Source: AGRICULTURAL RESEARCH SERVICE submitted to
DEVELOPING MOLECULAR MARKERS FOR ENHANCING RESISTANCE TO DROUGHT AND HIGH SALINITY IN ALFALFA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1007133
Grant No.
2015-70005-24071
Project No.
CALW-2015-08210
Proposal No.
2015-08210
Multistate No.
(N/A)
Program Code
AFRP
Project Start Date
Sep 1, 2015
Project End Date
Aug 31, 2018
Grant Year
2015
Project Director
Yu, L.
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
800 BUCHANAN ST, RM 2020
BERKELEY,CA 94710-1105
Performing Department
Agriculture Research Service
Non Technical Summary
Alfalfa is the most widely cultivated forage legume worldwide and the fourth most widely produced crop in the US. Drought and salinity are two major environmental factors determining alfalfa productivity. In the western United States, nearly all alfalfa hay acreage is irrigated. The costs associated with irrigation are significant. These costs are likely to increase as populations rise in these states. In addition, climate change may adversely impact water availability. The water use efficiency of alfalfa cultivars needs to be improved to help meet the challenges of finite water resources. The proposed research is to identify molecular markers (genes) and germplasm for drought and salt tolerance for developing superior alfalfa cultivars with enhanced water use efficiency. The first objective is to identify germplasm with drought and salt resistance in the primary gene pool and develop populations with a high percentage of resistant plants in elite genetic backgrounds through recurrent selection. Quantitative genetic analysis of inheritance of resistance will be carried out. The second objective is to map QTL associated with drought and salt resistance. Finally, the third objective will develop molecular markers tightly linked to the resistance loci for rapid selection of drought and salt tolerant alfalfa to accelerate breeding programs. Quantitative traits such as biotic and abiotic stress resistance are likely under the control of multiple genes and influenced by environmental factors. Identification of resistance loci that contribute to variation in such complex traits, is a primary challenge in plant breeding and population genetics. In the proposed study we will use an integrated framework that merges a QTL mapping approach called ''genome-wide association'' with high-throughput genome sequencing methodologies called "genotyping by sequencing (GBS)" in order to map traits quickly, efficiently, and in a relatively inexpensive manner. This framework provides a statistical basis for analyzing marker-trait association using linkage disequilibrium. It will help to identify molecular markers associated with resistance to biotic and abiotic stresses in alfalfa. In general, segregation population or association panel will be used for mapping genes/QTL. Parents or progenies will be phenotyped for traits of interest. The SNP markers will be used for mapping genes/QTL associated with drought/salt tolerance. SNPs tightly linked to the specific trait will then be used for marker-assisted selection in breeding programs. This project will provide new tools and strategies for improving resistance to abiotic stress that threatens alfalfa grown in the US and will train members of a new generation of alfalfa breeders in conventional and molecular breeding techniques.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
30%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20304301081100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
0430 - Climate;

Field Of Science
1081 - Breeding;
Goals / Objectives
Objective 1: Develop diverse alfalfa populations for identifying genes conditioning drought/salt tolerance traits.Objective 2: Map QTLs associated with drought and salt resistance using genome-wide association studies.Objective 3: Develop molecular markers and strategies for selection of drought and salt tolerant alfalfa and breeding for superior varieties with drought and salt resistance.The long-term goal of this research is to develop new methods for evaluating alfalfa for rapid identification of resistance to yield-limiting stresses caused by environmental factors and to improve understanding of the genetic factors controlling resistance to these stresses in order to develop superior alfalfa cultivars with enhanced drought/salt resistance and water use efficiency.
Project Methods
We used a greenhouse procedure for screening a panel of 200 accessions and identified 27 putative drought resistance accessions. We are currently crossing them to make synthetic populations. Several cycles of selection will be followed to identify top resistance lines while maintaining relatively high yield and quality. Meanwhile, we selected the high resistance accessions to cross with elite cultivars to generate populations for further analysis. However, as alfalfa is an autotetraploid species, phenotypic variance may be significant among plants within an accession. We select individual plants based on the performance in the drought screening and use them as parents for developing a mapping population for identifying major genes associated with drought and salt tolerance. Selected plants with drought tolerance and high yield will be used as parents to cross with an elite drought susceptible variety with high yield and quality and disease resistance, but not for drought or salt resistance to produce the F1 population. Back crosses will be made to create BC1. The segregated populations will be used for the mapping genes associated with drought and salt resistance. Individual plants will be cloned by cutting and replicated clones will be used for phenotyping. Original plants will be used for DNA extraction and genotyping by sequencing.We will evaluate these drought and salt tolerant alfalfa populations in different field locations to determine relative forage production, fall dormancy, flowering date, seed production, forage quality and plant morphological and physiological traits including root length, root thickness, root penetration ability, drought resistance index, relative leaf water content, osmotic adjustment of these lines compared with standard checks. We hypothesize that alfalfa genotypes will be identified from among these populations that would meet commercial standards for forage production under drought and saline conditions.The same populations will be used for salt stress. We will use the modified protocol developed by CoPI Dr. Peel's group (Peel et al. 2004). This will involve substitution of small cones with larger cones to increase soil capacity. The EC of the solution will be much lower (typically 9.0 dS M-1), which is sufficient for selecting salt tolerance genotypes. Plants will be cut to 5 cm at the onset of salt treatment and allow growing for five weeks before harvesting. This protocol can be repeated on the same plant with non-stress and followed by salt treatment. Reliable results have been obtained by using this protocol (Peel et al. 2004).Data analysis: Regularly spaced check plots planted at both sites will serve as covariates to adjust half-sib family biomass data for spatial variation in soil texture, and consequently water holding capacity, which subsequently influences forage productivity. Yield and other data from the C0-derived half-sib families and their respective covariate check will be subjected to ANOVA to assess variation for these traits in the base population. Least-square adjusted means for these traits will be computed and utilized for fine mapping of their associated loci in the AM study, and prediction of genomic estimated breeding values in the genomic selection study.Quantitative traits such as abiotic stress resistance are most likely under the control of multiple genes and interact with environmental factors. Identification of resistance loci that contribute to variation in such complex traits, is a primary challenge in plant breeding and population genetics. In the proposed study we will use an integrated framework that merges a QTL mapping approach called ''genome-wide association'' with high-throughput genome sequencing methodologies called "genotyping by sequencing (GBS)" in order to map traits quickly, efficiently, and in a relatively inexpensive manner. This framework provides a statistical basis for analyzing marker-trait association using linkage disequilibrium. It will help to identify molecular markers/genes associated with resistance to abiotic stresses in alfalfaMarkers tightly linked to the resistance loci will be used for developing diagnostic high throughput markers for MAS. Prior to use, DNA marker must be validated in a wide range of populations with diverse genetic backgrounds. Successful uses of DNA markers in MAS are largely rely on marker's reliability, quality and cost-effectiveness. First, markers should be tightly linked to target loci, preferably less than 5 cM genetic distance. The use of flanking markers or intragenic markers will greatly increase the reliability of the markers to predict phenotype. Secondly, the marker should be highly polymorphic in breeding material. The level of simplicity and the time required for the technique are critical considerations. High-throughput simple and quick methods are highly desirable. Finally, the marker assay should be cost-effective in order for MAS to be feasible. Another important factor impeding the successful application of markers for line development is the low reliability of markers to determine phenotype. This is often attributable to the accuracy of the primary QTL mapping study. Although QTLs have high LOD scores or low p-values and explain a large proportion of the phenotypic variance, they may be affected by sampling bias (such as small populations), and therefore may not be useful for MAS. Furthermore, the effect of a QTL may depend on the genetic background. Therefore maker validation is important for the successful use of the marker for MAS.Phenotypic effects of the resistance loci identified in the mapping populations will be validated in a Cycle 0 base population (n=200) possessing all of the above QTL in a 50% resistance parent alfalfa cultivar background. Marker-assisted selection will then be imposed for present and against absent of specific combinations of marker alleles associated with drought and salt resistance. Selected plants within each marker group category will be mated with each other to develop F1 MAS populations. Of those, half populations will be expected to be positive for specific alleles and another half will be negative. Further introgression of some QTL into elite backgrounds will be carried out. This will involve mating the C0 control and the positive populations with two or three cultivars with drought tolerance or salt tolerance and a drought sensitive high forage quality population available in CoPI, Dr. Viands group. These second-generation MAS-derived populations possessed a 75% elite background. Agronomic tarits such forage yield under drought and salt stress will be evaluated using the second-generation MAS-derived populations.High throughput platforms such as KASP (lgcgenomics.com) or Taqman (lifetechnologies.com) assays will be used to test the diagnostics of marker loci. Two Real-time PCR machines, Roche lightcycle 480 and Quantstudio 7 are available in PD Dr. Yu's group. Flank sequences to the SNP site of significant markers will be used for designing specific primers for array based genotyping platforms (KASP or Taqman). Multiplex primers will be used for evaluating the resistance locus or candidate gene. All markers will be scored in a given genotype. Single markers with two character states will be tested for significant phenotypic differences between genotype groups by the t test for each trait, and Mann-Whitney U test for marker quality. Marker combinations will be analyzed using analysis of variance (ANOVA) for each trait, and Kruskal-Wallis test for chip quality. Statistical analyses will use SAS software (SAS Institute Inc. 2011, SAS OnlineDoc 9.3, Cary, NC, USA).

Progress 09/01/16 to 08/31/17

Outputs
Target Audience:Progress Report for NIFA-AFRP_GRANT11918787 Project Title: Developing molecular markers for enhancing resistance to drought and high salinity in alfalfa PD: Long-Xi Yu Location: USDA-ARS, PROSSER, WASHINGTON Reporting period: 09/01/2016 to 08/31/2017 1. Objectives This Progress Report covers the second year of funding of the NIFA-ARS GRANT11918787. The overall goal of this project is to develop improved alfalfa breeding strategies, germplasm, and molecular tools to enhance resistance to drought and high salinity. The desired outcomes are molecular markers and high throughput strategies that can be used in marker-assisted breeding to develop improved alfalfa varieties with resistance to abiotic stress to increase alfalfa production and reduce costs. To achieve the long-term goal, the research has been focusing on three objectives: 1) Develop diverse alfalfa populations for identifying genes conditioning drought/salt tolerance traits. 2) Map QTLs associated with drought and salt resistance using genome-wide association studies. 3) Develop molecular markers and strategies for selection of drought and salt tolerant alfalfa and breeding for superior varieties with drought and salt resistance. 2. Approach Two hundred alfalfa accessions with potential drought tolerance were selected were used for screening drought tolerance. A split plot design will be used with three irrigation treatments as main plot treatments. Since field conditions are difficult to control, a highly controlled greenhouse assay was also used for screening drought/salt tolerance alfalfa. In the first phase, we developed a greenhouse protocol for measuring water usage and biomass. Agronomic and physiological traits including biomass, root characteristics, flowering time, relative leaf water content and osmotic adjustment are highly correlated with drought tolerance and they were measured in the mapping populations. We used an integrated framework that merges a QTL mapping approach called ''genome-wide association'' with high-throughput genome sequencing methodologies called "genotyping by sequencing (GBS)" in order to map traits quickly, efficiently, and in a relatively inexpensive manner. This framework provides a statistical basis for analyzing marker-trait association using linkage disequilibrium. 3. Progress Report Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. A panel of 200 alfalfa accessions with potential drought tolerance was evaluated for drought resistance in field and greenhouse in dry season of 2013 to 2016. Twenty agronomic, physiological and quality traits were evaluated. An integrated procedure for phenotyping drought resistance index (DRI) was used for evaluating 1,800 Individuals. We have identified 27 accessions with a higher level of resistance than the known drought resistant control that are useful in alfalfa breeding. Our study of marker-trait association identified twenty and fifteen loci associated with DRI and relative water content (RWC), respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. Markers associated with DRI are located on chromosomes 1, 2, 3, 4, 5, 6 and 7, while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5 and 7. Co-localizations of significant markers between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated with DRI in this work overlap with the reported QTLs associated with biomass under drought in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. BLAST search using their flanking sequences revealed homology to several annotated genes with functions in stress tolerance. A greenhouse procedure was used for phenotyping four agronomic and physiological traits affected by salt stress, including dry weight (DW), plant height (PH), leaf chlorophyll content (LCC) and stomatal conductance (SC). For each trait, a stress susceptibility index (SSI) was used to evaluate plant performance under stressed and non-stressed conditions. Marker-trait association identified a total of 42 markers significantly associated with salt tolerance. They were located on all chromosomes except chromosome 2 based on the alignment of their flanking sequences to the reference genome (Medicago truncatula). Of those identified, 13 were associated with multiple traits. Several loci identified in the present study were also identified in previous reports. BLAST search revealed that 19 putative candidate genes linked to 24 significant markers. Among them, B3 DNA-binding protein, Thiaminepyrophosphokinase and IQ calmodulin-binding motif protein were identified among multiple traits in the present and previous studies. With further validation, these markers may be used for marker-assisted breeding new alfalfa varieties with drought resistance and enhanced water use efficiency. The results have been reported at professional conferences and in peer-reviewed journals. 4. Accomplishments Germplasm and molecular markers for improving alfalfa drought tolerance. Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. An ARS Research Geneticist at Prosser, Washington, conducted a replicated trial in both greenhouse and field and identified 27 accessions with higher level of resistance than the known drought resistant control. A laboratory study on marker-trait association identified 20 and 15 loci associated with drought resistance index and relative water content, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. The same panel of germplasm was also used for salt tolerance during seed germination. Markers associated with salt tolerance during germination have been identified and they were located on chromosomes 1, 2 and 4.These markers are potentially useful in marker-assisted breeding new alfalfa varieties with drought/salt resistance and enhanced water use efficiency. 5. Technology Transfer Based on this research, a new MTA has been signed for delivery drought resistant lines to a seed company. Molecular markers developed by this research have been entered to the NCBI web site and the Alfalfa toolbox for public users. 6. Publications Zhang T, Yu L-X, Zheng P, Li Y, Rivera M, Main D and Greene S (2015) Identification of Loci Associated with Drought Resistance Traits in Heterozygous Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies with Genotyping by Sequencing. PLoS ONE 10(9): e0138931. doi:10.1371/journal.pone.0138931. Yu L-X, Liu X, Boge W and Liu X-P (2016) Genome-Wide Association Study Identifies Loci for Salt Tolerance during Germination in Autotetraploid Alfalfa (Medicago sativa L.) Using Genotyping-by-Sequencing. Front. Plant Sci. 7:956. doi: 10.3389/fpls.2016.00956. Liu X-P and Yu L-X (2017) Genome-Wide Association Mapping of Loci Associated with Plant Growth and Forage Production under Salt Stress in Alfalfa (Medicago sativa L.). Front. Plant Sci. 8:853. doi: 10.3389/fpls.2017.00853 Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We have developed additionalpopulations for the project and will use them for genome-wide association studies in next year to accomplish the goals.

Impacts
What was accomplished under these goals? Accomplishments Germplasm and molecular markers for improving alfalfa drought tolerance. Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. An ARS Research Geneticist at Prosser, Washington, conducted a replicated trial in both greenhouse and field and identified 27 accessions with higher level of resistance than the known drought resistant control. A laboratory study on marker-trait association identified 20 and 15 loci associated with drought resistance index and relative water content, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. The same panel of germplasm was also used for salt tolerance during seed germination. Markers associated with salt tolerance during germination have been identified and they were located on chromosomes 1, 2 and 4.These markers are potentially useful in marker-assisted breeding new alfalfa varieties with drought/salt resistance and enhanced water use efficiency.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang T, Yu L-X, Zheng P, Li Y, Rivera M, Main D and Greene S (2015) Identification of Loci Associated with Drought Resistance Traits in Heterozygous Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies with Genotyping by Sequencing. PLoS ONE 10(9): e0138931. doi:10.1371/journal.pone.0138931. Yu L-X, Liu X, Boge W and Liu X-P (2016) Genome-Wide Association Study Identifies Loci for Salt Tolerance during Germination in Autotetraploid Alfalfa (Medicago sativa L.) Using Genotyping-by-Sequencing. Front. Plant Sci. 7:956. doi: 10.3389/fpls.2016.00956. Liu X-P and Yu L-X (2017) Genome-Wide Association Mapping of Loci Associated with Plant Growth and Forage Production under Salt Stress in Alfalfa (Medicago sativa L.). Front. Plant Sci. 8:853. doi: 10.3389/fpls.2017.00853


Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The audiences are industry professionals, consultants, extension specialists, educators, and research scientists. Area and regional hay producers are fully engaged in several aspects of educational development and outreach from this project. Traditional modes of outreach to clientele will be implemented through presentations at the NAAIC conferences, hay growers meetings, field days and Extension bulletins. We will also report the results and methodology with various aspects into the NAAIC webpage. Presentations to national meetings are also planned. Our collaboratorshave signed a material transfer agreement todeliver our germplasm to industrial seed companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have trained 3 undergrdate students for field and greenhouse procedureson evaluation of alfalfa plants for drought and salt tolerance. How have the results been disseminated to communities of interest?Technology Transfer Based on this research, a new MTA has been signed for delivery drought resistant lines to a seed company. Molecular markers developed by this research have been entered to the NCBI web site and the Alfalfa toolbox for public users. What do you plan to do during the next reporting period to accomplish the goals?Markers tightly linked to the resistance loci will be used for developing diagnostic high throughput markers for MAS. Prior to use, DNA marker must be validated in a wide range of populations with diverse genetic backgrounds. Successful uses of DNA markers in MAS are largely rely on marker's reliability, quality and cost-effectiveness. First, markers should be tightly linked to target loci, preferably less than 5 cM genetic distance. The use of flanking markers or intragenic markers will greatly increase the reliability of the markers to predict phenotype. Secondly, the marker should be highly polymorphic in breeding material. The level of simplicity and the time required for the technique are critical considerations. High-throughput simple and quick methods are highly desirable. Finally, the marker assay should be cost-effective in order for MAS to be feasible. Another important factor impeding the successful application of markers for line development is the low reliability of markers to determine phenotype. This is often attributable to the accuracy of the primary QTL mapping study. Although QTLs have high LOD scores or low p-values and explain a large proportion of the phenotypic variance, they may be affected by sampling bias (such as small populations), and therefore may not be useful for MAS. Furthermore, the effect of a QTL may depend on the genetic background. Therefore maker validation is important for the successful use of the marker for MAS.

Impacts
What was accomplished under these goals? Progress Report Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. A panel of 200 alfalfa accessions with potential drought tolerance was evaluated for drought resistance in field and greenhouse in dry season of 2013 to 2016. Twenty agronomic, physiological and quality traits were evaluated. An integrated procedure for phenotyping drought resistance index (DRI) was used for evaluating 1,800 Individuals. We have identified 27 accessions with a higher level of resistance than the known drought resistant control that are useful in alfalfa breeding. Our study of marker-trait association identified twenty and fifteen loci associated with DRI and relative water content (RWC), respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. Markers associated with DRI are located on chromosomes 1, 2, 3, 4, 5, 6 and 7, while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5 and 7. Co-localizations of significant markers between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated with DRI in this work overlap with the reported QTLs associated with biomass under drought in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. BLAST search using their flanking sequences revealed homology to several annotated genes with functions in stress tolerance. With further validation, these markers may be used for marker-assisted breeding new alfalfa varieties with drought resistance and enhanced water use efficiency. The results have been reported at professional conferences and in peer-reviewed journals. 4. Accomplishments Germplasm and molecular markers for improving alfalfa drought tolerance. Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. An ARS Research Geneticist at Prosser, Washington, conducted a replicated trial in both greenhouse and field and identified 27 accessions with higher level of resistance than the known drought resistant control. A laboratory study on marker-trait association identified 20 and 15 loci associated with drought resistance index and relative water content, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. The same panel of germplasm was also used for salt tolerance during seed germination. Markers associated with salt tolerance during germination have been identified and they were located on chromosomes 1, 2 and 4.These markers are potentially useful in marker-assisted breeding new alfalfa varieties with drought/salt resistance and enhanced water use efficiency.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang T, Yu L-X, Zheng P, Li Y, Rivera M, Main D and Greene S (2015) Identification of Loci Associated with Drought Resistance Traits in Heterozygous Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies with Genotyping by Sequencing. PLoS ONE 10(9): e0138931. doi:10.1371/journal.pone.0138931. Yu L-X, Liu X, Boge W and Liu X-P (2016) Genome-Wide Association Study Identifies Loci for Salt Tolerance during Germination in Autotetraploid Alfalfa (Medicago sativa L.) Using Genotyping-by-Sequencing. Front. Plant Sci. 7:956. doi: 10.3389/fpls.2016.00956.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: http://www.naaic.org/Meetings/National/2016meeting/Presentations/Long-xi%20Yu.pdf