Final report for GS23-296
Project Information
Southern pea (Vigna unguiculata L. Walp.) is an important part of the diet of some subpopulations of the southeastern United States. These communities and demographics within these communities experience malnutrition and micronutrient deficiencies. This study will attempt to identify genetic resources in a panel of 192 American southern pea accessions previously genotyped under low-input conditions. Inductively coupled plasma mass spectroscopy (ICP-MS) will be used to quantify the micronutrient content of grains, and plants will be grown under field greenhouse conditions for two consecutive years to evaluate the heritability of these traits. Single nucleotide polymorphism (SNP) markers generated in this population will be used to find quantitative trait loci (QTLs) associated with micronutrient content in grains, particularly iron, zinc, and magnesium. Candidate genes will be identified and testing/development of single sequence repeat (SSR) or Kompetitive Allele Specific PCR (KASP) markers associated with causal QTLs for the accumulation of these micronutrients.
Objective 1: Identify American southern pea cultivars or landraces with high micronutrient content and evaluate the biofortification potential for iron, magnesium, and zinc in this population grown with low-inputs.
Objective 2: Identify causal quantitative trait loci and/or genes in American southern pea germplasm associated with high levels of the iron, magnesium, and zinc.
Objective 3: Validate SNPs/QTLs/genes associated with seed micronutrient content using SSR or KASP markers and evaluate their potential use in breeding programs.
Research
- Field planting of 192 previously genotyped American southern pea accessions in an augmented design in May of 2023 and 2024. There will be 12 blocks containing 16 accessions out of the 192 genotyped accessions and 4 checks. Checks can be analyzed as RCBD.
- Seed were collected upon maturity and 2g were digested in concentrated nitric acid by microwave then diluted to a working solution of 2.0% nitric acid. This sample was sent to the Arizona State University analytical lab for inductively coupled plasma - mass spectrometry (ICP-MS) analysis of the "major suite" of elements (P, K, Ca, Mg, S, Al, B, Cu, Fe, Mn, Mo, Na, Zn).
- Analysis of data was with R coding language (dplyer, purrr, lme4, lmerTest, emmeans, multcompView packages). Broad Sense Heritability was calculated and cultivars with high levels of seed micronutrients were identified. Population distributions and correlations were graphed.
- With previously generated genotypic SNP data for this American southern pea population, a genome-wide association study (GWAS) was conducted to identify causal QTLs / regions of the genome responsible for high levels of micronutrient content in seeds. TASSEL software in R were used to identify marker-trait associations in the 192 genotypes for seed content of minerals. Identification of these SNPs can allow development of new SNP-based markers for potential use in southern pea breeding programs
Mean values for Ca, Cu, Fe, Mg, Mn, and Zn concentrations in 2023 were 700.992, 6.11, 46.66, 1943.95, 16.36, and 36.98 ppm, respectively. Concentrations of the same respective mineral elements in 2024 were 599.78, 6.50, 44.64, 1853.72, 15.36, and 36.66 ppm. Ranges of concentrations were slightly greater in 2023 than in 2024, particularly for Ca and Mg. The distribution of nutrient concentrations were similar across years for some elements but were for others. Ca, Mg, and Mn were especially different in their distribution, while Cu, Fe, and Zn showed more similar distributions regardless of year. Correlation between all nutrients was generally highly positive in 2023 but was much less positive overall in 2024. ANOVA on seed nutrient concentrations showed significant differences by genotype, year, and blocks within years except for Zn by year. These results show temporal and spatial factors heavily influence seed nutrient accumulation. Broad-sense heritability (H²) of seed nutrient concentrations were also calculated and ranged from 0.49 for Fe to 0.71 for Mn, indicating moderate to high genetic control. Ca, Cu, Mg, Mn, and Zn showed moderate to high heritability (0.60–0.71), whereas Fe had lower heritability (0.49). This suggests a greater environmental influence on Fe concentration. Genotype with the highest 10 seed nutrient concentrations per year are shown in Table 4. Many genotypes had high seed nutrient concentrations for multiple elements, especially within a single year. However, some genotypes of interest maintained high concentrations of at least one element across both years. Genotypes PI663045 and PI663035 had high concentrations of Ca and Cu, respectively, during both years. Three other genotypes PI663072, PI663074, and PI663032 had high concentrations of multiple micronutrients across multiple years. PI663072 had the highest concentrations of both Mn and Zn for both years, while also having high concentrations of Mg in 2023 and Cu in 2023 and 2024. Mn concentrations in seeds of PI663074 were high in both 2023 and 2024, while Cu was high in 2024 and Mg was high in 2023. PI663032 was high in Mg for both years and had high Ca and Mn in 2023. There were no genotypes with similarly high Fe concentrations in both years, and none of the genotypes previously described were observed to have Fe ppm values in the top 10. This aligns with the relatively low H2 of Fe (0.49) compared to the other nutrients. Additionally, the presence of high nutrient concentrations in seeds of genotypes primarily within the same year align with ANOVA results and the positive correlation between nutrient concentrations observed in each year. Most mineral concentrations had relatively high H2 with only Fe having a value less than 0.5. Fe was also the only element that did not have a genotype with a high concentration of in both 2023 and 2024. Previously generated single-nucleotide polymorphism (SNP) markers were used to carry out genome-wide association studies (GWAS) on the six seed mineral concentrations. Out of these approximately 1,900,000 SNPs, 10,000 randomly selected SNPs were retained on each chromosome for use in the study. Thus, a total of 110,000 SNPs in total were used. The Fixed and random model Circulating Probability Unification (FarmCPU) and Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) models were used to identify significant marker-trait associations (MTAs) executed through GAPIT version 3 in R software. Manhattan plots were generated by the GAPIT software. Results of the GWAS using BLUPs for genotype seed mineral concentrations identified significant marker-trait associations (MTAs) for Ca, Cu, and Zn. There were no significant MATs for Fe, Mn, or Mg. Significant MTAs identified for Ca included one on Vu03 and two on Vu04. Only a single SNP was significantly associated with Ca content using both FarmCPU and BLINK models. Five SNPs (two on Vu01, one on Vu03, one on Vu09, and one on Vu011) were significantly associated with seed Cu concentration as identified using the FarmCPU model, but these were not detected using BLINK. Finally, two significant markers associated with seed Zn concentrations on Vu04 were identified using the BLINK model, but none were detected via FarmCPU.
Educational & Outreach Activities
Participation Summary:
The project was part of PhD dissertation research for Mr. Max Miller (current ABD) and included grow outs over two years at the Agricultural Research and Education Center (AREC) of Tennessee State University (TSU) in Nashville, TN. The field plots were on display at two Small Farm Expo events held by TSU, where grain legume production including the cowpea GWAS population was a highlight stop for a trolley tour for visitors to TSU. These folks included approximately 20 farmers per year and 10 to 15 researchers, graduate students and professors visiting per year. A flyer on cowpea cultivation and diversity along with black eyed pea cooking recipes was handed out to participants. Dr. Blair led approximately 3 tours each year for visiting growers and non-governmental agency (NGO) staff to understand the potential of reviving black eyed pea production in Tennessee. Consultations for growers were provided one-on-one at the Small Farm Expo's which continues after morning trolley tours with a luncheon and meetings. Mr. Miller presented his results on cowpea diversity at a University of Tennessee/TSU collaborative meeting, at the National Plant Breeding Workshop for USDA, at the HortScience meeting in New Orleans 2025 and for his dissertation defense. A journal article is in preparation on nutritional results from the GWAS analysis and will be submitted to Journal of American Society of Horticultural Sciences.
Project Outcomes
Knowledge about seed minerals is important for both plant nutrition and human nutrition for the grains and products consumed or the seedlings grown from them. Elemental / Mineral ICP concentrations were the basis for our linking nutritional traits in terms of required dietary micronutrients with abiotic stress tolerance in the selection and breeding of black-eyed peas. These would relate to longer term benefits to farmers and consumers as varieties bred with this basic information start to get into the marketplace. Fortunately, we used adapted American germplasm for our study of cowpeas. All the genotypes had either historical or current uses in various states across the Southern USA so are the basis for what we consume in terms of black-eyed, crowder and pink-eyed types of Southern Peas.
Although no new funding was obtained for the project, a no-cost extension was granted by USDA-NIFA on a related black-eyed pea project on abiotic stress tolerance (Aluminum-stress) that Mr. Miller was heavily involved in. Dr. Blair, his advisor, built on the results of that phenotyping project with a Capacity Building grant for additional abiotic stress tolerance (drought physiology). The ICP nutrient project allowed the training of undergraduate students in the field and exposing them to agricultural research with a specialty crop. Growers were exposed to our cowpea research at two small farm expo events for future working collaborations. The new institutional collaborations included Arizona State University for the ICP mineral analysis of 192 cowpea accessions, and the Donald Danforth Center for the drought comparisons which were done on two contrasting cowpea lines and other grain legume controls. Sustainable agriculture involves the balancing of many demands for quality and productivity characteristics of varieties used in farming systems and this is especially the case for a food / pulse legume like black-eyed peas.