WO2016191293A1 - Prédiction de la vigueur hybride à l'aide de l'expression génique sensible au stress à régulation circadienne - Google Patents

Prédiction de la vigueur hybride à l'aide de l'expression génique sensible au stress à régulation circadienne Download PDF

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WO2016191293A1
WO2016191293A1 PCT/US2016/033580 US2016033580W WO2016191293A1 WO 2016191293 A1 WO2016191293 A1 WO 2016191293A1 US 2016033580 W US2016033580 W US 2016033580W WO 2016191293 A1 WO2016191293 A1 WO 2016191293A1
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stress
plants
gene
responsive
expression level
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Z. Jeffrey Chen
Marisa MILLER
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Board Of Regents, The University Of Texas System
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield

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  • Hybrid plants and animals grow larger and more vigorously than their parents, a common phenomenon known as hybrid vigor or heterosis. Since Charles Darwin systematically described the phenomenon, heterosis has been widely applied in agriculture to dramatically improve the production of crops and farm animals. Heterosis in food crops often refers to grain yield, while biomass heterosis is commonly measured in other crops including vegetable and energy crops.
  • the molecular basis for heterosis remains elusive, and traditional dominance and overdominance genetic models cannot adequately explain heterosis in yeast and many crop plants.
  • the conventional technology uses phenotypes and DNA sequence polymorphism as selection criteria to make high-yield hybrids. The predictability resulting from this conventional technology is relatively low.
  • the present invention provides methods of producing a hybrid plant with increased biomass comprising the steps of: (a) detecting an expression level of a stress- responsive gene in a plurality of plants; (b) selecting a first parent plant having a first expression level of the stress-responsive gene from the plurality of plants; (c) selecting a second parent plant of a different genotype having a second expression level of the stress- responsive gene from the plurality of plants, wherein the second expression level is different from the first expression level; and (d) crossing the first parent plant with the second parent plant to produce a progeny plant.
  • the stress-responsive gene is an abiotic stress gene, for example LSR3, ERD11/GSTF6, COR47/RD17, LTI45/ERD10, COR413-TM1, ALDH7B4, ATGRP3, PIP2B, RD28, CAX1/RCI4, COR15A, PIP1B, LTI30, PIP1A, PIP1;4, COR78/RD29A, COR6.6/KIN2, RD22, ERD1, GRP8, GRP2, GRP7/CCR2, MDH, HSP70 family, CAT2, LOS2, ATAVP1, LTP4, TCH2, TCH4, mtHsc70-l, COR15B, MT1A, LTP3, LOS1, Salt-stress responsive protein, FIB, KIN1, or homologs thereof.
  • abiotic stress gene for example LSR3, ERD11/GSTF6, COR47/RD17, LTI45/ERD10, COR413-TM1, ALDH7B4, ATGRP3, P
  • the stress-responsive gene is a biotic stress gene, for example PR1, PR5, NUDT6, HSPR02, CNGC11, EBP/ERF72, PCC1, HR3/MLA10, CAX3, PAD4, WRKY70, PR2/BGL2, ACD6, NIA2, NHL3, Protease inhibitor, WAK1, WAK2, EP1, RVE2, BG3, LURP1, DMR6, SAG21, SUR2, ANK, or homologs thereof.
  • a biotic stress gene for example PR1, PR5, NUDT6, HSPR02, CNGC11, EBP/ERF72, PCC1, HR3/MLA10, CAX3, PAD4, WRKY70, PR2/BGL2, ACD6, NIA2, NHL3, Protease inhibitor, WAK1, WAK2, EP1, RVE2, BG3, LURP1, DMR6, SAG21, SUR2, ANK, or homologs thereof.
  • the expression level of the stress-responsive gene is detected at one or more time points selected from the group consisting of zeitgeber time (ZT) 0, ZT6, ZT12, ZT15, ZT18, and ZT21.
  • the ratio of the first gene expression level to the second gene expression level may be greater than 1.00, for example greater than 1.5, or greater than 3.0.
  • the expression levels of stress-responsive genes are detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR).
  • the invention provides methods for producing hybrid progeny plants wherein the progeny plant exhibits and improved agronomic trait compared with the parent plants, for example biomass, yield, disease tolerance, insect resistance, pathogen resistance, plant growth and development, starch content, oil content, fatty acid content, protein content, fruit ripening, and stress resistance.
  • Embodiments of the present invention include methods for use with monocot plants or dicot plants, for example plants is selected from the group consisting of: Arabidopsis thaliana, maize (corn; Zea mays), soybean (Glycine max), cotton (Gossypium hirsutum; Gossypium sp.), peanut (Arachis hypogaea), barley (Hordeum vulgar e); oats (Avena sativa); orchard grass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp.); tall fescue (Festuca arundinacea); turfgrass species (e.g.
  • oilseed crops may include soybean, canola, oil seed rape, oil palm, sunflower, olive, corn, cottonseed, peanut, flaxseed, safflower, and coconut.
  • the invention provides a progeny plant produced by the methods of the invention, or a plant part, plant cell, or seed of such a progeny plant.
  • the invention provides methods of producing a hybrid plant with increased biomass comprising the steps of: (a) detecting an expression level of at least two stress-responsive genes in a plurality of plants; (b) selecting a first parent plant having a first expression level of each of the stress-responsive genes from the population of plants; (c) selecting a second parent plant of a different genotype having a second expression level of each of the stress-responsive gene from the population of plants, wherein the first expression level for each gene is different than the second expression level for that gene; and (d) crossing the first parent plant with the second parent plant to produce a progeny plant.
  • a method for producing a hybrid plant with high-yield biomass comprising the following steps of measuring gene expression for a stress-responsive gene in a population of plants under both a stress condition and a non-stress condition. Based on the gene expression of the stress responsive gene, a normal gene expression profile is then determined for the population of plants.
  • the normal gene expression profile comprises a first expression level based on the expression of the stress-responsive gene during the non-stress condition and a second expression level based on the expression of the stress-responsive gene during the stress condition.
  • a first plant is then selected based on display of a lower expression of the stress-responsive gene during the non-stress condition as compared to the first expression level and the same or higher expression of the stress-responsive gene during the stress condition as compared to the second expression level. This first plant is then crossed with a second plant to produce the hybrid plant.
  • the second plant can be selected in a similar fashion as the first plant based on expression of a second stress-responsive gene.
  • gene expression for a second stress-responsive gene is measured in a second population of plants under both a second stress condition and a second non-stress condition.
  • a second normal gene expression profile based on the second population of plants collectively is then developed, wherein the second normal gene expression profile comprises a third expression level based on the expression of the second stress-responsive gene during the second non- stress condition and a fourth expression level based on the expression of the second stress- responsive gene during the second stress condition.
  • the second plant is selected based on its lower expression of the second stress-responsive gene during the second non- stress condition as compared to the third expression level and the same or higher expression of the second stress-responsive gene during the second stress condition as compared to the fourth expression level.
  • the first plant may display a higher expression of the second stress-responsive gene during the second non-stress condition as compared to the second plant and the same or lower expression of the second stress-responsive gene during the second stress condition as compared to the second plant.
  • the second plant may display a higher expression of the first stress-responsive gene during the first non- stress condition as compared to the first plant and the same or lower expression of the first stress-responsive gene during the first stress condition as compared to the first plant.
  • the first stress condition is abiotic and the second stress condition is biotic.
  • the first stress condition is biotic and the second stress condition is abiotic.
  • a hybrid plant is also provided.
  • the hybrid plant is derived from a first parent and a second parent, wherein the first parent displays a first expression level of a abiotic stress-responsive gene and a second expression level of a biotic stress-responsive gene, wherein the second parent displays a third expression level of the abiotic stress- responsive gene and a fourth expression level of the biotic stress-responsive gene, wherein the first expression level is higher than the third expression level, and wherein the second expression level is lower than the fourth expression level.
  • Fold-change Log2-transformed values from low to high, (e-j) Percentage of down- regulated differentially expressed genes that are biotic or abiotic stress-responsive genes at ZTO (e, f), ZT6 (g, h), and ZT15 (i, j) in reciprocal Fl hybrids ColXC24 (e, g, i) and C24XCol (f, h, j).
  • Figure 3 provides data to support circadian clock regulation of the rhythmic expression of ACD6 and COR78.
  • Figure 4 provides data to demonstrate Stress-responsive gene expression levels as predictors for heterosis and effects of cold stress and salicylic acid (SA) on the growth rate in hybrids and their parents, (a, b).
  • C24 (dashed-box) has higher ACD6 and lower COR78 expression levels than Col, Ler, and Ws (grey-boxes), (c, d, e) Biomass increase relative to MPV (Y-axis) in various hybrids was plotted against absolute values of the log2-fold expression level changes (X-axis) in different stress genes at three time points (ZTO, 9, 18). Scatter plots for ACD6 at ZT18 (c), COR78 at ZT9 (d), and COR47 at ZT9 (e). Regression lines were statistically significant for ACD6 at ZT18 (c), COR78 at ZT9 (d) and COR47 at ZT9 (e).
  • Figure 5 provides data demonstrating the effects of knockdown or overexpression of abiotic and biotic genes on biomass.
  • (b) Rosettes of 3 week-old plants in Fl(C24xWs) and its parents C24 and Ws. Scale bars 1 cm for all images.
  • X-axis positions relative to the transcribed region.
  • Figure 7 provides a proposed model for altered stress-responsive gene expression in the promotion of growth vigor in Fl hybrids
  • Natural variation of circadian rhythms and stress responses is associated with the adaptation of each ecotype to local environments
  • Parent 1 and parent 2 that have adapted to different environments require high levels of gene expression in response to abiotic (parent 1) or biotic (parent 2) stress, which is partly mediated by circadian rhythms (clock symbols).
  • Parent 1 and parent 2 do not reach the full growth potential, probably because of the fitness cost in response to the stress in respective adaptive environments.
  • the expression of both biotic and abiotic stress-responsive genes is compromised, leading to increased levels of growth vigor.
  • FIG. 9 shows biotic and abiotic stress genes exhibiting altered expression in Fl hybrids, (a-h) Expression of PCC1 (a), HSPOR2 (b), PR1 (c), CPR5 (d), COR47 (e), COR15A (f), RD22 (g), and RD28 (h) in a 48-hour period starting from dawn.
  • R.E.L. relative expression levels to that of ACT7 in each sample. Values were averaged from three biological replicates (+ s.d.). Single and double asterisks indicate statistical significance levels at p ⁇ 0.05 and p ⁇ 0.01, respectively, using two-tailed student's t-test (compared to the mid-parent value, MPV). Arrows indicate down-regulation in hybrids compared to the MPV.
  • Figure 10 shows cis and trans effects on stress genes in Fl hybrids, (a) Number of genes with cis and trans effects in Fl hybrids, (b) Percentage of stress-responsive genes with cis or trans effects. Statistically significant enrichment of stress genes is indicated by asterisks, which are representative of a FDR adjusted p ⁇ 0.05 using the hypergeometric test (compared to the whole genome background), (c-h) Clustered heatmaps showing RPKM of trans -effected genes in parents and hybrid alleles, (i-p) RPKM values in Col, C24, and Col and C24 alleles in reciprocal hybrids for ACD6 (i), PR1Q), PR2/BGL2 (k), PAD4 (1), COR78 (m), COR15A (n), COR47 (o) and RD22 (p) at ZTO (i-1) or ZT6 (m-p).
  • Figure 13 shows responsive gene expression in higher and lower vigor Fl hybrids,
  • (e, f) Seedlings of 3-week-old plants (scale bars 1 cm for all images) in Fl(ColXWs) and its parents Col and Ws (e) and in (ColXL ⁇ ?r) and its parents Col and Ler (f).
  • Single and double asterisks indicate statistical significance levels at p ⁇ 0.05 and p ⁇ 0.01, respectively, using two-tailed student's t-test (compared to MPV). Upward and downward arrows indicate up- and down-regulation in the hybrids compared to the MPV.
  • Figure 14 shows an experimental scheme for stress treatments. All experiments were performed in a 16-hour light/8-hour dark regime. The white box represents light and the black box represents dark,
  • Figure 15 shows performance and stress-responsive gene expression in Fl(ColXC24) hybrids and their parents (Col and C24).
  • (a-d) Relative expression ratios (R.E.R.) of COR78 (a, b) and COR15A (c, d) between treated and untreated samples across 5 time points after the cold-treatment at ZTO (a, c) or ZT15 (b, d).
  • Relative expression ratios (R.E.R.) of COR78 (k), COR15A (1), ACD6 (m), and PR1 (n) between treated and control samples across 3 time points after 15 days of stress treatment. Dashed lines indicate no expression change between treated and untreated samples. Expression values are averaged from three biological replicates (+ s.d.). Asterisks indicate statistical significance levels at p ⁇ 0.05 using two-tailed student's t-test, compared to the mid-parent value (MPV). Values were averaged from three biological replicates (+ s.d.).
  • Hybrid plants exhibit enhanced agronomic traits due to the varied genetic contribution of their parents, a phenomenon known as heterosis or outbreeding enhancement. While useful hybrids have been identified through extensive breeding and evaluation of the resulting phenotypes, this process is costly and time-consuming. Efforts to develop methods of selecting parents for producing hybrid plants with beneficial traits have been hampered by an incomplete understanding of the factors contributing to heterosis. A need therefore remains for improved methods of producing hybrid plants with useful phenotypes. In the absence of improved methods for selecting parent plants for hybrid breeding programs, it may not be practical to attempt to produce certain new hybrid crop plants.
  • the present invention provides previously unknown methods for producing hybrid plants with significantly improved agronomic traits.
  • methods are provided for selecting parent plants for crossing based on expression levels of stress- responsive genes, which result in hybrid plants exhibiting enhanced traits due to heterosis.
  • the methods of the present invention can therefore be used to accurately predict advantageous traits in hybrid plants, including increased biomass or increased yield.
  • the present invention provides methods of producing hybrid plants with improved agronomic traits comprising selecting parent plants based on expression levels of stress- responsive or photosynthetic genes.
  • the invention provides methods comprising detecting expression levels of one or more stress-responsive genes in a plurality of plants, and selecting parent plants for a cross which express the stress-responsive genes at different levels. For example, a first parent plant may express high levels of a stress- responsive gene, while a second parent plant expresses low levels of the stress-responsive gene. In another example, a first parent plant may express low levels of a stress-responsive gene, while a second parent plant expresses high levels of the stress-responsive gene.
  • the plurality of plants from which the parent plants are selected according to the methods of the present invention may be from the same species of plant, and may comprise different genotypes.
  • parent plants for a hybrid cross may be selected by determining a difference in expression level of a particular gene between prospective parent plants.
  • Genes useful in selecting parent plants for crossing according to the invention include any stress-responsive gene.
  • parent plants may be selected by detecting a difference in expression levels of abiotic stress genes, biotic stress genes, or photosynthetic genes. Gene expression levels may be detected and compared between prospective parent plants at any timepoint, including zeitgeber time (ZT) 0, ZT6, ZT12, ZT15, ZT18, and ZT21.
  • parent plants having ratios of stress-responsive gene expression of 1.00, 1.50, or 2.00 are predictive of improved agronomic traits in progeny plants.
  • the present invention further provides methods of producing hybrid plants having beneficial traits by detecting and comparing expression levels of one or more stress- responsive genes to select parent plants for a cross. A difference in expression levels of one or more stress-responsive genes can be used to select parent plants for a cross resulting in hybrid plants with advantageous traits.
  • the methods of the present invention may comprise detecting expression levels of any stress-responsive gene, including biotic stress genes and abiotic stress genes.
  • biotic stress genes include PRl (AT2G14610), PR5 (AT1G75040), NUDT6 (AT2G04450), HSPR02 (AT2G40000), CNGC11 (AT2G46440), EBP/ERF72 (AT3G16770), PCC1 (AT3G22231), HR3/MLA10 (AT3G50470), CAX3 (AT3G51860), PAD4 (AT3G52430), WRKY70 (AT3G56400), PR2/BGL2 (AT3G57260), ACD6 (AT4G14400), NIA2 (AT1G37130), NHL3 (AT5G06320), Protease inhibitor (AT5G55450), WAK1 (AT1G21250), WAK2 (AT1G21270), EP1 (AT4G23170), RVE2 (AT5G37260),
  • PRl AT2G
  • Non-limiting examples of abiotic stress genes useful in the present invention include LSR3 (AT1G01470), ERD11/GSTF6 (AT1G02930), COR47/RD17 (AT1G20440), LTI45/ERD10 (AT1G20450), COR413-TM1 (AT1G29395), ALDH7B4 (AT1G54100), ATGRP3 (AT2G05520), PIP2B (AT2G37170), RD28 (AT2G37180), CAX1/RCI4 (AT2G38170), COR15A (AT2G42540), PIP1B (AT2G45960), LTI30 (AT3G50970), PIP1A (AT3G61430), PIP1;4 (AT4G00430), COR78/RD29A (AT5G52310), COR6.6/KIN2 (AT5G15970), RD22 (AT5G25610), ERD1 (AT5G51070), GRP8 (AT
  • Hybrid plants produced by the novel methods of the present invention therefore exhibit enhanced agronomic traits including modified biomass, modified yield, improved disease tolerance, improved insect resistance, improved pathogen resistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, and improved stress resistance.
  • the methods provided by the invention may be used to produce hybrid plants exhibiting advantageous traits resulting from heterosis in monocotyledonous or dicotyledonous plants including, but not limited to, Arabidopsis thaliana, maize (corn; Zea mays), soybean (Glycine max), cotton (Gossypium hirsutum; Gossypium sp.), peanut (Arachis hypogaea), barley (Hordeum vulgare); oats (Avena sativa); orchard grass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp.); tall fescue (Festuca arundinacea); turfgrass species (e.g.
  • oilseed crops may include soybean, canola, oil seed rape, oil palm, sunflower, olive, corn, cottonseed, peanut, flaxseed, safflower, and coconut, among others.
  • Expression levels of stress-responsive genes may be detected by various methods known to one of skill in the art. For example, expression levels of a gene in a tissue sample may be detected by isolating mRNA from the sample and quantitating mRNA transcripts by reverse transcription polymerase chain reaction (RT-PCR) using primers directed to the mRNA to be detected.
  • RT-PCR reverse transcription polymerase chain reaction
  • RNA-sequencing or microarray technologies can be used to identify genome-wide gene expression changes, including all stress-responsive genes, at different times of day. The data can be analyzed to find optimal times of day to detect expression level differences in stress-responsive genes among parental strains.
  • Detection of stress-response gene expression according to methods of the present invention may be carried out at any time of day.
  • stress-responsive gene expression levels may be detected in ACD6 at ZT18, in COR78 at ZT9, and in COR47 at ZT9.
  • the optimal time of day in which the stress-responsive gene expression differences between the parents are predictive of hybrid performance which corresponds to the peak of gene expression for a given gene in the diurnal cycle. Therefore, to find optimal times of day for hybrid performance prediction, gene expression of stress-responsive genes should be assayed for at least 24 hours to identify the peak times of gene expression in the desired parental types.
  • the present invention provides methods for identifying parent plants which can be crossed to produce a hybrid plant having a particular agronomic trait.
  • Hybrid plants resulting from a cross according to the invention may exhibit an improved trait compared with either of the parent plants, or compared with the mid-parent value (MPV) for a trait of interest.
  • the improved trait is increase biomass or increased yield.
  • One application of the present methods is therefore in breeding programs aimed at increasing the presence or degree of favorable traits in plants. This can be accomplished through a single round of breeding, or by repeated crossing of progeny plants with other suitable parent plants identified using the methods of the invention. Multiple rounds of crossing, including backcrossing, using the selection methods provided herein are contemplated by the invention.
  • Plant materials include the following Arabidopsis thaliana ecotypes, which were used to generate Fl hybrids; C24, Columbia (Col), Cvi-0 (CS22614), Est-1 (CS22629), Ler, Nd-1 (CS22619), Oy-0 (CS22658), Sorbo (CS22653), Wei-0 (CS22622), and Ws. Crossing was carried out as previously described (Miller, et al. G3 (Bethesda) 2:505-513, 2012).
  • acd6- 1 hyperactive mutant (Max Planck Institute for Developmental Biology, Tubingen, Germany), and ccal-11 (CS9378), lhy21 (CS9379), and ccal-l llhy21 (CS9380) T-DNA insertion mutants in the Ws background (Arabidopsis Biological Resource Center) were also used. All plants were grown under a 16/8-h light/dark cycle with temperatures of 22 °C (light) and 20°C (dark) on soil, and rosette leaves from ⁇ 3 week old plants before flowering were collected for RNA analysis, unless otherwise noted. For qRT-PCR validation, plants were grown in three biological replicates, and leaves were harvested every three hours for two diurnal cycles (48 hours). For plant transformation, 4-5 week old plants were used for Agrobacterium tumefaciens- mediated transformation through floral dipping.
  • Biomass was measured after drying rosettes before bolting at 80°C for 24 hours as previously described (Miller, et al. G3 (Bethesda) 2:505-513, 2012).
  • Trypan blue staining solution was prepared by adding 20 mg of Trypan blue to 40 ml of lactophenol solution for a final concentration of 0.5 mg rnl -1 . Leaves were incubated for 3 h in a sufficient amount of solution to immerse the tissue. The tissue was then cleared in a sufficient amount of chloral hydrate solution to cover the tissue (25 g of chloral hydrate per 10 ml water) for 30 min.
  • mRNA-seq reads from 12 libraries (Col, C24, ColxC24 and C24xCol at three time points) were mapped to the TAIR9 genome and cDNA sequence using BFAST (Blat-like Fast Accurate Search Tool, publically available on the internet).
  • Transcript levels were quantified by counting reads per kilobase per million mapped reads (RPKM).
  • RPKM kilobase per million mapped reads
  • SNP data for C24 was obtained from the Arabidopsis 1001 Genomes database (available on the internet). Reads mapped to regions containing SNPs were extracted and then assigned to reads from Col or C24 according to the SNP database. The total RPKM value in hybrids was split to Col and C24 alleles based on the ratio between the number of reads mapped to either Col and C24. To calculate cis and trans effects, a previously published strategy was employed (Shi, et al. Nat. Commun. 3, 950, 2012).
  • Integrative Genomics Viewer was used to display publically available mRNA- seq, sRNA-seq, and methylation data (Shen, et al. Plant Cell 24, 875-892, 2012) (GSE34658). Genes were identified as having a higher methylation level than the MPV if average methylation differences were 0.1 or greater for CG and CHG contexts and 0.05 or greater for CHH contexts, similar to the criteria used by Shen, et al.
  • genes present in QTL intervals were tested for significant overlap with DEGs using the GeneSect Tool, which implements a non-parametric randomization test which can determine whether the overlap between two gene lists is higher or lower than expected by chance, from the Virtual Plant website, as well as the hypergeometric test in R.
  • PCC1-F 19 ACAAATCTCACATCCTCACTCC PCC1 (At3g22231) qPCR
  • Plasmid constructs [0055] For luciferase reporter constructs, genomic DNA from Col, C24, and Ws was used to amplify ACD6 and COR78 promoter regions. SNPs between Col and C24 promoter and coding regions were identified using Polymorph (Ossowski, et al , publically available on the internet). The amplified fragments were cloned into pGEM-T vector (Promega) for sequence verification. The Promoter :LUC plasmid constructs were generated by inserting luciferase gene between the restriction enzyme sites Ncol and BamHI in the pFAMIR plasmid (Yadegari, University of Arizona).
  • the transcribed region of COR78 from Col or C24 was cloned into the pF35SE vector (A vector for 35S- driven gene expression, Yadegari, University of Arizona) between RsrII and Aatll restriction sites.
  • Artificial miRNAs were designed using the WMD3 web app (Ossowski and Fitz, publically available on the internet) against a conserved region in Col and C24 and then amplified using the pRS300 vector as a template. The amplified fragments were cloned into pGEM-T vector (Promega) for sequence verification.
  • the artificial miRNAs were then cloned into the pF35SE vector between RsrII and Aatll restriction sites for amiACD6 and between Xmal and Aatll restriction sites for amiCOR78. All constructs were individually cloned into Agrobacterium strain GV3101 for plant transformation and seeds were screened on 1 % (w/v) agar with Murashige and Skoog (MS) media containing 7.5 mg/L phosphinothricin. The primer sequences are listed in Table 1.
  • Plants containing either ACD6:LUC or COR78:LUC constructs were analyzed using a Packard TopCount luminometer as previously described. Seeds were sterilized with bleach and 75% ethanol and plated on 1 % (w/v) agar with MS media containing 7.5 mg/L phosphinothricin. Seeds were stratified 2 days in the dark at 4°C and then transferred into 16- h light and 8-h dark cycles for 7 days, and then transferred to MS containing no selection for 3 days. Seedlings were transferred to white microtiter plates (Nunc, Denmark) containing agar MS medium plus 30g sucrose/L and 30 iL of 0.5 mM luciferin (Gold Biotechnology).
  • Microtiter plates were covered with clear plastic MicroAmp sealing film (Applied Biosystems, Foster City, CA) in which holes were placed above each well for seedling gas exchange.
  • Applied Biosystems Foster City, CA
  • luciferin One day after addition of luciferin, plates were moved to the TopCount and interleaved with two clear plates to allow light diffusion to the seedlings. All luciferase data were analyzed using the Biological Rhythm Analysis Software System (BRASS, publically available on the internet). All period estimates were performed on rhythms from 24-120 hours using fast Fourier Transform-nonlinear least squares (FFT-NLLS) analysis.
  • FFT-NLLS fast Fourier Transform-nonlinear least squares
  • Fig. 14 The scheme for stress experiments is shown in Fig. 14.
  • 6 two week old seedlings (3 seedlings were used per replicate) were removed from agar media and were placed into 5 ml culture tubes containing room- temperature liquid MS media with 3% sucrose 24 hours before cold- treatment.
  • Cold- treatment was performed by placing the tubes in ice or leaving at 22°C for the control. After 60 minutes, tubes containing seedlings were placed back into 22°C and then whole seedlings (excluding roots) were snap frozen in liquid nitrogen at the ZT times listed in Fig. 14.
  • Example 1 Diurnal repression of stress-responsive genes in hybrids
  • Biotic stress-responsive genes such as ACCELERATED CELL DEATH6 (ACD6, At4gl4400), PATHOGEN AND ORCADIAN CONTROLLED 1 (PCC1, At3g22231), and HOMOLOG of SUGAR BEET HSl PRO-2 (HSPR02, At2g40000) were primarily repressed to below MPV levels between ZTO and ZT6 (Fig. 2a and Fig. 9a,b), and PATHOGENESIS-RELATED GENES 1 (PR1, At2gl4610) and PR5 (Atlg75040) were repressed at all times (Fig. 9c,d).
  • cold stress-responsive genes including COLD -REGULATED78 (COR78, At5g52310), COR47 (Atlg20440), COR15A (At2g42540), RESPONSIVE-TO-DESICCATION22 (RD22, At5g25610), and RD28 (At2g37180), were repressed in the middle and later parts of the day (Fig. 2b and Fig. 9e-h).
  • COLD -REGULATED78 COR78, At5g52310
  • COR47 Alg20440
  • COR15A At2g42540
  • RESPONSIVE-TO-DESICCATION22 RESPONSIVE-TO-DESICCATION22
  • RD28 At2g37180
  • irans-regulated genes of either parental origin could be repressed to the low-parent level or lower (Fig.10c -h), including several known biotic (Fig. lOi-1) and abiotic (Fig. lOm-p) stress- responsive genes.
  • Fig. lOi-1 several known biotic
  • Fig. lOm-p abiotic stress- responsive genes.
  • the genome-wide data suggest a role for trans-acting factors in the diurnal repression of stress-responsive genes in these hybrids.
  • the circadian clock regulators, CCA1 and LATE ELONGATED HYPOCOTYL (LHY, Atlg01060) were repressed in the middle of the day and upregulated around dawn (Fig. l la,b), and their feedback regulator TOC1 (At5g61380) showed the opposite expression changes (Fig.
  • ACD6(C24):LUC in C24 was expressed at higher levels than ACD6(Col):LUC in Col (Fig. 2c).
  • COR78(Col):LUC in Col was expressed at higher levels than COR78(C24):LUC in C24 (Fig. 2e).
  • the expression differences between the ecotypes were amplified when they were expressed in reciprocal combinations.
  • ACD6(Col):LUC expression amplitudes in C24 were 10- 15 -fold higher than ACD6(C24):LUC levels in Col (Fig. 2d).
  • COR78(C24):LUC amplitudes in Col were 7-8 fold higher than COR78(Col):LUC in C24 (Fig. 2f).
  • genetic backgrounds could act as transacting factors to mediate rhythmic expression peaks of these stress-responsive genes, probably through altered binding of regulatory factors such as the clock proteins or other upstream regulators to the promoters between the ecotypes (Table 3).
  • SNP data was obtained from the Arabidopsis 1001 genomes database (hitp:// " i001 genomes. org/).
  • Example 3 Stress-responsive expression as a predictor for heterosis
  • Fig. 4a,b, Table 4, and Fig. 12a-d The diurnal regulation of stress-responsive genes could provide a basis for natural variation among diverse ecotypes tested (Fig. 4a,b, Table 4, and Fig. 12a-d), which is consistent with their wide-geographical distributions.
  • abiotic genes were poorly expressed (Fig. 4a,b and Fig. 12c,d), but biotic genes were highly expressed at ZT18, which correlated with higher SA levels and more necrotic lesions on mature leaves in C24 than in Col 15 (Fig. lc). This is because these ecotypes are adapted to warmer environments with relatively more pathogens 40.
  • the table shows the overlap between all DEGs in hybrids (982 total) and QTL regions for dry weight at day 15 (DW15) from Meyer et al 2010. QTL regions were identified in the RILs and verified in the ILs (See Supplementary Table 5 in Meyer et al 2010).
  • hybrids Under long-term stress (cold or SA) conditions (see Methods and Fig. 14), hybrids maintained higher relative growth rates (RGR) than their parents (Fig. 4f-i). After removal from stress conditions the RGR of hybrids accelerated more than that of the parents, consistent with increased protection from freezing damage during cold stress. As a result, hybrids accumulated more biomass than parents (Fig. 15i,j). Hybrids also showed higher than MPV induction of cold-responsive genes at certain times after two weeks growing in the cold, although this trend was less obvious after the long-term treatment with SA (Fig. 15k-n).
  • Hybrid necrosis as previously reported, was likely caused by the induction of stress-responsive genes. Indeed, expression of several stress-responsive genes including PR1, PR2, and PRS is elevated in these necrotic hybrids. However, the higher- vigor hybrids have a better-timed stress-response strategy whereby stress-responsive genes are generally repressed under non-stress conditions and selectively induced at certain times under the stress, thus balancing the tradeoff between a rapid requirement for stress responses and long- term maintenance of growth vigor.

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Abstract

L'invention concerne des procédés de production de plantes hybrides présentant l'hétérosis, notamment des procédés permettant de sélectionner des plantes parentales pour un croisement permettant de produire une plante de descendance hybride ayant des propriétés avantageuses telles qu'une biomasse accrue et un rendement accru. L'invention concerne en outre des plantes, des parties de plantes, des cellules végétales, et des graines obtenues par ces procédés.
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