CN1248296A - Means for identifying nucleotide sequences involved in apomixis - Google Patents
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Abstract
The invention concerns a method for identifying in Gramineae, more particularly in maize, a nucleotide sequence involved in the apomixis in apomictic plants. It is characterised in that it consists in: identifying in the genome of the Gramineae, by phenotypic analysis, genetic mapping and marking by means of transposons, of the meiotic mutations whereof the corresponding gene is shown to be orthologous to genes involved in the expression of apomixis. The invention also concerns the use of a cloned gene in the Gramineae to identify and isolate the orthologous gene sequence in apomictic plants. The invention further concerns the use or modification of the isolated sequence in apomictic forms for inducing an apomictic development in sexual plants.
Description
Target of the present invention is to differentiate, separate and identify the method for related nucleotide sequence in the apomixis.
The present invention relates more specifically to differentiate these sequences in the genome of apospecies, then with the Method and kit for of its separation and evaluation.
The invention still further relates to the transgenosis of utilizing these sequences to realize uses and the product that is obtained.
Under its modern notion, apomixis or agamospermy comprise through agamic all phenomenons of seed.In 300 kinds of angiosperms of 35 sections, apospecies have been found.Apomictic multi-form (generally only being used for female reproduction) grows with the parthenogenesis of ameiosis, the fertilization of no ossphere and embryo is feature.Apomixis thereby cause producing heredity and go up the filial generation identical with its parental generation plant.
With apomixis natural corresponding be that syngenesis or both sexes are merged.With apomixis antithesis, syngenesis comprises and relates to reduction division and process syngamous simultaneously.The homologous chromosomes that reduction division will be derived from parental generation randomly is assigned in the different gametes.Reduction division also allows to carry out reorganization between homologous chromosomes by exchange.The gamete cooperation is the fusion between gamete.Gamete cooperates to allow meets again in body one by one from the special combination of two parents' genetic information.Thereby the reorganization that both sexes are merged by the parental gene group produces the filial generation that uniqueness is gone up in heredity.
In the cycle of development of plants, people also observe two kinds of replacing of generation in succession, and division of their minuends and fertilization are separated.First is from generation to generation corresponding to asexual generation.One or more cells of sporophyte carry out reduction division, produce meiospore.Meiospore develops into gametophyte, and the latter represents gametophytic generation (originating in gamete).Gametogamy produces zygote, and this representative has turned back to asexual generation again.
In sexual angiosperm, female gamete body (embryo of silkworms capsule) develops into the multi-cellular structure very inequality with sporophyte (ovule).In the ovule growth course, a kind of special cell is two continuous stages of sporogonium experience: megaspore takes place (to form the megaspore or the meiospore of subtrahend, from sporogonium) and megagamete generation (forming the female gamete body from megaspore), the many cells gametophyte (ossphere) that contains single gamete produced.Nearly 80% angiosperm relates to this growth type (Polygonum type).Apomictic multi-form corresponding to a series of versions under this theme.
First kind of classification of apomixis according to the origin of embryo is two kinds of basic forms.For adventitious embryony, embryo directly comes from the megarchidium or the differentiation of integument somatocyte of ovule.Thereby there are not sporophyte and a gametophytic digenesis.And gametophytic apomixis is characterised in that the unreduced female gamete body of formation, and embryo is from the parthenogenesis sexual development of ossphere.Hereinafter, allly mention the equal assignment daughter of apomixis apomixis.
According to the source difference of female gamete body, gametophytic apomixis has two big classes.For the apospory form, unreduced embryo of silkworms capsule generally is a megarchidium from the somatocyte of ovule.For multiple sporulation form, the embryo of silkworms capsule is from sexual cell, i.e. sporogonium.The ameiosis reproduction comprises apospory and multiple sporulation simultaneously.In fact exist a large amount of different causing to form the process of unreduced gamete body.
In sexual angiosperm, male gametophyte (pollen granule) contains two kinds of sexual cell.A kind of ossphere fertilization generation embryo that makes, another kind combines with the nuclear of the centrocyte that is derived from endosperm.Embryo and endosperm thereby all be sexual.This is called double fertilization, is angiospermous characteristic feature.In most of apospecies, embryo is grown not having under the fertilization, and that endosperm still keeps is sexual.When centrocyte fertilization is endosperm development when necessary, be called false the cooperation or false conformability apomixis; All under no fertilization situation, grow when embryo and endosperm, then be called independently apomixis.
Say the actual monogony that is equivalent to through seed of apomixis in the embryo level.This is to know that by following the factor of identification draws jointly: ameiosis or forming embryo of silkworms capsule, parthenogenesis under the ameiotic situation or form embryo under the situation of ossphere nonfertilization.Ameiosis and parthenogenesis have guaranteed sporophyte and gametophytic digenesis, but do not have replacing of different IPs phase: sporophyte and gametophyte remain on the level of times body.
But in the plant level, apomixis is actually a kind of mixing mode of reproduction, comprises both sexes fusion and monogony.In fact, generally speaking apomixis is a kind of non-imposed phenomenon: it appears in " abnormal type " individual apospecies filial generation, i.e. heredity is gone up different with maternal plant.Grow for proper apomixis, need in conjunction with two conditions of subtrahend and nonfertilization not.If one or two condition can not satisfy, may produce abnormal type.According to the success or the failure of reduction division and fertilization, the filial generation possibility type in the non-imposed apospecies is as follows:
| Fertilization | There is not fertilization | |
| Reduction division | ????n+n | ????n+0 |
| Countless divisions | ????2n+n | ????2n+0 |
In the definition of the heterozygote of " n+n " or types such as " 2n+n ", the state of first assignment daughter, i.e. subtrahend (n) or not subtrahend (2n).Whether the existence of second reflection ossphere fertilization." 2n+0 " class is represented proper apomixis, and on behalf of both sexes, " n+n " class merge." 2n+n " class causes genomic accumulation, and " n+0 " class causes the haploidization of maternal plant.These separately different classes of ratios are different because of species, even also different between different plant in given kind.Specification sheets with the lower section, the apomixis of mentioning relates to these non-imposed apomixis forms, also relates to the mandatory apomixis form that only produces the filial generation of 2n+0 type.
Apomictic hereditary decision mechanism is also understood seldom.Recognize that now agamic angiosperm is derived from amphigenetic ancestors, a kind of reproductive forms will manifest hereditary decision mechanism to alternative transformation.Therefore, apomixis is the result that apomixis gene or allelotrope are expressed, and promptly exists in apospecies and expresses, but do not exist in sexual plant or do not have a function.
The genetically controlled overwhelming majority work of apomixis is related to apomeiosis, relate to apospory more.Now aposporous Mendelian inheritance principle there are enough common recognitions (document that sees reference (1) and (2), reference are listed in the last of specification sheets).People are familiar with seldom the multiple sporulation, but existing result (3,4) shows that received hypothesis can be used for multiple sporulation plant undoubtedly in the apospory plant.In these different models, relevant gene action mode is still a mystery.But these analyses tend to show that the gene of being responsible for apomeiosis can start whole apomixis program separately.
Because it improves the potentiality of plant, apomixis has caused the very big interest of people.Using apomixis on staple crop will be a kind of heterotic short-cut method of stablizing.This will be the potential revolution to the reproductive system working method.
Many programs that have been born its objective is evaluation " apomictic gene " and it are imported in crop.
The earliest may also be that state-of-the-art work is to utilize natural apospecies.Its corresponding allelotrope existence also has function.To the transfer of important crop or by the species hybridization between crop and apomictic marriage plant, or by separating corresponding gene to import in the target species ((5) by transgenosis subsequently; (6); (7) and (8)).
Another kind develops rapidly, and method is to produce apomixis by different mutafacient system in sexual plant.Arabidopis thaliana A.thalina is the most frequently used model ((9), (10) and (8)).Similar research also sees petunia (Petunia hybrida) (10) and Herba Hieracii Umbellati (Hieracium) (8).In all these examples, basic hypothesis is that this only relates to simple Genetic Control, and shifts or modify enough conditions that some allelotrope seldom can provide apomixis to express.Here apomixis is interpreted as the result of aforementioned incident: maiotic failure and parthenogenesis.
The work of the inventor in this field is sought them to belong in the species of (Tripsacum) (with the genus of corn marriage) apomixis with Tripsacum and express genes involved directly to the homologous gene in corn.In this specification sheets and claim, " orthologous gene " refers to from the derivative different genes of common gene, or paralogous gene, refers to contain the species of these genes simultaneously.Described gene has identical functions aspect apomixis.
Tripsacum belongs to bluestem grass family.This is unique marriage object of known Zea on the American continent.(4) and (11) the Tripsacum mode of reproduction has been carried out the most completely research.It is what time following that these work are clear and definite:
-all polyploid strains (accessions) are passed through the reproduction of multiple spore apomixis mode,
Not subtrahend in the-apomixis form mainly is the Antennaria type, the Taraxacum type seldom,
Embryo of silkworms capsule in the-multiple sporulation form passes through three successive mitotic division directly from macrosporinite,
Do not have around reduction division failure in the-multiple sporulation form and the macrosporinite callosity sacrum body occur relevant,
The analysis of the F1 colony between the multiple sporulation form of-corn and Tripsacum shows a kind of simple inheritance of apomeiosis,
-by molecule corn probe in detecting to the various allelic genetic mapping and the locus of responsible apomeiosis approaching; These probes are umc28, csu68 and umc62,
-these probes are determined to be responsible for the karyomit(e) of Tripsacum apomeiosis and the portion homologous sexual intercourse between No. 6 long-armed far-ends of karyomit(e) of corn.
Present inventor's work proposes them and proves that apomictic gene has one or more orthologous genes in the responsible Tripsacum in the genome of sexual grass (particularly corn).This notion makes to have developed differentiates that the clone is responsible for the general policies of apomictic nucleotide sequence and implements its new tool then.
Therefore, the purpose of this invention is to provide a kind of in Gramineae, particularly corn, differentiate with the related gene of apomixis directly to the method for homologous gene.
The present invention also aims to provide a kind of method of separating this gene order.
The present invention also aims to respective straight application and the application in transgenosis of these sequences in homogenic sequence in separating apospecies of this sequence.
The present invention also aims in a kind of confirmation apospecies emic method between isolating sequence of institute and apomixis phenotypic expression.
Be used for discerning Gramineae, particularly corn and the gene of being responsible for all or part apomixis growth in a kind of apomixis form directly to the inventive method of homologous nucleotide sequence, it is characterized in that phenotypic expression approached or in the apomixis form sudden change of observed phenotype at Gramineae, particularly map in the corn gene group so that differentiate those show with the related gene of apomixis directly to those sudden changes of homologous.
Here differentiate relevant phenotype based on four features of multiple sporulation form, these features can be observed separately or be observed jointly: (a) sudden change takes place special to megaspore, do not influence the arrenotoky function, (b) they cause forming unreduced gamete from sporogonium, (c) they do not appear as feature to have callosity sacrum body around the megasporocyte, (d) reference mark of working in embryo of silkworms capsule formation process usually shows inactivation, although a stage failure embryo of silkworms capsule in the megaspore generating process normally forms.
The discriminating of the nucleotide sequence of mentioning in the method for above definition is included in sldh gene seat or gene in one embodiment of the invention, or with meiotic mutants at Gramineae, particularly map in the corn gene group, with differentiate show with the related gene of apomixis directly to homologous those.
The present invention be more particularly directed to a kind of in Gramineae, particularly corn, differentiate with the apomixis form in the gene of control apomeiosis directly to the method for homologous gene order, it is characterized in that studying phenotypic expression, and, in the genome of Gramineae, particularly corn, determine the position of various meiotic mutants by means of locating the molecular marker of being responsible for the locus of apomeiosis in the described apomixis form.
Generally, will be by localized mutant clon of the present invention and order-checking.
The present inventor proves especially in the Tripsacum that apomixis expresses related gene and have one or more orthologous genes in corn.
The present invention in corn and Tripsacum of described serial sign uses and makes that having identified candidate gene (1) with following feature and the gene of controlling the multiple sporulation has homologous genes group position, be the identical chromosomal region that they are arranged in corn and Tripsacum multiple sporulation controlling gene homologous sections, the relevant phenotype of determining according to above-mentioned standard with (2).Preferably, described position relates to elongate and the afd locus in the corn gene group.
The feature of the inventive method is that also it comprises with the localized meiotic mutants of transposon tagging.The present invention be more particularly directed to locus with transposon tagging elongate.
Utilize the transposon of known array can be to the gene manufacturing sudden change of known phenotypic expression only.The insertion Chang Yiqi afunction of transposon in gene to be separated is feature.For Recessive alleles, this is often with the feature that appears as of recessive phenotype in the heterozygosis plant.But significant especially transposon comprises Mutator or Ac/Ds type transposable element.
Preferably carry out the clone and the order-checking of institute's positional mutation.
Mutator gene can be separated in insertion site by the mark transposon, has inserted the range gene seat of transposon and has analyzed by Mendelian and molecular biological routine techniques (12) and (13) clone, also order-checking if desired.
More specifically corresponding to the site of elongate locus, at first phenotypic evaluation is carried out in the site that allelotrope el1 is expressed for for example.Then it is located by genetic mapping, the locus position of control multiple sporulation in this position and the Tripsacum is compared.By means of this locus of transposon tagging, separate then, then order-checking.
This sequence in the apomixis form or the application in the orthologous gene are being differentiated, separated then at least a portion that the invention still further relates to the sequence of above definition.
The present invention relates to isolating nucleotide sequence.These sequences are characterised in that all or part is grown in they and the responsible apomixis form sequence is directly to homology.The invention still further relates to sequence homologous sequence on functional meaning with above-mentioned discriminating.
The present invention be more particularly directed to such nucleotide sequence corresponding to sudden change elongate gene.
The invention still further relates to the nucleic acid that contains one or more sequences as defined above and in vegetable material, express required adjusting sequence.
The invention still further relates to the cloning vector and the expression vector that contain this nucleic acid, also relate to the cell host that contains these carriers, for example agrobacterium tumefaciens (Agrobacterium tumefaciens).
The invention still further relates to this sequence, with other feature allelotrope of apomixis form, transforming vegetable material, vegetable cell, the Plants and Seeds of various etap in the time of suitably so that they carry out the developmental application of apomixis.Described allelotrope is corresponding to other genes except that orthologous gene provided by the invention.
The present invention be more particularly directed to a kind of method that produces apospecies, it is characterized in that using the sequence of elongate gene of suddenling change as defined above.
The plant transformed material itself comprises within the scope of the invention, it is characterized in that it contains described sequence in its genome apomixis at least grows related part, in the time of suitably with other characteristic allelotrope of apomixis form.
Imagine described cell, Plants and Seeds belong to Gramineae.They are corn particularly.
The conversion of vegetable material, cell, Plants and Seeds preferably utilizes genetically modified routine techniques to carry out.
Be example now to obtain rotaring gene corn plant.
A. maize calli obtain and as the application of genetic transformation target
No matter adopt which kind of method (electroporation, Agrobacterium, microfilament, particle gun), the genetic transformation of corn generally need use the undifferentiated cell in the rapid division that keeps regeneration whole plants ability.Such cell has been formed the friable embryogenic generation callus (so-called II type) of corn.
The method and the substratum that utilize Armstrong (1994) to describe are that the immature embryo of H1 II or (A188x B73) obtains these callus from genotype.Cultivate by on initial substratum, carrying out branch in per 15 days continuously, with the callus propagation that obtains like this and keep.
The method of describing by (1989) such as Vain is regulated the hormone of cell and osmotic pressure balance from these callus regeneration plantlets then.These plants are tamed in the greenhouse, and they can be hybridized or selfing there.
B. utilize particle gun to carry out the genetic transformation of corn
Epimere has been described and has been transformed obtaining and regenerating of required clone; A kind of genetic transforming method that causes modifying factor stable integration in Plant Genome is described here.This method depends on the use of particle gun; Target cell is the fragment of the callus of description in the epimere 1.These are had 10 to the fragment of 20mm2 surface-area in bombardment preceding 4 hours, be placed on the central authorities of Petri plate with the amount of 16/plate, this plate contains the substratum identical with initial substratum, but has wherein added 0.2M mannitol+0.2M Sorbitol Powder.Explanation by manufacturers will have plasmid purifying on the Qiagen post of waiting to introduce gene.Press Klein etc. then, nature (Nature), 1987,327, the working method that the 70-73 page or leaf is described is deposited in it on tungsten particle (M10).The particle of bag quilt press the method that J.Finer (1992) describes and is launched to target cell with rifle like this.
The callus plate that bombarded like this with Scellofrais sealing, is cultivated 27 ℃ of lucifuges then then.Carry out during after this 24 hours dividing the first time and cultivate, go up at the substratum identical (reagent has wherein brought Selection In) then and carried out once 3 totally months in per 15 days with initial substratum.Operable selective reagents generally is activeconstituents (Basta , Round up ) or some microbiotic (Totomycin, kantlex etc.) of some sterilant.
Behind 3 months or the shorter time, obtain the callus that its not selected reagent place of growth suppresses, select the cell that cell fission produced of genes to form by in its gene genetic, having integrated one or more copies usually and in most cases.The probability that obtains this callus is about 0.8 callus/bombardment plate.
With these callus discriminatings, single separation, amplification, cultivate then with the regeneration plantlet.For fear of the interference of no transformed cells, all these operate in the substratum that contains selective reagents carries out.
The regenerated plant is tamed growth then in the greenhouse like this, and this moment, they can be hybridized or selfing.
C. utilize agrobacterium tumefaciens to carry out the genetic transformation of corn
Used this technology Ishida etc. (Nature Biotechnol (Nature Biotechnology), 1996,14:745-750) or Horsch etc. " science " (Science), 1984,223, the 496-498 page or leaf has report.
But therefore the present invention provides the method that produces the apospecies population with transposable element.
Specifically, the invention provides the method that induced apomixis is grown in sexual plant, particularly corn.
In another kind of application the of the present invention, more than at least a portion of Ding Yi sequence is used for differentiating and the orthologous gene seat sequence of separating the apomixis form.
Therefore the present invention relates to the hybridization probe and the primer that can be used for round pcr from described sequence establishment.
This probe and primer are especially corresponding to from those of elongate sequence establishment.
Hybridization or round pcr preferably carry out according to a conventional method.
The invention still further relates to a kind of the discriminating and the method for separating the gene of being responsible for the sporulation of apomixis Tripsacum medium multiple, it is characterized in that using at least a portion of elongate locus sequence.
The feature of the inventive method also be in the apomixis Tripsacum isolating sequence be used for this sequence and apomixis express between the functional analysis of relation.As illustrating among the embodiment, mutafacient system is used in particular for confirming the relation between elongate gene order isolating apomixis Tripsacum sequence and apomixis phenotypic expression.
According to the present invention, confirmed the relation between described sequence and apomeiosis especially.
Other features of the present invention and advantage provide in following examples.1-5 with reference to the accompanying drawings in these embodiments:
-Fig. 1: the genetic mapping of the karyomit(e) sections of control apomixis tetraploid Tripsacum medium multiple sporulation, and with the comparison of sexual diplont Tripsacum and corn,
The structure of the mapping population of-Fig. 2: el1 sudden change (elongate),
-Fig. 3: be used for the structure of the mutagenesis colony of mark elongate locus,
-Fig. 4: in the plant of isozygotying of allelotrope el1 megasporocyte and the elongate locus have wild allelic maize plant phenotypic evaluation and with the comparison of the sexual of Tripsacum and apomixis form,
-Fig. 5: the structure that is used for the mutagenesis population of the corn-Tripsacum hybrid of the functional analysis of relation between apospecies institute's separation sequence and apomixis expression.
Embodiment 1: the genetic mapping of apomeiosis in the Tripsacum
Following description control Tripsacum belongs to the generation of the genetic map of the karyomit(e) sections of apomeiosis in apomixis form and the sexual form.
1) material
The mapping of-diploid Tripsacum: two used parents are the sexual diplonts (2n=36) at ORSTOM-CIMMYT preservation center, are kept at Tlaltizapan testing station, Mexico Morelos state.They are Tripsacum maizar Hern.and Randolph, and preserving number is CIMMYT#99-1114 and Tripsacum dactyloides var.meridionale de Wet andTimothy, and preserving number is CIMMYT #575-5136.Population comprises 175 strain F1 plants, and wherein 56 strains are used for mapping.
The mapping of-apomeiosis: the mapping population comprises 232 strain F1 corn-Tripsacum plants.Parental maize (H1) is the corn hybrid (2n=2x=20) between two CIMMYT strains (CML135-CML139).Another is the apomictic Tripsacum T.dactyloides of tetraploid (2n=4x=72), and preserving number is CIMMYT #65-1234.Apospecies are used as male.
2) method
. the analysis of mode of reproduction: this is to carry out with the method for (4) such as Leblanc.
. the detection of the RFLP type molecular marker relevant with apomeiosis:
Following strategy detects with Michelmore etc. (14) being used to of describing generally that to reply the method for relevant molecular marker suitable with special phenotype.
Probe obtains from Missouri university (Columbia).
About 100 kinds of RFLP probes on existing maize genetic figure, have been selected, with the density (seeing (4), annex 4) that reaches about 20-30cM between two marks.Used various: UMC collection of illustrative plates (Missouri university, Columbia with reference to collection of illustrative plates; The corn database, the UMC95 collection of illustrative plates), a kind of collection of illustrative plates (15) of Cornell university and the various collection of illustrative plates (16) that provide by CIMMYT.It is chain that the chi2 check is used to measure potential, estimates recombination value with the method for Allard (17).Because the donor parents of the sections of control apomeiosis is the tetraploid plant of heterozygosis, detect three conditions of chain needs between multiple sporulation and RFLP allelotrope: have the RFLP polymorphism at this locus between (1) two parent, (2) this equipotential gene is heterozygosis in Tripsacum, (3) this equipotential gene must be the combination of single dominance in tetraploid, can with other 3 distinguish.
-diplontic mapping
Used and belonged to two F1 mapping populations between two heterozygosis parents not of the same race.Drawing method is as described in (18) such as Ritter.
3) result
The discriminating of-relevant RFLP sign with apomeiosis
Fig. 1 shown the control apomeiosis the karyomit(e) sections genetic mapping and with the comparison of Tripsacum and the sexual diplont of corn." Apo " is corresponding to the locus of being responsible for apomeiosis.Roughly marked the umc71 position on No. 6 karyomit(e)s of corn, this equipotential gene on No. 6 karyomit(e) gets from the UMC collection of illustrative plates of latest edition.This collection of illustrative plates is that 52 strain plants from F1 colony between corn and Tripsacum draw.In this population, reduction division and multiple sporulation be 1: 1 isolating (24 strain apospecies, the sexual plant of 28 strains, chi2=0.31, p=0.6).Tested 84 probes, probe is selected on the UMC collection of illustrative plates, covers the corn gene group to try one's best widely.90% polymorphism that has detected between at least a corn and Tripsacum wherein.Three Represents polymorphism and allelic three probes special on whole F1 population, have been tested to apomict.Verified relevant by the detected allelotrope of probe umc28 with apomeiosis.In second step, 14 probes that approach umc28 locus on the UMC collection of illustrative plates have been tested.Wherein 4, umc71, umc62, csu68 and cdo202 detect a plurality of RFLP allelotrope, and these allelotrope are not only relevant with the multiple sporulation but also be total to-separate fully between them.
Relatively mapping between-sexual diploid and apomixis tetraploid
5 signs relevant with the multiple sporulation are mapped on the diploid population.They can be on same parent (575-5136).Notice that these 5 signs are all strict chain in tetraploid plant, but all be isolating with significant recombination value at diploid parents with in corn.
The relatively mapping of-corn-Tripsacum:
The allelic probe but the karyomit(e) sections of Detection ﹠ Controling Tripsacum medium multiple sporulation is correlated with detects all allelotrope that belong to identical chain group in the corn collection of illustrative plates.This be No. 6 chromosomal long-armed.Some also detects other regional allelotrope of genome in them, particularly No. 3 and No. 8 karyomit(e)s.The position of various relevant probes with apomeiosis on the corn collection of illustrative plates is shown in following table:
The clone position
UMC38*?????????????6L；8L；3L；
UMC62??????????????6L
UMC71??????????????6L；8L；
UMC28??????????????6L
CSU68??????????????6L；8L；3L
CD0202?????????????6L；8L；3L
*: uncorrelated with the multiple sporulation, but belong to same chain group.
Embodiment 2: the discriminating of orthologous gene in the corn
In corn, studied and caused the gene that apomeiosis is expressed in the Tripsacum.Purpose is to differentiate the candidate gene with the relevant phenotype in (1) genome position identical with apomeiosis and (2) and apomeiosis in corn.
A lot of meiotic mutants bodies are arranged in corn.Select the phenotype standard of candidate gene as follows: there is the obviously sporogonium of differentiation in (1), (2) in these cells, there be not maiotic inducing fully, or thisly induce in early days the stage failure, (3) with the irrelevant functional gametophytic ability of generation of reduction division failure, there is not callosity sacrum body to occur or the decline of highly significant at least around (4) megaspore parental cell.In the potential candidate, promptly have all above-mentioned features or the part those, wherein unknown or coarse those utilizations in the position in the corn gene group are mapped with reference to locus, are with those that used probe in detecting arrives of mapping of apomeiosis in the Tripsacum with reference to locus.
-material and method
The result is reported in following work recessive meiotic mutants body elongate (el1) (19) is carried out.In the plant that the el1 locus isozygotys, karyomit(e) keeps the depolymerization state in the mid-term and the late period of first division, causes various chromosome abnormalties, and wherein considerable part is unreduced gametophyte (30 to 70%, depend on the residing genetic background of sudden change especially).The pollen fertilization of normal plants forms triploid embryo and defective pentaploid endosperm.
The exact position of elongate locus is unknown before the present invention.But known in the past it to belong to No. 8, corn chromosomal long-armed.Therefore it directly is not positioned to be accredited as and to control on the arm of maize chromosome of apomeiosis dna homolog by Leblanc etc.Though be positioned on No. 8 karyomit(e), it may belong to the sections (15) that duplicate is arranged between No. 6 long-armed far-ends of karyomit(e) and No. 8 karyomit(e) parts.
Fig. 2 has described the structure of el1 (elongate) sudden change mapping population.Having the 3 strain F1 plants of heterozygous genes type El1/el1 and the three strains el1/el1 plant of isozygotying backcrosses.For each single kind, cultivate 50 strain plants, selfing is also estimated their phenotype.Expection El1/el1 seed is normal, and the el1/el1 seed has the odd-shaped endosperm.In order to confirm the elongate phenotype, get that intraseminal 10-20 embryo with defective endosperm make sample, the method flow cytometry analysis that proposes by (20) such as Galbraith.From Maize Genetic Stock Center, Urbana, Illinois obtains the el1 mutant, and its form is the seed that the plant that el1 allelotrope isozygotys in the genetic background (further information is indeterminate) selfing produces.The strain W23 that isozygotys with wildtype phenotype is used to make up population.Utilize Mapmaker 2.0 softwares (being used for the Mackintosh machine) to detect chain and the evaluation recombination value.
The comparison of-apomeiosis plant and elongate phenotypic expression
The cytoembryology technical Analysis that the phenotypic expression of elongate sudden change in the el1/el1 plant sporogonium that isozygotys described with (11) such as Leblanc.On four kinds of materials, collect the prematurity inflorescence: (1) sexual diploid Tripsacum, (2) apomixis tetraploid Tripsacum, the strain that Leblanc etc. (11) describe, isozygoty El1/El1 corn strain (W23) and (4) a kind of el1/el1 of isozygotying strain of (3) wildtype phenotype.
But. utilize the transposable element mark and separate elongate locus sequence
With transposon tagging is to make sudden change with the known transposon of sequence in the gene of only knowing phenotypic expression.Inserting transposon in gene to be separated is feature to lose its function often.For the situation of Recessive alleles, its feature often is the appearance of recessive phenotype in the heterozygosis plant.By the insertion site of mark transposon, can clonal mutation gene itself.Like this, mutator gene itself separates by the insertion site of mark transposon, and the various locus that inserted transposon are analyzed by Mendelian and molecular biological routine techniques clone.Below experiment is carried out with Mutator system (21).
The population that is used for transposon tagging elongate locus is at the form demonstration (f: the frequency of occurrences of accompanying drawing 3 with chart; [EL] and [el]: dominance and recessive phenotype; El*: the allelotrope of mark.The plant hybridization that plant that recessive mutation is isozygotied and wild allelotrope El1 isozygoty.In the gamete population that produces by the El1/El1 parent, on the elongate locus, find one or more sudden changes.Insertion has caused this allelic afunction, thereby has found some el1/El1 genotype but be the F1 plant of el1 phenotype.This gene thereby be labeled.
The result:
The mapping of-elongate locus:
In the population of Separation Research, el1 separates (Chi2=0.5 with the El1 phenotype in 1: 1 population; P=0.6).Belonging to No. 3, No. 6, major part belongs to No. 8 chromosomal various RFLP probes and with three kinds of limiting enzyme EcoRIs, BamHI and HindIII the 50 strain plants of this population is tested.Can detect the probe of significant polymorphism analyzes in other 100 strains then.Utilize these three kinds experiment enzymes, Umc28, umc62 do not show the polymorphism allelotrope relevant with elongate with umc71.And Csu68 detects the allelotrope relevant with elongate with cdo202.Chain as follows between three locus estimating with reorganization percentage ratio:
| ??cdo?202?????csu68 | |
| ????elongate ????csu68 | ??9.3?????????7.4 ??1.9 |
-phenotypic evaluation:
Fig. 4 shows that (parent cell of megaspore among the A:W23, W23 are the El1/El1 genotype of corn in the comparison that sporogonium is grown in various types of materials; B: the parent cell of megaspore in the el1/el1 corn that isozygotys; C: the parent cell of megaspore in the sexual diploid Tripsacum; D: the parent cell of megaspore in the apomixis tetraploid Tripsacum).Differentiate viewed each etap, and outside ovule, mated between observed various forms (11) on size and the morphological basis.Sexual form for corn and Tripsacum: observed on all four development characteristics: the same modality of cell and nuclear (particularly: the parent cell of rectangle megaspore, thick remarkable wall, on cell walls the callosity sacrum body of highly significant occur, the formation from the parent cell of megaspore to megaspore).Observed same similarity in Tripsacum multiple sporulation form relatively with when isozygotying the El1/El1 maize plant: the same modality of nuclear and cell (direct development of megasporocyte in the embryo of silkworms capsule, thin cell walls, very obviously be different from nothing or the appearance of very rare callosity sacrum body in the closely similar and megasporocyte between nuclear, Tripsacum multiple spore form and the el1/el1 maize plant of sexual form).
The transposon tagging of-elongate locus:
The colony of pressing 12,500 strain F1 plants of Fig. 3 generation cultivates in the CIMMYT testing station of Mexico Tlatizapan.This 12,500 strain plant is fertilized with the corn hybrid (CML135*CML62 hybrid) of the Mutator that loses activity.The grain ear that monitors this 12,500 strain plant is to ripe, though so that find those heterozygosis on this locus to express those of elongate sudden change.For plant, with the corresponding embryo of flow cytometry analysis with defective endosperm.The two strain plants that are called TTEl-5 and TTEl-7 contain the defective endosperm relevant with the triploid embryo through evaluation.These plants are expressed the elongate phenotype in heterozygosis el1/El1 plant.In these plants, a kind of mark of the wild allelotrope of this locus in the Mutator type transposon.The seed that the hybridization of this two strains plant and CML135*CML62 hybrid produces is formed on the isolating population of this marker allele on the elongate locus: half has the plant that this hybridization produces the el1/El genotype (el1 is from the TTEl plant, El is from CML135*CML62), second half is the El1*/El genotype, and wherein El1* is by the allelotrope of transposon tagging and from the TTEl plant.But like this this gene of elongate locus can by analyze various copy transposable elements in these plants and with below the elongate locus that finds of the RFLP probe mentioned be divided into from, and differentiated and cloned.
-embodiment 3: be used to confirm that candidate's allelotrope and all or part of apomixis grows generation and the using method of the population that concerns between phenotypic expression
Overall plan and used material are shown in Fig. 5.The strain that contains the Mutator element obtains from California (Berkeley) Mr. Freeling of university.Double haploid BC2-28 plant used herein is (6) previous those that report such as Leblanc.They are apospecies, have corn and Tripsacum parents monoploid genome separately.We use these plants is in order to introduce the Mutator element in the apomixis material.
The colony of the apomictic BC2-28 plant of 1000 strains makes up from the apomictic doubled haploid plant of a strain the earliest, selects 2n+0 type plant from its offspring.We have created the identical apomixis polyhaploid genotype of 1000 copies like this.This 1000 strain plant and the hybridization of Mutator agamospecies.In the filial generation that obtains, we have selected atypical 2n+n plant, promptly mixed from the Mutator agamospecies genomic those.These agamospecies have various types of Mutator of about 200 copies.Thereby the apospecies of wishing to gather in the crops average 100 copies.The selection of BC2-28 plant and atypical apomixis BC3-38 plant is carried out with a simple morphological criteria.In fact double haploid has phenotype very easy to identify, and it is different from the phenotype that accumulative total caused (6) by extra corn gene group very much.About 35,000 seeds (BC2-28 xMutators) have been obtained altogether.They all are grown in the CIMMYT testing station of Morelos state Tlaltizapan in the summer in 1996.Germinate and selected atypical 2n+n in back one month.About 7,500 strain 2n+n plants have been obtained, i.e. 20% atypical plant almost.This colony with do not have the corn hybrid (CML135*CML62) of active Mutator element to hybridize again, produced the colony of about 150,000 seeds, represent reverse genetic colony.Given sequence is expressed influence to apomixis ideal material is analyzed in this colony's representative.In fact, the Tripsacum allelotrope of being responsible for this feature representation is to be single dominance assembled state (being single copy in genome) at this.Thereby in order to confirm allelotrope-function relationship, as long as check the phenotype effect of inserting the transposon in this sequence.If sudden change causes afunction, just can affirm this allelotrope-functional relationship.Because the sequence of the transposon and the gene of studying is all known, can differentiate by conventional round pcr the plant that this equipotential gene is suddenlyd change.
Reference
(1) Savidan, Y., apomictic natural and hereditary among the 1982 Panicum maximum Jacq., Paris 11 universities.
(2) Nogler, G.A., the apospory heredity among the apomictic Ranunculus auricomus of the 1984a V.Conclusions., Bot Helvetica 94:411-422.
(3) Mogie, M., 1988 estimate and control apomictic model, Biol J LinneanSoc 35:127-153.
(4) Leblanc, O., M.D.Peel, J.G.Carman and Y.Savidan, the megaspore in the several Tripsacum kinds of 1995a (Poaceae) is taken place and megagamete takes place, and american plant is learned magazine (Am J Bot) 82:57-63.
(5) Hanna, W., M.Dujardin, P.Ozias-Akins, E.Lubbers and L.Arthur, reproduction, cytology and the fertilization of 1993 pearl Millet X Pennisetum squamulatum BC4 plants.J?Hered?84：213-216。
(6) Leblanc, O., D.Grimanelli, N.Islam-Faridi, J.Berthaud and Y.Savidan, the reproductive behavior of 1996 corns-Tripsacum polyhaploid plant; The transfer process of apomixis in corn.J?Hered?87：108-111。
(7) Kindiger, B., V.Sokolov and I.V.Khatypova, the apomictic evaluation .Crop sci 36:1108-of 1,996 one groups of 39 karyomit(e)s corn-Tripsacum backcross hybrids.
(8) Jefferson, R.A., and R.Bicknell, 1996 apomictic potential impacts: a kind of molecular genetic approach, be stated from " the genetic impact of plant molecular (The impact ofplant molecular genetics) ", B.W.S.Sobral.Birkhauser compiles, Boston.
(9) Koltunow, A.M., R.A.Bicknel and A.M.Chaudhury, 1995 apomixiss: heredity is equal to the molecular strategy of seed nonfertilization reproduction.Plant physiology (PlantPhysiol) 108:1345-1352.
(10) Ramulu, K.S., P.Dijkhuis, A.Pereira, G.C.Angenent, M.M.Van Lookeren Campagne and J.J.M.Dons, 1997 are used for isolating EMS of plant apogamy mutant and transposon mutagenesis.Be stated from " being used to improve somaclonal variation and the induced mutation (Somaclonal variation and induced mutations in cropimprovement) of crop ".The Kluwers academic press, Amsterdam.
(11) Leblanc, O., D.Grimanelli, D.Gonzalez de Leon and Y.Savidan, 1995b utilize the apomixis mode in corn-RFLP Mark Detection corn-Tripsacum hybrid.Theory and applied genetics (Theor Appl Genet) 90:1198-1203.
(12) Walbot, V., 1992 utilize the mutagenesis and the gene clone strategy of transposon tagging and T-DNA mutagenesis.Ann?Rev?Plant?Physiol?Plant?Mol?Biol?43：49-82。
(13) Freeling, M. and Walbot, V. (volume), 1994 corn handbooks (The MaizeHandbook), Springer Verlag, New York.
(14) Michelmore, R.W., I.Paran and R.V.Kesseli, 1991 signs: utilize segregating population in specific gene group zone, to detect the fast method of sign by colony's compartment analysis discriminating and disease resistance gene linkage.Institute of NAS newspaper (Proc Natl AcadSci USA).88：9828-9832。
(15) Ahn, S.N., and S.D.Tanksley, the comparison linkage map of 1993 rice and corn gene group, institute of NAS reports 90:7980-7984.
(16) Ribaut, J.M., D.A.Hoisington, J.A.Deutsch, C.Jiang and D.Gonzalez-de-Leon, 1996 differentiate the quantitative trait locus under the drought condition in tropical corn.1. become the flower parameter and bloom-ear interval.Theory and applied genetics 92:905-914.
(17) Allard, R.W., 1956 help to calculate the formula and the form of genetic recombination value.Hilgardia?24：235-278。
(18) Ritter, E., C.Gebhardt and F.Salamini, 1990 heterozygosis parents are hybridized the estimation and the RFLP linkage maps of recombination frequency in the gained plant.Genetics (Genetics) 125:645-654.
(19) Rhoades, M.M., and E.Dempsey, what 1966 usefulness elongate gene pairs corn karyomit(e)s meiophase doubled induces.Genetics 54:505-522.
(20) Galbraith, D.W., K.R.Harkins, J.M.Maddox.N.M.Ayres, D.P.Sharma and E.Firoozabady, 1983 in complete plant tissue the quick fluidic cell numeration of cell cycle analyze.Science 220:1049-1051.
(21) Robertson .D.S., the active timing of Mu in 1980 corns.Genetics 94:969-978.
Claims (24)
1, in Gramineae, particularly corn, differentiate with the apomixis form in be responsible for sequence that all or part of apomixis grows directly to the method for homologous nucleotide sequence, it is characterized in that with phenotype near or in the apomixis form sudden change of observed phenotype at the Gramineae genome, particularly map in the corn gene group, with differentiate show with apomixis in related gene directly to homologous those.
2, according to the method for claim 1, it is characterized in that meiotic mutants at the Gramineae genome, particularly map in the corn gene group, with differentiate show with apomixis in related gene directly to homologous those.
3, according to the method for claim 2, it is characterized in that utilizing the molecular marker that can locate in the described apomixis form locus of being responsible for apomeiosis, with various meiotic mutants at the Gramineae genome, particularly locate in the corn gene group.
4,, it is characterized in that using the molecular marker of the locus that can locate the sporulation of Tripsacum medium multiple according to the method for claim 3.
5,, it is characterized in that described location relates to elongate and afd locus according to the method for claim 4.
6,, it is characterized in that it also comprises with the localized meiotic mutants of transposon tagging according to each method among the claim 1-5.
7,, but it is characterized in that the mark with transposon is to utilize the transposable element of Mutator or Ac/Ds type to carry out at the elongate locus according to the method for claim 4.
8,, it is characterized in that localized mutant clon of institute and order-checking according to each method of above claim.
9,, it is characterized in that when also need being it being checked order at gene by the sudden change of compartment analysis mark transposon insertion site rear clone according to the method for claim 6 or 7.
10, nucleotide sequence, it is characterized in that they be with the apomixis form in be responsible for sequence that all or part of apomixis grows directly to homologous, and homologous sequence.
11,, it is characterized in that its elongate gene corresponding to sudden change according to the nucleotide sequence of claim 10.
12, contain one or more as sequence that defines in claim 10 or 11 and the nucleic acid of in vegetable material, expressing the adjusting sequence of needs.
13, the clone and the expression vector that contain the nucleic acid of claim 12.
14, the cell host that contains the carrier of claim 13.
15, claim 10 or 11 sequence, in the time of suitably with other characteristic allelotrope of apomixis form, in introduced plant material genome, vegetable cell, the plant of various etap and seed so that make them carry out the developmental application of apomixis.
16, Gramineae, particularly corn plant cell is characterized in that it contains claim 10 or 11 sequences at least in its genome apomixis grows related part.
17, grass, particularly corn is characterized in that it contains claim 10 or 11 sequences at least in its genome apomixis grows related part.
18, the seed of Gramineae, particularly corn is characterized in that it contains claim 10 or 11 sequences at least in its genome apomixis grows related part.
19, produce the method for apospecies, it is characterized in that using the nucleotide sequence of claim 11.
20, at least a portion of claim 10 or 11 sequences differentiate with separates the apomixis form in the straight application of locus to homologous sequence.
21, hybridization probe and molecule primer is characterized in that they are sequence establishments of Accessory Right requirement 10 or 11.
22,, it is characterized in that they are to work out from the sequence of elongate according to the hybridization probe and the molecule primer of claim 18.
23, differentiate with separate apomictic Tripsacum in be responsible for the method for the gene of apomeiosis, it is characterized in that using the partial sequence at least of elongate locus.
24, the using method of relation between mutagenesis population institute's separation sequence and apomixis in confirming the Tripsacum of claim 20 is expressed.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR97/01821 | 1997-02-17 | ||
| FR9701821A FR2759708B1 (en) | 1997-02-17 | 1997-02-17 | MEANS FOR IDENTIFYING, ISOLATING AND CHARACTERIZING NUCLEOTIDE SEQUENCES INVOLVED IN APOMIXIA |
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| CN1248296A true CN1248296A (en) | 2000-03-22 |
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| EP (1) | EP0975799A1 (en) |
| CN (1) | CN1248296A (en) |
| AU (1) | AU6405498A (en) |
| CA (1) | CA2282085A1 (en) |
| DE (1) | DE975799T1 (en) |
| ES (1) | ES2146561T1 (en) |
| FR (1) | FR2759708B1 (en) |
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| US10131919B2 (en) | 2011-05-30 | 2018-11-20 | Leibniz-Institut Fur Pflanzengenetik Und Kul Turpflanzenforschung Gatersleben (Ipk) | Means and methods to induce apomixis in plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| IL149335A0 (en) * | 1999-10-29 | 2002-11-10 | Univ Utah State | Methods for stabilizing and controlling apomixis |
| US7264964B2 (en) | 2001-06-22 | 2007-09-04 | Ceres, Inc. | Chimeric histone acetyltransferase polypeptides |
| US7476777B2 (en) | 2002-09-17 | 2009-01-13 | Ceres, Inc. | Biological containment system |
| US8878002B2 (en) | 2005-12-09 | 2014-11-04 | Council Of Scientific And Industrial Research | Nucleic acids and methods for producing seeds with a full diploid complement of the maternal genome in the embryo |
| FR2952276A1 (en) * | 2009-11-09 | 2011-05-13 | Inst Rech Developpement Ird | INDUCTION OF APOMIXY IN CULTIVATED PLANTS WITH SEXUAL REPRODUCTION AND USE FOR REPRODUCTION OF TOTALLY OR PARTIALLY APOMICALLY ACTIVE PLANTS |
| WO2017039452A1 (en) * | 2015-09-04 | 2017-03-09 | Keygene N.V. | Diplospory gene |
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| US5811636A (en) * | 1995-09-22 | 1998-09-22 | The United States Of America As Represented By The Secretary Of Agriculture | Apomixis for producing true-breeding plant progenies |
| US5710367A (en) * | 1995-09-22 | 1998-01-20 | The United States Of America As Represented By The Secretary Of Agriculture | Apomictic maize |
-
1997
- 1997-02-17 FR FR9701821A patent/FR2759708B1/en not_active Expired - Fee Related
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1998
- 1998-02-17 CN CN98802588A patent/CN1248296A/en active Pending
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| US10131919B2 (en) | 2011-05-30 | 2018-11-20 | Leibniz-Institut Fur Pflanzengenetik Und Kul Turpflanzenforschung Gatersleben (Ipk) | Means and methods to induce apomixis in plants |
| US10907174B2 (en) | 2011-05-30 | 2021-02-02 | Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben (IPK) | Means and methods to induce apomixis in plants |
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| ES2146561T1 (en) | 2000-08-16 |
| FR2759708A1 (en) | 1998-08-21 |
| WO1998036090A1 (en) | 1998-08-20 |
| FR2759708B1 (en) | 1999-08-27 |
| CA2282085A1 (en) | 1998-08-20 |
| DE975799T1 (en) | 2000-08-31 |
| EP0975799A1 (en) | 2000-02-02 |
| AU6405498A (en) | 1998-09-08 |
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