Genome-wide SNP discovery and genetic linkage map construction in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) using a genotyping by sequencing (GBS) approach

Recently developed plant genomics approaches (LD mapping and genome-wide selection) require many molecular markers distributed throughout the plant genome. As a result, the availability of an increasing number of markers is essential for maintaining highly efficient and accurate plant breeding programs. In this study, we identified SNP loci in sunflower using a genotyping by sequencing (GBS) approach in an intraspecific F₂ mapping population. A total of 271,445,770 reads were generated by the Genome Analyzer II next-generation sequencing platform and 29.2 % of the reads were aligned to unique locations in the genome. A total of 46,278 SNP loci were identified and 7646 SNP loci were validated in an F₂ population. In addition, a SNP-based linkage map was constructed. This is the first report of SNP discovery in sunflower by GBS. The SNP markers and SNP-based linkage map will be valuable molecular genetics tools for sunflower breeding.     

Genetic diversity, linkage disequilibrium, and association mapping analyses of peach (Prunus persica) landraces in China

The genetic diversity, population structure, and linkage disequilibrium (LD) of peaches are greatly important in genome-wide association mapping. In the current study, 104 peach landrace accessions from six Chinese geographical regions were evaluated for fruit and phenological period. The accessions were genotyped with 53 genome-wide simple sequence repeat (SSR) markers. All SSR markers were highly polymorphic across the accessions, and a total of 340 alleles were detected, including 59 private alleles. Of the six regions studied, the northern part of China as well as the middle and lower reaches of the Changjiang River were found to be the most highly diverse genetically. Based on population structure analysis, the peaches were divided into five groups, which well agreed with the geographical distribution. Of the SSR pairs in these accessions, 18.07% (P?<?0.05) were in LD. The mean r ² value for all intrachromosomal loci pairs was 0.0149, and LD decayed at 6.01?cM. The general linear model was used to calculate the genome-wide marker-trait associations of 10 complex traits. The traits include flesh color around the stone, red pigment in the flesh, flesh texture, flesh adhesion, flesh firmness, fruit weight, chilling requirement, flowering time, ripening time, and fruit development period. These traits were estimated by analyzing the 104 landraces. Many of the associated markers were located in regions where quantitative trait loci (QTLs) were previously identified. Peach association mapping is an effective approach for identifying QTLs and may be an alternative to QTL mapping based on crosses between different lines.     

A first insight into peach [Prunus persica (L.) Batsch] SNP variability

Three factors may have reduced the diversity at both individual gene and whole genome levels in cultivated peach: its self-compatible mating system, the narrow genetic basis of most commercial cultivars, and the recent strong selection towards agronomically interesting traits. Previous diversity analyses with markers such as simple sequence repeats (SSRs) have revealed low levels of genetic variability. Here, we sequenced 23 genome-wide distributed DNA fragments in 47 occidental peach varieties, also observing reduced variability levels. On average, there was one single nucleotide polymorphism (SNP) every 598 bp and one indel every 4,189 bp. As expected, variability was higher in non-coding than in coding regions (one SNP every 390 non-coding bp versus one in 1,850 bp in coding DNA). In general, SNPs were observed at relatively high frequency, mean minor allele frequency?=?0.225, meaning that a large proportion of the SNPs discovered by sequencing similar germplasm will be useful for other purposes, such as association mapping. The average heterozygosity of the varieties was 0.28, with a low correlation between SSR and SNP heterozygosity. The whole sequence of two candidate genes, a pectate lyase 1 candidate for fruit firmness (CGPAA2668) and a sucrose synthase 1 candidate for sugar content (CGPPB6189), in the 47 varieties revealed that they both may have suffered a process of balancing selection.     

Identifying SNP markers tightly associated with six major genes in peach [<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch] using a high-density SNP array with an objective of marker-assisted selection (MAS)

One of the applications of genomics is to identify genetic markers linked to loci responsible for variation in phenotypic traits, which could be used in breeding programs to select individuals with favorable alleles, particularly at the seedling stage. With this aim, in the framework of the European project FruitBreedomics, we selected five main peach fruit characters and a resistance trait, controlled by major genes with Mendelian inheritance: fruit flesh color Y, fruit skin pubescence G, fruit shape S, sub-acid fruit D, stone adhesion-flesh texture F-M, and resistance to green peach aphid Rm2. They were all previously mapped in Prunus. We then selected three F₁ and three F₂ progenies segregating for these characters and developed genetic maps of the linkage groups including the major genes, using the single nucleotide polymorphism (SNP) genome-wide scans obtained with the International Peach SNP Consortium (IPSC) 9K SNP array v1. We identified SNPs co-segregating with the characters in all cases. Their positions were in agreement with the known positions of the major genes. The number of SNPs linked to each of these, as well as the size of the physical regions encompassing them, varied depending on the maps. As a result, the number of useful SNPs for marker-assisted selection varied accordingly. As a whole, this study establishes a sound basis for further development of MAS on these characters. Additionally, we also discussed some limitations that were observed regarding the SNP array efficiency.     

Genome-wide association study-based identification genes influencing agronomic traits in rice (Oryza sativa L.)

Rice is one of the most important cereal crops, providing the daily dietary intake for approximately 50% of the global human population. Here, we re-sequenced 259 rice accessions, generating 1371.65 Gb of raw data. Furthermore, we performed genome-wide association studies (GWAS) on 13 agronomic traits using 2.8 million single nucleotide polymorphisms (SNPs) characterized in 259 rice accessions. Phenotypic data and best linear unbiased prediction (BLUP) values of each of the 13 traits over two years of each trait were used for the GWAS. The results showed that 816 SNP signals were significantly associated with the 13 agronomic traits. Then we detected candidate genes related to target traits within 200 kb upstream and downstream of the associated SNP loci, based on linkage disequilibrium (LD) blocks in the whole rice genome. These candidate genes were further identified through haplotype block constructions. This comprehensive study provides a timely and important genomic resource for breeding high yielding rice cultivars.     

Development and evaluation of a 9K SNP array for peach by internationally coordinated SNP detection and validation in breeding germplasm

Although a large number of single nucleotide polymorphism (SNP) markers covering the entire genome are needed to enable molecular breeding efforts such as genome wide association studies, fine mapping, genomic selection and marker-assisted selection in peach [Prunus persica (L.) Batsch] and related Prunus species, only a limited number of genetic markers, including simple sequence repeats (SSRs), have been available to date. To address this need, an international consortium (The International Peach SNP Consortium; IPSC) has pursued a coordinated effort to perform genome-scale SNP discovery in peach using next generation sequencing platforms to develop and characterize a high-throughput Illumina Infinium® SNP genotyping array platform. We performed whole genome re-sequencing of 56 peach breeding accessions using the Illumina and Roche/454 sequencing technologies. Polymorphism detection algorithms identified a total of 1,022,354 SNPs. Validation with the Illumina GoldenGate® assay was performed on a subset of the predicted SNPs, verifying ∼75% of genic (exonic and intronic) SNPs, whereas only about a third of intergenic SNPs were verified. Conservative filtering was applied to arrive at a set of 8,144 SNPs that were included on the IPSC peach SNP array v1, distributed over all eight peach chromosomes with an average spacing of 26.7 kb between SNPs. Use of this platform to screen a total of 709 accessions of peach in two separate evaluation panels identified a total of 6,869 (84.3%) polymorphic SNPs.The almost 7,000 SNPs verified as polymorphic through extensive empirical evaluation represent an excellent source of markers for future studies in genetic relatedness, genetic mapping, and dissecting the genetic architecture of complex agricultural traits. The IPSC peach SNP array v1 is commercially available and we expect that it will be used worldwide for genetic studies in peach and related stone fruit and nut species.     

A DNA test for routine prediction in breeding of peach blush,Ppe-R<Subscript>f</Subscript>-SSR

Blush, the proportion of red overcolor on the skin surface of fruit, is highly variable in peach breeding germplasm and is important in the marketing of peach fruit. The fresh market peach industry demands a high level of blush to entice consumers, while the processing peach industry requires minimal blush. Therefore, blush is a major selection criterion in breeding programs. The use of DNA-based information could improve breeding efficiency and accuracy for fruit blush coverage, but a predictive DNA test is required. The objective of this study was to develop a DNA test for the prediction of blush coverage by targeting the major locus, R _f , associated with blush variation. Initially, haplotypes were developed based on five SNP markers associated with variation in blush coverage. To convert the 5-SNP haplotype test into a single, simple PCR-based assay, 11 simple sequence repeat markers were designed and used to screen individuals representing all SNP haplotypes. The most informative assay, named Ppe-R_f-SSR, was chosen to screen 200 individuals of the RosBREED peach reference germplasm set that incorporated germplasm from four breeding programs. Ppe-R_f-SSR accurately differentiated individuals with high-, medium-, and low-blush coverage in most lineages. Outcomes highlighted that DNA tests can be quite predictive for some breeding programs or specific germplasm sets, while for others the predictiveness can falter. Therefore, the confirmation of genotype effects for any DNA test is recommended in new germplasm before routine use. The prediction accuracy and breeding utility of Ppe-R_f-SSR in the University of Arkansas breeding program were subsequently confirmed by screening 443 seedlings, independent of the initial DNA test development process, derived from 18 cross-combinations of 28 parents. Ppe-R_f-SSR can be used to efficiently and accurately predict fruit blush coverage, especially in fresh market germplasm, and has been deployed for routine use in the University of Arkansas peach breeding program.     

Haplotype diversity in the endosperm specific β-amylase gene Bmy1 of cultivated barley (Hordeum vulgare L.)

Five single nucleotide polymorphism (SNP) sites corresponding to substitutions in the protein sequence of the β-amylase gene Bmy1 at amino acid (AA) positions 115, 165, 233, 347 and 430 were genotyped in 493 cultivated barley accessions by Pyrosequencing and a CAPS assay. A total of 6 different haplotypes for the Bmy1 gene were discovered of which 4 haplotypes were identified as previously described alleles Bmy1-Sd1, Bmy1-Sd2L, Bmy1-Sd2H and Bmy1-Sd3, while 2 haplotypes were new. A broad spectrum of haplotypes was found in spring barleys, while the winter barleys were dominated by the newly described haplotype Bmy1-Sd4. Individual haplotype frequencies varied between the geographic regions.Three pairs of SNP loci within the gene showed highly significant (P<0.0001) elevated values of linkage disequilibrium (LD) with r ² > 0.6. In the European and Asian subpopulations different loci were in linkage disequilibrium due to the differences in haplotype frequency distributions. By applying LD data to select haplotype tagging SNPs, three SNP sites corresponding to AA positions 115, 233 and 347 were identified that allowed to discriminate 4 haplotypes and to capture 91.6% of the available diversity by distinguishing 452 out of 493 accessions. In a subset of 2-rowed German spring barley varieties 4 SNPs and 2 haplotypes had a significant association with the malting quality parameter final attenuation limit which is related to the total amylolytic enzymatic activity.     

Characterization and identification of ISSR markers associated with resistance to dried-shrink disease in sea buckthorn

Sea buckthorn (Hippophae L.) is an economically important, globally distributed shrub with unique biochemical properties. This long-juvenile and long-lived woody plant can be more difficult to cultivate than other crop plants. Dried-shrink disease is a dangerous pathogen that destroys sea buckthorn and halts commercial production. We estimated variability of ISSR molecular markers in 52 accessions of 16 sea buckthorn varieties and lines to seek markers associated with dried-shrink resistance and help identify potential breeding varieties and lines. Fifteen ISSR primers generated 346 bands, with an average of 23 bands per primer. Genetic similarities (Jaccard coefficient) between pairs of accessions ranged from 0.216 to 0.781. At a Jaccard coefficient of 0.39, the dendrogram generated with 342 polymorphic bands clustered 51 accessions of H. rhamnoides into five groups and one H. salicifolia isolate. Four ISSR markers (887₁₉₀, 835₇₀₀, 809₂₉₀, and 811₂₈₀) were significantly correlated with resistance to dried-shrink disease (P < 0.001). These markers provide a potential method for breeding programs that select lineages resistant to dried-shrink disease, especially when no other genetic information such as linkage maps and Quantitative Trait Loci are available.     

Genetic diversity of maize kernel starch-synthesis genes with SNAPs.

Measuring genetic diversity in populations of a crop species is very important for understanding the genetic structure of and subsequently improving the crop species by genetic manipulation. Single-nucleotide amplified polymorphisms (SNAPs) among and within maize populations of waxy, dent, and sweet corns at 25 single-nucleotide polymorphism (SNP) sites in 6 kernel starch-synthesis genes (sh2, bt2, su1, ae1, wx1, and sh1) were determined. Because of the intensive selection of some favorable alleles in starch-synthesis genes during the breeding process, and the resultant strong linkage disequilibrium (LD), the number of haplotypes in each population was far less than expected. Subsequent phenetic clustering analysis with the SNAPs indicated that the dent, waxy, and sweet corns formed distinct subclusters, except in a few incidences. LD was surveyed among SNAPs of intragenic, intergenic, and intrachromosomal SNPs in whole and subpopulations, which revealed that some SNAPs showed high LD with many other SNAPs, but some SNAPs showed low or no significant LD with others, depending on the subpopulation, indicating that these starch genes have undergone different selection in each subpopulation during the breeding process. Because the starch synthesis genes used in this study are important in maize breeding, the genetic diversity, LD, and accessions having rare SNAP alleles might be valuable in maize improvement programs.     

Evolution and association analysis of Ghd7 in rice

Plant height, heading date, and yield are the main targets for rice genetic improvement. Ghd7 is a pleiotropic gene that controls the aforementioned traits simultaneously. In this study, a rice germplasm collection of 104 accessions (Oryza sativa) and 3 wild rice varieties (O.rufipogon) was used to analyze the evolution and association of Ghd7 with plant height, heading date, and yield. Among the 104 accessions, 76 single nucleotide polymorphisms (SNPs) and six insertions and deletions were found within a 3932-bp DNA fragment of Ghd7. A higher pairwise π and θ in the promoter indicated a highly diversified promoter of Ghd7. Sixteen haplotypes and 8 types of Ghd7 protein were detected. SNP changes between haplotypes indicated that Ghd7 evolved from two distinct ancestral gene pools, and independent domestication processes were detected in indica and japonica varietals respectively. In addition to the previously reported premature stop mutation in the first exon of Ghd7, which caused phenotypic changes of multiple traits, we found another functional C/T mutation (SNP S_555) by structure-based association analysis. SNP S_555 is located in the promoter and was related to plant height probably by altering gene expression. Moreover, another seven SNP mutations in complete linkage were found to be associated with the number of spikelets per panicle, regardless of the photoperiod. These associations provide the potential for flexibility of Ghd7 application in rice breeding programs.     

The USDA Barley Core Collection: Genetic Diversity,Population Structure,and Potential for Genome-Wide Association Studies

New sources of genetic diversity must be incorporated into plant breeding programs if they are to continue increasing grain yield and quality, and tolerance to abiotic and biotic stresses. Germplasm collections provide a source of genetic and phenotypic diversity, but characterization of these resources is required to increase their utility for breeding programs. We used a barley SNP iSelect platform with 7,842 SNPs to genotype 2,417 barley accessions sampled from the USDA National Small Grains Collection of 33,176 accessions. Most of the accessions in this core collection are categorized as landraces or cultivars/breeding lines and were obtained from more than 100 countries. Both STRUCTURE and principal component analysis identified five major subpopulations within the core collection, mainly differentiated by geographical origin and spike row number (an inflorescence architecture trait). Different patterns of linkage disequilibrium (LD) were found across the barley genome and many regions of high LD contained traits involved in domestication and breeding selection. The genotype data were used to define ‘mini-core’ sets of accessions capturing the majority of the allelic diversity present in the core collection. These ‘mini-core’ sets can be used for evaluating traits that are difficult or expensive to score. Genome-wide association studies (GWAS) of ‘hull cover’, ‘spike row number’, and ‘heading date’ demonstrate the utility of the core collection for locating genetic factors determining important phenotypes. The GWAS results were referenced to a new barley consensus map containing 5,665 SNPs. Our results demonstrate that GWAS and high-density SNP genotyping are effective tools for plant breeders interested in accessing genetic diversity in large germplasm collections.     

Genomic selection and complex trait prediction using a fast EM algorithm applied to genome-wide markers

Background  

The information provided by dense genome-wide markers using high throughput technology is of considerable potential in human disease studies and livestock breeding programs. Genome-wide association studies relate individual single nucleotide polymorphisms (SNP) from dense SNP panels to individual measurements of complex traits, with the underlying assumption being that any association is caused by linkage disequilibrium (LD) between SNP and quantitative trait loci (QTL) affecting the trait. Often SNP are in genomic regions of no trait variation. Whole genome Bayesian models are an effective way of incorporating this and other important prior information into modelling. However a full Bayesian analysis is often not feasible due to the large computational time involved.     

Association Mapping for Fruit,Plant and Leaf Morphology Traits in Eggplant

An eggplant (Solanum melongena) association panel of 191 accessions, comprising a mixture of breeding lines, old varieties and landrace selections was SNP genotyped and phenotyped for key breeding fruit and plant traits at two locations over two seasons. A genome-wide association (GWA) analysis was performed using the mixed linear model, which takes into account both a kinship matrix and the sub-population membership of the accessions. Overall, 194 phenotype/genotype associations were uncovered, relating to 30 of the 33 measured traits. These associations involved 79 SNP loci mapping to 39 distinct chromosomal regions distributed over all 12 eggplant chromosomes. A comparison of the map positions of these SNPs with those of loci derived from conventional linkage mapping showed that GWA analysis both validated many of the known controlling loci and detected a large number of new marker/trait associations. Exploiting established syntenic relationships between eggplant chromosomes and those of tomato and pepper recognized orthologous regions in ten eggplant chromosomes harbouring genes influencing breeders’ traits.     

Evaluation of Linkage Disequilibrium Pattern and Association Study on Seed Oil Content in Brassica napus Using ddRAD Sequencing

High-density genetic markers are the prerequisite for understanding linkage disequilibrium (LD) and genome-wide association studies (GWASs) of complex traits in crops. To evaluate the LD pattern in oilseed rape, we sequenced a previous association panel containing 189 B. napus inbred lines using double-digested restriction-site associated DNA (ddRAD) and genotyped 19,327 RAD tags. A total of 15,921 RAD tags were assigned to a published genetic linkage map and the majority (71.1%) of these tags was uniquely mapped to the draft reference genome “Darmor-bzh.” The distance of LD decay was 1,214 kb across the genome at the background level (r² = 0.26), with the distances of LD decay being 405 kb and 2,111 kb in the A and C subgenomes, respectively. A total of 361 haplotype blocks with length > 100 kb were identified in the entire genome. The association panel could be classified into two groups, P1 and P2, which are essentially consistent with the geographical origins of varieties. A large number of group-specific haplotypes were identified, reflecting that varieties in the P1 and P2 groups experienced distinct selection in breeding programs to adapt their different growth habitats. GWAS repeatedly detected two loci significantly associated with oil content of seeds based on the developed SNPs, suggesting that the high-density SNPs were useful for understanding the genetic determinants of complex traits in GWAS.     

High-Density SNP Genotyping of Tomato (Solanum lycopersicum L.) Reveals Patterns of Genetic Variation Due to Breeding

The effects of selection on genome variation were investigated and visualized in tomato using a high-density single nucleotide polymorphism (SNP) array. 7,720 SNPs were genotyped on a collection of 426 tomato accessions (410 inbreds and 16 hybrids) and over 97% of the markers were polymorphic in the entire collection. Principal component analysis (PCA) and pairwise estimates of F _st supported that the inbred accessions represented seven sub-populations including processing, large-fruited fresh market, large-fruited vintage, cultivated cherry, landrace, wild cherry, and S. pimpinellifolium. Further divisions were found within both the contemporary processing and fresh market sub-populations. These sub-populations showed higher levels of genetic diversity relative to the vintage sub-population. The array provided a large number of polymorphic SNP markers across each sub-population, ranging from 3,159 in the vintage accessions to 6,234 in the cultivated cherry accessions. Visualization of minor allele frequency revealed regions of the genome that distinguished three representative sub-populations of cultivated tomato (processing, fresh market, and vintage), particularly on chromosomes 2, 4, 5, 6, and 11. The PCA loadings and F _st outlier analysis between these three sub-populations identified a large number of candidate loci under positive selection on chromosomes 4, 5, and 11. The extent of linkage disequilibrium (LD) was examined within each chromosome for these sub-populations. LD decay varied between chromosomes and sub-populations, with large differences reflective of breeding history. For example, on chromosome 11, decay occurred over 0.8 cM for processing accessions and over 19.7 cM for fresh market accessions. The observed SNP variation and LD decay suggest that different patterns of genetic variation in cultivated tomato are due to introgression from wild species and selection for market specialization.     

Accuracy of marker-assisted selection with single markers and marker haplotypes in cattle

A key question for the implementation of marker-assisted selection (MAS) using markers in linkage disequilibrium with quantitative trait loci (QTLs) is how many markers surrounding each QTL should be used to ensure the marker or marker haplotypes are in sufficient linkage disequilibrium (LD) with the QTL. In this paper we compare the accuracy of MAS using either single markers or marker haplotypes in an Angus cattle data set consisting of 9323 genome-wide single nucleotide polymorphisms (SNPs) genotyped in 379 Angus cattle. The extent of LD in the data set was such that the average marker-marker r2 was 0.2 at 200 kb. The accuracy of MAS increased as the number of markers in the haplotype surrounding the QTL increased, although only when the number of markers in the haplotype was 4 or greater did the accuracy exceed that achieved when the SNP in the highest LD with the QTL was used. A large number of phenotypic records (>1000) were required to accurately estimate the effects of the haplotypes.     

Rosaceae conserved orthologous sequences marker polymorphism in sweet cherry germplasm and construction of a SNP-based map

The Rosaceae Conserved Orthologous Set (RosCOS) provides a gene-based genome-wide set of markers that have been used in comparative analyses of peach (Prunus persica), apple (Malus × domestica), and strawberry (Fragaria spp.). In order to extend the use of these RosCOS to sweet cherry (Prunus avium L.), we identified markers that are polymorphic in breeding germplasm. Ninety-five percent (595/627) of previously designed RosCOS primer pairs amplified a product in six sweet cherry cultivars predicted to represent the range of genetic diversity in breeding germplasm. A total of 45% (282/627) RosCOS were polymorphic among the six cultivars, and allele number ranged from 2 to 6, with a genome-wide mean of 2.35. A subset of 92 genome-wide single nucleotide polymorphisms (SNPs) corresponding to 76 RosCOS was analyzed in 36 founder accessions and progeny. The expected and observed heterozygosity suggested that 83% of the RosCOS were in Hardy–Weinberg equilibrium, implying that most RosCOS behave as neutral markers. Principal coordinate analysis (PCO) identified one wild accession and two Spanish landraces that clustered differently from the other accessions. The relatively high number of unique alleles found in the three differentially clustered selections suggested that their use as parents has potential to increase the genetic diversity in future US-bred cultivars. Of the 92 RosCOS SNPs, 81 SNPs that represented 68 genome-wide RosCOS segregated in four mapping populations. These RosCOS were mapped in four F₁ populations, thereby greatly improving the genetic linkage map of sweet cherry.