Progress 09/01/08 to 08/31/11
Outputs
OUTPUTS: We identified a quantitative trait locus (QTL) (Htg6.1) associated with thermoinhibition of seed germination at high temperatures in a recombinant inbred line population derived from lettuce (Lactuca sativa) cultivar (Salinas, thermosensitive) and L. serriola accession UC96US23 (UC, thermotolerant). Through genetic mapping and physiological evidence, we identified LsNCED4, a regulated gene in the abscisic acid (ABA) biosynthetic pathway, as a strong candidate to be responsible for Htg6.1. Using transgenic gene-transfer and silencing (RNAi) approaches, we demonstrated that the allele of LsNCED4 from the UC parent was necessary and sufficient to prevent the induction of thermoinhibition when lettuce seeds were imbibed at high temperature. We also used TILLING to isolate mutants in the LsNCED4 gene in a different cultivated lettuce background (cv. Desert Storm), two of which also resulted in a thermoinhibition-resistant phenotype. Together, these data provide convincing evidence that LsNCED4 is the gene responsible for the Htg6.1 QTL, which was the objective of the project. Thus, a primary output from the project is the knowledge that a single gene in the ABA biosynthetic pathway serves as a critical component of the signaling pathway connecting an environmental signal (temperature) with the biological response (germination). Using microarray data of the transcriptomes of seeds of the UC and Salinas genotypes imbibed at high and low temperatures for different times or in ABA, we identified genes whose transcription is altered by genotype/temperature/ABA combinations consistent with the germination phenotypes. Gene expression modules associated with different types of seed germination behavior were characterized and network diagrams were constructed that reveal gene expression correlations and interactions during seed thermoinhibition. This project output provides a basis for understanding the transcriptional networks through which hormonal regulation of seed germination occurs. Biological outputs from the project include RNAi lines in which LsNCED4 has been silenced. Seeds from this line provide useful experimental material for investigating the role of ABA in multiple aspects of seed biology. Similarly, lines in which LsNCED4 has been inactivated by mutation can be used experimentally, and are also being distributed for breeding into commercial lettuce cultivars. As they are not transgenic, but have a similar phenotype to RNAi lines, they can be utilized directly by breeding programs to incorporate high temperature germination tolerance into lettuce cultivars. These results have been disseminated at major international scientific meetings (see abstracts) and through peer-reviewed publications (additional manuscripts in final preparation now). They have also been disseminated directly to lettuce breeding companies through individual meetings, annual meetings with industry consortia, and through newsletters from the UC Davis Seed Biotechnology Center. Near-isogenic lines carrying the UC allele of LsNCED4 have been made available to breeders. PARTICIPANTS: Dr. Kent J. Bradford is the Project Director who manages the project and supervises the personnel and research program. Dr. Heqiang Huo (Postdoctoral Researcher) has conducted gene expression assays, constructed vectors for both Arabidopsis and lettuce transformation, and assayed the phenotypes resulting from transgenic modification. Dr. Peetambar Dahal (Staff Research Associate) has grown seed populations for fine-mapping, conducted genotyping assays to screen for recombinants, and propagated and analyzed TILLING mutants. Dr. Keshavulu Kunusoth (Visiting Scientist, BOYSCAST Fellowship, India) assisted in growing populations, developing Taqman assays for specific alleles, and genotyping of plants and progeny. Mr. Sebastian Reyes Chin-Wo (Undergraduate intern, Costa Rica Institute of Technology) conducted the bioinformatic and correlation analyses of microarray transcriptome data. Mr. Abdul Kadir Abd Wahab (Undergraduate assistant, Malaysia) assisted in plant propagation, seed germination, DNA/RNA extraction and other project activities. Mr. Miguel Macias (Undergraduate intern) contributed to fine mapping of the LsNCED4 alleles and to the mutant analyses. He is currently a MS student at UC Davis. Ms. Mandy Ding (Undergraduate intern) contributed to plant propagation, seed germination, DNA/RNA extraction and other project activities. Mr. Khalis Afnan Abdul Rahman (Undergraduate assistant, Malaysia) assisted in plant propagation, seed germination, DNA/RNA extraction and other project activities. Mr. Muhammad Faris Abd Rahim (Undergraduate assistant, Malaysia) assisted in plant propagation, seed germination, DNA/RNA extraction and other project activities. Dr. Richard Michelmore (UC Davis Genome Center) and Dr. Allen Van Deynze and Dr. Hamid Ashrafi (UC Davis Seed Biotechnology Center) are collaborators who assisted with the mapping and microarray components of the project. Dr. David Still (California Polytechnic and State University, Pomona) collaborates on transfer of the high temperature germination trait into cultivated lettuce varieties for the desert growing regions. Arcadia Biosciences, Davis, CA, and Dr. Claire McCallum collaborated on mutant identification through TILLING. This project provided training and professional development for a postdoc (China), a visiting scientist (India), and six undergraduates (Costa Rica, Malaysia and USA). TARGET AUDIENCES: The genetic and physiological results pinpointing a candidate gene responsible for the Htg6.1 QTL have been presented in a number of venues, including scientific conferences (see ABSTRACTS), industry meetings (California Seed Association) and presentations to numerous visitors to the Seed Biotechnology Center. In late 2010 and 2011, results were presented at four international universities and five seed companies (list of presentations below). A paper describing part of this work was published in 2011, and manuscripts describing the transgenic and mutant results and the transcriptome analyses will be submitted for publication soon. These results are being disseminated directly to seed companies and breeders who can incorporate greater thermotolerance into their commercial lettuce cultivars. Presentations made in 2010-11 describing results of this project (others reported in prior annual reports and in ABSTRACTS above): Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition and priming. Plantum Seed Technology Workgroup, Wageningen University, The Netherlands, September 21, 2010. Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition and priming. Monsanto Vegetable Seeds, Bergschenhoek, The Netherlands, September 27, 2010. Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition and priming. INCOTEC, Enkhuizen, The Netherlands, September 28, 2010. Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition and priming. Syngenta, Enkhuizen, The Netherlands, September 28, 2010. Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition and priming. Nunhems Seed (Bayer), Nunhem, The Netherlands, September 29, 2010. Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition and priming. Rijk Zwaan Seed, Fijnart, The Netherlands, September 30, 2010. Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition and longevity. University of Pierre and Marie Curie, Jussieu, Paris, France, October 21, 2010. Bradford, K.J. Genetic and hormonal regulation of lettuce seed thermoinhibition. Department of Horticulture, Taiwan National University, Taipei, Taiwan, Nov. 11, 2010. Bradford, K.J. UC Davis Seed Biotechnology Center and Bradford Laboratory Research. Department of Plant Science, Pontificia Catholic University, Santiago, Chile, May 2, 2011. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have confirmed that LsNCED4 is associated with lettuce thermoinhibition and that reducing its expression by natural variation, mutation or RNAi improves germination at high temperatures. Genetic markers have been developed that will enable the use of marker-assisted breeding to introgress the natural allele from L. serriola into L. sativa cultivars. Near-isogenic lines are being disseminated to plant breeding companies as sources of this high temperature-tolerant allele. Discussions are in progress for similar releases of the mutant lines along with markers to facilitate introgression into existing cultivars. Seeds with these alleles also appear to respond more strongly to pre-planting treatments such as priming that are currently used to alleviate thermoinhibition. The reduction or elimination of the high temperature sensitivity of lettuce seed germination will improve seed quality and crop stand establishment under stressful conditions. As the mutant alleles and the knowledge generated in this project are incorporated into lettuce breeding programs, we anticipate major impacts in improving seed performance and stand establishment for lettuce growers planting in the warm season to supply the nation's winter lettuce. Knowledge from the transcriptome analyses will provide insight into the interconnections among hormonal biosynthesis and signaling pathways in seeds and identify additional targets for modification to improve seed germination stress tolerance. This project achieved all of its proposed benchmarks. It is one of only relatively few examples where a QTL of a natural variant of a key phenotype in a crop plant has been traced back to the causal gene, variants of which can then be incorporated in cultivars to overcome a specific production problem. The human and technical resources made available through this project were essential to achieving these objectives. As indicated below under Participants, undergraduate and graduate students, a postdoctoral researcher, visiting scientists and collaborators from other universities were all involved in this project at various times. This project enabled training in genetics, molecular biology, biotechnology and seed physiology for a range of participants.
Publications
- ABSTRACTS Huo, H., Dahal, P., Kunusoth, K., Zuchi, J., Yoong, F.-Y., McCallum, C., Bradford, K. J. (2011) Expression of LsNCED4 encoding an ABA biosynthetic enzyme is required for thermoinhibition of lettuce seeds. 10th International Conference of the International Society for Seed Science, Salvador, Brazil, April 10-15, 2011.
- Huo, H., Bradford, K. J. (2011) A DOG1-like gene in lettuce is up-regulated by ABA during thermoinhibition of germination. 10th International Conference of the International Society for Seed Science, Salvador, Brazil, April 10-15, 2011.
- Reyes-Chin-Wo, S., Dahal, P., Kunusoth, K., Bradford, KJ. (2011) Global transcriptomic analyses of seeds of two lettuce species identify gene clusters associated with germination, thermoinhibition or ABA. 10th International Conference of the International Society for Seed Science, Salvador, Brazil, April 10-15, 2011.
- Groot, S.P.C., Bradford, K.J. (2011) Genetics and lettuce seed quality. Eucarpia Leafy Vegetables 2011, Lille (France) 24-26 August, 2011
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: We previously demonstrated that LsNCED4, a regulated gene in the abscisic acid (ABA) biosynthetic pathway, is a strong candidate to be responsible for a QTL (Htg6.1) associated with thermoinhibition of seed germination in a recombinant inbred line population derived from lettuce (Lactuca sativa) cultivar Salinas (thermosensitive) and L. serriola accession UC96US23 (thermotolerant). Transgenic expression of the LsNCED4 gene from both parents complemented Arabidopsis nced6 and nced9 mutants in multiple assays, indicating that LsNCED4 coding regions from both parents produced functional proteins. Expression of the promoter and coding region of LsNCED4 from the Salinas parent in the UC96US23 background caused thermoinhibition of UC96US23 seeds, confirming that this allele is sufficient to reproduce the effects of Htg6.1. Silencing of LsNCED4 expression in Salinas seeds using RNAi increased the upper temperature limit for germination. In addition, seeds of mutants isolated by TILLING that had stop or missense codons in LsNCED4 germinated at higher temperatures than did control seeds. Together, these data provide convincing evidence that LsNCED4 is the gene responsible for the Htg6.1 QTL. This gene is expressed highly late in seed development and is expressed constitutively in other plant tissues (confirmed by promoter-GUS expression in Arabidopsis), but is not responsive to water stress in leaves. Other NCED family members (LsNCED2 and LsNCED3) are responsive to water stress. BC4S2 progeny from two introgression lines were screened for recombination close to LsNCED4. Markers 0.7 cM apart flanking LsNCED4 as well as markers on a BAC containing LeNCED4 were assayed in ~3000 progeny to date. Recombinants have been identified and are being grown for seed production and phenotyping. Microarray data of the transcriptomes of seeds of the two genotypes imbibed at high and low temperatures for different times or in ABA were analyzed to identify gene sets whose transcription is altered by genotype/temperature/ABA combinations consistent with germination phenotypes. In addition to standard cluster and principal components analyses, weighted gene coexpression network analysis has been applied to the data. Gene expression modules associated with different types of seed germination behavior have been identified and network diagrams have been constructed that reveal gene expression correlations and interactions during seed thermoinhibition. These results have been disseminated via a presentation at the 3rd International Workshop on the Molecular Aspects of Seed Dormancy and Germination, York, UK, July 18-21, 2010 and presentations to seed company representatives in the Western Regional Seed Physiology Research Group. PARTICIPANTS: Dr. Kent J. Bradford is the Project Director who manages the project and supervises the personnel and research program. Dr. Heqiang Huo (Postdoctoral Researcher) has conducted gene expression assays, constructed vectors for both Arabidopsis and lettuce transformation, and assayed the phenotypes resulting from transgenic modification. Dr. Peetambar Dahal (Staff Research Associate) has grown seed populations for fine-mapping, conducted Taqman assays to screen for recombinants, and propagated and analyzed TILLING mutants. Dr. Keshavulu Kunusoth (Visiting Scientist, BOYSCAST Fellowship, India) assisted in growing populations, developing Taqman assays for specific alleles, and genotyping of plants and progeny. Mr. Sebastian Reyes Chin-Wo (Undergraduate intern, Costa Rica Institute of Technology) conducted the bioinformatic and correlation analyses of microarray transcriptome data. Mr. Abdul Kadir Abd Wahab (Undergraduate assistant, Malaysia) has assisted in plant propagation, seed germination, DNA/RNA extraction and other project activities. Dr. David Still (California Polytechnic and State University, Pomona) collaborates on transfer of the high temperature germination trait into cultivated lettuce varieties for the desert growing regions. This project is providing training and professional development for a postdoc (China), a visiting scientist (India), and two undergraduates (Costa Rica and Malaysia). TARGET AUDIENCES: The genetic and physiological results pinpointing a candidate gene responsible for the Htg6.1 QTL have been presented in a number of venues, including scientific conferences (see publications), industry meetings (California Seed Association) and presentations to numerous visitors to the Seed Biotechnology Center. In late 2010, results were presented at three international universities and five seed companies (details to be included in 2010-11 report). A manuscript is in press describing the genetic data (see publications), and manuscripts describing the transgenic and mutant results and the transcriptome analyses are in preparation for publication. These results are being disseminated directly to seed companies and breeders who can incorporate greater thermotolerance into their commercial lettuce cultivars. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In this year, we have confirmed that LsNCED4 is associated with lettuce thermoinhibition and that reducing its expression, by mutation, RNAi or natural variation, improves germination at high temperatures. Markers have been developed that will enable the use of marker-assisted breeding to introgress the natural allele from L. serriola into L. sativa cultivars. Recombinants are being identified that will isolate a small introgression carrying LsNCED4 from UC96US23 in a Salinas background. This material will subsequently be released for breeding programs also. Near-isogenic lines are already being disseminated to plant breeding companies as sources of this high temperature-tolerant allele. The incorporation of the UC96US23 allele of LsNCED4 into lettuce cultivars will improving seed quality and crop stand establishment under stressful conditions. Knowledge from the transcriptome analyses will provide insight into the interconnections among hormonal biosynthesis and signaling pathways in seeds and identify additional targets for modification to improve seed germination stress tolerance.
Publications
- ABSTRACTS Bradford, K.J., Huo, H., Reyes Chin-Wo, S., Dahal, P., Kunusoth, K., Still, D.W. (2010) Transcriptomic analyses of lettuce (Lactuca sativa L.) seeds in relation to thermodormancy. 3rd International Workshop on the Molecular Aspects of Seed Dormancy and Germination, York, UK, July 18-21, 2010.
- JOURNAL ARTICLES: Argyris, J., Truco, M.J., Ochoa, O., McHale, L., Dahal, P., Van Deynze, A., Michelmore, R.W., Bradford, K.J. 2010. A gene encoding an abscisic acid biosynthetic enzyme (LsNCED4) collocates with the high temperature germination locus Htg6.1 in lettuce (Lactuca sp.). Theor. Appl. Genet.: DOI 10.1007/s00122-010-1425-3
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: To fine map Htg6.1 QTL and candidate genes, a high-density map developed from microarray single position polymorphisms (SPPs) was used to show that the QTL coincided exactly with candidate gene LeNCED4. Selfed BC4 progeny were produced and markers are being developed from the map and from a BAC containing LeNCED4 to screen for recombinants to further isolate the candidate gene. Expression assays were conducted on developing and imbibed seeds and droughted leaves to determine differential expression of different LsNCED alleles. Promoter and coding region sequences of LsNCED4 were determined from 9 genotypes differing in seed thermoinhibition and polymorphisms are being analyzed for functional significance. Vectors were constructed to test for functionality (complementation) of the Salinas and UC96US23 LsNCED4 coding regions in Arabidopsis nced6/nced9 double mutants. Vectors for GUS reporter expression for Salinas and UC96US23 promoter analyses in Arabidopsis were also constructed. Transgenic Arabidopsis plants were generated and are being analyzed. RNAi vectors were constructed for transformation into lettuce to silence LsNCED4 specifically in Salinas variety. Vectors to overexpress LsNCED4 in the UC96US23 genotype were also constructed. Four potential mutants were identified by TILLING and selfed and backcrossed seeds were produced for further analysis. Microarray data have been collected for transcriptome analyses of seeds of two genotypes imbibed at high and low temperatures, in ABA, and for two times. These data are being analyzed to identify gene sets whose transcription is altered by genotype/temperature/ABA combinations consistent with germination phenotypes. In addition to standard cluster and principal components analyses, weighted gene coexpression network analysis has been applied to the data. Gene expression modules have been identified and network diagrams constructed. Analysis is continuing to determine whether transcriptional interactions between ABA and GA hormonal pathways can be identified. These results have been disseminated via abstracts and presentations at two meetings, the American Society of Plant Biologists Annual Meeting, Honolulu, HI (July 18-22, 2009) and 5th International Symposium on Seed, Transplant and Seedling Establishment of Horticultural Crops: Integrating Methods for Producing More with Less, Murcia, Spain (Sept. 27-Oct. 1, 2009). PARTICIPANTS: Dr. Kent J. Bradford is the Project Director who manages the project and supervises the personnel and research program. Dr. Heqiang Huo (Postdoctoral Researcher) has conducted gene expression assays, isolated and sequenced promoters and constructed vectors for both Arabidopsis and lettuce transformation. Dr. Peetambar Dahal (Staff Research Associate) has performed the microarray hybridizations, grown populations for fine-mapping, developed Taqman assays for specific alleles, and propagated potential TILLING mutants. Dr. Keshavulu Kunusoth (Visiting Scientist, BOYSCAST Fellowship, India) has assisted in growing populations, developing Taqman assays for specific alleles, and genotyping of plants and progeny. Mr. Sebastian Reyes Chin-Wo (Undergraduate intern, Costa Rica Institute of Technology,) has conducted the bioinformatic analyses of mapping and microarray data. Mr. Abdul Kadir Abd Wahab (Undergraduate assistant, Malaysia) has assisted in plant propagation, seed germination, DNA/RNA extraction and other project activities. Dr. Richard Michelmore (UC Davis Genome Center) and Dr. Allen Van Deynze and Dr. Hamid Ashrafi (UC Davis Seed Biotechnology Center) are collaborators who assisted with the mapping and microarray components of the project. Dr. David Still (California Polytechnic and State University, Pomona) collaborates on transfer of the high temperature germination trait into cultivated lettuce varieties for the desert growing regions. This project is providing training and professional development for a postdoc (China), a visiting scientist (India), and two undergraduates (Costa Rica and Malaysia). TARGET AUDIENCES: While early in this project, the fine mapping results pinpointing a candidate gene using a dense map developed from microarray polymorphism data have been presented in a number of venues, including scientific conferences (see publications), industry meetings (California Seed Association) and presentations to numerous visitors to the Seed Biotechnology Center. They are the first illustration of the power of this high-density map to identify candidate genes directly from mapping data. A manuscript describing these data is in final preparation for publication. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
As this is the first year of the project, most outcomes and impacts are yet to come. Knowledge from this project is being incorporated into lettuce germplasm development programs with the expected outcome of improving seed quality and crop stand establishment under stressful conditions.
Publications
- ABSTRACTS Dahal, P., Argyris, J., Huo, A., and Bradford, K.J. (2009) A gene encoding an ABA biosynthetic enzyme (LsNCED4) maps precisely with a QTL (Htg6.1) for thermodormancy of lettuce seeds. American Society of Plant Biologists Annual Meeting, Honolulu, HI, July 18-22, 2009
- Bradford, K.J., Argyris, J., Dahal, P., Huo, H., Truco, M.J., Ochoa, O., Still, D.W., Hayashi, E., Michelmore, R.W. (2009) Genetic and hormonal regulation of lettuce seed germination at high temperatures. 5th International Symposium on Seed, Transplant and Seedling Establishment of Horticultural Crops: Integrating Methods for Producing More with Less. Murcia, Spain, Sept. 27-Oct. 1, 2009
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