Source: TEXAS A&M UNIVERSITY submitted to
MOLECULAR MAPPING OF GENES FOR DROUGHT AND HEAT TOLERANCE IN COWPEA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1000977
Grant No.
2014-67013-21590
Project No.
TEX09569
Proposal No.
2013-02238
Multistate No.
(N/A)
Program Code
A1101
Project Start Date
Dec 1, 2013
Project End Date
Nov 30, 2016
Grant Year
2014
Project Director
Zhang, M.
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Soil and Crop Science
Non Technical Summary
Cowpea (blackeye pea) is a high-protein grain, vegetable, fodder and high nitrogen-fixing legume intercrop in the cropping systems of many crops such as corn and cotton in many countries, including the USA. It is highly tolerant to drought, heat and several other biotic/abiotic stresses. This research is to take the advantage of recently-developed high-throughput sequencing technology-based restriction site-associated DNA sequencing (RAD-seq) to map the genes controlling drought and heat tolerance and to develop SNP markers, the latest DNA marker technology, enabling efficient manipulation of heat and drought tolerances in cowpea and related species. We previously developed the methods efficient for high-throughput assay of drought and heat tolerance in cowpea, identified a number of drought and/or heat highly-tolerant lines and developed a recombinant inbred line (RIL) mapping population for genetically analyzing drought and heat tolerance. In this research, we will genetically analyze the drought and heat tolerance of the population, map the related genes and develop tools enabling effectively manipulating the traits. This research will not only map the drought and heat tolerant genes in cowpea, but also provide a platform for mapping the genes controlling several other biotic and abiotic stress tolerances such as aphid resistance and low-phosphorus tolerance, both of which are also of extreme significance for agricultural production of many crops. The drought and heat tolerant genes, once defined and cloned, will significantly advance our understanding of molecular basis underlying plant drought and heat tolerances, and help design tools enabling effectively combining multiple adaptive traits into new cultivars adapted to global changing climate in this and related crops, thus supporting the long-term genetic improvement in and sustainability of U.S. agriculture and food systems.
Animal Health Component
0%
Research Effort Categories
Basic
40%
Applied
20%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011410108040%
2031410108040%
2021410108120%
Knowledge Area
202 - Plant Genetic Resources; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1410 - Beans (dry);

Field Of Science
1080 - Genetics; 1081 - Breeding;
Goals / Objectives
Drought and heat have been major production constraints in many crops for centuries. As the problems are becoming more serious and new powerful technologies are becoming available and more economical, there is a renewed worldwide interest in identification and incorporation of drought and heat tolerant genes in improved crop varieties. Cowpea, also known as blackeye pea in the USA, could be an excellent system and a source of genes to address the issue of plant drought and heat tolerance due to the availability of genetic resources unique in these stress tolerances and genetics. The goal of this proposal is to take the advantage of recently-developed next generation high-throughput sequencing technology-based Restriction site-Associated DNA sequencing (RAD-seq) to rapidly map the genes controlling drought and heat tolerance in cowpea and develop SNP (single nucleotide polymorphism) markers enabling efficient genetic manipulation and breeding of the traits within cowpea and potentially in other related crop species. Toward this end, we have developed a mapping population of 110 recombinant inbred lines (RILs) (F2:6) from a cross of two cowpea lines (IT98K-476-8 and GEC) that are highly tolerant or susceptible to both drought and high temperature (see below). Currently, we are augmenting the population into more than 200 RILs which will be available before this project commences (hereafter the population is defined as the ITG RIL population). In this research, we will especially accomplish the following three specific research objectives: Phenotypically analyze the drought and heat tolerances of the ITG RIL population; Genetically map the genes controlling the drought and heat tolerances using RAD-seq; and Identify SNP markers closely linked to the genes of drought and heat tolerances, and develop molecular tools enabling enhanced breeding of the traits in cowpea and related species.
Project Methods
This research is to map the genes or QTLs controlling drought and heat tolerances using the state-of-the-art high-throughput sequencing technology based-RAD-seq, isolate SNP markers closely linked to the genes, and to develop genomic toolkits enabling enhanced drought and heat tolerance breeding and ultimately, cloning the genes controlling the traits. Particularly, we will accomplish the following three research objectives: Objective 1: Phenotypically analyze the drought and heat tolerances of the ITG RIL population, 09/01/2013 - 08/31/2016 The ITG RIL population consisting of over 200 RILs (F2:6) developed in our preliminary research will be used in this research. This population has been shown to genetically segregate for both drought and heat tolerances and several other traits of agronomic importance. Nevertheless, in this project we will only focus on genetics and mapping of the drought and heat tolerant genes. In the next phase of the project, we will use the SNP genotype dataset to be created in this research to map the genes for low-P tolerance and resistance to aphid and Striga. The protocols for phenotyping these traits have been well established and will be used for this study. Drought tolerance: A simple wooden box screening method previously developed by this group (Singh et al. 1999) will be used to phenotype the drought tolerance of the ITG RIL population at the seedling stage which has been shown to be highly correlated with the drought tolerance at all the stages of cowpea growth including reproductive stage. This method has been well established by evaluating the cowpea test lines in boxes, pots as well as in the field (Singh and Matsui 2002). The plants will be watered daily using a small watering can until the partial emergence of the first trifoliate leaf, after which watering is stopped. Thereafter, when the water stress becomes visible, a daily record of wilted plants in each line (DAS, days after stress) will be made until all the plants of the susceptible lines appear dead. Based on the days taken to wilting and permanent death of plants, each line will be rated drought tolerant or susceptible, on the RIL basis with 10 plants per RIL. The experiment will follow a complete randomized block design and be repeated three times to ensure the accuracy of the phenotyping. Heat tolerance: The early studies on screening for heat tolerance were based on the observations under naturally occurring hot environments, with a day temperature of about 40oC and a night temperature of above 25oC. In this research, the heat tolerance phenotyping method that we developed in the preliminary research will be used to phenotype the heat tolerance of the ITG RIL population in an environmentally-controlled greenhouse. A day temperature regime of 40oC - 43oC and a night temperature regime of 25oC - 28oC that are the same as those of our field trial in 2011, Texas will be used. Flowering, flower drop, pollen sterility, pod set and grain yield per plant will be assayed, relative to the controls of the lines growing in the normal condition (non-heat stressed). The experiment will follow a complete randomized block design and be repeated three times to ensure the accuracy of the phenotyping, with 10 plants per RIL. Expected Outputs: Three times-repeated phenomic data of heat and drought tolerances of 200 RILs and 2 parents collected from an environmentally-controlled greenhouse. Objective 2: Genetically map the genes controlling the drought and heat tolerances using RAD-seq, 09/01/2013 - 08/31/2015 To map the genes controlling the drought and heat tolerances in cowpea with SNP markers and to develop the toolkits enabling enhanced drought and heat tolerance breeding, we will genotype the ITG RIL population and construct a high-density SNP map by RAD-seq using the next-generation sequencing technology. The ddRADseq method and associated data analysis pipeline developed by Peterson et al. (2012) will be used. Plant tissues will be collected at the seedling stage of plant growth and nuclear DNA will be isolated. The ddRAD-seq libraries will be constructed with EcoR I and Msp I (Peterson et al. 2012) and subjected to HiSeq 2000. As cowpea has a genome size of 620 Mb/1C, there are theoretically 151,000 EcoR I sites in its genome. If both sequences flanking the EcoR I sites are sequenced, with an average read length of 100 bp, the collective unique sequences will be 151,000 EcoR I sites x 100 bp per read x 2 sides = 30,200 kb. Since ~3 billion 100-base reads of high-quality sequences could be generated per HiSeq 2000 run and if all 200 RILs and 2 parents of the population are sequenced in one flow cell of the Hiseq 2000, the genome coverage per site per RIL will be >49x, which is well-suited for the SNP discovery and genotyping purposes in this research (Byers et al. 2012; Peterson et al. 2012). The sequencing data will be analyzed using the cowpea genome sequence (Chen et al. 2007; Close et al. 2011) as a reference. SNPs will be identified and used for genotyping the RILs and parents. We will first construct an SNP genetic map for cowpea from the SNPs as the standard SNP mapping using JoinMap5 (Kyazma, Holland). Then, we will map the phenotypic data of drought tolerance (DAS - days after stress) and heat tolerance (flowering, flower drop, pollen sterility, pod set and grain yield per plant) using multiple interval mapping (MIM) (Zeng et al. 1999) as implemented in QTL Cartographer and Windows QTL Cartographer (http://statgen.ncsu.edu/qtlcart/). Furthermore, the research results will be comparatively analyzed against the QTLs previously mapped (for review, see Zhang et al. 2008), whenever possible. Expected Outputs: 1) >15-fold RAD sequences of 200 RILs and 2 parents; 2) a high-density SNP genetic map for cowpea; and 3) mapped genes or QTLs of cowpea drought and heat tolerances and their interactions. Objective 3: Identify SNP markers closely linked to the genes of drought and heat tolerance and develop molecular tools enabling enhanced breeding of the traits in cowpea and related species, 09/01/2014 - 08/31/2016 Because SNPs are abundant in plant genomes, including cowpea, with a range of 1 - 20 SNPs/kb, it is expected that several closely-linked SNPs will be identified for each gene or locus controlling the cowpea drought or heat tolerance. We will first identify the SNPs most closely linked with and flanking the genes for drought or heat tolerance from the resulting SNP genetic map and use their sequence tags as anchors to conduct alignments against the ESTs and genome sequences of cowpea (Chen et al. 2007; Close et al. 2011) and other legume species whose genome sequences are available such as soybean (Schmutz et al. 2010) and M. truncatula (Young et al. 2011). This work could allow identifying the ESTs (if the SNP tags are part of ESTs) and/or genomic loci of the SNPs in the cowpea genome or existing genetic map(s) and their corresponding orthologous loci in the other legume genomes. This will help validate the mapping results of the genes, if it happens they were also mapped in the genomes. The SNP-containing ESTs and/or genomic loci will also help design assay primers for each of the SNP markers. We will validate the SNP markers for each of the drought or heat tolerant genes or QTLs using the KASPar (KBioscience Ltd., Hoddesdon, UK) assay among the 41 cowpea lines that we screened in the Preliminary Research. The primers for each SNP closely linked to the genes or QTLs of the drought or heat tolerance will be designed using PrimerPicker (KBioscience 2009) with default parameters. Expected Outputs: SNP-based breeding toolkits that enable effective manipulation of the drought and heat tolerant genes and enhanced stress tolerance breeding in cowpea.

Progress 12/01/13 to 11/30/16

Outputs
Target Audience:The target audience of this project has been breeders, students in plant breeding and researchers in genetics, molecular biology, genomics and molecular breeding. The major research goals of this project are to map the genes controlling heat and drought tolerance using the modern SNP markers generated with the RAD-seq technology and to develop SNP haplotype markers that enable to enhance breeding for heat and drought tolerance in cowpea and related species. We have made significant efforts to transfer and disseminate the technologies and toolkits developed in the project: We transferred the findings of this project and SNP marker tools developed through this project to cowpea breeders, including local, national and international, such as several cowpea breeders of the International Institute of Tropical Agriculture (IITA), Nigeria. We have trained 1 undergraduate student and 2 Ph.D. graduate students in plant breeding and science. The two Ph.D. students, Laura Masor and Brijesh Angira, did their dissertation research through this project, with Laura Masor focusing on genetic analysis and mapping of cowpea drought tolerance and Brijesh Angira focusing on genetic analysis and mapping of cowpea heat tolerance. The RAD-seq data and cowpea SNP genetic map developed through this project has been made available to the public and disseminated to several cowpea genomics and genetics researchers such as Dr. Timothy Close, University of California at Riverside, CA. Moreover, the genetic map is also being used by colleagues to map the genes controlling serval other traits important to agriculture such as low-P tolerance and to clone the genes controlling heat and drought tolerance. The RAD-seq data and phenotypic data generated through this project are being used to develop models and methods of genomic selection, a rising technology for enhanced plant breeding, in cowpea. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained 1 undergraduate student (Marco Roque) and 2 Ph.D. graduate students (Laura Masor and Brijesh Angira) in plant breeding, genetics, genomics and molecular breeding. All of these students were paid through this project. Both of the Ph.D. graduate students have their research and dissertation sponsored by this project. We have also trained a postdoc, Dr. Yun-Hua Liu, in large-scale RAD seq data analysis, SNP discovery and SNP genotyping. Moreover, the pipeline developed for construction of RAD-seq DNA libraries, large-scale RAD-seq data analysis, large-scale SNP discovery and genotyping in this project has been applied to a cotton project (PI: Hongbin Zhang) sponsored by USDA/NIFA (2013-01839) and used to train students in our programs and class rooms, and visiting scientists for germplasm analysis, SNP discovery, genetic map development, gene and QTL mapping, and genome-wide association study (GWAS). How have the results been disseminated to communities of interest? One of the investigators of this project, Dr. B. B. Singh, is a cowpea breeder at the Texas A&M AgriLife Research (formerly, Texas Agricultural Experiment Station). This has allowed the results and toolkits developed in this project to be immediately transferred to cowpea breeding in his program. The ITG RIL population has allowed him to develop three super early mature lines, with a maturity of only 45 days. The project has been reported in AgriLife TODAY with the title "Texas A&M researchers to study cowpea drought and heat tolerance". The research results and findings have been presented at a number of local, national and international conferences. The sequence data and the genetic map has been disseminated through networks to several laboratories in the USA. The dissertations of the two Ph.D. students have been made accessible to the public through TAMU libraries. Four manuscripts are currently in preparation from the results of this project, which will be submitted for publication in peer-reviewed professional journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have accomplished analysis of all data, including genotypic data and phenotypic data. The two Ph.D. students, Laura Masor and Brijesh Angira, sponsored by this project have written their dissertations and passed their defenses. We have also had the second version of the manuscripts (four) from the results of this project. We will submit them for peer-reviewed publications of these results as soon as we have the final versions of the manuscripts (within a month).

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Singh, B.B. 2016. Genetic enhancement for yield and nutritional quality in cowpea [Vigna unguiculata (L.) Walp.]. Indian J. Genet., 76(4): 568-582
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Angira, Brijesh. 2016. Genetic and physiological studies of heat tolerance in cowpea. Ph.D. dissertation, Texas A&M University, College Station, Texas.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Masor, Laura. 2016. Molecular mapping of traits in cowpea. Ph.D. dissertation, Texas A&M University, College Station, Texas.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zhang Y, Scheuring CF, Angira B, Masor LL, Liu Y-H, Zhang YD, Singh BB, Zhang H-B, Hays D, Zhang MP. 2016. Molecular mapping of the genome and agronomic traits in cowpea. International Plant & Animal Genome Conference XXIV, W1014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Singh BB, Angira B, Masor LL,Zhang MP, Zhang H-B, Foster JL, Asiwe JA, Singh YV, Hays D. 2016. Breeding next generation cowpea varieties for adaptation to changing climates and cropping systems. Joint Pan-African Grain Legume and World Cowpea Conference 2016, Feb. 28  March 4, 2016, Livingstone, Zambia (poster presentation) http://gl2016conf.iita.org/index.php/presentations/.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Angira B, Masor L, Zhang Y, Scheuring CF, Zhang YD, Singh BB, Zhang H-B, Hays DB, Zhang MP. 2016. Molecular mapping of heat tolerance in cowpea. Joint Pan-African Grain Legume and World Cowpea Conference 2016, Feb. 28  March 4, 2016, Livingstone, Zambia (invited oral presentation) http://gl2016conf.iita.org/index.php/presentations/.


Progress 12/01/14 to 11/30/15

Outputs
Target Audience:The major research goals of this project are to map the genes controlling heat and drought tolerance using the modern SNP markers with the RAD-seq technology and to develop SNP markers that enable enhanced breeding for heat and drought tolerance in cowpea and related species. One of the investigators of this project, Dr. B. B. Singh, is an internationally leading cowpea breeder. In this project, he has not only conducted the proposed research activities, he has also made significant efforts to transfer and disseminate the technologies and toolkits developed in the project to cowpea breeders and graduate students who are majoring in plant breeding such as Ms. Laura Masor (Ph.D. student) and Mr. Brijesh Angira (Ph.D. student). The genetic map will be also used to map the genes controlling several other traits important to agriculture such as Aphid resistance and low-P tolerance, and to clone the genes controlling heat and drought tolerance. Once the genes for these traits are cloned, they will provide tools for deciphering the molecular mechanisms underlying plant heat and drought tolerance and aid in effective breeding for plant heat and drought tolerance. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A simple, rapid and economical protocol has been developed for construction of RAD-seq DNA libraries in this project. The protocol has been shared with a cotton project (PI: Hongbin Zhang) sponsored by USDA/NIFA (2013-01839) and used to train students and visiting scientists for germplasm analysis, SNP discovery, genetic map development, gene and QTL mapping, and genome-wide association study (GWAS). How have the results been disseminated to communities of interest?1) One of the investigators of this project, Dr. B. B. Singh, is a cowpea breeder at the Texas A&M AgriLife Research (formerly, Texas Agricultural Experiment Station). This has allowed the results and toolkits developed in this project to be immediately transferred to cowpea breeding in his program. The ITG RIL population has allowed him to develop three super early mature lines, with a maturity of only 45 days. 2) The project has been reported in AgriLife TODAY with the title "Texas A&M researchers to study cowpea drought and heat tolerance". What do you plan to do during the next reporting period to accomplish the goals?As proposed in the original proposal, we have nearly accomplished or exceeded the proposed research objectives. In 2016, we plan to accomplish the followings: 1) Continue to phenotype the ITG RIL population in both field and greenhouse conditions (Objective 1), with an emphasis on heat and drought tolerance; 2) Continue to map the QTLs controlling agronomic traits, with an emphasis on heat and drought tolerance (Objective 2); and 3) Write manuscripts from the results and publish them in peer-reviewed journals.

Impacts
What was accomplished under these goals? This is a three-year project started on December 1, 2013. We have nearly accomplished or exceeded the proposed research objectives: Objective 1 - Phenotypically analyze the drought and heat tolerance of the ITG RIL population (Years 1 - 3) We planted the 184RILs of the ITG RIL population, along with parents in greenhouse on campus and at the Texas A&M AgriLife Research Farm in College Station, Weslaco and Corpus Christi, Texas in 2014 and 2015, with two replications under both irrigated and drought-stressed conditions. Observations were recorded on tolerance to heat and drought, plant height, plant type, joint pigmentation, date to first flower, date to first pod maturity, and date to 95% maturity. Furthermore, five plant samples were harvested from both replicates to measure biomass, pod weight, seed weight, number of seeds per pod, number of pods, and 100-seed weight. Days to first flower of the ITG RILs ranged from 30 to 60 days and maturity ranged from 45 to 89 days in the field. Statistical analysis showed a significant difference among the ITG RILs for days to flowering, days to maturity, biomass. 100-seed weight and plant height. Plant height of the population ranged from 8 to 34 cm, which confirmed a wide variation within the population. Correlation between maturity and height was found to be significant, with a correlation coefficient of 0.74. Similar results were found in the greenhouse study. Days to flowering and days to maturity in greenhouse ranged 23 to 61 days and 45 to 92 days, respectively, showing a transgressive segregation, as the parent GEC matured in about 65 days and the parent IT98K-476 matured in about 75 days. Objective 2 -Genetically map the genes controlling the drought and heat tolerance using RAD-seq (Years 1 - 2) Leaf tissues were sampled from all 184RILs and parents (GEC and IT98K-476-8) of the ITG population (F2:8). Nuclear DNA was isolated from the entire ITG population and parents, and constructed into RAD-seq libraries with a combination of restriction enzymes, BamHI and MluCI. The flanking sequences of the BamHI sites were sequenced for all 184RILs and parents of the ITG population using a 100SE module with HiSeq 2000. A range of 380,399 - 2,267,979 100-nucleotide clean reads were obtained for each line, with an average of 1,449,966 100-nucleotide clean reads per line. Over 97% of the sequences had a quality of ≥ Q20. Given that cowpea has a genome size of 620 Mb/1C and an estimation of one BamHI site in approximately 10 kb, there are approximately 62,000 BamHI sites in the cowpea genome. Because we sequenced both flanking sides of all BamHI sites, the coverage of the sequences for each BamHI site ranged from 6.1x to 36.6x for each line, with an average coverage of 11.7x for each site in the genome of each line. Therefore, the RAD sequences of the population should be sufficient for construction of a SNP genetic map for cowpea and for mapping of the genes controlling drought and heat tolerances in the species. In 2015, we analyzed the RAD sequences using our RAD-seq sequence analysis pipeline and identified 6,001 SNPs for the ITG RIL population. Then, we examined the SNPs one by one and filtered those significantly distorted in genetic segregation. Consequently, we obtained a total of 4,154 high-quality SNPs. We constructed an SNP genetic map for cowpea from the 4,154 SNPs using a software named using QTL IciMapping (Meng et al. 2015, Crop J., 3:269-283). The software first grouped the SNPs that are genetically co-segregating into bins and then constructed them into the genetic map. A total of 531 bins, with each bin consisting of an average of 7.8 SNPs, resulted. We constructed the SNP genetic map using four different sets of parameters. The results showed that the SNP genetic maps constructed using all four sets of parameters were almost identical. Therefore, we selected the genetic map constructed with the fourth set of parameters, named v4.0, for trait QTL mapping. The map v4.0 consists of 11 linkage groups (LGs) and 531 bins containing 4,154 SNPs. The map collectively spans 1,084.65 cM, having a density of one SNP marker in approximately 0.26 cM or 149 kb. We mapped the proposed heat tolerance and drought tolerance using the SNP genetic map constructed in this project. For heat tolerance mapping, the phenotypic data were collected from both greenhouse (2014) and the field condition (2015) in Corpus Christi and Weslaco, Texas. All experiments had 2 replicates. A ranking scale ranging from 0.00 for highly heat tolerance to 5.00 for highly heat sensitivity was used to phenotype the heat tolerance. Therefore, G x E interaction was taken into account in its QTL mapping. As a result, seven QTLs were identified, distributing in four LGs, 2, 3, 4 and 8, when a LOD score of >3.00 was applied. Of these seven QTLs, one on LG 3 had a LOD score of 5.8 that was significantly higher than the LOD scores of other QTLs, agreeing with the previous study showing that heat tolerance in the genetic background was likely controlled by one major gene. For drought tolerance mapping, the percentages of 100-seed weight, number of pods per plant and biomass of the RILs under drought-stressed condition relative to irrigated conditions were used. The phenotypic data were collected from three locations, College Station (2014 and 2015), Corpus Christi (2015) and Weslaco (2015), Texas. When G x E interaction was taken into account and LOD ≥ 3.00 was applied, we identified 14 QTLs (LODs = 3.0 - 8.0) for the percentage of 100-seed weight, 2 QTLs (LODs = 3.3 or 3.4) for the percentage of number of pods per plant and 19 QTLs (LODs = 3.0 - 12.1) for the percentage of biomass yield. In addition, we mapped the biomass yield, plant height, flowering date (days to flower) and 100-seed weight that varied significantly (P ≤ 0.05) in the irrigated and drought-stressed conditions using LOD scores ≥ 3.00. In the drought stressed condition, we identified 29 QTLs (LOD scores = 3.0 - 9.3) for flowering date, 13 QTLs (LOD scores = 3.5 - 5.8) for biomass yield, 3 QTLs (LOD scores = 3.0 - 3.5) for 100-seed weight and 24 QTLs (LOD scores = 3.1 - 7.2) for plant height. In the irrigated condition, we identified 6 QTLs (LOD scores = 3.6 - 4.4) for biomass yield, 7 QTLs (LOD scores = 3.1 - 4.5) for flowering date, no QTL for 100-seed weight and 8 QTLs (LOD scores = 3.0 - 7.5) for plant height. The difference in number of QTLs identified between the two conditions suggests the importance of G x E interaction in the trait performance. We have also estimated the effects of additive (A) and G x E interaction (A by E) on the performance of all the traits mapped, including the heat tolerance and drought tolerance. Objective 3 Identify SNP markers closely linked to the drought and heat tolerant genes, and develop molecular tools enabling enhanced breeding of the traits in cowpea and related species (Years 2 - 3) Based on the above QTL mapping results, we have identified the SNP bins flanking every QTL mapped in this project. Each bin contains an average of 7.8 SNPs. Because the SNPs in every bin are genetically co-segregating, any of them could be used for marker-assisted selection of the trait QTLs for enhanced breeding. The multiple SNPs flanking each QTL significantly broaden the utility of the markers in different breeding programs because as long as one of them is polymorphic in a breeding population, it will be applicable to the breeding population for marker-assisted selection. Therefore, the SNP bins are much more applicable for different breeding programs than a single marker mapped for a QTL.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Masor LL, Hays DB, Singh BB, Zhang H-B, Zhang MP. 2015. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). Oregon State University Plant Breeding Seminar (Oral), Corvallis, OR. June 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Masor LL, Hays DB, Singh BB, Zhang H-B, Zhang MP. 2015. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). Texas A&M Plant Breeding Symposium. Poster presentation, College Station, TX. Feb. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Angira B, Masor L, Zhang Y, Scheuring CF, Zhang YD, Singh BB, Zhang H-B, Hays DB, Zhang MP. 2015. Molecular Mapping of Heat Tolerance in Cowpea. American Society of Agronomy Annual Meeting. Crop Breeding and Genetics: II. Poster 521. Minneapolis, MN. Nov. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Masor LL, Scheuring CF, Zhang Y, Angira B, Zhang MP, Zhang H-B, Singh BB. 2015. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). National Association of Plant Breeders Annual Meeting. Poster presentation, Pullman, WA. Aug. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Masor LL, Scheuring CF, Zhang Y, Angira B, Zhang MP, Zhang H-B, Singh BB. 2015. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). American Society of Agronomy Annual Meeting. Oral presentation, Minneapolis, MN. Nov. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Masor LL, Scheuring CF, Zhang Y, Angira B, Zhang MP, Zhang H-B, Singh BB. 2015. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). Student Organic Seed Symposium. Oral presentation, Madison, WI. Aug. 2015.


Progress 12/01/13 to 11/30/14

Outputs
Target Audience: The major research goals of this project are to map the genes controlling heat and drought tolerance using the modern SNP markers with the RAD-seq technology and to develop SNP haplotype markers that enable enhanced breeding for heat and drought tolerance in cowpea and related species. One of the investigators of this project, Dr. B. B. Singh, is a cowpea breeder. He has not only conducted the proposed research activities of the project, he has also made significant efforts to transfer and disseminate the technologies and toolkits developed in the project to cowpea breeders and graduate students who are majoring in plant breeding such as Ms. Laura Masor (Ph.D. student) and Mr. Brijesh Angira (Ph.D. student). The genetic map will be also used to map the genes controlling serval other traits important to agriculture such as Aphid resistance and low-P tolerance, and to clone the genes controlling heat and drought tolerance. Once the genes for these traits are cloned, they will provide tools for deciphering the molecular mechanisms underlying plant heat and drought tolerance and aid in effective breeding for plant heat and drought tolerance. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A simple, rapid and economical protocol has been developed for construction of RAD-seq DNA libraries in this project. The protocol has been shared with a cotton project (PI: Hongbin Zhang) sponsored by USDA/NIFA (2013-01839) and used to train students and visiting scientists for germplasm analysis, genetic map development, gene and QTL mapping, and genome-wide association study (GWAS). How have the results been disseminated to communities of interest? One of the investigators of this project, Dr. B. B. Singh, is a cowpea breeder at the Texas A&M AgriLife Research (formerly, Texas Agricultural Experiment Station). This has allowed the results and toolkits developed in this project to be immediately transferred to cowpea breeding in his program. The ITG RIL population has allowed him to develop a few of super early mature lines, with a maturity of only 45 days. The project has been reported in AgriLife TODAY with the title "Texas A&M researchers to study cowpea drought and heat tolerance". What do you plan to do during the next reporting period to accomplish the goals? As proposed in the original proposal, we plan to accomplish the following experiments in the next reporting period: Continue to phenotype the ITG RIL population in both field and greenhouse (Objective 1), with an emphasis on heat and drought tolerance; Continue to construct a SNP genetic map for cowpea and map the QTLs controlling agronomic traits, with an emphasis on heat and drought tolerance (Objective 2); and Develop SNP-based molecular toolkits enabling enhanced cowpea breeding, especially for heat and drought tolerance (Objective 3).

Impacts
What was accomplished under these goals? This is a three-year project started on Decembery 1, 2013. We have accomplished or exceeded the proposed research objectives according to the project timetable (see the original proposal). Objective 1 - Phenotypically analyze the drought and heat tolerance of the ITG RIL population (Years 1 - 3) We have accomplished the development of the ITG RIL pollution (F2:8) from the cross GEC x IT98K-476-8 and development of a new RIL population (F2:7) from the cross IT99K-241-2 x IT97K-556-4 (named ITIT RIL population) by the single seed descent method. The ITG population consists of 180 RILs and was shown to segregate for both heat and drought tolerance, whereas the ITIT population consists of 192 RILs and was shown to be excellent for drought tolerance analysis. The ITG RIL population, along with parents, was planted in greenhouse and at the College Station Research Farm in 2014 with two replications under both irrigation and drought stress. Observations were recorded on tolerance to heat and drought, plant height, plant type, joint pigmentation, date to first flower, date to first pod maturity, and date to 95% maturity. Furthermore, five plant samples were harvested from both replicates to measure biomass, pod weight, seed weight, number of seeds per pod, number of pods, and 100-seed weight. Days to first flower of the ITG RILs ranged from 30 to 60 days and maturity ranged from 45 to 89 days in the field. Statistical analysis showed a significant difference among the ITG RILs for days to flowering, days to maturity, and plant height. Plant height of the population ranged from 8 to 34 cm, which confirmed a wide variation within the population. Correlation between maturity and height was found to be significant, with a correlation coefficient of 0.74. Similar results were found in the greenhouse study. Days to flowering and days to maturity in greenhouse ranged 23 to 61 days and 45 to 92 days, respectively, showing a transgressive segregation, as the parent GEC matured in about 65 days and the parent IT98K-476 matured in about 75 days. Plant samples are being processed and other agronomical traits are being documented and analyzed. Objective 2 -Genetically map the genes controlling the drought and heat tolerance using RAD-seq (Years 1 - 2) Leaf tissues were sampled from all 180 RILs and parents (GEC and IT98K-476-8) of the ITG population (F2:8). Nuclear DNA was isolated from the entire ITG population and parents, and constructed into RAD-seq libraries with a combination of restriction enzymes, BamHI and MluCI. The flanking sequences of the BamHI sites were sequenced for all 180 RILs and parents of the ITG population using a 100SE module with HiSeq 2000. A range of 380,399 - 2,267,979 100-nucleotide clean reads were obtained for each line, with an average of 1,449,966 100-nucleotide clean reads per line. Over 97% of the sequences had a quality of ≥ Q20. Given that cowpea has a genome size of 620 Mb/1C and an estimation of one BamHI site in approximately 10 kb, there are approximately 62,000 BamHI sites in the cowpea genome. Because we sequenced both flanking sides of all BamHI sites, the coverage of the sequences for each BamHI site ranged from 6.1x to 36.6x for each line, with an average coverage of 11.7x for each site in the genome of each line. Therefore, the RAD sequences of the population should be sufficient for construction of a SNP genetic map for cowpea and for mapping of the genes controlling drought and heat tolerance in the species. Currently, we are analyzing the RAD sequences and constructing a SNP genetic map of the cowpea genome that will be used as a framework to map the genes controlling drought and heat tolerance. Objective 3 Identify SNP markers closely linked to the drought and heat tolerant genes, and develop molecular tools enabling enhanced breeding of the traits in cowpea and related species (Years 2 - 3) This experiment was proposed to begin in late Year 2. As soon as the genes controlling drought and heat tolerance are genetically mapped, SNP markers closely linked to the genes of drought and heat tolerance and breeding toolkits will be developed.

Publications

  • Type: Books Status: Published Year Published: 2014 Citation: Singh BB. 2014. Cowpea: The Food Legume of the 21st Century. Crop Science Society of America, Madison, WI.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Masor LL, Hays DB, Singh BB, Zhang H-B, Zhang MP. 2014. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). 8th Annual Plant Breeding Coordinating Committee Meeting and 4th Annual National Association of Plant Breeders Meeting. August 5-8, 2014 in Minneapolis, MN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Masor L. 2013. Screening for drought tolerance among Cowpea (Vigna unguiculata) genotypes. 25th Annual Texas Plant Protection Conference. December 10-11, 2013, Bryan, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Angira B, Singh BB, Alexander T, Hays DB, 2013. Genetic Variability for Heat Tolerance in Cowpea. 25th Annual Texas Plant Protection Conference. December 10-11, 2013, Bryan, TX.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Angira B, Hays DB, Singh BB, 2014. Genetic Variability and Number of Genes Conditioning Heat Tolerance in Cowpea. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America Annual meeting 2014. November 2-5, 2014, Long Beach, CA. P542. https://scisoc.confex.com/scisoc/2014am/webprogram/Paper89467.html
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Masor L, Hays DB, Zhang HB, Zhang M. 2014. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). 26th Annual Texas Plant Protection Conference. December 10-11, 2014, Bryan, TX.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Masor L, Hays DB, Zhang HB, Zhang M. 2014. Molecular mapping of drought tolerance genes in cowpea (Vigna unguiculata L. Walp). American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America Annual meeting 2014. November 2-5, 2014, Long Beach, CA. P603. https://scisoc.confex.com/scisoc/2014am/webprogram/Paper87662.html