Identification of sources of resistance to scald (Rhynchosporium commune) and of related genomic regions using genome-wide association in a mapping panel of spring barley.

Barley is an important crop worldwide known for its adaptation to harsh environments and used in multiple forms as feed, food and beverages. Its productivity is affected by major abiotic and biotic stresses. Scald caused by hemibiotrophic fungus Rhynchosporium commune is a major foliar disease in many parts of the world. Host plant resistance is targeted by breeders to efficiently control this disease. An association mapping panel of 316 spring barley genotypes (AM2017) was screened for seedling resistance in greenhouse against three R. commune isolates and for adult plant resistance in three field locations in Morocco. The phenotyping results showed different numbers of entries with resistant and moderately resistant reactions at both seedling and adult plant stages. The reactions differed between the isolates with the highest percentage of resistant genotypes observed for isolate SC-S611 (49.4%) and highest percentage of susceptible genotypes (73.8%) for isolate SC-1122. At adult plant stage, the highest percentage of scald resistant genotypes (64.5%) was observed at Rommani site compared to 56% at Guich site and only 28.8% at Marchouch site. Seven genotypes were resistant at the seedling and adult plant stages. Genome wide association study (GWAS) revealed 102 MTA (15 QTL) at the seedling stage, and 25 MTA (12 QTL) associated with scald resistance at the adult plant stage. In addition, the sequences of 92 out of 102 at SRT, and 24 out of 25 significant SNP markers at APR were located in genomic regions enriched with functional proteins involved in diverse cellular processes including disease resistance. These markers span over all chromosomes with the majority of SNPs located on 3H and 7H. This study has verified 18 QTL reported in previous studies. In addition, it was successful in identifying new sources of resistance and novel genomic regions which could help in enhancing scald resistance in barley breeding programs.

CGIAR Barley Breeding Toolbox: A diversity panel to facilitate breeding and genomic research in the developing world.

Breeding programs in developing countries still cannot afford the new genotyping technologies, hindering their research. We aimed to assemble an Association Mapping panel to serve as CGIAR Barley Breeding Toolbox (CBBT), especially for the Developing World. The germplasm had to be representative of the one grown in the Developing World; with high genetic variability and be of public domain. For it, we genotyped with the Infinium iSelect 50K chip, a Global Barley Panel (GBP) of 530 genotypes representing a wide range of row-types, end-uses, growth habits, geographical origins and environments. 40,342 markers were polymorphic with an average polymorphism information content of 0.35 and 66% of them exceeding 0.25. The analysis of the population structure identified 8 subpopulations mostly linked to geographical origin, four of them with significant ICARDA origin. The 16 allele combinations at 4 major flowering genes (HvVRN-H3, HvPPD-H1, HvVRN-H1 and HvCEN) explained 11.07% genetic variation and were linked to the geographic origins of the lines. ICARDA material showed the widest diversity as revealed by the highest number of polymorphic loci (99.76% of all polymorphic SNPs in GBP), number of private alleles and the fact that ICARDA lines were present in all 8 subpopulations and carried all 16 allelic combinations. Due to their genetic diversity and their representativity of the germplasm adapted to the Developing World, ICARDA-derived lines and cultivated landraces were pre-selected to form the CBBT. Using the Mean of Transformed Kinships method, we assembled a panel capturing most of the allelic diversity in the GBP. The CBBT (N=250) preserves good balance between row-types and good representation of both phenology allelic combinations and subpopulations of the GBP. The CBBT and its genotypic data is available to researchers worldwide as a collaborative tool to underpin the genetic mechanisms of traits of interest for barley cultivation.

Shedding Light on Races of the Spinach Fusarium Wilt Pathogen, Fusarium oxysporum f. sp. spinaciae.

Two pathogenicity groups of Fusarium oxysporum f. sp. spinaciae, the causal agent of Fusarium wilt of spinach (Spinacia oleracea), were described recently based on virulence of isolates on proprietary spinach inbreds. In this study, a wide range in severity of wilt was observed for 68 spinach cultivars inoculated with an isolate of each pathogenicity group, with 22 (32.4%) cultivars displaying differential responses to the isolates. In a second set of trials, seven spinach cultivars were inoculated with five isolates of each pathogenicity group. The cultivars had similar wilt responses to isolates within each group. In both sets of trials, the most severe wilt developed on cultivars inoculated with pathogenicity group 2 isolates when daylength was shorter and light intensity lower. To test whether light intensity exacerbates severity of Fusarium wilt, three spinach cultivars were inoculated with two isolates of each pathogenicity group and grown with or without shading. Shaded plants developed more severe wilt than nonshaded plants. This difference in wilt severity was greatest for plants inoculated with pathogenicity group 2 isolates. We propose naming isolates of pathogenicity groups 1 and 2 as races 1 and 2 of F. oxysporum f. sp. spinaciae, respectively, and recommend the cultivars Kiowa (susceptible to both races) and Magnetic (susceptible to race 2 and highly resistant to race 1) as differentials. Results of this study should help breeders screen spinach germplasm for resistance to both races of F. oxysporum f. sp. spinaciae.

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.

Genome Wide Association Mapping of Spot Blotch Resistance at Seedling and Adult Plant Stages in Barley.

Barley spot blotch (SB) caused by Cochliobolus sativus is one of the major constrains to barley production in warmer regions worldwide. The study was undertaken to identify and estimate effects of loci underlying quantitative resistance to SB at the seedling and adult plant stages. A panel of 261 high input (HI-AM) barley genotypes consisting of released cultivars, advanced breeding lines, and landraces, was screened for resistance to SB. The seedling resistance screening was conducted using two virulent isolates from Morocco (ICSB3 and SB54) while the adult plant stage resistance was evaluated at two hot spot locations, Faizabad and Varanasi, in India under artificial inoculation using a mixture of prevalent virulent isolates. The HI-AM panel was genotyped using DArT-Seq high-throughput genotyping platform. Genome wide association mapping (GWAM) was conducted using 13,182 PAV and 6,311 SNP markers, for seedling and adult plant resistance. Both GLM and MLM model were employed in TASSEL (v 5.0) using principal component analysis and Kinship Matrix as covariates. Final disease rating and Area Under Disease Progress Curve (AUDPC) were used for the evaluation of adult stage plant resistance. The GWAM analysis indicated 23 QTL at the seedling stage (14 for isolate ICSB3 and 9 for isolate SB54), while 15 QTL were detected at the adult plant stage resistance (6 at Faizabad and 9 at Varanasi) and 5 for AUDPC based resistance at Varanasi. Common QTL at seedling and adult plant stages were found across all barley chromosomes. Seedling stage QTL explained together 73.24% of the variance for seedling resistance to isolate ICSB3 and 49.26% for isolate SB54, whereas, QTL for adult plant stage resistance explained together 38.32%, 44.09% and 26.42% of the variance at Faizabad and Varanasi and AUDPC at Varanasi, respectively. Several QTL identified in this study were also reported in previous studies using bi-parental and association mapping populations, corroborating our results. The promising QTL detected at both stages, once validated, can be used for marker assisted selection (MAS) in SB resistance barley breeding program.

Genome Wide Association Mapping of Seedling and Adult Plant Resistance to Barley Stripe Rust (Puccinia striiformis f. sp. hordei) in India.

Barley stripe rust is caused by Puccinia striiformis f.sp. hordei, (Psh), occurs worldwide, and is a major disease in South Asia. The aim of this work was to identify and estimate effects of loci underlying quantitative resistance to rust at seedling and adult plant stages. HI-AM panel of 261 barley genotypes consisting of released cultivars from North and South America, Europe, Australia, advanced breeding lines, and local landraces from ICARDA barley program were screened at seedling and adult plant stages for resistance to Psh. Seedling resistance was evaluated with the five prevalent Psh races in India. Screening for the adult plant stage resistance was also performed in two different locations by inoculating with a mixture of the five races used for seedling screeing. The panel was genotyped using DaRT-Seq high-throughput genotyping platform. The genome-wide association mapping (GWAM) showed a total of 45 QTL located across the seven barley chromosomes for seedling resistance to the five races and 18 QTL for adult plant stage resistance. Common QTL for different races at seedling stage were found on all chromosomes except on chromosome 1H. Four common QTL associated with seedling and adult plant stage resistance were found on chromosomes 2, 5, and 6H. Moreover, one of the QTL located on the long arm of chromosome 5H showed stable effects across environments for adult plant stage resistance. Several QTL identified in this study were also reported before in bi-parental and association mapping populations studies validating current GWAM. However 15 new QTL were found at adult plant stage on all chromosomes except the 4H, explaining up to 36.79% of the variance. The promising QTL detected at both stages, once validated, can be used for MAS in Psh resistance breeding program globally.

The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion.

Mitogen-activated protein kinases (MAPKs) play a central role in transferring signals and regulating gene expression in response to extracellular stimuli. An ortholog of the Saccharomyces cerevisiae cell wall integrity MAPK was identified in the phytopathogenic fungus Sclerotinia sclerotiorum. Disruption of the S. sclerotiorum Smk3 gene severely reduced virulence on intact host plant leaves but not on leaves stripped of cuticle wax. This was attributed to alterations in hyphal apical dominance leading to the inability to aggregate and form infection cushions. The mutation also caused loss of the ability to produce sclerotia, increased aerial hyphae formation, and altered hyphal hydrophobicity and cell wall integrity. Mutants had slower radial expansion rates on solid media but more tolerance to elevated temperatures. Loss of the SMK3 cell wall integrity MAPK appears to have impaired the ability of S. sclerotiorum to sense its surrounding environment, leading to misregulation of a variety of functions. Many of the phenotypes were similar to those observed in S. sclerotiorum adenylate cyclase and SMK1 MAPK mutants, suggesting that these signaling pathways co-regulate aspects of fungal growth, physiology, and pathogenicity.

Microsatellite markers used for genome-wide association mapping of partial resistance to Sclerotinia sclerotiorum in a world collection of Brassica napus.

The fungal pathogen Sclerotinia sclerotiorum causes stem rot of oilseed rape (Brassica napus) worldwide. In preparation for genome-wide association mapping (GWAM) of sclerotinia resistance in B. napus, 152 accessions from diverse geographical regions were screened with a single Canadian isolate, #321. Plants were inoculated by attaching mycelium plugs to the main stem at full flower. Lesion lengths measured 7, 14 and 21 days after inoculation were used to calculate the area under the disease progress curve (AUDPC). Depth of penetration was noted and used to calculate percent soft and collapsed lesions (% s + c). The two disease traits were highly correlated (r = 0.93). Partially resistant accessions (AUDPC <7 and % s + c <2) were identified primarily from South Korea and Japan with a few from Pakistan, China and Europe. Genotyping of accessions with 84 simple sequence repeat markers provided 690 polymorphic loci for GWAM. The general linear model in TASSEL best fitted the data when adjusted for population structure (STRUCTURE), GLM + Q. After correction for positive false discovery rate, 34 loci were significantly associated with both disease traits of which 21 alleles contributed to resistance, while the remaining enhanced susceptibility. The phenotypic variation explained by the loci ranged from 6 to 25 %. Five loci mapped to published quantitative trait loci conferring sclerotinia resistance in Chinese lines.

Plant breeding can be made more efficient by having fewer, better crosses.

BACKGROUND: Crop yields have to increase to provide food security for the world's growing population. To achieve these yield increases there will have to be a significant contribution from genetic gains made by conventional plant breeding. However, the breeding process is not efficient because crosses made between parental combinations that fail to produce useful varieties consume over 99% of the resources. RESULTS: We tested in a rice-breeding programme if its efficiency could be improved by using many fewer, but more judiciously chosen crosses than usual. In a 15-year programme in Nepal, with varietal testing also in India and Bangladesh, we made only six crosses that were stringently chosen on complementary parental performance. We evaluated their success by the adoption and official release of the varieties they produced. We then modelled optimum cross number using assumptions based on our experimental results.Four of the six crosses succeeded. This was a fifty-fold improvement over breeding programmes that employ many crosses where only about one, or fewer, crosses in 200 succeed. Based on these results, we modelled the optimum number of crosses by assuming there would be a decline in the reliability of the breeder's prediction of the value of each cross as more crosses were made (because there is progressively less information on the traits of the parents). Fewer-cross programmes were more likely to succeed and did so using fewer resources. Making more crosses reduced the overall probability of success of the breeding programme. CONCLUSIONS: The efficiency of national and international breeding programmes would be increased by making fewer crosses among more carefully chosen parents. This would increase the number of higher yielding varieties that are delivered to farmers and hence help to improve food security.

Phenotypic and Molecular Diversity of Cochliobolus sativus Populations from Wheat.

Spot blotch, caused by Cochliobolus sativus, is a devastating foliar disease of wheat in Nepal and in the Northern Great Plains of the United States. However, limited information on variation in virulence and genetic structure of C. sativus from wheat is available. In this study, pathogenic variation of 96 isolates of C. sativus from the Hill and Plain areas in Nepal (n = 48) and in the Central and Northern areas in North Dakota (n = 48) were evaluated on 12 differential wheat lines. DNA polymorphisms in all isolates were analyzed using eight selected amplified fragment length polymorphism primer combinations. Phenotypic data analysis showed the isolates varied greatly and were classified into 47 pathotypes. Cluster analysis indicated the isolates fell into three distinct groups with low, intermediate, and high virulence. Population genetic analysis revealed significant linkage disequilibrium ( = 0.066 to 0.292), indicating that sexual reproduction plays little or no role in evolution and disease epidemiology in wheat fields. Furthermore, the corrected standardized fixation index (G″ST = 0.05 and 0.02) showed no evidence of genetic differentiation in C. sativus populations. Collectively, these results confirmed high pathogenic and molecular diversity in the C. sativus populations collected from wheat foliar infections and will be useful to assist in developing resistant cultivars to manage this disease.

Trichothecene profiling and population genetic analysis of Gibberella zeae from barley in North Dakota and Minnesota.

Gibberella zeae, the principal cause of Fusarium head blight (FHB) of barley, contaminates grains with several mycotoxins, which creates a serious problem for the malting barley industry in the United States, China, and Europe. However, limited studies have been conducted on the trichothecene profiles and population genetic structure of G. zeae isolates collected from barley in the United States. Trichothecene biosynthesis gene (TRI)-based polymerase chain reaction (PCR) assays and 10 variable number tandem repeat (VNTR) markers were used to determine the genetic diversity and compare the trichothecene profiles of an older population (n = 115 isolates) of G. zeae collected in 1997 to 2000 with a newer population (n = 147 isolates) collected in 2008. Samples were from across the major barley-growing regions in North Dakota and Minnesota. The results of TRI-based PCR assays were further validated using a subset of 32 and 28 isolates of G. zeae by sequence analysis and gas chromatography, respectively. TRI-based PCR assays revealed that all the G. zeae isolates in both populations had markers for deoxynivalenol (DON), and the frequencies of isolates with a 3-acetyldeoxynivalenol (3-ADON) marker in the newer population were ≈11-fold higher than those among isolates in the older population. G. zeae populations from barley in the Midwest of the United States showed no spatial structure, and all the isolates were solidly in clade 7 of G. zeae, which is quite different from other barley-growing areas of world, where multiple species of G. zeae are commonly found in close proximity and display spatial structure. VNTR analysis showed high gene diversity (H = 0.82 to 0.83) and genotypic diversity but low linkage disequilibrium (LD = 0.02 to 0.07) in both populations. Low genetic differentiation (F(ST) = 0.013) and high gene flow (Nm = 36.84) was observed between the two populations and among subpopulations within the same population (Nm = 12.77 to 29.97), suggesting that temporal and spatial variations had little influence on population differentiation in the Upper Midwest. Similarly, low F(ST) (0.02) was observed between 3-ADON and 15-acetyldeoxynivalenol populations, indicating minor influence of the chemotype of G. zeae isolates on population subdivision, although there was a rapid increase in the frequencies of isolates with the 3-ADON marker in the Upper Midwest between the older collection made in 1997 to 2000 and the newer collection made in 2008. This study provides information to barley-breeding programs for their selection of isolates of G. zeae for evaluating barley genotypes for resistance to FHB and DON accumulation.

A global perspective of the richness and evenness of traditional crop-variety diversity maintained by farming communities.

Varietal data from 27 crop species from five continents were drawn together to determine overall trends in crop varietal diversity on farm. Measurements of richness, evenness, and divergence showed that considerable crop genetic diversity continues to be maintained on farm, in the form of traditional crop varieties. Major staples had higher richness and evenness than nonstaples. Variety richness for clonal species was much higher than that of other breeding systems. A close linear relationship between traditional variety richness and evenness (both transformed), empirically derived from data spanning a wide range of crops and countries, was found both at household and community levels. Fitting a neutral "function" to traditional variety diversity relationships, comparable to a species abundance distribution of "neutral ecology," provided a benchmark to assess the standing diversity on farm. In some cases, high dominance occurred, with much of the variety richness held at low frequencies. This suggested that diversity may be maintained as an insurance to meet future environmental changes or social and economic needs. In other cases, a more even frequency distribution of varieties was found, possibly implying that farmers are selecting varieties to service a diversity of current needs and purposes. Divergence estimates, measured as the proportion of community evenness displayed among farmers, underscore the importance of a large number of small farms adopting distinctly diverse varietal strategies as a major force that maintains crop genetic diversity on farm.