Genome-Wide Association Study and Genomic Prediction of Fusarium Wilt Resistance in Common Bean Core Collection.

The common bean (Phaseolus vulgaris L.) is a globally cultivated leguminous crop. Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. phaseoli (Fop), is a significant disease leading to substantial yield loss in common beans. Disease-resistant cultivars are recommended to counteract this. The objective of this investigation was to identify single nucleotide polymorphism (SNP) markers associated with FW resistance and to pinpoint potential resistant common bean accessions within a core collection, utilizing a panel of 157 accessions through the Genome-wide association study (GWAS) approach with TASSEL 5 and GAPIT 3. Phenotypes for Fop race 1 and race 4 were matched with genotypic data from 4740 SNPs of BARCBean6K_3 Infinium Bea Chips. After ranking the 157-accession panel and revealing 21 Fusarium wilt-resistant accessions, the GWAS pinpointed 16 SNPs on chromosomes Pv04, Pv05, Pv07, Pv8, and Pv09 linked to Fop race 1 resistance, 23 SNPs on chromosomes Pv03, Pv04, Pv05, Pv07, Pv09, Pv10, and Pv11 associated with Fop race 4 resistance, and 7 SNPs on chromosomes Pv04 and Pv09 correlated with both Fop race 1 and race 4 resistances. Furthermore, within a 30 kb flanking region of these associated SNPs, a total of 17 candidate genes were identified. Some of these genes were annotated as classical disease resistance protein/enzymes, including NB-ARC domain proteins, Leucine-rich repeat protein kinase family proteins, zinc finger family proteins, P-loopcontaining nucleoside triphosphate hydrolase superfamily, etc. Genomic prediction (GP) accuracy for Fop race resistances ranged from 0.26 to 0.55. This study advanced common bean genetic enhancement through marker-assisted selection (MAS) and genomic selection (GS) strategies, paving the way for improved Fop resistance.

A Genome-Wide Association Study Reveals Region Associated with Seed Protein Content in Cowpea.

Cowpea (Vigna unguiculata L. Walp., 2n = 2x = 22) is a protein-rich crop that complements staple cereals for humans and serves as fodder for livestock. It is widely grown in Africa and other developing countries as the primary source of protein in the diet; therefore, it is necessary to identify the protein-related loci to improve cowpea breeding. In the current study, we conducted a genome-wide association study (GWAS) on 161 cowpea accessions (151 USDA germplasm plus 10 Arkansas breeding lines) with a wide range of seed protein contents (21.8~28.9%) with 110,155 high-quality whole-genome single-nucleotide polymorphisms (SNPs) to identify markers associated with protein content, then performed genomic prediction (GP) for future breeding. A total of seven significant SNP markers were identified using five GWAS models (single-marker regression (SMR), the general linear model (GLM), Mixed Linear Model (MLM), Fixed and Random Model Circulating Probability Unification (FarmCPU), and Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK), which are located at the same locus on chromosome 8 for seed protein content. This locus was associated with the gene Vigun08g039200, which was annotated as the protein of the thioredoxin superfamily, playing a critical function for protein content increase and nutritional quality improvement. In this study, a genomic prediction (GP) approach was employed to assess the accuracy of predicting seed protein content in cowpea. The GP was conducted using cross-prediction with five models, namely ridge regression best linear unbiased prediction (rrBLUP), Bayesian ridge regression (BRR), Bayesian A (BA), Bayesian B (BB), and Bayesian least absolute shrinkage and selection operator (BL), applied to seven random whole genome marker sets with different densities (10 k, 5 k, 2 k, 1 k, 500, 200, and 7), as well as significant markers identified through GWAS. The accuracies of the GP varied between 42.9% and 52.1% across the seven SNPs considered, depending on the model used. These findings not only have the potential to expedite the breeding cycle through early prediction of individual performance prior to phenotyping, but also offer practical implications for cowpea breeding programs striving to enhance seed protein content and nutritional quality.

Skim resequencing finely maps the downy mildew resistance loci RPF2 and RPF3 in spinach cultivars whale and Lazio.

Commercial production of spinach (Spinacia oleracea L.) is centered in California and Arizona in the US, where downy mildew caused by Peronospora effusa is the most destructive disease. Nineteen typical races of P. effusa have been reported to infect spinach, with 16 identified after 1990. The regular appearance of new pathogen races breaks the resistance gene introgressed in spinach. We attempted to map and delineate the RPF2 locus at a finer resolution, identify linked single nucleotide polymorphism (SNP) markers, and report candidate downy mildew resistance (R) genes. Progeny populations segregating for RPF2 locus derived from resistant differential cultivar Lazio were infected using race 5 of P. effusa and were used to study for genetic transmission and mapping analysis in this study. Association analysis performed with low coverage whole genome resequencing-generated SNP markers mapped the RPF2 locus between 0.47 to 1.46 Mb of chromosome 3 with peak SNP (Chr3_1, 221, 009) showing a LOD value of 61.6 in the GLM model in TASSEL, which was within 1.08 Kb from Spo12821, a gene that encodes CC-NBS-LRR plant disease resistance protein. In addition, a combined analysis of progeny panels of Lazio and Whale segregating for RPF2 and RPF3 loci delineated the resistance section in chromosome 3 between 1.18-1.23 and 1.75-1.76 Mb. This study provides valuable information on the RPF2 resistance region in the spinach cultivar Lazio compared to RPF3 loci in the cultivar Whale. The RPF2 and RPF3 specific SNP markers, plus the resistant genes reported here, could add value to breeding efforts to develop downy mildew resistant cultivars in the future.

Resequencing worldwide spinach germplasm for identification of field resistance QTLs to downy mildew and assessment of genomic selection methods.

Downy mildew, commercially the most important disease of spinach, is caused by the obligate oomycete Peronospora effusa. In the past two decades, new pathogen races have repeatedly overcome the resistance used in newly released cultivars, urging the need for more durable resistance. Commercial spinach cultivars are bred with major R genes to impart resistance to downy mildew pathogens and are effective against some pathogen races/isolates. This work aimed to evaluate the worldwide USDA spinach germplasm collections and commercial cultivars for resistance to downy mildew pathogen in the field condition under natural inoculum pressure and conduct genome wide association analysis (GWAS) to identify resistance-associated genomic regions (alleles). Another objective was to evaluate the prediction accuracy (PA) using several genomic prediction (GP) methods to assess the potential implementation of genomic selection (GS) to improve spinach breeding for resistance to downy mildew pathogen. More than four hundred diverse spinach genotypes comprising USDA germplasm accessions and commercial cultivars were evaluated for resistance to downy mildew pathogen between 2017-2019 in Salinas Valley, California and Yuma, Arizona. GWAS was performed using single nucleotide polymorphism (SNP) markers identified via whole genome resequencing (WGR) in GAPIT and TASSEL programs; detected 14, 12, 5, and 10 significantly associated SNP markers with the resistance from four tested environments, respectively; and the QTL alleles were detected at the previously reported region of chromosome 3 in three of the four experiments. In parallel, PA was assessed using six GP models and seven unique marker datasets for field resistance to downy mildew pathogen across four tested environments. The results suggest the suitability of GS to improve field resistance to downy mildew pathogen. The QTL, SNP markers, and PA estimates provide new information in spinach breeding to select resistant plants and breeding lines through marker-assisted selection (MAS) and GS, eventually helping to accumulate beneficial alleles for durable disease resistance.

Mapping and selection of downy mildew resistance in spinach cv. whale by low coverage whole genome sequencing.

Spinach (Spinacia oleracea) is a popular leafy vegetable crop and commercial production is centered in California and Arizona in the US. The oomycete Peronospora effusa causes the most important disease in spinach, downy mildew. A total of nineteen races of P. effusa are known, with more than 15 documented in the last three decades, and the regular emergence of new races is continually overcoming the genetic resistance to the pathogen. This study aimed to finely map the downy mildew resistance locus RPF3 in spinach, identify single nucleotide polymorphism (SNP) markers associated with the resistance, refine the candidate genes responsible for the resistance, and evaluate the prediction performance using multiple machine learning genomic prediction (GP) methods. Segregating progeny population developed from a cross of resistant cultivar Whale and susceptible cultivar Viroflay to race 5 of P. effusa was inoculated under greenhouse conditions to determine downy mildew disease response across the panel. The progeny panel and the parents were resequenced at low coverage (1x) to identify genome wide SNP markers. Association analysis was performed using disease response phenotype data and SNP markers in TASSEL, GAPIT, and GENESIS programs and mapped the race 5 resistance loci (RPF3) to 1.25 and 2.73 Mb of Monoe-Viroflay chromosome 3 with the associated SNP in the 1.25 Mb region was 0.9 Kb from the NBS-LRR gene SOV3g001250. The RPF3 locus in the 1.22-1.23 Mb region of Sp75 chromosome 3 is 2.41-3.65 Kb from the gene Spo12821 annotated as NBS-LRR disease resistance protein. This study extended our understanding of the genetic basis of downy mildew resistance in spinach cultivar Whale and mapped the RPF3 resistance loci close to the NBS-LRR gene providing a target to pursue functional validation. Three SNP markers efficiently selected resistance based on multiple genomic selection (GS) models. The results from this study have added new genomic resources, generated an informed basis of the RPF3 locus resistant to spinach downy mildew pathogen, and developed markers and prediction methods to select resistant lines.

Genome-wide association study and genomic prediction of white rust resistance in USDA GRIN spinach germplasm.

White rust, caused by Albugo occidentalis, is one of the major yield-limiting diseases of spinach (Spinacia oleracea) in some major commercial production areas, particularly in southern Texas in the United States. The use of host resistance is the most economical and environment-friendly approach to managing white rust in spinach production. The objectives of this study were to conduct a genome-wide associating study (GWAS), to identify single nucleotide polymorphism (SNP) markers associated with white rust resistance in spinach, and to perform genomic prediction (GP) to estimate the prediction accuracy (PA). A GWAS panel of 346 USDA (US Dept. of Agriculture) germplasm accessions was phenotyped for white rust resistance under field conditions and GWAS was performed using 13 235 whole-genome resequencing (WGR) generated SNPs. Nine SNPs, chr2_53 049 132, chr3_58 479 501, chr3_95 114 909, chr4_9 176 069, chr4_17 807 168, chr4_83 938 338, chr4_87 601 768, chr6_1 877 096, and chr6_31 287 118, located on chromosomes 2, 3, 4, and 6 were associated with white rust resistance in this GWAS panel. Four scenarios were tested for PA using Pearson's correlation coefficient (r) between the genomic estimation breeding value (GEBV) and the observed values: (1) different ratios between the training set and testing set (fold), (2) different GP models, (3) different SNP numbers in three different SNP sets, and (4) the use of GWAS-derived significant SNP markers. The results indicated that a 2- to 10-fold difference in the various GP models had similar, although not identical, averaged r values in each SNP set; using GWAS-derived significant SNP markers would increase PA with a high r-value up to 0.84. The SNP markers and the high PA can provide valuable information for breeders to improve spinach by marker-assisted selection (MAS) and genomic selection (GS).

Genetic architecture of salt tolerance in a Multi-Parent Advanced Generation Inter-Cross (MAGIC) cowpea population.

BACKGROUND: Previous reports have shown that soil salinity is a growing threat to cowpea production, and thus the need for breeding salt-tolerant cowpea cultivars. A total of 234 Multi-Parent Advanced Generation Inter-Cross (MAGIC) lines along with their 8 founders were evaluated for salt tolerance under greenhouse conditions. The objectives of this study were to evaluate salt tolerance in a multi-parent advanced generation inter-cross (MAGIC) cowpea population, to identify single nucleotide polymorphism (SNP) markers associated with salt tolerance, and to assess the accuracy of genomic selection (GS) in predicting salt tolerance, and to explore possible epistatic interactions affecting salt tolerance in cowpea. Phenotyping was validated through the use of salt-tolerant and salt-susceptible controls that were previously reported. Genome-wide association study (GWAS) was conducted using a total of 32,047 filtered SNPs. The epistatic interaction analysis was conducted using the PLINK platform. RESULTS: Results indicated that: (1) large variation in traits evaluated for salt tolerance was identified among the MAGIC lines, (2) a total of 7, 2, 18, 18, 3, 2, 5, 1, and 23 were associated with number of dead plants, salt injury score, leaf SPAD chlorophyll under salt treatment, relative tolerance index for leaf SPAD chlorophyll, fresh leaf biomass under salt treatment, relative tolerance index for fresh leaf biomass, relative tolerance index for fresh stem biomass, relative tolerance index for the total above-ground fresh biomass, and relative tolerance index for plant height, respectively, with overlapping SNP markers between traits, (3) candidate genes encoding for proteins involved in ion transport such as Na+/Ca2+ K+ independent exchanger and H+/oligopeptide symporter were identified, and (4) epistatic interactions were identified. CONCLUSIONS: These results will have direct applications in breeding programs aiming at improving salt tolerance in cowpea through marker-assisted selection. To the best of our knowledge, this study was one of the earliest reports using a MAGIC population to investigate the genetic architecture of salt tolerance in cowpea.

Genetic Diversity, Structure, and Selective Sweeps in Spinacia turkestanica Associated With the Domestication of Cultivated Spinach.

Genotype-by-sequencing (GBS) was used to explore the genetic diversity and structure of Spinacia turkestanica, and the selective sweeps involved in domestication of cultivated spinach, S. oleracea, from S. turkestanica. A total 7,065 single nucleotide polymorphisms (SNPs) generated for 16 Spinacia oleracea and 76 S. turkestanica accessions placed the S. oleracea accessions in one group, Q1, and the 76 S. turkestanica accessions, which originated from Central Asia, in two distinct groups, Q2 and Q3. The Q2 group shared greater genetic identity with the S. oleracea accessions, Q1, than the Q3 S. turkestanica group. Likewise, the S. oleracea Q1 group had a smaller Fst (0.008) with the Q2 group than with the Q3 group (Fst = 0.012), and a greater gene flow (Nm = 30.13) with the Q2 group than with the Q3 group (Nm = 21.83). The Q2 accessions originated primarily from Uzbekistan while the Q3 accessions originated mostly from Tajikistan. The Zarafshan Mountain Range appears to have served as a physical barrier that largely separated members of the Q2 and Q3 groups of S. turkestanica. Accessions with admixtures of Q2 and Q3 were collected primarily from lower elevations at the southern end of the Zarafshan Mountain Range in Uzbekistan. Selective sweep regions identified at 32, 49, and 52 Mb on chromosomes 1, 2, and 3, respectively, appear to have played a vital role in the domestication of S. oleracea as they are correlated with important domestication traits, including day length sensitivity for bolting (flowering). High XP-CLR scores at the 52 Mb genomic region of chromosome three suggest that a selective sweep at this region was responsible for early differentiation of S. turkestanica into two groups in Central Asia.

High resolution mapping and candidate gene identification of downy mildew race 16 resistance in spinach.

BACKGROUND: Downy mildew, the most devastating disease of spinach (Spinacia oleracea L.), is caused by the oomycete Peronospora effusa [=P. farinosa f. sp. spinaciae]. The P. effusa shows race specificities to the resistant host and comprises 19 reported races and many novel isolates. Sixteen new P. effusa races were identified during the past three decades, and the new pathogen races are continually overcoming the genetic resistances used in commercial cultivars. A spinach breeding population derived from the cross between cultivars Whale and Lazio was inoculated with P. effusa race 16 in an environment-controlled facility; disease response was recorded and genotyped using genotyping by sequencing (GBS). The main objective of this study was to identify resistance-associated single nucleotide polymorphism (SNP) markers from the cultivar Whale against the P. effusa race 16. RESULTS: Association analysis conducted using GBS markers identified six significant SNPs (S3_658,306, S3_692697, S3_1050601, S3_1227787, S3_1227802, S3_1231197). The downy mildew resistance locus from cultivar Whale was mapped to a 0.57 Mb region on chromosome 3, including four disease resistance candidate genes (Spo12736, Spo12784, Spo12908, and Spo12821) within 2.69-11.28 Kb of the peak SNP. CONCLUSIONS: Genomewide association analysis approach was used to map the P. effusa race 16 resistance loci and identify associated SNP markers and the candidate genes. The results from this study could be valuable in understanding the genetic basis of downy mildew resistance, and the SNP marker will be useful in spinach breeding to select resistant lines.

Genome-wide simple sequence repeats (SSR) markers discovered from whole-genome sequence comparisons of multiple spinach accessions.

The availability of well-assembled genome sequences and reduced sequencing costs have enabled the resequencing of many additional accessions in several crops, thus facilitating the rapid discovery and development of simple sequence repeat (SSR) markers. Although the genome sequence of inbred spinach line Sp75 is available, previous efforts have resulted in a limited number of useful SSR markers. Identification of additional polymorphic SSR markers will support genetics and breeding research in spinach. This study aimed to use the available genomic resources to mine and catalog a large number of polymorphic SSR markers. A search for SSR loci on six chromosome sequences of spinach line Sp75 using GMATA identified a total of 42,155 loci with repeat motifs of two to six nucleotides in the Sp75 reference genome. Whole-genome sequences (30x) of additional 21 accessions were aligned against the chromosome sequences of the reference genome and in silico genotyped using the HipSTR program by comparing and counting repeat numbers variation across the SSR loci among the accessions. The HipSTR program generated SSR genotype data were filtered for monomorphic and high missing loci, and a final set of the 5986 polymorphic SSR loci were identified. The polymorphic SSR loci were present at a density of 12.9 SSRs/Mb and were physically mapped. Out of 36 randomly selected SSR loci for validation, two failed to amplify, while the remaining were all polymorphic in a set of 48 spinach accessions from 34 countries. Genetic diversity analysis performed using the SSRs allele score data on the 48 spinach accessions showed three main population groups. This strategy to mine and develop polymorphic SSR markers by a comparative analysis of the genome sequences of multiple accessions and computational genotyping of the candidate SSR loci eliminates the need for laborious experimental screening. Our approach increased the efficiency of discovering a large set of novel polymorphic SSR markers, as demonstrated in this report.

Genome Wide Association Studies in Multiple Spinach Breeding Populations Refine Downy Mildew Race 13 Resistance Genes.

Downy mildew, caused by the oomycete Peronospora effusa, is the most economically important disease on spinach. Fourteen new races of P. effusa have been identified in the last three decades. The frequent emergence of new races of P. effusa continually overcome the genetic resistance to the pathogen. The objectives of this research were to more clearly map the downy mildew resistance locus RPF1 in spinach, to identify single nucleotide polymorphism (SNP) markers associated with the resistance, and to refine the candidate genes responsible for the resistance. Progeny from populations generated from crosses of cultivars resistant (due to RPF1) to race 13 of P. effusa (Swan, T-Bird, Squirrel, and Tonga) with race 13 susceptible cultivars (Whale and Polka) were inoculated and the downy mildew disease response determined. Association analysis was performed in TASSEL, GAPIT, PLINK, and GENESIS programs using SNP markers identified from genotyping by sequencing (GBS). Association analysis mapped the race 13 resistance loci (RPF1) to positions 0.39, 0.69, 0.94-0.98, and 1.2 Mb of chromosome 3. The associated SNPs were within 1-7 kb of the disease resistance genes Spo12784, Spo12719, Spo12905, and Spo12821, and 11-18 Kb from Spo12903. This study extended our understanding of the genetic basis of downy mildew resistance in spinach and provided the most promising candidate genes Spo12784 and Spo12903 near the RPF1 locus, to pursue functional validation. The SNP markers may be used to select for the resistant lines to improve genetic resistance against the downy mildew pathogen and in developing durably resistant cultivars.

Sporangiospore Viability and Oospore Production in the Spinach Downy Mildew Pathogen, Peronospora effusa.

Downy mildew of spinach, caused by the obligate pathogen Peronospora effusa, remains the most important constraint in the major spinach production areas in the United States. This disease can potentially be initiated by asexual sporangiospores via "green bridges", sexually derived oospores from seed or soil, or dormant mycelium. However, the relative importance of the various types of primary inoculum is not well known. The ability of P. effusa sporangiospores to withstand abiotic stress, such as desiccation, and remain viable during short- and long-distance dispersal and the ability of oospores to germinate and infect seedlings remain unclear. Thus, the primary objectives of this research were to evaluate the impact of desiccation on sporangiospore survival and infection efficiency and examine occurrence, production, and germination of oospores. Results indicate that desiccation significantly reduces sporangiospore viability as well as infection potential. Leaf wetness duration of 4 h was needed for disease establishment by spinach downy mildew sporangiospores. Oospores were observed in leaves of numerous commercial spinach cultivars grown in California in 2018 and Arizona in 2019. Frequency of occurrence varied between the two states-years. The presence of opposite mating types in spinach production areas in the United States was demonstrated by pairing isolates in controlled crosses and producing oospores on detached leaves as well as intact plants. Information from the study of variables that affect sporangiospore viability and oospore production will help in improving our understanding of the epidemiology of this important pathogen, which has implications for management of spinach downy mildew.

Transcript profiling for regulation of sweet potato skin color in Sushu8 and its mutant Zhengshu20.

Sweet potato [Ipomoea batatas (L.) Lam.] (2n = 6x = 90) is an economic important autopolyploid species and its varieties differ regarding storage root skin and flesh colors. Two sweet potato genetic lines, Sushu8 (with red skin) and its mutant Zhengshu20, which produced different colored storage roots, were used in this study. The total flavonoid, carotenoid, and anthocyanin contents of the two lines were analyzed and revealed that anthocyanin was primarily responsible for the skin color difference. In addition, the early storage root expanding stage was the key period for anthocyanin accumulation in Sushu8. A total of 24 samples, including the skins of the fibrous root and the storage root at the early and middle expanding stages as well as the flesh of the storage root at the middle expanding stage, were analyzed based on differentially expressed genes identified by transcriptome sequencing and a weighted gene co-expression network analysis. Two gene modules highly related with the regulation of sweet potato skin color through stress responses as well as starch synthesis and glucose metabolism were identified. Furthermore, the WRKY75 transcription factor gene, fructose-bisphosphate aldolase 2 gene, and other DEGs highly related to the regulation of anthocyanin metabolism were enriched in the brown and green modules.

Association analysis of salt tolerance in cowpea (Vigna unguiculata (L.) Walp) at germination and seedling stages.

KEY MESSAGE: This is the first report on association analysis of salt tolerance and identification of SNP markers associated with salt tolerance in cowpea. Cowpea (Vigna unguiculata (L.) Walp) is one of the most important cultivated legumes in Africa. The worldwide annual production in cowpea dry seed is 5.4 million metric tons. However, cowpea is unfavorably affected by salinity stress at germination and seedling stages, which is exacerbated by the effects of climate change. The lack of knowledge on the genetic underlying salt tolerance in cowpea limits the establishment of a breeding strategy for developing salt-tolerant cowpea cultivars. The objectives of this study were to conduct association mapping for salt tolerance at germination and seedling stages and to identify SNP markers associated with salt tolerance in cowpea. We analyzed the salt tolerance index of 116 and 155 cowpea accessions at germination and seedling stages, respectively. A total of 1049 SNPs postulated from genotyping-by-sequencing were used for association analysis. Population structure was inferred using Structure 2.3.4; K optimal was determined using Structure Harvester. TASSEL 5, GAPIT, and FarmCPU involving three models such as single marker regression, general linear model, and mixed linear model were used for the association study. Substantial variation in salt tolerance index for germination rate, plant height reduction, fresh and dry shoot biomass reduction, foliar leaf injury, and inhibition of the first trifoliate leaf was observed. The cowpea accessions were structured into two subpopulations. Three SNPs, Scaffold87490_622, Scaffold87490_630, and C35017374_128 were highly associated with salt tolerance at germination stage. Seven SNPs, Scaffold93827_270, Scaffold68489_600, Scaffold87490_633, Scaffold87490_640, Scaffold82042_3387, C35069468_1916, and Scaffold93942_1089 were found to be associated with salt tolerance at seedling stage. The SNP markers were consistent across the three models and could be used as a tool to select salt-tolerant lines for breeding improved cowpea tolerance to salinity.

Genetic diversity and association mapping of mineral element concentrations in spinach leaves.

BACKGROUND: Spinach is a useful source of dietary vitamins and mineral elements. Breeding new spinach cultivars with high nutritional value is one of the main goals in spinach breeding programs worldwide, and identification of single nucleotide polymorphism (SNP) markers for mineral element concentrations is necessary to support spinach molecular breeding. The purpose of this study was to conduct a genome-wide association study (GWAS) and to identify SNP markers associated with mineral elements in the USDA-GRIN spinach germplasm collection. RESULTS: A total of 14 mineral elements: boron (B), calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), nickel (Ni), phosphorus (P), sulfur (S), and zinc (Zn) were evaluated in 292 spinach accessions originally collected from 29 countries. Significant genetic variations were found among the tested genotypes as evidenced by the 2 to 42 times difference in mineral concentrations. A total of 2402 SNPs identified from genotyping by sequencing (GBS) approach were used for genetic diversity and GWAS. Six statistical methods were used for association analysis. Forty-five SNP markers were identified to be strongly associated with the concentrations of 13 mineral elements. Only two weakly associated SNP markers were associated with K concentration. Co-localized SNPs for different elemental concentrations were discovered in this research. Three SNP markers, AYZV02017731_40, AYZV02094133_57, and AYZV02281036_185 were identified to be associated with concentrations of four mineral components, Co, Mn, S, and Zn. There is a high validating correlation coefficient with r > 0.7 among concentrations of the four elements. Thirty-one spinach accessions, which rank in the top three highest concentrations in each of the 14 mineral elements, were identified as potential parents for spinach breeding programs in the future. CONCLUSIONS: The 45 SNP markers strongly associated with the concentrations of the 13 mineral elements: B, Ca, Co, Cu, Fe, Mg, Mn, Mo, Na, Ni, P, S, and Zn could be used in breeding programs to improve the nutritional quality of spinach through marker-assisted selection (MAS). The 31 spinach accessions with high concentrations of one to several mineral elements can be used as potential parents for spinach breeding programs.

Genetic diversity and population structure analysis of spinach by single-nucleotide polymorphisms identified through genotyping-by-sequencing.

Spinach (Spinacia oleracea L., 2n = 2x = 12) is an economically important vegetable crop worldwide and one of the healthiest vegetables due to its high concentrations of nutrients and minerals. The objective of this research was to conduct genetic diversity and population structure analysis of a collection of world-wide spinach genotypes using single nucleotide polymorphisms (SNPs) markers. Genotyping by sequencing (GBS) was used to discover SNPs in spinach genotypes. Three sets of spinach genotypes were used: 1) 268 USDA GRIN spinach germplasm accessions originally collected from 30 countries; 2) 45 commercial spinach F1 hybrids from three countries; and 3) 30 US Arkansas spinach cultivars/breeding lines. The results from this study indicated that there was genetic diversity among the 343 spinach genotypes tested. Furthermore, the genetic background in improved commercial F1 hybrids and in Arkansas cultivars/lines had a different structured populations from the USDA germplasm. In addition, the genetic diversity and population structures were associated with geographic origin and germplasm from the US Arkansas breeding program had a unique genetic background. These data could provide genetic diversity information and the molecular markers for selecting parents in spinach breeding programs.

In silico development and characterization of tri-nucleotide simple sequence repeat markers in hazelnut (Corylus avellana L.).

Plant genomes are now sequenced rapidly and inexpensively. In silico approaches allow efficient development of simple sequence repeat markers, also known as microsatellite markers, from these sequences. A search of the genome sequence of 'Jefferson' hazelnut (Corylus avellana L.) identified 8,708 tri-nucleotide simple sequence repeats with at least five repeat units, and stepwise removal of the less promising sequences led to the development of 150 polymorphic markers. Fragments in the 'Jefferson' sequence containing tri-nucleotide repeats were used as references and aligned with genomic sequences from seven other cultivars. Following in silico alignment, sequences that showed variation in number of repeat units were selected and primer pairs were designed for 243 of them. Screening on agarose gels identified 173 as polymorphic. Removal of duplicate and previously published sequences reduced the number to 150, for which fluorescent primers and capillary electrophoresis were used for amplicon sizing. These were characterized using 50 diverse hazelnut accessions. Of the 150, 132 generated the expected one or two alleles per accession while 18 amplified more than two amplicons in at least one accession. Diversity parameters of the 132 marker loci averaged 4.73 for number of alleles, 0.51 for expected heterozygosity (He), 0.49 for observed heterozygosity (Ho), 0.46 for polymorphism information content (PIC), and 0.04 for frequency of null alleles. The clustering of the 50 accessions in a dendrogram constructed from the 150 markers confirmed the wide genetic diversity and presence of three of the four major geographic groups: Central European, Black Sea, and Spanish-Italian. In the mapping population, 105 loci segregated, of which 101 were assigned to a linkage group (LG), with positions well-dispersed across all 11 LGs. These new markers will be useful for cultivar fingerprinting, diversity studies, genome comparisons, mapping, and alignment of the linkage map with the genome sequence and physical map.