technology that will rapidly accelerate new cultivar development that are non-transgenic resulting in varieties with improved biofuels traits which can be introduced into the market without the process of deregulation. In addition, the use of a linked transgenic male sterility trait with herbicide resistance used in conjunction with advanced tissue culture embryo rescue techniques can also be exploited to force recovery of rare wide crosses. When the herbicide resistance marker is linked to a dominant male sterility trait and used as the maternal parent, this will serve as: 1) an ample filter to facilitate forcing and recovery of rare wide cross progeny; but more importantly, (2) when pollinated by wild type pollen, hybrid plants (Ti or F 1 hybrids) containing bialaphos for herbicide resistance selection, whereby only hybrid plants derived from a fertilized embryo will be recovered. One of the benefits of this novel wide cross recovery method is that it can dramatically increase the numbers of wide crosses that can be recovered and the numbers of clones of each wide cross.
Therefore, the use of wide crosses to generate bridge intermediates for hybrid plant development may be an important method as illustrated here for switchgrass, but may also be useful for Miscanthus, Energy Cane, rye grass, sugar cane, sweet sorghum and bioenergy woody trees, such as Eucalyptus, Salix, Paulownia, and Populus. The much lower expense of using GAB now to follow new traits in new hybrid materials makes this approach achievable.
The Use of Transgenic Plants for Recovery of Non-Transgenic Hybrids from Wide Crosses The recovery of progeny from wide crosses in flowering plants using transgenic plants to recover hybrids for the production of non-genetically modified hybrids via backcrossing or outcrossing and conventional breeding has been recently demonstrated. The fact that wide crosses occur in nature is visible in many extant species of plants and animals.
However, the frequency of fertile progeny from wide crosses in nature is often low but exploitation of such events would be very useful for crop breeding purposes.
The introgression of genes from wide crosses will increase genetic diversity and allow trait introduction that does not include transgenes, which will shorten the breeding and commercialization processes. Various schemes have been developed for the establishment of efficient breeding platforms for agricultural improvement of bioenergy crops, exemplified here in switchgrass, but obviously more broadly applicable.
In one example, one of the monocot parental types are transgenic members of the Poacea, such as switchgrass (Panicum virgatum L. cv Alamo). The second parental type can be intervarietal. TABLE 1 below shows various heterotic groups and/or ecotypes of switchgrass (Panicum virgatum) and a related species, Altantic Coastal PanicGrass, also referred to as ACP (Panicum amarum.). Heterotic and ecotype groups and varietal differences in switchgrass. List of seventeen public switchgrass (Panicum virgatum L) cultivars and their corresponding State of Origin, and Plant Form (i.e. Upland or Lowland).
Corresponding sources shown in superscript. In TABLE 1 the list of seventeen public switchgrass (Panicum virgatum L) cultivars and their corresponding State of Origin, and Plant Form (i.e. Upland or Lowland). Corresponding sources shown in superscript. 1USDA
Soil Conservation Service Agricultural Handbook No. 170. Grass Varieties in the United States. 2 Plant Patent Application.3USDA-NRCS.Release documents from Brooksville, FL
Plant Materials Center. 4 Personal communication to Calvin Ernst. 5 USDA NRCS
Bismarck, ND. Switchgrass Biomass Trials in North Dakota, South Dakota, and Minnesota. 6 USDA-NRCS, Cape May Plant Materials Center "High Tide Switchgrass" release brochure. 7 USDA-NRCS, Rose Lake Plant Materials Center "Southlow Switchgrass" release brochure.
Atlantic Coastal Panicgrass (ACP) is a separate lowland plant form species, Panicum amarum, indigenous along the coastal regions of Eastern United States (NC, SC, MA, CT).

Cultivar State of Origin Plant Form Alamo TXI Lowland Blackwell OKI Upland Bowmaster AR, NC2 Lowland Carthage NC 1 Upland Cave In Rock ILI Upland Dacotah NDI Upland Fore stburg SDI Upland High Tide MD6 Lowland Kanlow OKI Lowland Miami FL3 Lowland Performer AR,OK,NC2 Lowland Shawnee IL4 Upland Shelter WVI Upland Southlow MI7 Upland Stuart FL3 Lowland Sunburst SD5 Upland Timber AR?, NC?4 Lowland TABLE 1 shows a list of the generally applicable public switchgrass varieties which would be useful for hybrid plant production. The second parental type may also be interspecific or intergeneric and maybe also a member of the Poacea, such as but not limited to, Andropogon sp., sp., Pennisetum sp., Sorghum sp. Zea sp., Saccharum sp., Miscanthus sp., a Saccharum sp. x Miscanthus sp. hybrids, Erianthus sp., Tripsicum sp., or Zea x Tripiscum sp. hybrids. Recent introduction of massively parallel DNA
sequencing platforms are capable of generating millions of reads from a given sample of genomic or cDNA. These technologies have dramatically accelerated biological research by enabling inexpensive and robust comprehensive profiling of mRNA for gene discovery and the measurements of absolute abundance mRNAs. Ultra-high throughput genome sequencing of the public switchgrass varieties has been applied to seventeen public varieties. TABLE 1 shows a list of the seventeen public switchgrass (Panicum virgatum L) cultivars and their corresponding State of Origin, and Plant Form (i.e., Upland or Lowland) which have been recently sequenced (Kausch and Dellaporta, unpublished). Switchgrass possesses a large polyploid genome comprised mostly of non-coding repetitive DNA, presenting numerous problems for genomic sequencing, marker development, and trait identification. Methods can be used to reduce complexity, such as sequencing transcribed DNA (cDNA), but suffer from data that is over-represented by highly expressed genes and under-represented for rare mRNAs.

Alternatively, then novel enrichment methods were applied for coding sequences (hypomethylated) that effectively reduce the fraction of sequenced genomic DNA
by at least 10-fold, while maximizing overlap between independent datasets needed for SNP
discovery.
Moreover, because genomic and not cDNA is used in sequencing, the data generated are normalized and enriched for coding regions regardless of expression representation. The opportunity to transfer large genetic components and sequence resources between wide crosses among grass species is provided by the large conserved synteny between grass genomes. Extensive and large chromosomal blocks are present in most grass genomes with different rearrangements which afford ample targets for genetic exchange in hybrids and wide crosses. Gaut shows that the levels of synteny among various grass genomes support evolutionary dynamics of speciation and is between 40% and 73% across distant clades.
Based on syntenic data, is likely that grass genomes evolved from a common ancestoral genome with a minimal size of 33.6 Mb. Bolot proposed that all extant grass species have evolved from this ancestral genome through a combination of whole genome or segmental duplications, diploidization, translocations, gene conversions and gene duplication events. Indeed the ability to generate new hybrids via wide crosses and hybrids is facilitated by the microsynteny present in genomic regions of grass species and can be now used via genomic assisted breeding to follow outcomes as genomic sequencing has become affordable.
In addition complete reference genome data is now (or soon will be) available from whole genome sequencing projects on a number of grasses including rice, maize, sorghum and Brachypodium, and progress is being made on such bioenergy crops as switchgrass, Miscanthus and ryegrass, which will great aid the breeding schemes described in this invention.
Perquisite to the embodiments in this invention, transgenic plants were created using a herbicide resistance selectable marker bar, as one example and used to establish a basic protocol. This procedure is outlined here below.
Making Expression Cassettes for Switchgrass Transformation The methodology for the construction of appropriate vectors consisted of making T-DNA vectors containing ligation-independent (LIC)-based expression cassettes to direct constitutive and tissue-specific transgene expression. These vectors are based on the publicly available pPZP backbone with the introduction of LIC expression cassettes for rapid transgene introduction. The pPZP vectors are subjected to site-directed mutagenesis for LIC

compatibility and engineered with a variety of plant selectable markers including bialaphos and hygromycin plant selectable markers.
These expression cassettes had been already constructed, sequenced, and introduced into swithgrass and pre-tested in transgenic plants that had been recovered.
Promoter regions of transgene cassettes were pre-tested using reporter gene coding regions. The following transgenes with the following tissue specific promoters driving GUG&GFP
reporters were pre-tested for: 1. Constitutive expression based on the maize ubiquitin gene promoter (as a control); 2. Anther-specific expression based on the maize Zm tap gene promoter; and, 3.
microspore specific Zm ms 1 and pollen specific expression based on maize Zm13 gene promoter. Specifically, the goal this is to use this series of vectors to test tissue specific gene expression and provide a basis for creating male and female sterile plants that can be used in breeding experiments in wide crosses. Promoter directed cell ablation employing either cytoxin gene expression (i.e. barnase) or RNAi approaches can be used to create male and female sterile lines. As tissue specificity is established, in parallel, we will use expression of either barnase or RNAi in place of the reporter genes for the generation of the proposed male and female sterile lines. Molecular analysis of all transgenics is conducted.
Each expression cassette includes >1 kb of promoter sequence, including the 5' UTR
region of the gene, and >500 bp of the terminator region, including the 3' UTR
region.
Transgenes are generated directly in T-DNA vectors by LIC-based insertion of full-length cDNA or genomic DNA as translational fusions with the expression cassettes.
The development of the expression set provides the switchgrass breeding efforts and in particular efforts to generate wide crosses with valuable germplasm. Agrobacterium-mediated transformation, using publically available strains such as EHA 101 or EHA 104, is used to test and evaluate these constructs in stable To transformants and their Ti backcrossed progeny.
Generating Transgenic Lines and Testing Transgene Expression A routine and high through-put method for switchgrass transformation was pre-tested (Figure 2) which was used to introduce molecular constructs designed to test expression of transgenes. In particular, Figure 2 shows the switchgrass transformation sequence: (A) Embryogenic callus initiation from mature caryopses; (B) followed by transfection of embryogenic callus with Agrobacterium carrying vectors; (C) selection of transformants for herbicide resistance; and (D) transgenic plant regeneration. Note: the same embryogenic callus induction media used here will be deployed for induction of callus from immature caryopses from the wide crosses.
The switchgrass transformation sequence typically begins with mature seed to generate embryogenic callus, however, the present invention teaches that embryogenic calli can also be produced from immature embryos both excised from the developing immature caryopsis or/and left in situ. Embryogenic callus initiation from mature caryopses is show in Figure 2 at A; followed by transfection of embryogenic callus with Agrobacterium carrying vectors (Figure 2 at B); selection of transformants for herbicide resistance (Figure 2 at C);
and transgenic TO plant regeneration (Figure 2 at D). Note: the same embryogenic callus induction media used here will be deployed for induction of callus from immature caryopses from the wide crosses.
Previously generation of hundreds of transgenic switchgrass plants and evaluation of their Ti and T2 progeny showed efficacy of the transformation protocol.
Three commercially available cultivars of switchgrass (Panicum virgatum L.);
cvs Alamo, Kanlow, Southlow, and Atlantic Coastal Panicgrass (Panicum amarum Ell.
var.
amarulum) are all now routinely transformable. This protocol exploits this expertise to generate the transgenics used in this invention. Approximately 50 independent randomly inserted events per construct were created and regenerated to 3-5 plantlets per clone and grown to maturity in the TO generation. TO plants with reporter genes and sterility constructs were analyzed for their phenotypes in the TO generation, whereas, all fertile trangenics, including reporter constructs, are backcrossed to wild type plants to recover Ti plants and seed for further use in wide cross recovery and analysis. Molecular analysis is by routine PCR and standard Southern blot analysis. In addition a genomics platform has been established to follow transgene introgression in subsequent crosses.
Analysis of the transgenic outcomes and wide crosses include both phenotypic and molecular investigations. First, reporter constructs (GUS and GFP) driven by the same promoters as the ablation constructs (SL) are analyzed in TO and Ti plants using microscopy to verify tissue specificity and the absence of ectopic expression.
Verification of intact inserts was conducted by, Southern blot analysis, RT-PCR and sequencing. The same analyses have been conducted for the other tissue specific constructs listed in Task 1.
Concurrently, analysis of the sterility of male and female lines was also by microscopy but with the addition of the results of controlled crosses conducted in growth chambers. Since switchgrass is an obligate outcrosser, this system cleanly identified sterile lines and was also be used for the recovery of wide crosses through the exploitation of the herbicide selectable marker. This data was used to determine the degree of sterility per event, aware of position effects, to identify adequate expressers for the introduced constructs. IKI
staining for pollen fertility was used on male and pollen specific ablations, but reliance on seed set and linked resistance in controlled crosses is required to evaluate female sterility.
Wide crosses were conducted with these transgenic lines then in both directions as described in the embodiments Test constructs comprising the 1.2-kb rice rts gene regulatory fragment, TAP
(Lee et at. 1996) fused with two different genes as described were evaluated in transgenic switchgrass events. One was the antisense of rice rts gene that is predominantly expressed in tapetum cells during meiosis (pTAP:arts-35S:bar). Another gene was the Bacillus amyloliquefaciens ribonuclease gene, barnase, which ablates tapetal cells by destruction of RNA (pT AP:barnase-Ubi:bar). Both approaches have been previously shown to be effective in various plant species. In transgenic creeping bentgrass (Agrostis stolonifera L.), more than 90% of the plants (20 out of 23) containing barnase and around 50% of the plants (40 out of 79) containing the antisense rts gene were completely male sterile, without viable pollen.
Therefore, transgenic nuclear male sterility, resulting in the lack of viable pollen grains in the bentgrass system provided male sterility.
Analysis of the same male sterility constructs (pTAP:barnase-Ubi:bar and pTAP:arts-35S:bar), was conducted in transgenic switchgrass (Figure 2). These results also underscore the importance of a functional male sterility system for switchgrass.
Primary Alamo transgenics were generated using Agrobacterium tumefaciens strain LBA4404 containing a ¨13 kb transformation vector derived from the intermediate binary vector pSB11 (Genbank accession: AB027256). The T-DNA region contains a selectable marker cassette comprised of the bar gene {Thompson, 1987 #28} under control of a rice ubiquitin (ubince) promoter and the nopaline synthase (nos) terminator sequence. The T-DNA
region was sequenced on an Applied Biosystems 3500x1 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The SEQ ID No.s 5-7 describe these vectors.
Wide cross analysis was done testing constitutive herbicide resistance alone, and linked to constitutive herbicide resistance for recovery of wide crosses Wide cross investigations generated results for recovery of inter-specific hybridization using herbicide resistance between Panicum amarum Ell. var.
amarulum X
Panicum virgatum cv Alamo. A diagrammatic representation of a crossing scheme to generate a non-transgenic population is shown in (Figures 4A and 4B) Genetic transformation of switchgrass cultivars using the a test construct: (Os rice) pOsTap-barnase-OsUbi-bar; plasmid which functionally coveys constitutive herbicide (3%
Finale) resistance according to Deresienski et at. (2010). This test construct was used to generate transgenic lines used in subsequent wide cross studies. Following transformation and selection for herbicide resistance, transgenic TO plants were regenerated and grown to maturity in 6 parts soil/1 part rice hulls in 12" pots. Wild-type individuals from the switchgrass (Panicum virgatum L.) cvs Cave-In- Rock, Shawnee, Sunburst, Alamo, Southlow and Atlantic Coastal Panicgrass (Panicum amarum Ell. var. amarulum) were germinated from seed and grown to maturity under identical soil conditions (Ernst Conservation Seeds, Meadville, PA USA).
Over twenty crosses were established between herbicide-resistant TO cv 'Alamo' transgenic events and the individual wild-type Panicum spp. varieties in pollen cages. Floral development was closely monitored and crosses were initiated using plants best synchronized in floral development; panicles will be chosen when approximately 75% fully emerged and flowers were pre-anthesis. Pollen cage frames have been constructed, wrapped with a double-layer of summer-weight AgribonTM row-cover and used to isolate and cross two plants in Conviron Walk-in Growth Chambers (Model # CG 108). Once crossing was complete and seed fully developed, the seed was carefully harvested from each plant individually.
Interspecific hybridization of Alamo Switchgrass and 'Atlantic' Coastal Panicgrass and generation of an F1BC1 Population The Fi hybrid population was made using a transgenic Alamo parent (T85-2) carrying a single T-DNA insertion as the pollen donor and a wild-type ACP parent as the recipient.
Pollen cage frames were constructed using 1" PVC pipe in the dimensions 45"x 25" x 25".
Fitted 3-way 1" PVC connectors were utilized to fit all PVC lengths together in a rectangular box. Each cage was wrapped with a double-layer of summer-weight AgribonTM row-cover.
In six independent pollen cage experiments, parental types were set up in interspecific combinations using three vegetative clones of T85-2 as a pollen donor with a wild-type ACP
plant. T85-2 plants were removed after pollen shed had ceased, and Fi seed was harvested from the ACP parent. Control crosses were similarly conducted with wild type plants.
Herbicide-resistant Fi plants were backcrossed to wild-type Alamo and screened for herbicide resistance/sensitivity to identify the putative bar-negative, herbicide-sensitive (Hbs) F1BC1 population. Screening was performed via leaf painting assay using 3%
Finale herbicide (Bayer).

These experiments show one of the basis for the present invention. The diagrammatic scheme (Figures 4A and 4B) illustrates the recovery of intergeneric crosses using herbicide selection as a marker. This diagrammatic scheme illustrates, as an example, the recovery of wide interspecific crosses using herbicide selection as a marker, however, this same or similar scheme also applies to wide intra- and inter-varietal, intra- and inter-specific, inter-generic and distant relative crosses. However, in this example, transgenic Panicum virgatum L. cv Alamo Switchgrass (4x) is herbicide resistant containing the bar gene, is resistant to bialphos and 3% Finale or Liberty and is used as the paternal pollen donor.
These plants may be hemizygous TO, or contain at least one copy of the transgene in T1,T2, or .... generations.
The maternal pollen recipient is wild -type Panicum amarum Ell. amarulum Atlantic Coastal Panicgrass (ACP) which in non-transgenic, hence herbicide sensitive to bialaphos and 3% Finale or Liberty). Pollinations may be most conveniently accomplished in pollen cages as described previously using one several clones of an event herbicide sensitive as a pollen donor and a single wild type plant as pollen recipient.
After pollination and seed maturation, seed is harvested only from the wild type maternal parent and germinated. Seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scoring for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant, herbicide sensitive and populations. At floral maturity, the Hbl herbicide resistant Alamo X ACP hybrid plant (s) are used preferably as paternal pollen donor (s) in a backcross to wild -type Panicum virgatum cv Alamo Switchgrass (4x) non-transgenic herbicide sensitive plants and the resultant seed is recovered and germinated. The recovered seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scoring for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant and herbicide sensitive populations. Seedlings are treated with 3%
Finale using one-several leaves in the viable paint assay and scoring for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant and herbicide sensitive populations. The non-transgenic hybrid plants contain Alamo X ACP X Alamo genomic contributions and are subsequently analyzed and scored for desirable traits correlated with genomic markers.
Desirable plants may enter into population block breeding plots, and mass selection and subsequent commercial development can proceed. These plants, can also serve as hybrids to cross with other compatible or incompatible parents.
In self-compatible varieties, species or genera, this same experiment is repeated and embryo rescue is deployed for the recovery of potential hybrids. Plants are monitored on a daily basis and samples taken for embryo rescue techniques. This protocol was repeated using the male sterility transgenics linked to constitutive herbicide resistance for recovery of wide crosses.
Ti plant from seed was grown to about 6 inches in height, sprayed with 3%
Finale solution, observed, scored for herbicide resistance after 7 d. and grown to floral maturity.
Task 4 . Molecular and Phenotypic analysis of TO paternal parents and Ti hybrid offspring;
(Figures 5A ¨ 5E). Molecular analysis shows interspecific nature of the Panicum virgatumL
x Panicum amarum Ell. var. amarulum, hybrids. Urea based extractions of total genomic DNA from approximately 1 g of lyophilized young leaf tissues were performed.
Polymerase chain reaction assays were performed on all herbicide resistant putative TO
plants used in crosses with primers designed for detection of the bar gene.
Specifically, primers that have been designed for amplification of a 202 bp fragment of the bar gene are 5 ' -ACTGGGCTCCACGCTCTAC-3' (forward)/5 '-GAAGTCCAGCTGCCAGAAAC-3 ' (reverse). Southern blot analyses were performed on the individual To transformants used in crosses. Southern blot analyses of TO
paternal parents and Ti putative hybrids were performed using 20i,ig of genomic DNA digested with Ncol, size-fractionated by agarose gel electrophoresis in a 0.8% (w/v) agarose gel and transferred to a HybondTM N+ positively charged nylon membrane through capillary DNA transfer according to the manufacturer's protocol (HybondTm). Positive and negative controls consisted of 20 pg of the approximately 4 kb bar cassette excised with EcoRI
and 2Ong of Ncol digested genomic wild-type cv Alamo DNA as the negative hybridization control. A
202 bp bar probe for hybridization to the NcoI-digested genomic DNA was generated by PCR amplification and purified using a Qiagen Gel Extraction KitTM. The probe was labeled using random priming with digoxigenin-11-dUTP (Roche DIG High Prime Random Labeling and DNA Detection Starter Kit IITm). Pre-hybridization, hybridization, and post-hybridization washes will be done according to the standard protocol of the Roche DIG High Prime Random Labeling and DNA Detection Starter Kit IITM. The digoxigenin-labeled hybridized fragments are detected by enzyme immunoassay with anti-DIG-alkaline phosphatase and an enzyme catalyzed chemiluminescent reaction (CSPD). Digital images of the membranes was captured using a Kodak Image Station 4000MM and viewed with molecular imaging software (Carestream Health, Inc.).

Molecular Analysis Southern blot analyses were performed using 20 i.ig of genomic DNA digested with the restriction endonuclease EcoRV for the published figure or NcoI for Fi single-copy validation. 20 lug of digested wild-type genomic DNA from Alamo switchgrass and Atlantic Coastal Panicgrass (ACP) were also included as negative hybridization controls. The digested DNA was size-fractionated by agarose gel electrophoresis in a 0.8% (w/v) agarose gel and transferred to either a positively charged Roche Nylon MembraneTM (Roche Applied Science, IN, USA, cat no 11417240001) or (NcoI only) a HybondTM N+ positively charged nylon membrane (GE Healthcare, Piscataway, NJ, USA) by high-salt capillary transfer.
The resulting membrane was hybridized to either a (EcoRV) 513 bp or (NcoI) a 213 bp digoxigenin (DIG)-labeled bar probe or a generated using the PCR DIG Probe Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA, cat no 11636090910). Probe-hybridized fragments were detected by enzyme immunoassay and an enzyme catalyzed chemiluminescent reaction (CSPD) according the manufacturer's instructions.
Digital images of the membranes were captured using a Kodak Image Station 4000MM and viewed with molecular imaging software (GE Healthcare, Piscataway, NJ, USA).
A 49 bp indel polymorphism in the tDNA-Leu (trnL) gene of the chloroplast was used as a maternal marker. Insertion state was present in ACP, deletion state was present in Alamo. Product was amplified in Alamo (T85-2) and ACP parentals, Fi, and four using primers 5' -GGTAATGGAACTCCCTCGAAATTA-3' (forward) / 5 '-GGACTCTCTCTTTATCCTCGTTCG-3' (reverse) at final concentration 0.504 and Phusion HF master mix (M0530, New England Biolabs). PCR conditions were 98 C 2 min, 30 cycles of 98 C 30 sec, 64 C 15 sec, 72 C 20 sec, followed by 72 C 2 min. Products were visualized using 2% Agarose Resolute GPG (AB00988, American Bio) gel and Sybr Green dye (5-7563, Life Technologies).
A 202 bp region of the bar transgene was amplified in Alamo (T85-2) and ACP
parentals, Fi, and four F1BC1 using primers 5 '-ACTGGGCTCCACGCTCTA-3' (forward) /5'-GAAGTCCAGCTGCCAGAAAC-3' (reverse) at final concentration 0.5 [iM and Phusion HF master mix (M0530, New England Biolabs). PCR conditions were 98 C 2 min, 30 cycles of 98 C 30 sec, 62 C 15 sec, 72 C 20 sec, followed by 72 C 2 min. Productions were visualized using 2% Agarose Resolute GPG (AB00988, American Bio) gel and Sybr Green dye (S-7563, Life Technologies).

To confirm segregation of the bar transgene in an Fl BC1 hybrid switchgrass population, PCR assays were performed on a population generated after the one primarily discussed in this study. This population was generated from the same Alamo transgenic event (T85-2), but contains different F1BC1 individuals than the population originally sequenced. A
513 bp fragment was amplified in transgenic Alamo (T85-2), ACP wild type, Fi, four Hbs and four HbR F1BC1 offspring. For the positive control, 100 pg of transformation construct was used. Primers were 5 '-GGATCTACCATGAGCCCAGA-3' (forward)/5'-GAAGTCCAGCTGCCAGAAAC-3' reverse. Samples were prepared via the Harris Unicore System (ThermoScientific) in conjunction with the KAPA 3G Plant PCR kit (KK7251, KAPA Biosystems) following manufacturer's instructions for crude sample 501AL
prep, and PCR conditions were 95 C 10 min, 30 cycles of 95 C 30 sec, 58 15 sec, 72 C 30 sec, followed by 72 C 1 min. Products were visualized on 1.2% agarose gel.
Figure 3 shows PCR and herbicide resistance correlate with the introduced bar gene construct. The top panel shows results in the viable 'paint assay' shown left to right wild-type Panicum virgatum L. cv Alamo leaf response to treatment with 3% finale followed by the responses of the corresponding primary Panicum 701 virgatum L. cv Alamo TO

transformants; respective Wild-type, H20 and TO event numbers, 16-3, 24-9, 26-8, 30-2, 34-1, and 38-4 correspond to the PCR results shown in the bottom panel. The bottom panel TO
transformants after herbicide application and their representative bar and barnase fragment amplifications. PCR amplifications of 202 bp bar fragment and 303 bp barnase fragment from Switchgrass cv Alamo primary TO transformants. Shown left to right PCR
marker; bar +: bar positive control, plasmid DNA; barnase +: barnase positive control, plasmid DNA;
wild-type -: negative control, wild-type Alamo DNA; H20: water control.
Transformation event numbers, 16-3, 24-9, 26-8, 30-2, 34-1, and 38-4 are listed above each lane corresponding to the leaf samples in the top panel. This shows that co-transformation of a male sterility gene co-integrates with the selectable marker gene.
Wide Cross Genotyping This part of the invention development was conducted as a collaboration with the Yale Genome Center in New Haven, CT using GBS to quantify our reults. One of the goals was to develop a robust genotyping platform for trait identification, identification of wide crosses with transgenic materials, marker-assisted breeding, and transgene introgression into regionally selected germplasm (Figure 7). Applied expression profiling and deep sequencing technologies were applied to characterize selected switchgrass germplasm.
Recent introduction of massively parallel DNA sequencing platforms are capable of generating millions of reads from a given sample of genomic or cDNA. These technologies have dramatically accelerated biological research by enabling inexpensive and robust comprehensive profiling of mRNA for gene discovery and the measurements of absolute abundance mRNAs. Ultra-high throughput sequencing of the switchgrass genome has been applied to seventeen public varieties. TABLE 1 shows a list of the seventeen public switchgrass (Panicum virgatum L) cultivars and their corresponding State of Origin, and Plant Form (i.e. Upland or Lowland). Switchgrass possesses a large polyploid genome comprised mostly of non-coding repetitive DNA, presenting numerous problems for genomic sequencing, marker development, and trait identification.
Methods can be used to reduce complexity, such as sequencing transcribed DNA
(cDNA), but suffer from data that is over-represented by highly expressed genes and under-represented for rare mRNAs. Alternatively, then novel enrichment methods were applied for coding sequences (hypomethylated) that effectively reduce the fraction of sequenced genomic DNA by at least 10-fold, while maximizing overlap between independent datasets needed for SNP discovery. Moreover, because genomic and not cDNA is used in sequencing, the data generated are normalized and enriched for coding regions regardless of expression representation. An extensive collection of public and elite regionally selected varieties of switchgrass ecotypes was grown and evaluated in isolation plots. Ernst Conservation Seeds is one of the world's largest producers of switchgrass seeds and developers of new cultivars and kindly provided elite germplasm, plant growth support, and breeding expertise.
Since switchgrass is largely cross-pollinated and self-incompatible, each isolation plot serves to regionally evaluate germplasm, while generating a deep sequencing sib-mated F2 mapping population and foundation seed source for subsequent germplasm mapping and cultivar development operations. All seventeen switchgrass varieties have genomic analyses for hypomethylated regions.
A database of protein-coding sequences as well as a thorough characterization of genetic variation found in regionally-selected cultivars was compiled. By using genomic DNA, the limitations of cDNA sequencing, such as over- and under-representation of transcript abundance, was avoided. Moreover, sequences included regulatory regions not found in the cDNA datasets. Sequencing was complemented by effective computational strategies for gene discovery, annotation and visualization. Variation within and between cultivars was uncovered using massively parallel sequencing methods (Solexa) of short reads that are mapped to the reference cv Alamo genome which was extensively sequenced and annotated. Visualization software was developed for comparative analysis as well as to locate variation for marker development. The intent for Task 6 then, was to use this data to develop a genotyping platform that can be applied to future characterization of wide cross analysis as well genome and trait association studies, trait mapping, wide crosses and marker-assisted breeding.
This genomics then is deeply integrated with the present invention of wide cross recovery and downstream breeding applications to hybrid plant development. The inventive approach of this component consists of generating 100 Mb of paired-end genomic sequences from each switchgrass cultivar using the latest generation of ultra high-throughput DNA
sequencing instrumentation and applying this against recovered hybrids. Custom algorithms are used to template the sequence datasets against genome scaffolds derived from comparative analysis of related grass reference genomes, such as maize, rice, and sorghum.
These custom scripts permit accurate base calling for identifying true allelic variation using low-read coverage, by minimizing nucleotide over-calls and under-calls that manifest as insertion and deletion errors. The comparative analysis of these sequenced switchgrass genomes identifies blocks of genomic synteny shared among grasses and easily compared to the maize genome.
The assembled data is then mapped to these syntenic blocks to assign genetic and physical map positions to the switchgrass variation. This analysis generates a set of "virtual genomic contigs", or VGCs, to serve as the genomic template for switchgrass SNP discovery and validation as well as provides a basis for identification of novel genetic materials transmitted through wide crosses. The identification and validation of SNPs is performed by deep genome re-sequencing technologies based on bead technologies. This method generates 35 nucleotide paired-end reads using Solexa Genetic Analyzers. Using this re-sequencing strategy, approximately 2 Gb of genomic sequence is generated for each switchgrass cultivar and compared with generated wide cross hybrids. A second set of custom algorithms is used for realignment of Solexa data against the VGCs for SNP discovery and validation purposes.
The SNP database was used to develop a high-throughput genotyping platform based on bead genotyping technology.
The data generated using this invention shows (1) the unique introgression of variation in the F 1 BC1 hybrids (2) the absence of all transgene sequences, Ti plasmid backbone and any Agrobacterium sequences, and (3) that all F 1BC1 progeny using this approach are all non-transgenic Task 6. Development of two genotyping platforms. Using this information, the present invention utilizes two genotyping platforms: 1) a 384-plex genotyping assay for our marker-assisted breeding and transgene introgression;
and, 2) a 1536-plex genotyping assay for trait identification and genome association studies. This data is essential to develop and characterize new germplasm, new vector construction and analysis of genetic introgression in recovered wide crosses. This robust sequencing/genotyping platform provides a broader genomic function for trait identification, association genetics, marker-assisted breeding, and introgression of genetic material though wide crosses into regionally selected germplasm.
Hybrids can be selected and identified for desirable phenotypes contributed by either parent; including bioenergy traits, such as carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus).
These examples illustrate a similar strategy which applies to further applications of this invention on a wide variety of plants and crops.
GBS library Preparation and sequencing Genomic DNA was isolated from leaf tissue using published methods {Chen, 1994 #31}. Approximately l[tg of gDNA from each sample was digested with RsaI (RO
167, New England Biolabs) according to conditions recommended by manufacturer and GBS
samples were processed for sequencing according to the method described in Heffelfinger et al {Heffelfinger, 2014 #57}. ACP and Alamo parentals, F 1, and eighty-three F1BC1 samples were paired-end sequenced on an Illumina HiSeq 2000 system by the Yale Center for Genome Analysis.

Bioinformatics Eighty-six samples consisting of male and female parents, an Fi Bar positive offspring, and 83 F1BC1 offspring were sequenced on the Illumina HiSeq 2000 (IIlumina, San Diego CA USA) to a mean of 1,965,020 958,961 2x75 bp reads per sample. Reads were aligned against the draft Panicum virgatum v. 1.1 reference genome (DOE-JGI, http://www.phytozome.net/panicumvirgatum) {Goodstein, 2012 #39} using Bowtie2, and variants were called using the mpileup function of Samtools.
Variants retained for further analysis from the original dataset were filtered based on several criteria. Filtering criteria included variants that were 1) homozygous within and polymorphic between Alamo and ACP parents 2) heterozygous in the Fi offspring 3) typed by at least two reads within parental plants and the Fi offspring as well as in at least twenty F1BC1 offspring 4) aligned with a mapping quality? 20 4) PHRED score > 30 .
These filtering steps were performed via custom Perl scripts.
Genotypes were imputed using the least-squares algorithm described in Heffelfinger et al 2014. Briefly, variants were placed in 5 Mbp bins across the genome and a "mean-genotype" was calculated. Proximal bins with matching genotypes were merged.
Bins with differing calls were reanalyzed using a sliding, 1 Mbp window in forward and reverse direction to identify breakpoints. While only homozygous Alamo and heterozygous calls were possible given the cross, homozygous ACP calls were maintained in the dataset to indicate error rate and error-prone regions of the genome. Circos {Krzywinski, 2009 #34}
was used to display raw and imputed genome-wide marker datasets for parents, Fi, and offspring.
SPECIFIC EXAMPLES
In a first example, transgenic herbicide resistant Panicum virgatum L. cv Alamo may used in an inter-specific cross with non-transgenic Atlantic Coastal Panicgrass (Panicum amarum, Ell. var. amarulum). This embodiment is schematically represented in Figures 4A
and 4B and detailed in the accomplishment as previously described. Note that directionality (maternal X paternal) does not matter to the practice of hybrid plant recovery. The F 1 progeny from the wide crosses may be fertile, producing viable seeds which germinate to produce healthy fertile plants that can be used in backcrosses to wild type non-transgenic Panicum virgatum L. cv Alamo. The subsequent F2 population is then germinated from the resultant seed. The F2 seedling are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications.
Hybrids can be selected for desirable phenotypes contributed by either parent;

including bioenergy traits, such as carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus).
Genomic and marker assisted breeding is deployed characterize parental genomic contribution and to follow traits in subsequent downstream breeding for varietal development. Hybrids can be sexually crossed and/or vegtetatively propagated.
Genomic and marker assisted breeding is deployed characterize parental genomic contribution and to follow traits in subsequent downstream breeding for varietal development.
Hybrids can be sexually crossed and/or vegtetatively propagated.
In the first example experiments have generated inter-specific hybrids between our reference cv Alamo line and Atlantic Coastal Panicgrass (Panicum amarum, Ell.
var.
amarulum.) using a novel herbicide selection method (FIGURE 4 A & B). The male parent, a transgenic Alamo line (Finale resistance) was used as a pollen donor with the female Atlantic Coastal Panicgrass plants.
In particular, Figure 4A shows, as an example, the recovery of wide inter-specific crosses using herbicide selection as a marker, however, this same or similar scheme also applies to wide intra- and inter-varietal, intra- and inter-specific, inter-generic and distant relative crosses. In this example, transgenic and hence, genetically modified (GMO) Panicum virgatum L. cv Alamo Switchgrass (4x), (at upper left, shown in dark red) is herbicide resistant (Hbl, bar+, containing the bar gene, resistant to bialphos and 3% Finale or Liberty) and is used as the paternal pollen donor in a wide cross. These plants may be hemizygous TO, or contain at least one copy of the transgene in Ti ,T2, or .... generations.
The maternal pollen recipient is wild -type Panicum amarum Ell. amarulum Atlantic Coastal Panicgrass (ACP) (4x, at upper right) which in non-transgenic and hence (shown in dark green) herbicide sensitive to bialaphos and 3% Finale or Liberty.
Pollinations may be most conveniently accomplished in pollen cages using one several clones of an event herbicide sensitive as a pollen donor and a single wild type plant as pollen recipient. After pollination and seed maturation, seed is harvested only from the wild type maternal parent and germinated (center, right). Seedlings are treated with 3%
Finale using one-several leaves in the viable paint assay and scored for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +, red) and herbicide sensitive (bar-, green) populations (lower right). At floral maturity the (bar+) Hb 1 Herbicide Resistant Alamo X ACP hybrid plant (s) are used preferably as paternal pollen donor (s) in a backcross to wild -type Panicum virgatum cv Alamo Switchgrass (4x) non-transgenic herbicide sensitive plants (lower center) and the resultant seed is recovered and germinated.
The resultant seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scoring for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +, red) and herbicide sensitive (bar-, green) and populations (lower left). Seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scoring for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +, red) and herbicide sensitive (bar-, green) and populations (lower right). The non-transgenic hybrid plants contain Alamo X ACP X Alamo (blue, lower left) genomic contributions and are subsequently analyzed and scored for desirable traits correlated with genomic markers. These plants, can also serve as hybrids to cross with other compatible or incompatible parents. Desirable plants may enter into population block breeding plots, and mass selection and subsequent commercial development can proceed.
Figure 4B shows the development of non-transgenic hybrids using an transgenic herbicide resistant as a hybrid to recover a F 1BC1 population that can be used in breeding.
The difference between this Figure 4B and the proceeding diagram (Figure 4A) is that here intervarietial (close relative) parents are used.
Herbicide resistance (3% Finale) was observed in 62 % of the seedlings recovered from the YPanicum amarum Ell. var. amarulum X 5\Panicum virgatum cv Alamo cross yielding 644 putative inter-specific hybrid plants. All Finale selected TO
plantlets retained the herbicide resistant phenotype to floral maturity. Molecular analysis of TO
plants indicated the presence of the Atlantic Coastal Panicgrass maternal chloroplast marker and the paternal BAR transgene . Atlantic Coastal Panicgrass shares this marker with all upland cultivars but not with cv Alamo, hence confirming the hybrid origin of these plants. PCR
amplifications of both nuclear and cytoplasmic markers have therefore verified the inter-specific hybrid nature of these plants.
To plants were backcrossed to WT reference Alamo and F 1BC1 seed was recovered, germinated and screened for herbicide resistance using the "leaf painting assay" (3% Finale).
A total of 83 herbicide sensitive plants were recovered.
In one experiment, wide cross results YPanicum amarum Ell. var. amarulum X
5Panicum virgatum cv Alamo with herbicide resistance recovered over 50 putative hybrid Fl (i.e Ti) seeds. Paternal cv 'Alamo' TO parents were evaluated by Southern blot analyses of NcoI-digested DNA using bar probe and showed (18/31, 58.1%) single gene copy events, (4/31, 12.9%) with two gene copies and (9/31, 29.0%) multiple gene copy events (Figures 5A
¨5H).
Clones of individual events consistently showed identical Southern blot results indicating single cell origins for all examined events. Seed from transgenic individuals and wild-type individuals in each cross were recovered and treated completely separately to avoid contamination between maternal parents. At about 6 inches in height, the plantlets were sprayed with 3% Finale and a definitive difference in response between Ti individuals from each cross and segregation ratios were calculated. Total plantlet counts prior to herbicide application and counts of herbicide resistant plantlets recovered were used to calculate percentages of herbicide resistant plantlets from each cross (Table 1). The percentage of herbicide resistant seedlings in control crosses ranged from 1.1% to 64%. Of the seed recovered from of the YPanicum virgatum cv `Southlow' X 5Panicum virgatum cv 'Alamo' cross 32.3% were herbicide resistant yielding 70 putative intraspecific hybrid plants.
Herbicide resistance was observed in 38.2% of the seed recovered from the YPanicum amarum Ell. var. amarulum X 5Panicum virgatum cv 'Alamo' cross yielding 644 putative interspecific hybrid plants. All 3% Finale selected plantlets retained the herbicide resistant phenotype to floral maturity.
Molecular evidence for wide hybrids is shown in FIGURE 5. Molecular analysis of Ti Individuals from the pool of Finale resistant Ti individuals recovered from maternal wild-type by paternal transgenic crosses, 10 were randomly selected from each cross for molecular analysis. PCR amplification was used to verify inheritance of the transgene and revealed the presence of the bar gene in all Ti individuals analyzed. Control crosses between To transgenic (5) and wild type cv 'Alamo' (y) were evaluated by Southern blot of individual To events and 10 randomly selected herbicide resistant Ti plantlets. Southern blots show the identical number and size of hybridized fragments between Ti individuals and their TO
transgenic cv 'Alamo' parents (FIGURE 5). All Ti herbicide resistant plantlets showed stable inheritance of the transgene without size rearrangements or duplications and correlated with the herbicide resistant phenotype. Results from the wild-type Panicum virgatum cv 'Alamo' (y) X transgenic Panicum virgatum cv 'Alamo' 6' individuals represent control crosses, show that transgenes can be transmitted sexually to offspring, conditions were suitable for crossing to occur solely between the desired plants and stable Ti generation transformants could be generated.
Therefore, these same conditions were used in interspecific and intraspecific crosses.
Intraspecific hybrids from crosses between wild-type cv `Southlow' (y) and TO
cv 'Alamo' (d) resulted in 70 intraspecific hybrid plants, which all showed herbicide resistance. A
sample Southern blot analysis (FIGURE 5) of NcoI-digested DNA from paternal parent TO
cv 'Alamo' transformant # 34-2 alongside 10 randomly selected herbicide resistant Ti plantlets from the intraspecific cross of wild type cv `Southlow' switchgrass (y) x cv 'Alamo' transformant #85-2 (d) using the bar probe all show stable inheritance of the transgene without duplications. Similarly, interspecific hybrids from crosses between To cv 'Alamo' (5) and wild-type 'Atlantic' coastal panicgrass (y) were also conducted using identical conditions as the control crosses. The 644 progeny seed recovered from wild-type ACP 'Atlantic' Coastal Panicgrass' (y) all showed stable herbicide resistance to floral maturity.
Sample Southern blot analysis (Figure 5A) of NcoI-digested DNA from paternal parent TO cv 'Alamo' transformant #85-2 alongside 10 randomly selected herbicide resistant Ti plantlets from the wide hybrid cross wild-type 'Atlantic' coastal panicgrass (y) x cv 'Alamo' transformant #85-2 (5) using bar probe all show stable inheritance of the transgene.
Figures 5A ¨ 5H shows molecular characteristics of the wild-type panicum virgatum cv. 'Alamo', and wild-type Panicum amarum Ell. var. amarulum, and Fl (i.e. Ti) Panicum virgatum cv. 'Alamo' X Panicum amarum Ell. var. amarulum hybrid. Molecular analysis shows interspecific nature of the Panicum virgatumL x Panicum amarum Ell. var.
amarulum, hybrids. Figure 5A. shows TO transgenic events of P. virgatum cv Almo. A
sample Southern blot analysis of NcoI-digested DNA from cv Alamo TO paternal parents using bar probe shows 7 independent single gene insertion events. Clones of events show identical results.
FIGURE 5B shows intra-specific hybrids from crosses between wild-type cv `Southlow' (y) and TO cv 'Alamo' (d). A sample Southern blot analysis of NcoI-digested DNA
from paternal parent To cv Alamo transformant # 34-2 alongside 10 randomly selected herbicide resistant Ti plantlets from the intraspecific cross of wild type cv 'Southlow' switchgrass (y) X cv 'Alamo' transformant #85-2(5) using the bar probe. All sampled Ti progeny show stable inheritance of the transgene.
Figure 5C shows interspecific hybrids from crosses between TO cv Alamo (5) and wild-type 'Atlantic' coastal panicgrass (y). Southern blot analysis of NcoI-digested DNA
from paternal parent TO cv 'Alamo' transformant #85-2 alongside 10 randomly selected herbicide resistant Ti plantlets from the wide hybrid cross wild-type 'Atlantic' coastal panicgrass (y) x cv Alamo transformant #85-2 (d) using bar probe. All sampled Ti progeny show stable inheritance of the transgene.
Figure 5D shows PCR amplifications Wild type (nontransgenic control) X
herbicide resistant transgenic and interspecifc cross with Atlantic Coastal Panicgrass.
PCR Results for the nuclear bar marker in (controls)wild-type Panicum virgatum cv. Alamo, wild-type Panicum amarum Ell. var. amarulum, and five randomly selected herbicide-resistant Ti plantlets from the cross wild-type Panicum virgatum cv. Alamo X PVA-34_3 (AXT001-AXT005) and five randomly selected herbicide-resistant Ti plantlets from the cross wild-type Panicum amarum Ell. var. amarulum X PVA. (CXT006, CXT015, etc.).
Figure 5E shows chloroplast deletion is in Alamo but not Atlantic Coastal Panicgrass and can be used as a diagnostic for hybrids. The tRNA-Leu (trnL) gene of the chloroplast contains an intron with a 49 bp deletion present in cv Alamo ( that is not present in 'Atlantic' Coastal Panicgrass. MUSCLE results of the amplified fragment containing this deletion.
Figure 5F shows chloroplast deletion detected and verifies interspecific hybrid. PCR
amplifications of chloroplast DNA tRNA-Leu (trnL) 49 bp deletion site in wild-type Panicum virgatum cv. Alamo, wild-type Panicum amarum Ell. var. amarulum, PVA-34-3, PVA
-85-2, five randomly selected herbicide-resistant Ti plantlets from the cross wild-type Panicum virgatum cv. Alamo X PVA- _34_3 (AXT001-AXT005) and five randomly selected herbicide-resistant Ti plantlets from the cross wild-type Panicum amarum Ell.
var.
amarulum X PVA. (CXT006, CXT015, etc.).
Figures 5G and 5H show the single gene copy insert of the transgene in an Alamo trangenic used in these studies, the abscence of the transgene in both wild type (non-transgenic) Alamo and the ACP parent, the inheritance of the transgene in the Fl hybrid and the segration of the trangene in the herbicide sensitive and herbicide resitant Fl BC1 population. Lane descriptions: 1. MWM Roche DIG-labeled molecular weight marker III

(21.2 kb, 5.1/5.0 kb, 4.3 kb, 3.5 kb, 2.0/1.9 kb, 1.6 kb, 1.4 kb, 0.95 kb, 0.83 kb, 0.56 kb.
respectively). 2. T85-2, transgenic Alamo parent 3. Wild type Alamo 4. Wild type ACP 5. Fl hybrid - T85-2 x ACP WT parent. F1BC1 herbicide-sensitive HS, PCR negative plants (lanes 6-9) 6. F1BC1HS 005 7. F1BC1HS 009 8. F1BC1HS 024 9. F1BC1HR 033 F1BC1 herbicide-resistant HR, PCR positive plants 10. F1BC1HR 0011. 11. F 1BC1HR
026. 12.
F1BC1HR 031. 13. F1BC1HR 035Figure 5H shows the PCR resutls for the presence of the trangene in the Alamo trangenic and its absence in the herbicide sensitve FlBC1 population.
A 513 bp fragment internal to the bar CDS was amplified using the following primers (1) bar JHF - GGATCTACCATGAGCCCAGA and (2) bar JHR -GAAGTCCAGCTGCCAGAAAC. The PCR was carried out using the Harris Uni-Core (thermoscientificbio.com/per-enzymes-master-mixes-and-reagents/harris-uni-core-and-cutting-mat/) in conjunction with the KAPA 3G Plant PCR kit (kapabiosystems.com/product-applications/products/per-2/kapa3g-plant-per-kits/) according to the manufacturer's instructions for crude sample PCR in a 50 uL reaction. For the PCR positive control, 1 uL of the transformation construct at 100 pg was used. Thermocycling conditions were an initial denaturation cycle at 95 C for 10 min, followed by 35 cycles of 30 s at 95 C, 15 s at 58 C, and 30 s at 72 C, followed by a final extension of 1 min at 72 C. 10 uL of reaction products were loaded on a 1.2% agarose gel.
Lane descriptions: 1. MWM NEBL PCR ladder - 0.3 ug (on left and right sides of the gel) 2. Negative control - master mix that the Harris Uni-Core has been dipped into after sampling of the experimental samples 3. PCR positive control 4. The parental transgenic Alamo plant - T85-2 5. ACP wild type plant. 6. the Flhybrid plant (T85-2 x WT
ACP).
Lanes 7-19: F1BC1 herbicide-sensitive HS, PCR negative plants 7. F 1BC1HS 005 8.
F1BC1HS 009 9. F1BC1HS 024. Lanes 10-13 F1BC1 herbicide resistant plants.

herbicide-resistant HR, PCR positive plants. 10. F1BC1HR 033 11. F1BC1HR 0011.
12.
F1BC1HR 026. 13. F1BC1HR 031. 14. F1BC1HR 035. Al plants testing positive in the PCR
screen were herbicide resistant in a "paint assay" using 3% Finale herbicide (tomirwin.com/pdf/labels/Finale.pdf) whereas all those testing negative were herbicide sensitive.
The hybrid nature of the Panicum virgatum L cv Alamo x Panicum amarum Ell.
amularum cross is demonstrated by the data shown in Figures 5A - 5E. In Figure 5A, PCR
amplifications show chloroplast DNA tRNA-Leu (trnL) 49 bp deletion polymorphism in wild-type Panicum virgatum cv. 'Alamo', wild-type Panicum amarum Ell. var.
amarulum, five Ti Panicum virgatum cv. 'Alamo' X PVAhr and five Panicum amarum Ell. var.

amarulum X PVAhr. In Figure 5 B, PCR show amplifications for the nuclear bar marker in (controls) wild-type Panicum virgatum cv. 'Alamo', wild-type Panicum amarum Ell. var.
amarulum, and five Ti Panicum virgatum cv. 'Alamo' X PVAhr and five Panicum amarum Ell. var. amarulum X PVAhr. The results show that the specific mutually pollinated crosses performed in isolation using pollen cages in conjunction with a transgenic herbicide selectable marker is an efficient method that can be used for production of both intraspecific and interspecific hybrid plants and simple recovery through screening by herbicide resistance.
The ability to create new hybrid plants from different varieties of switchgrass and through wide-crossing allows the development of new hybrids with increased variation in a shorten number of breeding cycles (see Figure 1).
Figure 5F shows Southern Blot evidence for the single gene copy insert of the transgene in an Alamo trangenic used in these studies, the abscence of the transgene in both wild type (non-transgenic) Alamo and the ACP parent, the inheritance of the transgene in the Fl hybrid and the segration of the trangene in the herbicide sensitive and herbicide resitant F 1 BC1 population.
Phenotypic characterization of the parental transgenic Panicum virgatum cv.
'Alamo', and parental wild-type Panicum amarum Ell. var. amarulum, and Fl (i.e. Ti) Panicum virgatum cv. 'Alamo' X Panicum amarum Ell. var. amarulum hybrid clearly shows the individual characteristics of these two species and a blend of many characteristics in the F 1 hybrid. Figures 6 A ¨ 6D show phenotypic Characteristics of the wild-type Panicum virgatum cv. 'Alamo', and wild-type Panicum amarum Ell. var. amarulum, and Fl (i.e. Ti) Panicum virgatum cv. 'Alamo' X Panicum amarum Ell. var. amarulum hybrid.
Characteristics of adaxial side of the mature leaf were sampled from the second leaf lower than a mature inflorescence and photographed 2 cm from the axial all with consistent lighting parameters and exposures. Characteristics of inflorescence sampled from the main axis 3 cm from the terminal apex photographed with consistent lighting parameters and exposures.
Left to Right:
the wild-type Panicum virgatum cv. 'Alamo', and wild-type Panicum amarum Ell.
var.
amarulum, and Ti Panicum virgatum cv. 'Alamo' X Panicum amarum Ell. var.
amarulum hybrid.
In particular, Figure 6A shows (top and bottom)n characteristics of adaxial and abaxial sides of the mature leaf Left to Right: the wild-type Panicum virgatum cv. 'Alamo', and wild-type Panicum amarum Ell. var. amarulum, and Fl Panicum virgatum cv.
'Alamo' X

Panicum amarum Ell. var. amarulum hybrid.
Figure 6B shows (top and bottom) characteristics of inflorescence sampled from the main axis 3 cm from the terminal apex and spikelets (respectively) Left to Right: the wild-type Panicum virgatum cv.
'Alamo', and wild-type Panicum amarum Ell. var. amarulum, and Fl Panicum virgatum cv. 'Alamo' X
Panicum amarum Ell. var. amarulum hybrid.
Figure 6C shows SEM images of leaf surface of axis 3 cm from the terminal apex and spikelets (respectively) Left to Right: cv. 'Alamo', and wild-type ACP, and Fl hybrid.
Epicuticular wax patterns are distinctive. FIGURE 6D shows (top) bundle sheath scanning electron micrographs of cryofractured leaves (SEMs). Left to Right: cv.
'Alamo', and wild-type ACP, and Fl hybrid, and shows (bottom) chlorophyll content results; Left to Right: cv.
'Alamo', and wild-type ACP, and Fl hybrid.Characteristics of the adaxial side of the mature leaf were sampled from the second leaf lower than a mature inflorescence and photographed 2 cm from the axial all with consistent lighting parameters and exposures. The leaves show clear differences in interveinal spacing, color and stomatal density (FIGURE 6 A).
Characteristics of inflorescence sampled from the main axis 3 cm from the terminal apex photographed with consistent lighting parameters and exposures. The floral characteristics of Panicum virgatum cv. 'Alamo' indicate a loose panicle in comparison to the more compact and dense panicle of the parental wild-type Panicum amarum Ell. var. amarulum, whereas the Fl (i.e. Ti) Panicum virgatum cv. 'Alamo' X Panicum amarum Ell. var.
amarulum hybrid is intermediate (FIGURE 6 B TOP).
Similarly spiklet characteristics of Panicum virgatum cv. 'Alamo' show a red stigma in comparison to the lighter (almost white) stigma of of the parental wild-type Panicum amarum Ell. var. amarulum, whereas the Fl (i.e. Ti) Panicum virgatum cv.
'Alamo' X
Panicum amarum Ell. var. amarulum hybrid is again an intermediate (FIGURE 6 B
Bottom).
Scanning electron microscopy (SEM) images were taken of samples of leaf surface of axis 3 cm from the terminal apex. Epicuticular wax patterns are distinctive for the Panicum virgatum cv. 'Alamo' and parental wild-type Panicum amarum Ell. var. amarulum, whereas the Fl (i.e. Ti) Panicum virgatum cv. 'Alamo' X Panicum amarum Ell. var.
amarulum hybrid is again appears as intermediate (Figure 6 C). In Figure 6D at the top.
Scanning electron micrographs (SEMs) of cryofractured leaves show moderate differences in bundle sheath betweenthe parents and the hybrids, but chlorophyll content results clearly the Panicum virgatum cv. 'Alamo' and parental wild-type Panicum amarum Ell. var.
amarulum, whereas the F 1 (i.e. Ti) Panicum virgatum cv. 'Alamo' X Panicum amarum Ell.
var.
amarulum hybrid is more similar to the Alamo parent(Figure 6D at the bottom).

As predicted in the schematic shown in Figures 4A and 4B, the herbicide sensitive plants no longer contained the transgene by PCR and Southern blot analyses.
These plants also contained the Alamo maternal and nuclear markers as expected. Hybrid caryopses have also been recovered from additional wide crosses between intra-specific varieties with various ploidy levels. The results of this study indicate that the specific mutually pollinated crosses performed in isolation using pollen cages combined with embryo rescue is an effective method for production of both intra-specific and inter-specific hybrid plants and simple recovery through screening by herbicide resistance. The ability to create new hybrid plants from different varieties of switchgrass and related species through wide-crossing and verify by Genotyping-By-Sequencing (GBS) (Figure 7) allows the recovery of new hybrids which can enter a breeding platform such as shown in Figure 1.
In particular, Figure 7 shows a diagrammatic scheme illustrating the development and characterization of a non-trangenic F 1 BC1 populations derived from a transgenic hybrid.
This diagrammatic scheme illustrates, as an example, the use of Genotyping-by-Sequencing (GBS) to characterize the hybrid nature of the Fl and F1BC1 population in comparison to the parentals and the reference A13 Panicum viragtum A13 genome from the Joint Genome Institute (JGI). Also, this diagrammatic scheme illustrates the recovery of wide interspecific crosses using herbicide selection as a marker as shown previously in Figures 4A and 4B, however, this same or similar scheme also applies to wide intra- and inter-varietal, intra- and inter-specific, inter-generic and distant relative crosses. In this example, transgenic Panicum virgatum L. cv Alamo Switchgrass (4x), (at upper left,) is herbicide resistant (bar+, containing the bar gene, resistant to bialphos and 3% Finale or Liberty) and is used as the paternal pollen donor in a wide cross. These plants may be hemizygous TO, or contain at least one copy of the transgene in T1,T2, or ... generations.
The maternal pollen recipient is wild -type Panicum amarum Ell. amarulum Atlantic Coastal Panicgrass (ACP) (4x, at upper right) which in non-transgenic and hence herbicide sensitive to bialaphos and 3% Finale or Liberty. Pollinations may be most conveniently accomplished in pollen cages using one several clones of an event herbicide sensitive as a pollen donor and a single wild type plant as pollen recipient. After pollination and seed maturation, seed is harvested only from the wild type maternal parent and germinated (center, right). Seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and identified as the F 1 hybrids At floral maturity the (bar+) Hbl Herbicide Resistant Alamo X ACP F 1 hybrid plant (s) are used preferably as paternal pollen donor (s) in a backcross to wild -type Panicum virgatum cv Alamo Switchgrass (4x). The non-transgenic herbicide sensitive plants (upper center) and the resultant seed is recovered and germinated.
The non-transgenic hybrid plants contain the F 1 BC1 Alamo X ACP X Alamo genomic contributions and are subsequently analyzed and scored for desirable traits correlated with genomic markers. These plants, can also serve as hybrids to cross with other compatible or incompatible parents. Desirable plants may enter into population block breeding plots, and mass selection and subsequent commercial development can proceed.
The F 1 BC1 population was analyzed using the genotyping technology. Also the phenotypes of 83 F1BC1 were observed as. Parental herbicide resistant Panicum virgatum L.
cv Alamo Switchgrass (4x) was used as the pollen donor with wild-type Panicum amarum Ell. amarulum Atlantic Coastal Panicgrass (ACP) and the Fl progeny were backcrossed to the wild type Panicum virgatum L. cv Alamo Switchgrass (4x) and planted in a field plot and individuals were numbered and sampled for DNA isolation and genotyping.
Examples show significant independent phenotypic variation including leaf angle, plant color, crown diameter, leaf width and other characteristics. All parental, Fl, and 83 F
1BC1 plants were subjected to Genotyping-By-Sequencing (GBS).
ACP fraction in F1BC1 offspring.
The ACP fraction of each F1BC1 offspring genome was determined and samples were binned in intervals of 0.025 between 0 and 1. The mean ACP fraction per genome was 31.62% ( 6.35% (SD)). This is slightly above the expected value of 25%, likely due in part to segregation distortion due to selection against the BAR allele in the Alamo parent of the Fi. A secondary factor may be some miscalled or misplaced markers due to the preliminary state of the genome assembly, which is supported by regions of miscalled ACP
homozygosity that cannot exist in an FiBC I (Figure 8) Characterization of parental and Fi hybrid plants Panicum virgatum cv Alamo and Panicum amarum Ell. var. amarulum (ACP) are taxonomically described as different species yet results here indicate that they cross to produce fertile offspring. Therefore, a characterization of parental and Fi hybrid molecular and phenotypic characteristics was conducted to describe and confirm their differences and the phenotypes of the Fi hybrids. All Finale resistant-selected Fi plants retained the herbicide resistant phenotype to floral maturity.

Plant phenotypes Plant morphological characteristics for the parental Alamo and ACP plants were compared with the Fi hybrid. All characteristics show individuation of Alamo, ACP and the Fi hybrid. Alamo is a taller plant with expanded panicles; ACP is shorter with compressed panicles; and, the Fi hybrid shows intermediate characteristics (Figure 2 at A
and B), except it has increased tillering and expanded and smaller panicles than either parent. Various additional plant characteristics were observed that categorize phenotypic differences between both parents and the Fi hybrid including: inflorescence architecture, individual spikelets, leaf structure, epicuticular wax patterns, and bundle sheath appearance.
The inflorescence of Alamo has dispersed spikelets, ACP is compact and the hybrid is intermediate. Individual spikelets of Alamo have deep red stigmas, ACP is white and the hybrid is blended. The spikelet and rachis number is similar in all three plants. The leaves of Alamo are a deep greenish blue; those of ACP have a distinctive bluish hue, and the Fi hybrid is intermediate in color under greenhouse conditions. All leaf observations and measurements were made on samples from the second flag leaf 2.5 cm from the axil during anthesis.
Epicuticular wax patterns as observed in SEM on the adaxial surface of Alamo leaves is relatively smooth in comparison to ACP and the hybrid is intermediate. Also there are two types of trichomes on the surface on Alamo leaves, those comprised of a single cell and those with two cells whereas, ACP has only the two celled trichomes. The hybrid leaves have only two celled trichomes. Additional phenotypic plant characteristics categorizing similarities and differences between both Alamo, ACP parents and the Fi hybrid including: leaf chlorophyll content , rachis , spikelet, and seed set number per inflorescence, as well as adaxial and abaxial leaf stomatal density were compared. The chlorophyll content of Alamo is similar to that of the hybrid and less than that of the ACP, and the bundle sheath cells of Alamo appear slightly smaller, the veinal architecture typically shows fewer xylem elements than ACP and the hybrid appears similar in these characteristics to Alamo.
Confirmation of bar segregation in F1BC1 Segregation of the bar transgene was confirmed in the F1BC1 via PCR assay and Southern blot. In the PCR assay, the bar transgene was amplified in the HbR
Alamo parent (T85-2), Hbs ACP parent, HbR Fi, four Hbs F1BC1, and four HbR F1BC1 (Figure 3). The Southern blot was performed on the transgenic HbR Alamo parent, a wild type Hbs Alamo individual, a wild type Hbs ACP individual, the HbR Fi, four Hbs F1BC1, and four HbR F1BC1 In both the PCR and the Southern blot assays, the bar transgene was only detected in HbR
individuals.
Chloroplast DNA and bar transgene markers A chloroplast DNA tRNA-Leu (trnL) 49 bp indel polymorphism (deletion in wild-type Alamo) served as a marker to confirm maternal contribution from ACP
whereas the presence of the bar transgene confirmed parental contribution from ACP PCR
amplification of the 49 bp indel identified the wild type state in the ACP maternal parent, the Fi, and four FiBCi. The deletion state was only observed in the Alamo parent. The bar transgene was observed, however, in both the Alamo parent and the Fi. This confirms genetic contribution from both the ACP and Alamo parents to the Fi, supporting its identity as a hybrid.
Identification of useful markers The first goal of this project was to identify a robust dataset of variants from both the ACP and Alamo parents that were transmitted to the interspecific Fi hybrid and then transmitted and distributed amongst the F1BC1 population. For this, we first mapped reads from the Alamo and ACP parents, the Fi offspring, and the 83 F1BC1 offspring to the ver 1.1 assembly of the P. virgatum genome. Sites were selected which were homozygous within and polymorphic between the ACP and Alamo parents and heterozygous in the Fi offspring and evaluated their segregation within the F1BC1 offspring. A mean 92.45% of the 170,553,076 paired-end 75bp reads per sample successfully mapped to the P. virgatum draft reference genome. A total of 3,646,353 non-reference calls resulted in 35,170 filtered markers.
Variants were filtered based on homozygous intra-parental calls and differing inter-parental calls, heterozygosity in the Fi, a call supposed by at least two reads in at least twenty F1BC1 samples, and a mapping quality of at least 20. All samples contained at least 3,779 called markers, with a per sample mean of 17,732 ( 6,484.2 (SD)) markers.
Post-imputation genotypes Genotypes were imputed using a least-squares methodology as described in Heffelfinger et al 2014 (submitted) from the raw, ordered variants in each F1BC1 offspring (Figure 3). Regions of the genome were called as either homozygous Alamo, ACP, or heterozygous. Due to the nature of the cross, only homozygous Alamo and heterozygous are possible as genotypes, but homozygous ACP calls are useful for identifying error rate and error prone regions of the genome. Observation of post-imputed genotypes identifies recombination in all F1BC1 samples. The estimate of genetic distance was high at 4595.7 cM.
ACP and Alamo genetic contribution to the F1BC1 The contribution of the ACP and Alamo parentals was measured in the F1BC1 offspring after imputation. The contribution from the ACP parent across all individuals was found to be 31.62% ( 6.35% (SD). This was slightly above the expected value of 25%. The fraction of samples with a heterozygous call tended to be slightly higher than the fraction of samples with an Alamo homozygous call in many regions of the genome.
Further, some regions of the genome, such as most of chromosome 6a, and parts of 7b and 9a had greatly enriched heterozygous fractions. This may be evidence of segregation distortion, and it was expected that at least one region would be under artificial distortion due to the integration of and selection against the BAR gene.
The fraction of samples with a post-imputation ACP homozygous call for a region was also considered. While a true homozygous ACP genotype was not possible in the F1BC1, this call may be indicative of error prone regions. Regions with a homozygous ACP call tended to be small and proximal to the telomeres. No region contained an ACP
homozygous fraction >20%, and in most it was <10% or absent entirely. Total ACP
homozygous fraction was 1.92% across samples.
Identification of bar transgene from sequencing data Sequencing data was used to test for the presence of the bar transgene and plasmid backbone in parents, Fi, and F1BC1 samples. To do this, 50 sequences ranging from 34 to 61 bp flanking RsaI restriction sites were identified from bar and plasmid backbone sequences, and were searched for in sequencing read datasets from all samples (TABLE 2).
Sequences were positively identified only in the Alamo parent (10 reads) and Fi offspring (11 reads).
Only sequence from the bar transgene was identified. No sequence from either the bar transgene or plasmid backbone was found in any FiBC I individual.
Figures 9A and 9B show ACP transmission frequencies in the F 1BC1 by GBS. All 19,208 sites meeting the criteria of transmission from the ACP and Alamo parents and heterozygosity in the Fl, as well as minimum number of samples called in the FlBC1, were grouped into bins of size 5% based on their ACP allele frequency. The mean ACP
allele frequency was 56.3% and the standard deviation was 19.3%. In order to determine if segregation distortion caused ACP alleles to disappear from the F 1 BC1, an artificial dataset simulating neutral transmission was produced.
In particular, the distribution of ACP alleles at polymorphic sites are shown.
Figure 9A shows the distribution of all sites showing variation between Alamo and ACP
parents.
The peak at 0.0 is due to ACP PCR errors (e.g. no offspring inherit the ACP
variant call), whereas the peak at 1.0 is due to Alamo PCR errors, as all offspring share the reference allele with ACP at the resulting sites. Figure 9B shows the distribution of sites after filtering variants for heterozygosity in the Fl in addition to variation between Alamo and ACP
parents. The red line represents the simulated distribution of sites where the recombining pairs of the tetraploid differ enough that the reference genome treats them as two separate diploids, whereas the green line represents the simulated distribution of sites where the recombining pairs of the tetraploid have been collapsed as a single region in the reference.
All 19,208 sites meeting the criteria of transmission from the ACP and Alamo parents and heterozygosity in the Fl, as well as minimum number of samples called in the F 1 BC1, were grouped into bins of size 5% based on their ACP allele frequency. The mean ACP allele frequency was 56.3% and the standard deviation was 19.3%. In order to determine if segregation distortion caused ACP alleles to disappear from the F 1 BC1, an artificial dataset simulating neutral transmission was produced. While transmission is the same for every polymorphic site, how they are detected relative to the reference differs due to some reference contigs being collapsed from a diploid pair (red line) and others being collapsed from the entire tetraploid (green line). A simulation of 8,604 sites was produced for each type of reference contig. It was expected that segregation distortion against ACP
would result in a distribution that fell below the simulation. This is not observed in the actual distribution;
however. Indeed, only twelve sites have an ACP allele frequency below 5% and only three had a frequency of 0%. There is a skew above the simulated distribution, likely caused by segregation distortion resulting in an increase of ACP alleles from selection against BAR as well as bias in reads towards sites called from the entire tetraploid, which have an actual ACP
allele frequency of 75% under neutral conditions.
Figure 10 shows the distribution of variant sites and synteny alignment with the Setaria italica genome. The 5130 contigs containing filtered variants identified by both transmission from the ACP and Alamo parents and heterozygosity in the F 1 show alignment (dark red lines) to the Setaria italica (foxtail millet) genome (white, outer circle). The percent contribution of ACP sites (green histogram) and the percent of FlBC1 samples with coverage at those sites (blue histogram) are shown in 1MB bloc histograms across the Setaria italica genome. Percent of covered samples with ACP alleles and overall number of samples showing coverage are between 50-75% for most blocs. Histogram heatmap reflects the numbers of sites within a bloc. Orange and yellow concentric circles under histograms indicate 25% intervals. Bottom shows enlargement of are around chromosome 2.
Distribution of variant sites and synteny alignment with the Setaria italica genome confirms the contribution of both switchgrass a Alamo and ACP genomes in the hybrid. The 5130 contigs containing filtered variants were identified by both transmission from the ACP
and Alamo parents and heterozygosity in the F 1 show alignment (dark red lines) to the Setaria italica (foxtail millet) genome (white, outer circle). The percent contribution of ACP
sites (green histogram) and the percent of F 1BC1 samples with coverage at those sites (blue histogram) are shown in 1MB bloc histograms across the Setaria italica genome.
Percent of covered samples with ACP alleles and overall number of samples showing coverage are between 50-75% for most blocs. Histogram heatmap reflects the numbers of sites within a bloc. Orange and yellow concentric circles under histograms indicate 25%
intervals. Bottom shows enlargement of are around chromosome 2.
Figure 11 shows a low resolution description showing the ACP (red) and Alamo (blue) variants in each of the 83 Fl BC1 lines individually as a heatmap for the total variant calls; blue = Alamo and green histogram = ACP, total variants red outer circle on a synteny map of foxtail millet. In both Figures 10 and 11, the variants are pooled in 1 MB bins when mapping. FIGURE 11 shows enlargement of are around chromosome 2 as an example.
In a separate data analysis the contribution of ACP alleles were determined across the Alamo switchgrass genome. Figure 12 shows contributions of ACP alleles across the Switchgrass genome. 5,951 sites in 3,718 contigs were successfully aligned against the genome of Setaria italica, a sister species of Panicum virgatum, then ACP
contribution was measured in 1MB bins. The outer histogram (black and grey) indicates the number of sites within each bin. The minimum number of sites within a given bin was zero, which occurs at three locations, and the maximum was forty eight, and the mean number of sites was 12.7.
The standard deviation was 8.3. The heatmap indicates ACP allele presence for the eighty three F 1 BC1 offspring across all bins. Cool colors indicate a skew towards Alamo-only alleles within a given bin, whereas warm colors indicate that ACP alleles were detected.
Comparing the site number histogram with the heatmap reveals bins that tend to be either high ACP or Alamo for each sample tend to have few sites in them. Bins with more sites in them tend to have a larger range of contributions across samples.

This is somewhat contrary to what one would expect due to recombination, but is possible for several reasons. First, the tetraploid nature of Panicum virgatum allows for both Alamo only and ACP alleles to be detected in a single bin within a sample.
Second, low coverage data results in a failure to detect the ACP allele even when it is present. Finally, since both genomes have been assembled in a somewhat preliminary state, some sites are likely to be misaligned. Nonetheless, trends of high or low ACP contribution across bins are visible, concordant with what we would expect from recombination. The inner histogram (red) indicates the overall frequency of the ACP allele in each bin.
Frequencies tend to fluctuate between the minimum and maximum frequencies were 21% and 89%
respectively with a mean value of 56.1%. This closely follows the overall mean frequency of 56.3% in the complete dataset of 19,208 sites. Genome Wide Sequencing data shows the absence of all transgene sequences, Ti plasmid backbone and any Agrobacterium sequences demonstrating that all 83 of the F1BC1 progeny are non-transgenic.
The 5,951 sites in 3,718 contigs were successfully aligned against the genome of Setaria italica, a sister species of Panicum virgatum, then ACP contribution was measured in 1MB bins. Comparison of the site number histogram with the heatmap reveals bins that tend to be either high ACP or Alamo for each sample tend to have few sites in them.
Bins with more sites in them tend to have a larger range of contributions across samples. This is somewhat contrary to what one would expect due to recombination, but is possible for several reasons. First, the tetraploid nature of Panicum virgatum allows for both Alamo only and ACP alleles to be detected in a single bin within a sample. Second, low coverage data results in a failure to detect the ACP allele even when it is present.
Finally, since both genomes have been assembled in a somewhat preliminary state, some sites are likely to be misaligned. Nonetheless, trends of high or low ACP
contribution across bins are visible, concordant with what we would expect from recombination. The inner histogram (red) indicates the overall frequency of the ACP allele in each bin.
Frequencies tend to fluctuate between the minimum and maximum frequencies were 21% and 89%

respectively with a mean value of 56.1%. This closely follows the overall mean frequency of 56.3% in the complete dataset of 19,208 sites. Genotyping-By-Sequencing data shows the absence of all transgene sequences, Ti plasmid backbone and any Agrobacterium sequences demonstrating that all 83 of the F 1BC1 progeny are non-transgenic. This embodiment demonstrates the basic applicability of this method as a simple and general strategy for plant breeding to broaden germplasm utility.

To those skilled in the art, this invention teaches the use of transgenics as a method for making new hybrid, these hybrids can be made intervarietially to introgress traits rapidly for making hybrid plants, interspecifically or intergenerically, to combine distant genomes, or in use in genome editing startegis emplying ZNF, TALENS, CRISPR (or othe such genome editing approaches) where the transgene used needs to be segragated.
While switchgrass and its relatives have the potential to be important bioenergy crops as feedstock, domestication issues remain a critical barrier to this goal. One path towards overcoming these problems would be through the development of new cultivars and hybrids.
The ability to combine the genomes of intervarietal ecotypes or related species of switchgrass would provide expanded genetic resources through the incorporation of isolated germplasm and traits. We demonstrate the recovery of interspecific crosses between transgenic herbicide-resistant Panicum virgatum cv Alamo switchgrass and wild type Panicum aramrum Ell. var. amarulum (Atlantic Coastal Panicgrass).
Phylogenetics in swtichgrass and related species.
There are approximately 500 species in the genus Panicum. Genomics on switchgrass and its relatives is relatively recent, yet there have been considerable clarifications in phylogenetic and taxanomic relationships between species from this work. More recently switchgrass genomic diversity, ploidy and evolution was explored using a network-based SNP discovery protocol. These studies yielded a linkage map, an EST database, a set of SNP
markers across 18 linkage groups and BAC libraries. These results find collinearity of the switchgrass genome with the genomes of rice, sorghum, and Brachypodium distachyon and illustrate varietal isolation-by-distance and isolation-by-ploidy between switchgrass populations. Phylogentic analyses indicate a tendency of south to north migration in North America. Hence there are significant barriers to the inter-breeding of these ecotypes. Ploidy levels vary within switchgrass ecotypes, ranging from diploid (2n=2x=18) to duodecaploid (2n=12x=108). All lowland ecotypes have been identified as tetraploids (2n=4x=36) while upland types can be tetraploids or octaploids (2n=8x=72).
It is believed that the upland tetraploids were derived from upland octoploids. Mixed ploidy levels among accessions and within cultivars has also been observed.
Both geographic isolation and sexual incompatibility related to ploidy have resulted in varietal and species specific diversification. Martinez-Reyna and Vogel have shown that Pancium virgatum display both pre-fertilization and post-fertilization self incompatibility and that post-fertilization abortion between octoploid x octoploid, octoploid x tetraploid, tetraploid x octoploid is common. Using SRAP and EST-SSR markers it has been shown that Panicum amarum is a sister taxa to Panicum virgatum. Close genetic proximity and multiple abiotic stress resistance and yield traits result in them being excellent candidates for hybridization.
The interspecific Fi hybrids generated in this study germinated and were fertile.
Phenotypic and molecular comparisons between parental and Fi hybrid characteristics showed clear differences between the parents and the Fi hybrids, confirming the hybrid origin of these plants. Molecular and genomic analysis of these plants indicated the presence of an ACP-specific maternal chloroplast marker, and the paternal bar transgene further substantiating that they were hybrids. A subset of the hybrid Fi plants were then backcrossed to the wild type Alamo and 83 F1BC1 progeny were recovered demonstrating the robustness to this approach for screening of the segregating population. Phenotypically each of these individuals exhibited unique characteristics and were stable to floral maturity. As predicted, the majority of herbicide sensitive plants no longer contained the bar transgene as evaluated by PCR assay and sequencing. Genomewide analysis of parental contribution via GBS
clearly indicated that F1BC1 hybrids were derived from a population segregating for herbicide resistance. With the application of further testing for transgenic sequence in select F1BC1 individuals, this method may allow the recovery of non-transgenic and, arguably, non-GMO
offspring from wide-crosses. This method provides a proof-of-concept for efficient selection of interspecific hybrids using a selectable transgenic as an intermediate. It is also likely that this approach will also extend to intervarietal crosses.
While well established methods for molecular marker analysis have been previously described for developing mapping populations, and phylogentic comparisons have been applied here to improved Genotyping-by-Sequencing (GBS) approaches for data analysis described in Heffelfinger et al 2014. A total of 170,553,076 2x75 bp paired-end reads were produced via Illumina Hiseq 2000 sequencing of genomic DNA samples isolated from two parental species, Alamo switchgrass and Atlantic Coastal Panicgrass, one interspecific Fi hybrid, and 83 F1BC1 offspring. Alignment to the draft Panicum virgatum v1.1 reference genome identified a total of 3,646,353 non-reference sites. After applying stringent filtering criteria, a total of 35,170 sites were identified. Imputation was performed across all F1BC1 samples using this dataset to quantify parental contribution to each individual and confirm the occurrence of recombination events. These results clearly show the hybrid contribution of both parents and recombination in the F1BC1 individuals.

Accuracy of variant identification and imputation Beyond the specific concerns of variant identification with GBS and low coverage sequencing in general, the switchgrass reference genome presents problems as well. The current state of the P. virgatum reference assembly is scaffolded contigs with synteny established against related species Panicum hallii genome (Panicum virgatum v1.1, DOE-JGI, http://www.phytozome.net/panicumvirgatum). Compounding this difficulty is that switchgrass is an allotetraploid, resulting in a highly repetitive genome. The result of this is that few variants achieve a high mapping quality (>30), and of those that do, the possibility of misalignment remains high due to the draft nature of the assembly.
To resolve confounding issues as best as possible, retained markers were required to be observed to be transmitted from the parents, through the F 1, and into the FiBCi. To explain, variants were required to be homozygous within and polymorphic between the parents and heterozygous in the F 1. The requirement that all three possible marker states be observed reduced the likelihood of PCR, sequencing, and mapping artifacts. No expectation for allele frequency or segregation was applied to the F 1BC1 however, as segregation distortion would be masked.
Another concern, not related to the reference genome but instead caused by the high degree of multiplexing was false homozygosity. False homozygosity results when only one allele of a heterozygous site is observed in sequencing data. False homozygosity was primarily solved through the imputation and error correction method, which is relatively insensitive to a single genotype call and instead determines a regional genotype based on a "mean" value from a set of calls. So long as homozygous miscalls did not randomly skew towards ACP or Alamo for a given region, the rate of erroneously imputed homozygote sites should be low.
A partial estimate of this rate can be obtained from the amount of the genome called across F1BC1 individuals as ACP homozygous. Due to the nature of the cross, this is not physically possible, but may nonetheless result from false homozygous calls.
Across all samples, approximately 1.92% of the genome is called as ACP homozygous.
Assuming this error rate results in the same percentage of the genome being miscalled as Alamo homozygous, the total post-imputation error rate due to false homozygosity is under 4%. In reality, the percent of the genome miscalled as Alamo homozygous or heterozygous is probably higher than 4%, however, as erroneously mapped reads may result in regions of the genome being "placed" incorrectly, even if the genotype is technically correct.

Evidence of segregation distortion in the F1BC1 offspring A primary concern was that ACP alleles would be lost in the F1BC1 due to segregation distortion. Regions with a fixed homozygous Alamo state would present severe obstacles to introgressive hybridization and trait mapping. No evidence is observed of any segregation distortion favoring a homozygous Alamo state in the post-imputation F1BC1 offspring.
We observe, however, evidence of segregation distortion favoring the heterozygous state in some regions of the genome. Switchgrass, exhibits both pre-fertilization and post-fertilization self-incompatibility systems. Gametophytic self-incompatibility in grasses is controlled by two loci, S and Z. Martinez-Reyna and Vogel in switchgrass show that in controlled crosses between octoploid x octoploid, octoploid x tetraploid, tetraploid x octoploid post-fertilization abortion occurs in many cases 20-40 days after pollination. Based on this study, self-compatibility is estimated to be between 0.35% and 1.39%.
None of the self-compatibility genes have been cloned.
The mean fraction of the genome with the ACP allele across all offspring is slightly higher than expected (-25%) at 31.62% ( 6.35% (SD)). Further, while across most of the genome the fraction of offspring with a heterozygous call is similar to that with an Alamo homozygous call, there are several regions with varying degrees of enrichment of the heterozygous fraction. This enrichment is especially pronounced on chromosome 6a, and on parts of 9a and 7b. Chromosome 4a, 5b, 8a, and 8b show more modest levels of heterozygous enrichment. One of these regions is likely due to artificial segregation distortion caused by selection against BAR in the FiBCi. Chromosome 6a is the most likely candidate, due to the absence of any homozygous Alamo calls in any of the offspring near the telomere. Self-incompatibility loci may be responsible for the other regions showing significant segregation distortion.
Recombination observed in all F1BC1 offspring Recombination was observed in all F1BC1 offspring based off the imputed dataset.
Across all eighty-three F1BC1 individuals, a total of 3166 recombination events were observed for a total genetic distance of 4595.7 cM. This estimate is high, especially considering previous switchgrass estimates placed the total genome size at 1,733 cM (female) and 1,508 cM (male). The likely reason for this considerably higher recombination rate observed in our dataset is a high rate of error resulting from both the early state of the reference assembly and the highly repetitive nature of the genome. The low-coverage nature of GBS data also contributed to this error rate. As evaluated by the presence of ACP
homozygous calls, a biologically unlikely genotype given the nature of the crosses used to generate this population, these errors primarily occurred in the subtelomeric regions and resulted in small, likely spurious recombination events. Nonetheless, we observe considerable amounts of recombination outside of these likely error-prone regions, supporting our claim of actual recombination in the offspring.
The question of non-transgenic F1BC1 offspring The ability to utilize transgenics to isolate and derive non-transgenic (and from a regulatory perspective, potentially non-GMO) hybrids would accelerate the breeding process, and allow the combination of genomes from related cultivars and species to provide new a germplasm base that can rapidly accelerate the breeding process.
The use of transgenic selectable markers provides a useful solution for identification and recovery of embryonic hybrids without time-consuming phenotyping or marker based validation. The approach we have demonstrated for recovery of Fi hybrids followed by removal of the transgene by backcrossing to a wild type parent and selection against the marker may have extended applications for producing heterogeneous breeding and production field populations. In addition, this approach may also provide a breeding solution for the combination of QTLs in hybrid populations that may find agronomic and agricultural significance (i.e. disease resistance, drought and abiotic stress tolerance, and yield) within a heterogeneous hybrid population.
This platform could serve as a method for combining desirable QTLs by exploiting additive genetic variation and provide a more timely approach to developing novel hybrid populations in various crop species. The method demonstrated here then presents a viable approach for the rapid creation of new hybrids using transgenic markers.
Selection against the resistance marker, alongside appropriate screening and analysis, may further allow for the creation of non-transgenic hybrid populations. In addition this approach may be applicable for removal of the transgene from populations where genome editing functions are the goal.
This approach can be extended to other crop species. Figure 13 shows a diagrammatic scheme illustrating a general strategy for commercial hybrid production for tomato (Lycoperscion esculteum L). The non-transgenic F 1BC 1 hybrid plants contain Parent Two X Parent One X Parent One genomic contributions and may be subsequently analyzed and scored for desirable traits correlated with genomic markers. This example greatly reduces the breeding cycle necessary for the combination of two sets of complicated genomes with various agronomic characteristics (eg. X,Y,Z by A, B, C) as a hybrid. The bar +
population plants, can also serve as hybrids to cross with other compatible or incompatible parents. Desirable plants may enter into population block breeding plots, and mass selection and subsequent commercial development can proceed. A color marker or other visible trait can be substituted for the herbicide resistance marker.
The general strategy shown in Figure 13 provides for the commercial hybrid production for tomato (Lycoperscion esculteum L). In this diagrammatic scheme the recovery of wide inter-varietal crosses using herbicide selection as a marker, however, is the same or similar scheme that would also apply to wide intra- and inter-varietal, intra- and inter-specific, inter-generic close and distant relative crosses. In this example, transgenic Parent Two (at upper right) is herbicide resistant (Hbl, bar+, containing the bar gene, resistant to bialphos and 3% Finale or Liberty and male sterile) This parent may, for example contain a single or a number of agronomic traits of interest (e.g. X,Y,Z) and is used as the maternal recipient in a hybrid cross. The paternal pollen donor is a wild -type non-trangenic parent (at upper left ) with similar or different agronomic traits (E.g.
A,B,C).
After pollination and seed maturation, the F 1 seed is harvested only from the parent two and will be male sterile and herbicide resistant (bottom right). Seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for resulting resistant and sensitive plants after ¨21 days to reveal herbicide sensitivity At floral maturity wild type (non-transgenic) from parent one are used preferably as paternal pollen donor (s) in a backcross to the Fl (bar+) Hbl Herbicide Resistant plant (s) The resultant seed is recovered and germinated. The resultant seedlings are treated with 3% Finale using one-several leaves in the viable paint assay and scored for resulting resistant and sensitive plants after 21 days to reveal herbicide resistant (bar +, red) and herbicide sensitive (bar-, green) and populations (lower left) The non-transgenic F 1 BC1 hybrid plants contain Parent Two X
Parent One X
Parent One genomic contributions and may be subsequently analyzed and scored for desirable traits correlated with genomic markers. The bar + population plants, can also serve as hybrids to cross with other compatible or incompatible parents. Desirable plants may enter into population block breeding plots, and mass selection and subsequent commercial development can proceed. A color marker or other visible trait can be substituted for the herbicide resistance marker.
A list of target genes for 5\ and y sterility expression cassettes. Promoters from these genes may be operably linked to cytoxic genes, including barnase or RNAi, or comparable technology to direct cell specific ablation leading to the developmental disruption of male or female floral structures. A detailed meta-analysis of known male- and female-specific genes has identified several suitable genes and/or their promoters that can be used for the purpose of floral organ ablation. These genes have been used to create "expression cassettes" using SLIC technology, a method borrowed from synthetic genomics to construct reporter constructs for expression analysis as well as ablation constructs to create staminate and pistillate lines of switchgrass using cv Alamo. These genes or their orthologues, could also be the target for ZFN or TAL modifications to direct sterility functions.
Figures 14A and 14B show diagrammatic schemes following a similar approach to Figure 13 illustrating a practical strategy for commercial hybrid production for tomato (Lycoperscion esculteum L) with closely related parents (A), and similar parents (B).
The strategy shown in Figure 14A follows a similar plan to the general strategy described in Figure 13 and the parents are closely related genetically and phenotypically. In this example, transgenic Parent Two (cv GINAN, at upper right) is herbicide resistant (Hbl, bar+, containing the bar gene, resistant to bialaphos and 3% Finale or Liberty and male sterile) This parent may, for example is a fresh market indeterminate variety with genes for disease resistance to Fusarium (F1 and F2) and is used as the maternal recipient in a hybrid cross. The paternal pollen donor is a wild -type non-transgenic parent (cv TALA, at upper left ) which is a fresh market determinate variety with gene(s) for resistance to tomato yellow leaf curl virus (TYLCV). Following the strategy of Figure 13, the non-transgenic F 1 BC1 hybrid plants contain Parent Two X Parent One X Parent One genomic contributions and the population will contain individuals with resistance to Fusarium and tomato yellow leaf curl virus which may be subsequently analyzed and scored for the desirable traits correlated with genomic markers and propagated or bred. Here the two parents In this diagrammatic scheme the recovery of wide inter-varietal crosses using herbicide selection as a marker, however, is the same or similar scheme that would also apply to wide intra- and inter-varietal, intra- and inter-specific, inter-generic close and distant relative crosses. A color marker or other visible trait can be substituted for the herbicide resistance marker.
The strategy shown in Figure 14B follows a similar plan to the strategy described in Figure 13 except the parents are similar but less closely related genetically and phenotypically than in Figure 14A. In this example, transgenic Parent Two (cv GINAN, at upper right) is herbicide resistant (Hbl, bar+, containing the bar gene, resistant to bialaphos and 3% Finale or Liberty and male sterile) This parent may, for example is a fresh market indeterminate variety with genes for disease resistance to fusarium (F1 and F2) and is used as the maternal recipient in a hybrid cross. The paternal pollen donor is a wild -type non-transgenic (cv FLINT, at upper left) which is a saladette indeterminate variety with smaller fruits and gene(s) for disease resistance to F1,2,3 N,V, and ToMV. Following the strategy of Figure 13, the non-trangenic F 1 BC1 hybrid plants contain Parent Two X Parent One X
Parent One genomic contributions and the population will contain individuals with combined disease resistance which may be subsequently analyzed and scored for the desirable traits correlated with genomic markers and propagated or bred. A color marker or other visible trait can be substituted for the herbicide resistance marker.
Using this approach the non-trangenic F 1 BC1 hybrid plants contain Parent Two X
Parent One X Parent One genomic contributions and the population will contain individuals with combined disease resistance and phenotypic characteristics which can be scored for the desirable traits and correlated with genomic markers and propagated or bred. A
color marker or other visible trait can be substituted for the herbicide resistance marker.
Figures 15A and 15B illustrate diagrammatic schemes for a strategy for the creation of intergeneric hybrids such as crosses between (A) tomato ( Lycoperscion esculteum L) and pepper (Capsicum annum) and (B) tomato ( Lycoperscion esculteum L) and eggplant (Solananum melangena). This strategy follows a similar plan to the general strategy described in Figures 13, 14A and 14B except the parents are in different genera.
In the example shown in Figure 15A, transgenic Parent One (cv, at upper left) is a pepper (Capsicum annum) variety that is herbicide resistant (Hbl, bar+, containing the bar gene, resistant to bialaphos and 3% Finale or Liberty and male fertile) The maternal pollen recipient is a wild -type non-transgenic parent tomato (Lycoperscion esculteum L , variety (at upper left) Following fertilization the wide cross is selected by embryo rescue and cultured for the presence of the herbicide resistant marker. The embryo rescue can be performed traditionally by micro-surgery or by in situ embryo rescue by culturing the immature ovule and selecting for embryogenic callus production. The hybrids in some cases will be sterile (lower left) and may have various utility, including recovery to fertility via chromosome doubling or gene confinement; or, they will be, in some cases fertile and can be entered into backcrosses programs.
In the example shown in Figure 15B, transgenic Parent One (cv, at upper left) is a eggplant (Solananum melangena) variety which follows a similar strategy to that described in Figure 15A.
The tomato variety Solanum lycopersicum cv Buffalois known for superior taste characteristics of significant market value and Solanum lycopersicum cv Geronimo has certain production characteristic of value. Figure 16 shows at (A) flowers of wild type Solanum lycopersicum cv Buffalo at anthesis with full developed anthers and fertile pollen.
Figure 16 shows at (B) an inflorescence of wild type Solanum lycopersicum cv Buffalo with immature fruits at a stage suitable for in situ embryo rescue. Figure 16 shows at (C) a vegetative leaf from a mature wild type Solanum lycopersicum cv Buffalo with characteristic shape and deep green color (mature flower is shown at the lower left). The hybrid tomato variety Solanum lycopersicum cv Buffalo X Solanum lycopersicum cv Geronimo is recovered by a wide cross via a transgenic hybrid and backcrossed to either wild type parent to segregate away the transgene.
These examples indicate broad utility for: vegetable crops such as (but not limited to) tomato, pepper, eggplants, squash, melons and certain allium species; cereal crops, including (but not limited to) rice, corn, wheat, barley, rye, sorghum, triticale, millets; legumes including (but not limited to) soybean, peanuts, and beans; fruits including (but not limited to) apples, peaches, pears, plums, and nectarines; and, nuts including (but not limited to) walnuts, almonds, brazil nuts, and cashews, between these crops and closely related species.
In a second embodiment, a similar strategy is deployed to make rapid hybrids via intervarietial crosses. In this embodiment, transgenic herbicide resistant Panicum virgatum L.
cv Alamo may used in difficult to recover interspecific and/or intervarietal crosses to identify and define progeny useful for production of fertile non-transgenic hybrids. In this example, using a self-compatible intervarietial cross between transgenic herbicide resistant Panicum virgatum L. cv Alamo (4x) and non-transgenic Panicum virgatum L. cv Kanlow (4x); where (x) is the basal number of chromosomes, and in Panicum sp. x=9, therefore, 4x refers to a tetraploid where 2n=4x=36 chromosomes, and 8x refers to an octaploid where 2n=
8x = 72 chromosomes. In this embodiment, octoploid (8x) varieties can also be used.
This method will extend the breeding capabilities for ectotypes. In this example directionality (maternal X
paternal) also does not matter to the practice of hybrid plant recovery. The F
1 progeny from the wide crosses may be fertile, producing viable seeds which germinate to produce healthy fertile plants that can be used in backcrosses to wild type non-transgenic Panicum virgatum L. cv Alamo. The subsequent F2 population is then germinated from the resultant seed. The F2 seedlings are screened for the segregating presence or absence of the selectable marker transgene. The non-transgenic F2 hybrid population is then used in downstream varietal and breeding applications. Hybrid plant distinctiveness can be phenotypically and genetically characterized as described in the first embodiment.
This embodiment demonstrates the wide applicability of this method as a simple and general strategy for plant breeding. This example shows utility for; vegetable crops such as (but not limited to) tomato, pepper, eggplants, squash, melons and certain allium species;
cereal crops, including (but not limited to) rice, corn, wheat, barley, rye, sorghum, triticale, millets; legumes including (but not limited to) soybean, peanuts, and beans;
fruits including (but not limited to) apples, peaches, pears, plums, and nectarines; and, nuts including (but not limited to) walnuts, almonds, brazil nuts, and cashews between these crops and closely related cultivars and species. Desirable non-transgenic plants from F 1BC1 or subsequent populations may enter into breeding plots, and using genomic assisted breeding and selection can enter subsequent commercial development.
Breeding strategies are extended to near and wide cross hybrid production.
This embodiment demonstrates the wide applicability of this method as a simple and general strategy for plant breeding.
In this embodiment, transgenic herbicide resistant plants may used in to recover rare intra-, inter-specific or intergeneric crosses between self-incompatible, marginally compatible or comaptible parents to identify and define progeny useful for production of fertile hybrids. These parental plants may be hemizygous TO, or contain at least one copy of the transgene in Ti ,T2, or more generations. As an example, the recovery of wide inter-specific crosses using herbicide selection as a marker in Panicum virgatum L.
cv Alamo, however, this same or similar scheme also applies to wide inter-varietal, inter-specific, inter-generic and distant relative crosses as a method to enhance recovery of fertile hybrids. In this embodiment, a series of intra- and inter-varietal, intra- and inter-specific or intra- and inter-generic wide crosses within and between related species are generated. These can be used directly in breeding programs or used as hybrids to generate new cultivars and hybrids.
In a third embodiment, this breeding platform may also utilize transgenic male and/or female sterile lines which can be utilized in examples, such as those in the second embodiment from a reference or non-reference genotype that is linked to herbicide resistance.
The sterility characteristic is used to 'force' rare wide crosses and herbicide selection is used for recovery of wide Ti (or Fl) crosses In some cases this may require embryo rescue of the intermediates. T 1 (or F 1) hybrids can be backcrossed to the reference genotype, in this embodiment, to segregate away the transgene to generate a non-transgenic BC1 mapping population. One improvement on hybrid recovery in this embodiment is this method to 'force' outcrossing between parental lines. In this embodiment the generation of exclusively staminate and pistillate lines are made specifically for this purpose.

A detailed meta-analysis of known male- and female-specific genes has identified several suitable genes and/or their promoters that can be used for the purpose of floral organ ablation (TABLE 2 below).
GGene Target cells Reference AMS developing anthers Sorensen et al. 2003 BEL1 developing Robinson-Beers et al.
megaspores 1992 DDE2 developing anthers von Malek et al. 2002 EA1 developing NA
megaspores EA1 developing Marton et al. 2005 megaspores MS1 developing anthers Wilson et al. 2001 SIN! developing Robinson-Beers et al.
megaspores 1992 TDF1 developing anthers Zhu et al. 2008 As shown above, TABLE 2 includes a list of target genes for 5 and y sterility expression cassettes. Promoters from these genes may be operably linked to cytoxic genes, including barnase or RNAi, or comparable technology to direct cell specific ablation leading to the developmental disruption of male or female floral structures. A
detailed meta-analysis of known male- and female-specific genes has identified several suitable genes and/or their promoters that can be used for the purpose of floral organ ablation. These genes have been used to create "expression cassettes" using SLIC technology, a method borrowed from synthetic genomics to construct reporter constructs for expression analysis as well as ablation constructs to create staminate and pistillate lines of switchgrass using cv Alamo. These genes or their orthologues, could also be the target for ZFN,TALENS, CRISPR or a similar genome editing modifications to direct sterility functions.
These genes have been used to create "expression cassettes" such as using SLIC

technology (for example). SLIC technology is a method borrowed from synthetic genomics to construct reporter constructs for expression analysis as well as ablation constructs to create staminate and pistillate lines of switchgrass using cv Alamo. Other cloning methods can also be used . These genes could also be the target forgenome editing modifications to direct sterility functions. Alamo was chosen as the reference for several reasons including its ability to transform with Agrobacterium (Figure 2 ) and extensive genomic resources developed for the genome mapping component of this invention. It is referred to as the "reference" genome in this embodiment. Transformation with these cassettes has been conducted using reporter gene expression (GUS and GFP) as well as male and female specific cell ablation phenotypes and evaluated in mature TO florets. The observed reporter and ablation phenotypes demonstrate exclusively staminate (female sterile) or pistillate (male sterile) and are dependent on the appropriate expression cassette in the transgenic.
The benefit of sterility lines is the ability to force efficient hybrid production. In this embodiment, the scheme for generating and recovery of rare wide crosses and hybrids relies on the use of pistillate Panicum virgatum L. cv Alamo plants as a pollen recipient in wide-crosses as an example. [Note: that staminate reference plants can be used in reciprocal crosses as needed]. The floral ablation phenotype, physically linked to an herbicide selectable marker, can be used in embryo rescue experiments to select and recover F 1 hybrids. Interestingly, in both strategies only 50% of the hybrid offspring produced by these methods are transgenic due to the hemizygous nature of the transgene.
Therefore, non-transgenic hybrids can be recovered readily by screening for herbicide sensitivity in the Ti (F1) or advanced generations. The simple non-destructive leaf painting assay has been used for this purpose. Herbicide-resistant F 1 hybrids are unisexual and backcrossed with non-transgenic reference Alamo plants. The first generation resultant from the backcross (BC1) progeny can be assayed and selected for segregation of the transgene. Both non-transgenic (herbicide-sensitive) and transgenic, unisexuals (herbicide-resistant) BC1 progeny will be identified.
Alternatively, and similarly male sterility lines can be used to for recovery of rare wide crosses. One target for male-sterility is the tapetum, the innermost layer of the anther wall that surrounds the pollen sac, which is needed for pollen development. A
variety of anther and tapetum-specific genes have been identified that are involved in normal pollen development in many plant species, including maize, rice, tomato, Brassica campestri, and Arabidopsis thaliana. Selective ablation of tapetal cells by cell-specific expression of nuclear genes encoding cytotoxic molecules or an antisense gene essential for pollen development blocks pollen development, giving rise to stable male sterility. To induce male sterility in the turfgrass, Agrostis stoloniferia L (creeping bemntgrass) the 1.2-kb rice rts gene regulatory fragment, tap was fused with two different genes. One was the antisense of rice rts gene that is predominantly expressed in tapetum cells during meiosis. Another gene was the Bacillus amyloliquefaciens ribonuclease gene, barnase, which ablates tapetal cells by destruction of RNA. Both approaches have been shown to be effective in various plant species.
Figure 17 shows male sterile lines are generated through the introduction specific promoters are used to drive (A) cytotoxic genes such as barnase or (B) specific synthetic lethality genes, such as RNAi. These genes or their orthologues, could be the target for ZFN, TALENS, CRISPR or a similar genome editing modifications to direct sterility functions.
Female sterile lines are generated through the introduction specific promoters are used to drive (A) cytotoxic genes such as barnase or (B) specific synthetic lethality genes, such as RNAi. These genes or their orthologues, could also be the target for ZFN or TAL
modifications to direct sterility functions. Diagrammatic representations of transgenic cassettes for induction of male sterility. Nuclear male sterility is induced by: (A) tapetal ablation using a tapetum specific promoter from maize (Zm tap) driving expression of either (Coding), a cytoxic gene (i.e. barnase) or (RNAi) the antisense of the native gene with selection via herbicide resistance; or using a similar strategy driven by (B) a microspore specific promoter (Zm ms1); or, (C) a pollen specific promoter (Zm 13).
Transgenic lines from both (B) and (C) can be subjected to colchicine treatments to recover homozygous lines.
Orthologues of these genes could also be the target for ZFN or TAL
modifications to direct sterility functions.
Therefore by supplying wild type pollen as a donor male sterile maternal lines such as these can provide important breeding tools and perhaps function as a filter for forcing generation and recovery of rare wide crosses. The use of Zm ms1 (for microspore abortion) zml3 (for pollen sterility) can also be used for this purpose. These genes could also be the target for ZFN TALENS, CRISPR or similar technologies to create modifications to direct sterility functions. Transgenic recovered by both (Figure 17 shown at B) and (Figure 17 shown at C) can be subjected to colchicine treatments to recover homozygous lines to be used for future breeding and cultivar development.
Another important strategy in this inventive design is to physically link herbicide resistance (HR1 and HR2; i.e. bar or glyphosate) with male- and female-sterility transgenes, respectively. This permits a single herbicide for single sex sterility in parental lines and progeny. Figure 18 shows a schematic for transgene cassette design to generate male and female sterile lines under different selectable markers, designated Hb 1 and Hb2. Note that double herbicide selection can be used for complete sterility in Fls and that this accomplishes a separate and useful different objective, (i.e. namely gene confinement and trait stacking for GMO plant populations). Any other trait gene of interest (GOI) or series of GOIs, can be combined through this strategy into said hybrid. Physical linkage of herbicide resistance (HR1 and HR2) with male- and female-sterility transgenes, respectively. This permits a single herbicide for single sex sterility in parental lines and progeny. Note that double herbicide selection can be used for complete sterility in F is and that this accomplishes a separate and useful different objective. Two lines are created that, when crossed, would give rise to a fully sterile individual.
Male and female lines are created through the application of the promoters and/or the coding sequences such as those (but not limited to those) described in TABLE
2. Male sterile lines (top, line A-Male Sterility) are generated through the introduction specific promoters are used to drive (A) cytotoxic genes such as barnase or (B) specific synthetic lethality genes, such as RNAi. These genes or their orthologues, could be the target for ZFN, TALENS
CRISPR or similar modifications to direct sterility functions. Female sterile lines (bottom, line B-Female Sterility) are generated through the introduction specific promoters are used to drive (A) cytotoxic genes such as barnase or (B) specific synthetic lethality genes, such as RNAi. These genes or their orthologues, could also be the target for ZFN, TALENS<
CRISPR of othe genome editing modifications to direct sterility functions.
In particular, Figure 18 shows physical linkage of herbicide resistance (HR1 and HR2) with male- and female-sterility transgenes can be used for creation of hybridhybrid breeding populations. Physical linkage of herbicide resistance (HR1 and HR2) with male-and female-sterility transgenes, respectively, can also be used to create total sterile outcomes.
This permits a single herbicide for single sex sterility in parental lines and progeny. Note that double herbicide selection can be used for complete sterility in Ti (F 1)s and that this accomplishes a gene confinement strategy. Two lines are created that, when crossed, would give rise to a fully sterile individual.
The final transgene contains the target promoter translationally fused or operably linked to a selected CDS or open reading frame (ORF) and 3' non-translated region (3 '-UTR) with compatible 5' and 3' ends which are readily cloned into the LIC-adapted T-DNA vector.
The SLIC-LIC method is highly scalable and permits construction of many independent versions of promoter elements fused to reporter CDS, such as GUS and GFP, as well as cell ablation genes (barnase) or RNAi. Transgenic cv. Alamo (sequenced reference line) have been generated for male and female test vectors (10-20 independent single gene insertion events per vector) and have been analyzed molecularly for single-copy insertions and phenotypically for reporter gene expression and floral phenotypes characterized in our greenhouses. Single copy insertions have been detected using a Taqman qPCR
assay, to detect low copy insertions (1-2 copies), followed by genomic Southerns for verification.
In this strategy, as shown in this example, single-copy transgenics are backcrossed to wild type cv Alamo reference plants to test for stability and inheritance of the transgene phenotype. Stable single copy lines are used, sometimes in conjunction with embryo rescue, to create inter-varietial, inter-specific and inter-generic hybrids of switchgrass and related species. The breeding platform for efficient wide-cross production produces important hybrids. Success at using a dominant herbicide marker to create inter-specific hybrids in switchgrass form the basis of establishing an efficient breeding platform.
This embodiment teaches a greatly improved efficiency of hybrid production as well as the rescue of hybrid embryos by incorporating staminate and pistillate lines and herbicide selection into this program. The basic design is to use the pistillate reference plants as pollen recipient with a wide variety of cultivars and species. Reciprocal crosses, using the staminate reference plant are also possible. In closed pollen cage experiments parental types are set up in pairwise combinations. Seed set is monitored and collected for subsequent analysis.
using this novel technique, this intermixing to produce developing caryopses (F1 progeny) on the pistillate plants that are the result of pollen flow from the staminate plants but not vice versa.
Note that in most cases of wide crosses, Ti (F1) sterility, caused by embryo-endosperm incompatibility, is common and this may require the use of embryo rescue techniques, as described in the previous embodiments, to recover Fl progeny or reciprocal Fl crosses to avoid incompatibility. Recovery of rare wide cross progeny can be forced.
Immature, isolated caryopses can be excised and grown in vitro to recover plantlets. In rare wide cross cases, it may be necessary to generate embryogenic callus that will be regenerated to whole plants.
All recovered Ti (F1) hybrid plants are grown in the greenhouse and characterized molecularly. For instance in one direction of the cross, initially one can use a female cytoplasmic (chloroplast) marker and a male nuclear marker (transgene) to detect hybrids. A
more detailed phenotypic and genomic analysis can follow in the BC1 population. Hybrids are then examined for fertility and seed set in backcrosses to wild-type Panicum virgatum cv Alamo reference plants. For instance, since the Fl hybrids will retain the pistillate phenotype when selected for herbicide resistance, these Fl will be mated to wild-type reference plants in cage experiments to recover BC1 population.
The problem often seen with sterility of the Fl hybrids in wide crosses is not necessarily a disadvantage for many crops that are grown vegetatively. In fact, this may be an advantage in allocating resources to biomass production in the field.
Nevertheless, a breeding program may wish to introgress traits from one line into the genome of another, a process of recurrent backcrossing.
Tissue samples from hybrid plants are collected for genomic studies and the non-transgenic F 1 BC1 population can then transferred outdoors for field trial analysis where they can will be vegetatively propagated and entered into block breeding increases.
A set of clones are grown in several locations for regional selection and extensively characterized for biomass production and additional selected biofuels and agronomic traits such as above ground biomass, leaf number, inflorescence height and number, crown size per year and seed set. Characterized individuals can then be included in a downstream breeding process using genomic assisted breeding.
Hybrids can be selected for desirable phenotypes contributed by either parent;

including bioenergy traits, such as carbon allocation characteristics in root vs. shoot mass, cellulose content, low lignin, sugar content, photosynthetic efficiency, enhanced biomass yield acre, reduction of perception of nearest neighboring plant or tiller, biomass value added compounds, changes in photomorphogenic responses, including phytochrome red/far-red light perception and crypotchrome perception, optimized photoperiod, floral sterility, regulated dormancy, input requirements, such as fertilizers and pesticides, stratification characteristics, crown size, leaf phenotypes (including size, color, length width and angle), root mass and depth, tillering, stand development characteristics, seed set, inflorescence number, height and width, floral development; as well as biotic and abiotic stresses including water use efficiency, cold and freeze tolerance, pest resistance (including insect, nematode, fungus, bacterial, virus). Genomic and marker assisted breeding is deployed characterize parental genomic contribution and to follow traits in subsequent downstream breeding for varietal development. Hybrids can be sexually crossed and/or vegtetatively propagated.
SEQUENCE LISTING
The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
SEQ ID NO: 1 is a nucleic acid sequence of a corn ovule-specific gene -available form GenBank (see Tables 1 and 2).

SEQ ID NO: 2 is a nucleic acid sequence of a corn female inflorescence developmentally-specifically expressed gene.
SEQ ID NO: 3 is a nucleic acid sequence of a corn tapetum-specific gene SEQ ID NO: 4 is a nucleic acid sequence of a corn pollen-specific gene.
SEQ ID NO: 5 is a nucleic acid sequence for the bar transgene as:
LOCUS Seq5 6245 bp DNA linear 03-DEC-2014 DEFINITION Switchgrass transgene conferring herbicide resistance.
ACCESSION Seq5 SOURCE switchgrass transgene conferring herbicide resistance ORGANISM switchgrass transgene conferring herbicide resistance REFERENCE 1 (bases 1 to 6245) COMMENT Bankit Comment: Vecscreen Comment:Submitter says that this sequence represents a Cloning Vector. Bankit Comment: TAX: No, not new species/combinations;
Bankit Comment: TOTAL # OF SEQS:5.
Assembly Method :: DNASTAR SeqMan v. 12.1: Sequencing Technology Applied Biosystems 3500x1 Genetic Analyze:
Assembly-Data-END; FEATURES
Location/Qualifier: Source 1..6245/organism='switchgrass transgene conferring herbicide resistance' /mol type='other DNA' feature 1..25/note='T-DNA Right border (BR)' gene 229 ..1442/gene='RT S2' 229 ..1442/gene='RT S2'/note='RTS2 Promoter' gene1454 ..1892 /gene='barnase' CDS
1454 ..1789/gene='b arnas e'/co don start=1/product='b arnasetranslation='MAQVINTFDG
VADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKL
PGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR' terminator 1893..2169 /note='nos terminator'; gene 2185..4835/gene='RUBQ2' promoter 2185..4835/gene='RUBQ27note='RUBQ2 Promoter'gene4852..5324/gene='Os Actin 1 intron (japonica)'intron 4852..5324/gene='Os Actin 1 intron (japonica)'gene 5326 ..5898/gene='pho sp hinothricin N-acetyltransferase' 5340..5891/
gene='phosphinothricin N-acetyltransferaseYnote='Herbicide resistance -selection marker'\
/codon start=1/product='phosphinothricinNacetyltransferasetranslation='MSPERRPADI

RRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQEWTDDLVRLRERYPWLVAEV
DGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGSTLYTHLLKSLEAQ
GFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGFWQLDFS
LPVPPRPVLPVTEI' 5899..6163/note='nos terminator'misc feature 6221..6245/note='T-DNA left border (BL)' BASE COUNT 1630a 1398c 1459g 1758t ORIGIN
1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 61 aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 121 acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagcaag 181 cttgcatgcc tgcaggtcga ctctagagga tccgcgcgcg gatccgcgca ccggcgaggc 241 ggtgcgtctc ctcggagatg tggtagaagc tggcgcccca tttcatggcg gcgagcgggc 301 cgggcggcct cctgccgcag cgggattcgg tggctagacc gatctgtggg tggaggacgg 361 ggacgaggta gtggagacag aggcggcatt ggaagagggg aagaggagga ggaagtggtg 421 gcaggcagag gcggatgagg aacttgcgcc agcgacgtgg atatggaggg ggcgacggca 481 atggggaggc ggcgatggaa gcgaggagat gggcaggcgg cggaggcagc ggtggatttt 541 ttttttcttt ttctttttcg gaccctttca cctgctcggt gattcttctt ttttatacag 601 cacgacggct tctectattc acgacgcctc ggctggacca tggaccgttg gccactggag 661 cattcttcca tgatctagat tttttttttc actcaacttt actacttcac atctgatggc 721 tggtgttgaa ttcattgtgc atccaacggt cattattaaa ttgatgacgt ggcgcaatga 781 ggtgacgaaa cactttactt tttttactac tttagatctg tcggcaggag tcccagatat 841 gtatacttga gctggattag ttgggttttg gatggagtaa ctttctgcag actgcaacat 901 tctgacacac gtagcagcac aaaagagttg cgaacaaact tggactgtta acatgtcaac 961 gcataaaact gaaaaaaaaa acctgtcaaa atgcataata aataaaactg aaaaaaaata 1021 agaataaatg ttgagagtgg gatttgaacc cacgcccttt cggaccagaa ccttaatctg 1081 gcgccttaga ccaactcggc catctcaact ttttgctctg tcatccaaac aaagttataa 1141 gaaatcatat aataataact aagacttgat gcctcagtag tttagttaaa ctaatttgaa 1201 tttgttagta cagtttgcat ttcaaattgt tccaatttgg acgccacggc tggtttcagt 1261 tgctcacgac gcctcacaca catattttgc ttccttgctt gtgacactag ggcacaaaac 1321 tccaacactc aaacgacact tcacgcatct ctcctgaaat cttgcacccc ccaactctgc 1381 atcgtcgcgt ataaaatgca gaccaaaccc cagctcaact ctgcatcatc atcatcaact 1441 cgcgcggatc cccatggcac aggttatcaa cacgtttgac ggggttgcgg attatcttca 1501 gacatatcat aagctacctg ataattacat tacaaaatca gaagcacaag ccctcggctg 1561 ggtggcatca aaagggaacc ttgcagacgt cgctccgggg aaaagcatcg gcggagacat 1621 cttctcaaac agggaaggca aactcccggg caaaagcgga cgaacatggc gtgaagcgga 1681 tattaactat acatcaggct tcagaaattc agaccggatt ctttactcaa gcgactggct 1741 gatttacaaa acaacggacc attatcagac ctttacaaaa atcagataac gaaaaaaacg 1801 gcttccctgc gggaggccgt ttttttcagc tttacataaa gtgtgtaata aatttttctt 1861 caaactctga tcggtcaatt tcactttccg gcgagctcga atttccccga tcgttcaaac 1921 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 1981 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 2041 atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 2101 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 2161 cgggaattcc tcgagtctag aggagcatgc acttgtttat tgcaaagaat ggtgcgtagg 2221 gaacacgcat gatttttgaa ttgctggcac ataattttat cattagaaac tggaatgcaa 2281 catgtaccct ttgtcatggt ttctttccga gacattgcac tgtttttttt aatcctatca 2341 ttatcataat gccaagaact ggtcaccaac cagcattttg catcatggtt agttgagctg 2401 tccccatgta tcaataggtg cattgtattg gtccaaatat aaatgcagtg gatgcaacct 2461 atctcatggc cgtcaacaaa gaaatcaaaa gggaaatgca ccatcttata tctccagttt 2521 atatgaacag attggataag atcataagat caagtggttt atattatttt gaggaatata 2581 acatggattc atcctaatca ctcgtctagg cagtatgtgt attcatgatg gatatggtac 2641 tatactacgg agttttttct tcacaaaata acctgttatt ttgacctcca accaaacacg 2701 aattatacca aaaattgggt tatttcatct atagtacaac tctattataa acatgcagta 2761 aattatccta cacatatacc aaaattcaag tgtaataatc ctaatacaca gacttaaaaa 2821 acaaactatt tectifitaa gaaaaggaaa accattifit taacggaagg aaaacaaatt 2881 cgggtcaagg cggaagccag cgcgccaccc cacgtcagcg aatacggagg cgcggggttg 2941 acggcgtcac ccggtcctaa cggcgaccaa caaaccagcc agaagaaatt acagtaaaaa 3001 aaagtaaatt gcactttgat ccacctttta ttacccaagt ttcaatttgg accaccctta 3061 aacctatctt ttcaaattgg gccgggttgt ggtttggact accatgaaca acttttcgtc 3121 atgtctaact tccctttcgg caaacatatg aaccatatat agaggagatc ggccgtatac 3181 tagagctgat gtgtttaagg tcgttgattg cacgagaaaa aaaaatccaa atcgcaacaa 3241 tagcaaattt atctagttca aagtgaaaag atatgtttaa aggtagtcca aagtaaaact 3301 taggggctgt ttggttccca gccatacttt accattactt gccaacaaaa gttgccacac 3361 cttatctaag gtgaggtgat caaattgtta gccacaactt actaagccta agggaatctt 3421 gccacacttt tttgagccat tgacacgtgg gacttaattt gttagaggga aatcttgcca 3481 caactgtggc tacaaccaaa cacctgtcaa atttgcctaa ccttaggcgt ggcaaactgt 3541 ggcaaagtgt ggcttacaac caaacacacc cttagataat aaaatgtggt ccaaagcgta 3601 attcactaaa aaaaaatcaa cgagacgtgt accaaacgga gacaaacggc atcttctcga 3661 aatttcccaa ccgctcgctc gcccgcctcg tcttcccgga aaccgcggtg gtttcagcgt 3721 ggcggattct ccaagcagac ggagacgtca cggcacggga ctcctcccac cacccaaccg 3781 ccataaatac cagccccctc atctcctctc ctcgcatcag ctccaccccc gaaaaatttc 3841 tccccaatct cgcgaggctc tcgtcgtcga atcgaatcct ctcgcgtcct caaggtacgc 3901 tgcttctcct ctcctcgctt cgtttcgatt cgatttcgga cgggtgaggt tgttttgttg 3961 ctagatccga ttggtggtta gggttgtcga tgtgattatc gtgagatgtt taggggttgt 4021 agatctgatg gttgtgattt gggcacggtt ggttcgatag gtggaatcgt ggttaggttt 4081 tgggattgga tgttggttct gatgattggg gggaattttt acggttagat gaattgttgg 4141 atgattcgat tggggaaatc ggtgtagatc tgttggggaa ttgtggaact agtcatgcct 4201 gagtgattgg tgcgatttgt agcgtgttcc atcttgtagg ccttgttgcg agcatgttca 4261 gatctactgt tccgctcttg attgagttat tggtgccatg ggttggtgca aacacaggct 4321 ttaatatgtt atatctgttt tgtgtttgat gtagatctgt agggtagttc ttcttagaca 4381 tggttcaatt atgtagcttg tgcgtttcga tttgatttca tatgttcaca gattagataa 4441 tgatgaactc ttttaattaa ttgtcaatgg taaataggaa gtcttatcgc tatatctgtc 4501 ataatgatct catgttacta tctgccagta attttatgct aagaactata ttagaatatc 4561 atgttacaat ctgtagtaat atcatgttac aatctgtagt tcatctatat aatctattgt 4621 ggtaatttct ttttactatc tgtgtgaaga ttattgccac tagttcattc tacttatttc 4681 tgaagttcag gatacgtgtg ctgttactac ctatctgaat acatgtgtga tgtgcctgtt 4741 actatctttt tgaatacatg tatgttctgt tggaatatgt ttgctgtttg atccgttgtt 4801 gtgtccttaa tcttgtgcta gttcttaccc tatctagagg atccccatca tcggtaacca 4861 ccccgcccct ctcctctttc tttctccgtt ttttttttcc gtctcggtct cgatctttgg 4921 ccttggtagt ttgggtgggc gagaggcggc ttcgtgcgcg cccagatcgg tgcgcgggag 4981 gggcgggatc tcgcggctgg ggctctcgcc ggcgtggatc cggcccggat ctcgcgggga 5041 atggggctct cggatgtaga tctgcgatcc gccgttgttg ggggagatga tggggggttt 5101 aaaatttccg ccatgctaaa caagatcagg aagaggggaa aagggcacta tggtttatat 5161 ttttatatat ttctgctgct tcgtcaggct tagatgtgct agatctttct ttcttctttt 5221 tgtgggtaga atttgaatcc ctcagcattg ttcatcggta gtttttcttt tcatgatttg 5281 tgacaaatgc agcctcgtgc ggagcifitt tgtaggtaga cgatccccgg ggatctacca 5341 tgagcccaga acgacgcccg gccgacatcc gccgtgccac cgaggcggac atgccggcgg 5401 tctgcaccat cgtcaaccac tacatcgaga caagcacggt caacttccgt accgagccgc 5461 aggaaccgca ggagtggacg gacgacctcg tccgtctgcg ggagcgctat ccctggctcg 5521 tcgccgaggt ggacggcgag gtcgccggca tcgcctacgc gggcccctgg aaggcacgca 5581 acgcctacga ctggacggcc gagtcgaccg tgtacgtctc cccccgccac cagcggacgg 5641 gactgggctc cacgctctac acccacctgc tgaagtccct ggaggcacag ggcttcaaga 5701 gcgtggtcgc tgtcatcggg ctgcccaacg acccgagcgt gcgcatgcac gaggcgctcg 5761 gatatgcccc ccgcggcatg ctgcgggcgg ccggcttcaa gcacgggaac tggcatgacg 5821 tgggtttctg gcagctggac ttcagcctgc cggtaccgcc ccgtccggtc ctgcccgtca 5881 ccgagatctg atgacccgaa tttccccgat cgttcaaaca tttggcaata aagtttctta 5941 agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 6001 aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 6061 agagtcccgc aattatacat ttaatacgcg atagaa SEQ ID NO: 6 is a nucleic acid sequence ofthe cloning vector for pSB11 LOCUS AB027256 6323 bp DNA linear SYN 01-FEB-2005 DEFINITION Cloning vector pSB11 genes for streptomycin/spectinomycin nucleotydyltransferase, streptothricin acetyltransferase 3', cds.

VERSION AB027256.2 GI:58430930 KEYWORDS streptothricin acetyltransferase 3'; streptomycin/spectinomycin nucleotydyltransferase; aadA.
SOURCE Cloning vector pSB11 ORGANISM Cloning vector pSB11 other sequences; artificial sequences; vectors.

AUTHORS Komari,T., Hiei,Y., Saito,Y., Murai,N. and Kumashiro,T.
TITLE Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers JOURNAL Plant J. 10 (1), 165-174 (1996) REFERENCE 2 (bases 1 to 6323) AUTHORS Komari,T., Kuraya,Y., Murai,N., Ueki,J. and Saito,Y.
TITLE Direct Submission JOURNAL Submitted (10-MAY-1999) Jun Ueki, Japan Tobacco Inc., Plant Innovation Center; 700 Higashibara, Iwata, Shizuoka 438-0802, Japan (E-mail:jun.ueki@ims.jti.co.jp, Tel: 81 -538-32-7120, Fax:81-538-33-6046) COMMENT On Feb 2, 2005 this sequence version replaced gi:4930302.
FEATURES Location/Qualifiers source 1..
6323/organism=" Cloning vector pSB11"/mol type="other DNA"/db xref="taxon:94004"misc feature 1..51/note="multiple cloning sites"repeat region 104..128/note="left border repeat"gene complement(1304..2092) /gene="aadA" CDS
complement(1304..2092) /gene=" aadA"/co don start=ltransl tab le=l1pro duct=" streptomycin/sp ectinomycin "protein id="BAA78030.1"db xref="GI:4930303"
translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSD
IDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQ
DLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAM
ERLPAQYQPVILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
CDS
complement(2150..2674) /co don start=ltransl tab le=11/pro duct=" streptothricin acetyltransferase3'/protein id="BAA78031.1"/db xref="GI:4930304"
/translation="MKISVIPEQVAETLDAENHFIVREVFDVHLSDQGFELSTRSVSPYRK
DYISDDDSDEDSACYGAFIDQELVGKIELNSTWNDLASIEHIVVSHTHRGKGVAH
SLIEFAKKWALSRQLLGIRLETQTNNVPACNLYAKCGFTLGGIDLFTYKTRPQVS
NETAMYWYWFSGAQDDA"
ORIGIN
1 aagcttgcat gcctcgagtc tagaggatcc ccgggtaccg agctcgaatt cagtacatta 61 aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa tttgtttaca ccacaatata 121 tcctgccacc agccagccaa cagctccccg accggcagct cggcacaaaa tcaccactcg 181 atacaggcag cccatcagtc cgggacggcg tcagcgggag agccgttgta aggcggcaga 241 ctttgctcat gttaccgatg ctattcggaa gaacggcaac taagctgccg ggtttgaaac 301 acggatgatc tcgcggaggg tagcatgttg attgtaacga tgacagagcg ttgctgcctg 361 tgatcaaata tcatctccct cgcagagatc cgaattatca gccttcttat tcatttctcg 421 cttaaccgtg acaggctgtc gatcttgaga actatgccga cataatagga aatcgctgga 481 taaagccgct gaggaagctg agtggcgcta tttctttaga agtgaacgtt gacgatcgtc 541 gaccgtaccc cgatgaatta attcggacgt acgttctgaa cacagctgga tacttacttg 601 ggcgattgtc atacatgaca tcaacaatgt acccgtttgt gtaaccgtct cttggaggtt 661 cgtatgacac tagtggttcc cctcagcttg cgactagatg ttgaggccta acattttatt 721 agagagcagg ctagttgctt agatacatga tcttcaggcc gttatctgtc agggcaagcg 781 aaaattggcc atttatgacg accaatgccc cgcagaagct cccatctttg ccgccataga 841 cgccgcgccc cccttttggg gtgtagaaca tccttttgcc agatgtggaa aagaagttcg 901 ttgtcccatt gttggcaatg acgtagtagc cggcgaaagt gcgagaccca tttgcgctat 961 atataagcct acgatttccg ttgcgactat tgtcgtaatt ggatgaacta ttatcgtagt 1021 tgctctcaga gttgtcgtaa tttgatggac tattgtcgta attgcttatg gagttgtcgt 1081 agttgcttgg agaaatgtcg tagttggatg gggagtagtc atagggaaga cgagcttcat 1141 ccactaaaac aattggcagg tcagcaagtg cctgccccga tgccatcgca agtacgaggc 1201 ttagaaccac cttcaacaga tcgcgcatag tcttccccag ctctctaacg cttgagttaa 1261 gccgcgccgc gaagcggcgt cggcttgaac gaattgttag acattatttg ccgactacct 1321 tggtgatctc gcctttcacg tagtgaacaa attcttccaa ctgatctgcg cgcgaggcca 1381 agcgatcttc ttgtccaaga taagcctgcc tagcttcaag tatgacgggc tgatactggg 1441 ccggcaggcg ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta 1501 ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt 1561 cgggcggcga gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag 1621 gaaccggatc aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg 1681 cttttgtcag caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa 1741 gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg 1801 gaatgatgtc gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc 1861 caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa 1921 gccttacggt caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca 1981 ctgcggagcc gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc 2041 caactacctc tgatagttga gtcgatactt cggcgatcac cgcttccctc atgatgttta 2101 actcctgaat taagccgcgc cgcgaagcgg tgtcggcttg aatgaattgt taggcgtcat 2161 cctgtgctcc cgagaaccag taccagtaca tcgctgtttc gttcgagact tgaggtctag 2221 ttttatacgt gaacaggtca atgccgccga gagtaaagcc acattttgcg tacaaattgc 2281 aggcaggtac attgttcgtt tgtgtctcta atcgtatgcc aaggagctgt ctgcttagtg 2341 cccacttttt cgcaaattcg atgagactgt gcgcgactcc tttgcctcgg tgcgtgtgcg 2401 acacaacaat gtgttcgata gaggctagat cgttccatgt tgagttgagt tcaatcttcc 2461 cgacaagctc ttggtcgatg aatgcgccat agcaagcaga gtcttcatca gagtcatcat 2521 ccgagatgta atccttccgg taggggctca cacttctggt agatagttca aagccttggt 2581 cggataggtg cacatcgaac acttcacgaa caatgaaatg gttctcagca tccaatgttt 2641 ccgccacctg ctcagggatc accgaaatct tcatatgacg cctaacgcct ggcacagcgg 2701 atcgcaaacc tggcgcggct tttggcacaa aaggcgtgac aggtttgcga atccgttgct 2761 gccacttgtt aacccttttg ccagatttgg taactataat ttatgttaga ggcgaagtct 2821 tgggtaaaaa ctggcctaaa attgctgggg atttcaggaa agtaaacatc accttccggc 2881 tcgatgtcta ttgtagatat atgtagtgta tctacttgat cgggggatct gctgcctcgc 2941 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc 3001 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg 3061 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt 3121 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 3181 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac 3241 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 3301 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 3361 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 3421 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 3481 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 3541 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 3601 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 3661 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 3721 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 3781 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 3841 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 3901 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 3961 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 4021 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 4081 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 4141 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 4201 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 4261 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 4321 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 4381 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 4441 gttaatagtt tgcgcaacgt tgttgccatt gctgcagggg gggggggggg ggggttccat 4501 tgttcattcc acggacaaaa acagagaaag gaaacgacag aggccaaaaa gctcgctttc 4561 agcacctgtc gtttcctttc ttttcagagg gtattttaaa taaaaacatt aagttatgac 4621 gaagaagaac ggaaacgcct taaaccggaa aattttcata aatagcgaaa acccgcgagg 4681 tcgccgcccc gtaacctgtc ggatcaccgg aaaggacccg taaagtgata atgattatca 4741 tctacatatc acaacgtgcg tggaggccat caaaccacgt caaataatca attatgacgc 4801 aggtatcgta ttaattgatc tgcatcaact taacgtaaaa acaacttcag acaatacaaa 4861 tcagcgacac tgaatacggg gcaacctcat gtcccccccc ccccccccct gcaggcatcg 4921 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 4981 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 5041 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 5101 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 5161 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata 5221 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 5281 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 5341 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 5401 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 5461 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 5521 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 5581 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 5641 cgaggccctt tcgtcttcaa gaattggtcg acgatcttgc tgcgttcgga tattttcgtg 5701 gagttcccgc cacagacccg gattgaaggc gagatccagc aactcgcgcc agatcatcct 5761 gtgacggaac tttggcgcgt gatgactggc caggacgtcg gccgaaagag cgacaagcag 5821 atcacgcttt tcgacagcgt cggatttgcg atcgaggatt tttcggcgct gcgctacgtc 5881 cgcgaccgcg ttgagggatc aagccacagc agcccactcg accttctagc cgacccagac 5941 gagccaaggg atctttttgg aatgctgctc cgtcgtcagg ctttccgacg tttgggtggt 6001 tgaacagaag tcattatcgc acggaatgcc aagcactccc gaggggaacc ctgtggttgg 6061 catgcacata caaatggacg aacggataaa ccttttcacg ccettttaaa tatccgatta 6121 ttctaataaa cgctctific tcttaggttt acccgccaat atatcctgtc aaacactgat 6181 agtttaaact gaaggcggga aacgacaatc tgatcatgag cggagaatta agggagtcac 6241 gttatgaccc ccgccgatga cgcgggacaa gccgttttac gtttggaact gacagaaccg 6301 caacgttgaa ggagccactc age.
SEQ.ID. NO 7 Switchgrass transgene conferring herbicide resistance.
REFERENCE 1 (bases 1 to 6245) COMMENT Bankit Comment: Vecscreen Comment:Submitter says that this sequencerepresents a Cloning Vector. Bankit Comment: TAX: No, not new species/combinations;

Bankit Comment: TOTAL # OF SEQS:7. Assembly Method :: DNASTAR SeqMan v.
12.1S equencing Technology : : Applied Biosystems 3500x1 Genetic AnalyzerAssembly-Data-ENDFEATURES
Lo cation/Qualifiers sourc el.. 6245/note='T-DNA Right border (BR)'gene 229..1442/gene='RTS2'promoter229..1442/gene='RTS2'/note='RTS2Promoter' 1454 ..1892gene='barnase'CDS1454 ..1789/gene='barnase'/codon start=1/producWbarnase 7translation='MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTT
DHYQTFTKIR' 1893 ..2169/note='no sterminatoegene2185 ..4835/gene='RUBQ2'promoter 2185..4835/gene='RUBQ27note='RUBQ2 Promoter'gene 4852..5324/gene='Os Actin 1 intron (japonica)'intron4852..5324/gene='OsActinlintronjaponica)'gene 5326..5898/gene='phosphinothricin N-acetyltransferase'CDS5340..5891/gene='phosphinothricin N-acetyltransferase'note='Herbicideresistanceselection markee/codon start=1/product='phosphinothricinNacetyltransferase/translation='MSPER
RPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQEWTDDLVRLRERYPWL
VAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGSTLYTHLLKS
LEAQ GFKSVVAVIGLPNDP SVRMHEALGYAPRGMLRAAGFKHGNWHDVGFWQ
LDFSLPVPPRPVLPVTEI' terminator 5899..6163/note='nos terminator'misc feature 6221..6245/note='T-DNA
left border (BL)'BASE COUNT 1630 a 1398 c 1459 g 1758 t ORIGIN
1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 61 aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 121 acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagcaag 181 cttgcatgcc tgcaggtcga ctctagagga tccgcgcgcg gatccgcgca ccggcgaggc 241 ggtgcgtctc ctcggagatg tggtagaagc tggcgcccca tttcatggcg gcgagcgggc 301 cgggcggcct cctgccgcag cgggattcgg tggctagacc gatctgtggg tggaggacgg 361 ggacgaggta gtggagacag aggcggcatt ggaagagggg aagaggagga ggaagtggtg 421 gcaggcagag gcggatgagg aacttgcgcc agcgacgtgg atatggaggg ggcgacggca 481 atggggaggc ggcgatggaa gcgaggagat gggcaggcgg cggaggcagc ggtggatttt 541 ttttttcttt ttctttttcg gaccctttca cctgctcggt gattcttctt ttttatacag 601 cacgacggct tctcctattc acgacgcctc ggctggacca tggaccgttg gccactggag 661 cattcttcca tgatctagat tttttttttc actcaacttt actacttcac atctgatggc 721 tggtgttgaa ttcattgtgc atccaacggt cattattaaa ttgatgacgt ggcgcaatga 781 ggtgacgaaa cactttactt tttttactac tttagatctg tcggcaggag tcccagatat 841 gtatacttga gctggattag ttgggttttg gatggagtaa ctttctgcag actgcaacat 901 tctgacacac gtagcagcac aaaagagttg cgaacaaact tggactgtta acatgtcaac 961 gcataaaact gaaaaaaaaa acctgtcaaa atgcataata aataaaactg aaaaaaaata 1021 agaataaatg ttgagagtgg gatttgaacc cacgcccttt cggaccagaa ccttaatctg 1081 gcgccttaga ccaactcggc catctcaact ttttgctctg tcatccaaac aaagttataa 1141 gaaatcatat aataataact aagacttgat gcctcagtag tttagttaaa ctaatttgaa 1201 tttgttagta cagtttgcat ttcaaattgt tccaatttgg acgccacggc tggtttcagt 1261 tgctcacgac gcctcacaca catattttgc ttccttgctt gtgacactag ggcacaaaac 1321 tccaacactc aaacgacact tcacgcatct ctcctgaaat cttgcacccc ccaactctgc 1381 atcgtcgcgt ataaaatgca gaccaaaccc cagctcaact ctgcatcatc atcatcaact 1441 cgcgcggatc cccatggcac aggttatcaa cacgtttgac ggggttgcgg attatcttca 1501 gacatatcat aagctacctg ataattacat tacaaaatca gaagcacaag ccctcggctg 1561 ggtggcatca aaagggaacc ttgcagacgt cgctccgggg aaaagcatcg gcggagacat 1621 cttctcaaac agggaaggca aactcccggg caaaagcgga cgaacatggc gtgaagcgga 1681 tattaactat acatcaggct tcagaaattc agaccggatt ctttactcaa gcgactggct 1741 gatttacaaa acaacggacc attatcagac ctttacaaaa atcagataac gaaaaaaacg 1801 gcttccctgc gggaggccgt ttttttcagc tttacataaa gtgtgtaata aatttttctt 1861 caaactctga tcggtcaatt tcactttccg gcgagctcga atttccccga tcgttcaaac 1921 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 1981 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 2041 atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 2101 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 2161 cgggaattcc tcgagtctag aggagcatgc acttgtttat tgcaaagaat ggtgcgtagg 2221 gaacacgcat gatttttgaa ttgctggcac ataattttat cattagaaac tggaatgcaa 2281 catgtaccct ttgtcatggt ttctttccga gacattgcac tgtttttttt aatcctatca 2341 ttatcataat gccaagaact ggtcaccaac cagcattttg catcatggtt agttgagctg 2401 tccccatgta tcaataggtg cattgtattg gtccaaatat aaatgcagtg gatgcaacct 2461 atctcatggc cgtcaacaaa gaaatcaaaa gggaaatgca ccatcttata tctccagttt 2521 atatgaacag attggataag atcataagat caagtggttt atattatttt gaggaatata 2581 acatggattc atcctaatca ctcgtctagg cagtatgtgt attcatgatg gatatggtac 2641 tatactacgg agttttttct tcacaaaata acctgttatt ttgacctcca accaaacacg 2701 aattatacca aaaattgggt tatttcatct atagtacaac tctattataa acatgcagta 2761 aattatccta cacatatacc aaaattcaag tgtaataatc ctaatacaca gacttaaaaa 2821 acaaactatt tectifitaa gaaaaggaaa accattifit taacggaagg aaaacaaatt 2881 cgggtcaagg cggaagccag cgcgccaccc cacgtcagcg aatacggagg cgcggggttg 2941 acggcgtcac ccggtcctaa cggcgaccaa caaaccagcc agaagaaatt acagtaaaaa 3001 aaagtaaatt gcactttgat ccacctttta ttacccaagt ttcaatttgg accaccctta 3061 aacctatctt ttcaaattgg gccgggttgt ggtttggact accatgaaca acttttcgtc 3121 atgtctaact tccctttcgg caaacatatg aaccatatat agaggagatc ggccgtatac 3181 tagagctgat gtgtttaagg tcgttgattg cacgagaaaa aaaaatccaa atcgcaacaa 3241 tagcaaattt atctagttca aagtgaaaag atatgtttaa aggtagtcca aagtaaaact 3301 taggggctgt ttggttccca gccatacttt accattactt gccaacaaaa gttgccacac 3361 cttatctaag gtgaggtgat caaattgtta gccacaactt actaagccta agggaatctt 3421 gccacacttt tttgagccat tgacacgtgg gacttaattt gttagaggga aatcttgcca 3481 caactgtggc tacaaccaaa cacctgtcaa atttgcctaa ccttaggcgt ggcaaactgt 3541 ggcaaagtgt ggcttacaac caaacacacc cttagataat aaaatgtggt ccaaagcgta 3601 attcactaaa aaaaaatcaa cgagacgtgt accaaacgga gacaaacggc atcttctcga 3661 aatttcccaa ccgctcgctc gcccgcctcg tcttcccgga aaccgcggtg gtttcagcgt 3721 ggcggattct ccaagcagac ggagacgtca cggcacggga ctcctcccac cacccaaccg 3781 ccataaatac cagccccctc atctcctctc ctcgcatcag ctccaccccc gaaaaatttc 3841 tccccaatct cgcgaggctc tcgtcgtcga atcgaatcct ctcgcgtcct caaggtacgc 3901 tgcttctcct ctcctcgctt cgtttcgatt cgatttcgga cgggtgaggt tgttttgttg 3961 ctagatccga ttggtggtta gggttgtcga tgtgattatc gtgagatgtt taggggttgt 4021 agatctgatg gttgtgattt gggcacggtt ggttcgatag gtggaatcgt ggttaggttt 4081 tgggattgga tgttggttct gatgattggg gggaattttt acggttagat gaattgttgg 4141 atgattcgat tggggaaatc ggtgtagatc tgttggggaa ttgtggaact agtcatgcct 4201 gagtgattgg tgcgatttgt agcgtgttcc atcttgtagg ccttgttgcg agcatgttca 4261 gatctactgt tccgctcttg attgagttat tggtgccatg ggttggtgca aacacaggct 4321 ttaatatgtt atatctgttt tgtgtttgat gtagatctgt agggtagttc ttcttagaca 4381 tggttcaatt atgtagcttg tgcgtttcga tttgatttca tatgttcaca gattagataa 4441 tgatgaactc ttttaattaa ttgtcaatgg taaataggaa gtcttatcgc tatatctgtc 4501 ataatgatct catgttacta tctgccagta attttatgct aagaactata ttagaatatc 4561 atgttacaat ctgtagtaat atcatgttac aatctgtagt tcatctatat aatctattgt 4621 ggtaatttct ttttactatc tgtgtgaaga ttattgccac tagttcattc tacttatttc 4681 tgaagttcag gatacgtgtg ctgttactac ctatctgaat acatgtgtga tgtgcctgtt 4741 actatctttt tgaatacatg tatgttctgt tggaatatgt ttgctgtttg atccgttgtt 4801 gtgtccttaa tcttgtgcta gttcttaccc tatctagagg atccccatca tcggtaacca 4861 ccccgcccct ctcctctttc tttctccgtt ttttttttcc gtctcggtct cgatctttgg 4921 ccttggtagt ttgggtgggc gagaggcggc ttcgtgcgcg cccagatcgg tgcgcgggag 4981 gggcgggatc tcgcggctgg ggctctcgcc ggcgtggatc cggcccggat ctcgcgggga 5041 atggggctct cggatgtaga tctgcgatcc gccgttgttg ggggagatga tggggggttt 5101 aaaatttccg ccatgctaaa caagatcagg aagaggggaa aagggcacta tggtttatat 5161 ttttatatat ttctgctgct tcgtcaggct tagatgtgct agatctttct ttcttctttt 5221 tgtgggtaga atttgaatcc ctcagcattg ttcatcggta gtttttcttt tcatgatttg 5281 tgacaaatgc agcctcgtgc ggagcttttt tgtaggtaga cgatccccgg ggatctacca 5341 tgagcccaga acgacgcccg gccgacatcc gccgtgccac cgaggcggac atgccggcgg 5401 tctgcaccat cgtcaaccac tacatcgaga caagcacggt caacttccgt accgagccgc 5461 aggaaccgca ggagtggacg gacgacctcg tccgtctgcg ggagcgctat ccctggctcg 5521 tcgccgaggt ggacggcgag gtcgccggca tcgcctacgc gggcccctgg aaggcacgca 5581 acgcctacga ctggacggcc gagtcgaccg tgtacgtctc cccccgccac cagcggacgg 5641 gactgggctc cacgctctac acccacctgc tgaagtccct ggaggcacag ggcttcaaga 5701 gcgtggtcgc tgtcatcggg ctgcccaacg acccgagcgt gcgcatgcac gaggcgctcg 5761 gatatgcccc ccgcggcatg ctgcgggcgg ccggcttcaa gcacgggaac tggcatgacg 5821 tgggtttctg gcagctggac ttcagcctgc cggtaccgcc ccgtccggtc ctgcccgtca 5881 ccgagatctg atgacccgaa tttccccgat cgttcaaaca tttggcaata aagtttctta 5941 agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 6001 aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 6061 agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 6121 gataaattat cgcgcgcggt gtcatctatg ttactagatc gggaattcag tacattaaaa 6181 acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacacca caatatatcc 6241 tgcca '-Abbreviations and Terms The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. For example, reference to "a transgenic plant"
includes one or a plurality of such plants, and reference to "the floral-specific promoter"
includes reference to one or more floral-specific promoters or their homologues and equivalents thereof known to those skilled in the art, and so forth.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
ACP: Atlantic Coastal Panicgrass (Panicum amarum) Allele: One of the different forms of a gene that can exist at a single locus Anther-specific gene: A gene sequence that is primarily expressed in the anther, relative to expression in other plant tissues. Includes any anther-specific gene whose malfunction or functional deletion results in male-sterility. Examples include, but are not limited to: anther-specific gene from tobacco (GenBank Accession Nos. AF376772-AF376774), and Osg4B and Osg6B (GenBank Accession Nos. D21159 and 21160).
Anther-specific promoter: A DNA sequence that directs a higher level of transcription of an associated gene in anther tissue relative to the other tissues of the plant. Examples include, but are not limited to: anther-specific gene promoter from tobacco (GenBank Accession Nos. AF376772-AF376774), and the promoters of Osg4B and Osg6B
(GenBank Accession Nos. D21159 and D21160).
Asexual: A plant lacking floral structures such that it is incapable of participating either as a male or female parent in sexual reproduction and propagates vegetatively.
Bridge intermediate: refers to a genetic bridge for importing genes into hybrids providing a mechanism for importing any new genes not found in common breeding program materials, and any de novo genetic material that arises from these wide varietal, species or genera crosses using traditional plant breeding techniques.
Comprises: A term that means "including." For example, "comprising A or B"
means including A or B, or both A and B, unless clearly indicated otherwise.
Deletion: The removal of a sequence of a nucleic acid, for example DNA, the regions on either side being joined together.
Desirable trait: A characteristic which is beneficial to a plant, such as a commercially desirable, agronomically important trait. Examples include, but are not limited to: resistance to insects and other pests and disease-causing agents (such as viral, bacterial, fungal, and nematode agents); tolerance or resistance to herbicides; enhanced stability;
increased yield or shelf-life; environmental tolerances (such as tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress, or oxidative stress); male sterility; and nutritional enhancements (such as starch quantity and quality; oil quantity and quality;
protein quality and quantity; amino acid composition; and the like). On one example, a desirable trait is selected for through conventional breeding. In another example, a desirable trait is obtained by transfecting the plant with a transgene(s) encoding one or more genes that confer the desirable trait to the plant.
Egg: In seed plants an egg is an ovum (plural; ova, from t ovum meaning egg or egg cell) is a haploid female reproductive cell or gamete.
Floral deficient: A plant that is lacking, or is functionally deficient in, one or several parts of the male or female structures contained within a single flower or inflorescence effectively resulting in either male or female sterility.
Floral-specific gene: gene sequence that is primarily expressed in floral tissue or during the transition from a vegetative to floral meristem, such as the tapetum, anther, ovule, style, or stigma, relative to the other tissues of the plant. Includes any floral-specific gene whose malfunction or functional deletion results in sterility of the plant either directly or by preventing fertilization of gametes through floral deficiencies.
Floral-specific promoter: A DNA sequence that directs a higher level of transcription of an associated gene in floral tissues or during the transition from vegetative to floral meristem relative to the other tissues of the plant. Examples include, but are not limited to:
meristem transition-specific promoters, floral meristem-specific promoters, anther-specific promoters, pollen-specific promoters, tapetum-specific promoters, ovule-specific promoters, megasporocyte-specific promoters, megasporangium-specific promoter-0, integument-specific promoters, stigma-specific promoters, and style-specific promoters.
In one example, floral-specific promoters include an embryo-specific promoter or a late embryo-specific promoter, such as the late embryo specific promoter of DNH 1 or the HVA1 promoter, the GLB1 promoter from corn, and any of the Zein promoters (Z27). In another example, floral-specific promoters include the FLO/LFY promoter from switchgrass.
The determination of whether a sequence operates to confer floral specific expression in a particular system (taking into account the plant species into which the construct is being introduced, the level of expression required, etc.), is preformed using known methods, such as operably linking the promoter to a marker gene (e.g. GUS, and GFP), introducing such constructs into plants and then determining the level of expression of the marker gene in floral and other plant tissues. Sub-regions which confer only or predominantly floral expression, are considered to contain the necessary elements to confer floral specific expression.
Functionally equivalent: Nucleic acid sequence alterations in a vector that yield the same results described herein. Such sequence alterations can include, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, and insertions.
For example, in a nucleic acid including a barnase sequence that is cytotoxic, a functionally equivalent barnase sequence may differ from the exact barnase sequences disclosed herein, but maintains its cytotoxic activity. Methods for determining such activity are disclosed herein.
GB S : Genotyping-By-S equencing Genetic markers: Alleles used as experimental probes to keep track of an individual, a tissue, a cell, a nucleus, a chromosome, or a gene GMO : Genetically Modified Organism Gene of interest: (GOI) Any gene, or combination of functional nucleic acid sequences (such as comprised in plant expression cassettes such as with a promoter, coding sequence and termination sequence) in plants that may result in a desired phenotype Genotype: The allelic composition of a cell--either of the entire cell or, more commonly, for a certain gene or a set of genes of an individual.
Hybrid plant: An individual plant produced by crossing two parents of different genotypes or germplasm backgrounds.
Intergeneric (literally between/among genera) describes relationships, mating, breeding, behaviors, biochemical variations and other issues between individuals of separate genus thereby contrasting with interspecific.
Interspecific (literally between/among species) describes relationships, mating, breeding, behaviors, biochemical variations and other issues between individuals of separate species thereby contrasting with intraspecific.
Intervarietal (literally between varieties, or cultivars) is a term used to describe relationships, mating, breeding, behaviors, biochemical variations and other issues between individuals of a single variety, thereby contrasting with interspecific Intraspecific (literally within species) is a term used to describe relationships, mating, breeding, behaviors, biochemical variations and other issues within individuals of a single species, thereby contrasting with interspecific Intravarietal (literally within varieties, or cultivars) is a term used to describe relationships, mating, breeding, behaviors, biochemical variations and other issues within individuals of a single variety, thereby contrasting with interspecific Isolated: An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids, proteins and peptides.
Locus: The place on a chromosome where a gene is located.
Molecular genetics: The study of the molecular processes underlying gene structure and function Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
Oligonucleotide: A linear polynucleotide (such as DNA or RNA) sequence of at least 9-350 nucleotides, for example at least 15, 18, 24, 25, 27, 30, 50, 100 or even 200 nucleotides long.
ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
Ovule: In seed plants, the ovule is the structure that gives rise to and contains the female reproductive cells. It consists of three parts: The integument(s) forming its outer layer(s), the nucellus, and the megaspore-derived female gametophyte (or megagametophyte) in its center. The megagametophyte (also called embryo sac in flowering plants) produces an egg cell (or several egg cells in some groups) for. After fertilization, the ovule develops into a seed.
Peptide: A chain of amino acids of which is at least 4 amino acids in length.
In one example, a peptide is from about 4 to about 30 amino acids in length, for example about 8 to about 25 amino acids in length, such as from about 9 to about 15 amino acids in length, for example about 9-10 amino acids in length.
Perennial: A plant which grows to floral maturity for three seasons or more.
Whereas annual plants sprout from seeds, grow, flower, set seed and senesce in one growing season, perennial plants persist for several growing seasons. The persistent seasonal flowering of perennial plants may also, but not necessarily, include light and temperature requirements that result in vernalization. Examples include, but are not limited to:
certain grasses, such as members of the Poacea, such as switchgrass (Panicum virgatum L. cv Alamo).
Andropogon sp., Panicum, sp., Pennisetum sp., Zea sp., Saccharum sp., Miscanthus sp., a Saccharum sp. x Miscanthus sp. hybrids, Erianthus sp., Tripsicum sp., or Zea X Tripiscum sp.
hybrids, also including species of turfgrass, forage grass or various ornamental grasses;
trees, including poplar, willow, eucalyptus, Paulownia and also trees broadly known such as fruit and nut, and crop trees (for example bananas and papayas), forest and ornamental trees, rubber plants, and shrubs; grapes; roses.
Plant breeding: The application of genetic analysis to development of plant lines better suited for human purposes Pollen-specific gene: A DNA sequence that directs a higher level of transcription of an associated gene in microspores and/or pollen (i.e., after meiosis) relative to the other tissues of the plant. Examples include, but are not limited to: pollen-specific promoters LAT52, LAT56, and LAT59 from tomato (GenBank Accession Nos. BG642507, X56487 and X56488), rice pollen specific gene promoter PSI (GenBank Accession No.
Z16402), and pollen specific promoter from corn (GenBank Accession No. BD136635 and BD136636).
Pollen-specific promoter: A gene sequence that is primarily expressed in pollen relative to the other cells of the plant. Includes any pollen-specific gene whose malfunction or functional deletion results in male-sterility. Examples include, but are not limited to: LAT52, LAT56, and LAT59 from tomato (GenBank Accession Nos. BG642507, X56487 and X56488), PSI (GenBank Accession No. Z16402), and pollen specific gene from corn (GenBank Accession No. BD136635 and BD136636).
Promoter: An array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA
element. A
promoter also optionally includes distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included.
Specific, non-limiting examples of promoters that can be used to practice the disclosed methods include, but are not limited to, a floral-specific promoter, constitutive promoters, as well as inducible promoters for example a heat shock promoter, a glucocorticoid promoter, and a chemically inducible promoter. Promoters produced by recombinant DNA or synthetic techniques may also be used. A polynucleotide encoding a protein can be inserted into an expression vector that contains a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence of the host. In one example, an expression vector contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
Probe: Defined nucleic acid segment that can be used to identify specific molecules bearing the complementary DNA or RNA sequence, usually through autoradiography, chemiluminescence or color detection.
RFLP: Refers to restriction fragment length polymorphism that is a specific DNA
sequence revealed as a band of particular molecular weight size on a Southern blot probed with a radiolabelled RFLP probe and is considered to be an allele of a gene.
Selectable marker: A nucleic acid sequence that confers a selectable phenotype, such as in plant cells, that facilitates identification of cells containing the nucleic acid sequence.
Transgenic plants expressing a selectable marker can be screened for transmission of the gene(s) of interest. Examples include, but are not limited to: genes that confer resistance to toxic chemicals (e.g. ampicillin, spectinomycin, streptomycin, kanamycin, geneticin, hygromycin, glyphosate or tetracycline resistance, as well as bar and pat genes which confer herbicide resistance), complement a nutritional deficiency (e.g., uracil, histidine, leucine), or impart a visually distinguishing characteristic (e.g., color changes or fluorescence, such as 13-gal).
Southern blot: Transfer of electrophoretically separated fragments of DNA from the gel to an absorbent surface such as paper or a membrane which is then immersed in a solution containing a labeled probe that will bind to homologous DNA sequences.
SD: Standard Deviation Tapetum-specific gene: A gene sequence that is primarily expressed in the tapetum relative to the other tissues of the plant. Includes any tapetum cell-specific gene whose malfunction results in male-sterility. Examples include, but are not limited to: TA29 and TA13, pca55, pEl and pT72, Bcpl from Brassica and Arabidopsis (GenBank Accession Nos.
X68209 and X68211), A9 from Brassicaceae (GenBank Accession No. A26204), and TAZ1, a tapetum-specific zinc finger gene from petunia (GenBank Accession No.
AB063169).
Tapetum-specific promoter: A DNA sequence that directs a higher level of transcription of an associated gene in tapetal tissue relative to the other tissues of the plant.
Tapetum is nutritive tissue required for pollen development. Examples include, but are not limited to the promoters associated with the genes listed under tapetum-specific genes.
Transduced and transformed: A virus or vector "transduces" or transfects" a cell when it transfers nucleic acid into the cell. A cell is "transformed" by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to, transfection with viral vectors, transformation with plasmid vectors, electroporation, lipofection, Agrobacterium-mediated transfer, direct DNA uptake, and microprojectile bombardment.
Transgene: An exogenous nucleic acid sequence. In one example, a transgene is a gene sequence, for example a sequence that encodes a cytotoxic polypeptide. In yet another example, the transgene is an antisense nucleotide, wherein expression of the antisense nucleotide inhibits expression of a target nucleic acid sequence. A transgene can contain native regulatory sequences operably linked to the transgene (e.g. the wild-type promoter, found operably linked to the gene in a wild-type cell). Alternatively, a heterologous promoter can be operably linked to the transgene.
Transgenic Cell: Transformed cells that contain a transgene, which may or may not be native to the cell.
Vector: A nucleic acid molecule as introduced into a cell, thereby producing a transformed cell. A vector can include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. Examples include, but are not limited to a plasmid, cosmid, bacteriophage, or virus that carries exogenous DNA into a cell. A
vector can also include one or more cytotoxic genes, antisense molecules, and/or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express the nucleic acids and/or proteins encoded by the vector. A
vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a, liposome, protein coating or the like.
Wild type: refers to a reference and it can mean an organism, set of genes, gene or nucleotide sequence. For purposes herein the wild type refers to the parents of hybrid progeny.
In light of the foregoing, it will now be appreciated by those skilled in the art that various changes may be made to the embodiment herein chosen for purposes of disclosure without departing from the inventive concept defined by the appended claims.
What is claimed is:

Claims (42)

What is claimed is:

1. A method of producing a hybrid plant system for breeding purposes using a transgenic plant as a intermediate to create a non-transgenic plant.

2. The method of claim 1, wherein the method uses a transgenic plant containing one or more selectable markers.

3. The method of claim 1, wherein the transgene functions for genome editing.

4. The method of claim 1, wherein the method produces a hybrid plant.

5. The method of claim 1, wherein the method produces a hybrid plant from intra-varietial parents.

6. The method of claim 1, wherein the method produces a hybrid plant from inter-varietial parents

7. The method of claim 1, wherein the method produces a hybrid plant from intra-specific parents.

8. The method of claim 1, wherein the method produces a hybrid plant from inter-specific parents

9. The method of claim 1, wherein the method produces a hybrid plant from intra-generic parents.

10. The method of claim 1, wherein the method produces a hybrid plant from inter-generic parents.

11. The method of claim 1, wherein the method produces a fertile F1 hybrid embryo, seed or plant.

12. The method of claim 10, wherein the method produces a fertile FI hybrid plant used to backcross to its non-transgenic compatible parent.

13. The method of claim 10, wherein the method produces a fertile hybrid plant used to outcross to a non-transgenic compatible parent.

14. The methods of claims 11 and 12, wherein the method produces a F2 population of mature hybrid seed, seedlings or plants used to screen for one or more selectable markers.

15. The method of claim 13, wherein the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals where all sequences of the transgene cassette have segregated from the genome.

16. The method of claim 14, wherein the hybrid F2 individuals where all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses.

17. The method of claim 15, wherein the subsequent crosses involve inter- or intra- varietal, specific or generic parents.

18. The method of claim 16, wherein the F3 hybrid progeny are used in subsequent crosses.

19. The method of claim 1, wherein the method produces a infertile F1 hybrid embryo, seed or hybrid plant.

20. The method of claim 18, wherein the method producing a F1 hybrid embryo, seed or plant is recovered to produce a fertile F1 embryo, seed or hybrid plant.

21. The method of claim 20, wherein the seed or plant is recovered to produce a fertile F1 embryo, see or hybrid plant using embryo rescue and chromosomal doubling using colchicine.

22. The method of claim 19, wherein the method produces a fertile F2 hybrid embryo, seed or hybrid plan used to backcross to its non-transgenic parent.

23. The method of claim 18, wherein the method produces a fertile F2 hybrid embryo, seed or plant used to outcross to a non-transgenic parent.

24. The methods of claims 20 or 22, wherein the method produces a F2 population of mature hybrid seed, seedlings or plants used to screen for one or more selectable markers.

25. The method of claim 23, wherein the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals where all sequences of the transgene cassette have segregated from the genome.

26. The method of claim 24, wherein the hybrid F2 individuals where all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses.

27. The method of claim 25, wherein the subsequent crosses involve inter- or intra- varietal, specific or generic parents.

28. The method of claim 26, wherein the F3 hybrid progeny are used in subsequent crosses.

29. The method of claim 18, wherein the method produces a infertile F1 hybrid embryo, seed or plant is vegetatively propagated as a sterile population for gene confinement.

30. The method of claim 10, wherein the method produces a fertile FI hybrid plant used to outcross to non-transgenic incompatible inter- or intra- varietal, specific or generic parents.

31. The methods of claim 29, wherein the method produces a F2 population of mature hybrid seed, seedlings or plants used to screen for one or more selectable markers.

32. The method of claim 30, wherein the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals where all sequences of the transgene cassette have segregated from the genome.

33. The method of claim 31, wherein the hybrid F2 individuals where all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses.

34. The method of claim 32, wherein the subsequent crosses involve inter- or intra- varietal, specific or generic parents.

35. The method of claim 33, wherein the F3 hybrid progeny are used in subsequent crosses

36. The method of claim 29, wherein the method producing a F2 hybrid embryo, seed or plant is recovered to produce a fertile embryo, seed or hybrid plant.

37. The method of claim 36, wherein the F2 hybrid embryo, seed or hybrid plant is recovered to prodce a fertile embryo, seed or hybrid plant using embryo rescue and chromosomal doubling using colchicine.

38. The method of claim 35, wherein the hybrid F2 population contains individuals containing the intact or partial fragments of the transgene cassette and individuals where all sequences of the transgene cassette have segregated from the genome.

39. The method of claim 36, wherein the hybrid F2 individuals where all sequences of the transgene cassette have segregated from its genome are used in subsequent crosses.

40. The method of claim 37, wherein the subsequent crosses involve inter- or intra- varietal, specific or generic parents.

41. The method of claim 38, wherein the F3 hybrid progeny are used in subsequent crosses.

42. The method of claim 29, wherein the method produces a infertile F1 hybrid embryo, seed or plant is vegetatively propagated as a sterile population.