Population structure and marker–trait associations for pomological traits in peach and nectarine cultivars

Marker–trait associations based on populations from controlled crosses have been established in peach using markers mapped on the peach consensus map. In this study, we explored the utility of unstructured populations for association mapping to determine useful marker–trait associations in peach/nectarine cultivars. We used 94 peach cultivars representing local Spanish and modern cultivars from international breeding programs that are maintained at the Experimental Station of Aula Dei, Spain. This collection was characterized for pomological traits and was screened with 40 SSR markers that span the peach genome. Population structure analysis using STRUCTURE software identified two subpopulations, the local and modern cultivars, with admixture within both groups. The local Spanish cultivars were somewhat less diverse than modern cultivars. Marker–trait associations were determined in TASSEL with and without modelling coefficient of membership (Q) values as covariates. The results showed significant associations with pomological traits. We chose three markers on LG4 because of their proximity to the endoPG locus (freestone–melting flesh) that strongly affects pomological traits. Two genotypes of BPPCT015 marker showed significant associations with harvest date, flavonoids and sorbitol. Also, two genotypes of CPPCT028 showed associations with harvest date, total phenolics, RAC, and total sugars. Finally, two genotypes of endoPG1 showed associations with flesh firmness and total sugars. The analysis of linkage disequilibrium (LD) revealed a high level of LD up to 20 cM, and decay at farther distances. Therefore, association mapping could be a powerful tool for identifying marker–trait associations and would be useful for marker-assisted selection in peach breeding.     

Mapping of the genomic regions controlling seed storability in soybean (Glycine max L.)

Seed storability is especially important in the tropics due to high temperature and relative humidity of storage environment that cause rapid deterioration of seeds in storage. The objective of this study was to use SSR markers to identify genomic regions associated with quantitative trait loci (QTLs) controlling seed storability based on relative germination rate in the F_2:3 population derived from a cross between vegetable soybean line (MJ0004-6) with poor longevity and landrace cultivar from Myanmar (R18500) with good longevity. The F_2:4 seeds harvested in 2011 and 2012 were used to investigate seed storability. The F₂ population was genotyped with 148 markers and the genetic map consisted of 128 SSR loci which converged into 38 linkage groups covering 1664.3 cM of soybean genome. Single marker analysis revealed that 13 markers from six linkage groups (C1, D2, E, F, J and L) were associated with seed storability. Composite interval mapping identified a total of three QTLs on linkage groups C1, F and L with phenotypic variance explained ranging from 8.79 to 13.43%. The R18500 alleles increased seed storability at all of the detected QTLs. No common QTLs were found for storability of seeds harvested in 2011 and 2012. This study agreed with previous reports in other crops that genotype by environment interaction plays an important role in expression of seed storability.     

Discovery of QTLs for water mining and water use efficiency traits in rice under water-limited condition through association mapping

Developing trait introgressed rice cultivars is essential to sustain yield under aerobic conditions. Here, we report DNA markers governing variability in root traits, water use efficiency (WUE) and other biometric traits like total leaf area by association mapping. A set of 173 diverse rice germplasm accessions were phenotyped for root traits in specially designed root structures and WUE using carbon isotope discrimination (Δ¹³C) during the monsoon season (July to October) of two consecutive years (2007 and 2008). The panel was genotyped using 291 SSR markers spanning the entire genome of rice. Root biomass varied between 1.8 and 16.3 g plant^?1 while root length between 22 and 78 cm representing significant genetic variability. Similarly, Δ¹³C varied from 18 to 23 ‰. The SSR markers showed extensive polymorphism with around 73 % of all the markers revealing polymorphism information content values more than 0.5. Model-based structure analysis using the squared-allele frequency correlations revealed six subgroups among the panel with an average LD decay of about 10–20 cM. The Benjamini–Hochberg analysis was carried out to compute the false discovery rate combined with the analysis of effective LD. A total of 82 markers were involved in 175 significant (corrected P values and Q values <0.05) marker–trait associations (MTAs) across experiment 1 and experiment 2 and for the pooled data. Out of these, 22 markers were found to be associated with more than one trait. Common markers with significant associations were discovered for root biomass, total leaf area and total biomass suggesting the interdependency of these traits. Finally, 12 markers showed significant and stable MTAs across the experiments for different traits. An in silico analysis indicated that 45 % of the MTAs overlapped with previously reported QTLs and can be used for QTL introgression through breeding.     

Mapping and validation of simple sequence repeat markers linked to a major gene controlling seed cadmium accumulation in soybean [Glycine max (L.) Merr]

Daily consumption of cadmium (Cd) contaminated foods poses a risk to human health. Cultivar selection is an important method to limit Cd uptake and accumulation, however, analyzing grain Cd concentration is costly and time-consuming. Developing markers for low Cd accumulation will facilitate marker assisted selection (MAS). Inheritance studies using a threshold value of 0.2 mg kg^?1 for low and high and an F_2:3 population showed that low Cd accumulation in soybean seed is under the control of a major gene (Cda1, proposed name) with the allele for low accumulation being dominant. A recombinant inbred line (RIL) population (F_6:8) derived from the cross AC Hime (high Cd accumulation) and Westag-97 (low Cd accumulation) was used to identify the DNA markers linked to Cda gene(s) or quantitative trait loci (QTLs) controlling low Cd accumulation. We screened 171 simple sequence repeat (SSR) primers that showed polymorphism between parents on the 166 RILs. Of these, 40 primers were newly developed from the soybean genomic DNA sequence. Seven SSR markers, SatK138, SatK139, SatK140 (0.5 cM), SatK147, SacK149, SaatK150 and SattK152 (0.3 cM), were linked to Cda1 in soybean seed. All the linked markers were mapped to the same linkage group (LG) K. The closest flanking SSR markers linked to Cda1 were validated using a parallel population (RILs) involving Leo × Westag-97. Linked markers were also validated with diverse soybean genotypes differing in their seed Cd concentration and showed that SSR markers SatK147, SacK149, and SattK152 clearly differentiated the high and low Cd accumulating genotypes tested. To treat Cd uptake as a quantitative trait, QTL analysis using a linkage map constructed with 161 markers identified a major QTL associated with low Cd concentration in the seeds. The QTL was also mapped to the same location as Cda1 on LG-K. This QTL accounted for 57.3% of the phenotypic variation. Potential candidate genes (genes with known or predicted function that could influence the seed Cd concentration) like protein kinase, putative Adagio-like protein, and plasma membrane H⁺-ATPase were found to be located in the locus of interest. Of the four SSR markers located in the region, SattK152 was localized in the plasma membrane H⁺-ATPase gene. SSR markers closely linked to Cda1 in seeds of soybean were identified and have potential to be used for MAS to develop low Cd accumulating cultivars in a breeding program.     

A continental-scale study of seed lifespan in experimental storage examining seed,plant, and environmental traits associated with longevity

Management of seed banks conserving the biodiversity of phylogenetically diverse species requires insight into seed longevity. This study determined the seed longevity of 172 species sourced from across the mega-diverse flora of the Australia continent. Seeds were aged via a controlled ageing experiment through storage at 45 °C and 60 % RH, or 60 °C and 60 % RH, and regularly tested for germination. Relative seed longevity between species was determined by comparing the time to 50 % viability loss (p ₅₀), calculated via probit analysis of seed survival curves. Seed, plant, and environmental traits were examined for associations with longevity. The p ₅₀ values varied between species from 3.0 to 588.6 days. Serotinous species, and woody trees and shrubs, had significantly longer-lived seeds than geosporous species, and species of herbaceous habit. Seeds that possess physical dormancy, and seeds with large embryos with little endosperm, were also long-lived. There was a weak, but significant, positive correlation between seed mass and longevity. Seeds sourced from regions of higher mean annual temperature and rainfall were significantly longer-lived than seeds from cooler and drier regions, although both environmental factors were weakly associated with longevity. Compared with species from other regions of the world, prolonged longevity is a feature of many Australian species. Nevertheless, seed life-spans vary substantially between species and close consideration of seed traits along with biotic and abiotic components of the plants and their environment can assist to differentiate between potentially long- and short-lived seeds.     

Genetic diversity and association mapping of seed vigor in rice (Oryza sativa L.)

Key message

Twenty-seven QTLs were identified for rice seed vigor, in which 16 were novel QTLs. Fifteen elite parental combinations were designed for improving seed vigor in rice.

Abstract

Seed vigor is closely related to direct seeding in rice (Oryza sativa L.). Previous quantitative trait locus (QTL) studies for seed vigor were mainly derived from bi-parental segregating populations and no report from natural populations. In this study, association mapping for seed vigor was performed on a selected sample of 540 rice cultivars (419 from China and 121 from Vietnam). Population structure was estimated on the basis of 262 simple sequence repeat (SSR) markers. Seed vigor was evaluated by root length (RL), shoot length (SL) and shoot dry weight in 2011 and 2012. Abundant phenotypic and genetic diversities were found in the studied population. The population was divided into seven subpopulations, and the levels of linkage disequilibrium (LD) ranged from 10 to 80 cM. We identified 27 marker–trait associations involving 18 SSR markers for three traits. According to phenotypic effects for alleles of the detected QTLs, elite alleles were mined. These elite alleles could be used to design parental combinations and the expected results would be obtained by pyramiding or substituting the elite alleles per QTL (apart from possible epistatic effects). Our results demonstrate that association mapping can complement and enhance previous QTL information for marker-assisted selection and breeding by design.     

Wet-dry cycling extends seed persistence by re-instating antioxidant capacity

Seeds in the field experience wet-dry cycling that is akin to the well-studied commercial process of seed priming in which seeds are hydrated and then re-dried to standardise their germination characteristics. To investigate whether the persistence (defined as in situ longevity) and antioxidant capacity of seeds are influenced by wet-dry cycling, seeds of the global agronomic weed Avena sterilis ssp. ludoviciana were subjected to (1) controlled ageing at 60% relative humidity and 53.5°C for 31 days, (2) controlled ageing then priming, or (3) ageing in the field in three soils for 21 months. Changes in seed viability (total germination), mean germination time, seedling vigour (mean seedling length), and the concentrations of the glutathione (GSH) / glutathione disulphide (GSSG) redox couple were recorded over time. As controlled-aged seeds lost viability, GSH levels declined and the relative proportion of GSSG contributing to total glutathione increased, indicative of a failing antioxidant capacity. Subjecting seeds that were aged under controlled conditions to a wet-dry cycle (to ?1 MPa) prevented viability loss and increased GSH levels. Field-aged seeds that underwent numerous wet-dry cycles due to natural rainfall maintained high viability and high GSH levels. Thus wet-dry cycles in the field may enhance seed longevity and persistence coincident with re-synthesis of protective compounds such as GSH.     

Marker–trait association analysis of kernel hardness and related agronomic traits in a core collection of wheat lines

Identification of marker–trait associations is the first step towards marker-assisted selection in plant breeding. Here we assess genetic diversity and population structure of 94 diverse wheat elite lines and use genome-wide association mapping to identify marker–trait associations for five important traits: kernel hardness (KHA), thousand-kernel weight, grain protein content, test weight (TWT), and plant height (PHT). The 94 accessions employed in this study were grouped into three subpopulations based on the first three principal components, which accounted for 51.5 % of the variations. A mixed linear model was used to detect marker–trait associations incorporating covariance of population structure and relative kinship. A total of six marker loci was significantly associated with KHA, TWT, and PHT after the correction of false discovery rate (α _c  = 0.05). The gene pinB was found to be highly associated with KHA, and is reported to be a major determinant of KHA together with the gene pinA at the Ha locus on chromosome 5D. Marker XwPt-7187 on chromosome 2A was also significantly associated with KHA, two Diversity Arrays Technology markers XwPt-1250 and XwPt-4628 with TWT, and marker Xgwm512 with PHT, making the first report of marker–trait associations in these genomic regions.     

Linkage disequilibrium and population-structure analysis among Capsicum annuum L. cultivars for use in association mapping

Knowledge of population structure and linkage disequilibrium among the worldwide collections of peppers currently classified as hot, mild, sweet and ornamental types is indispensable for applying association mapping and genomic selection to improve pepper. The current study aimed to resolve the genetic diversity and relatedness of Capsicum annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes in samples collected in 2011 and 2012. The physical distance covered by the entire set of SSRs used was 2,265.9 Mb from the 3.48-Gb hot-pepper genome size. The model-based program STRUCTURE was used to infer five clusters, which was further confirmed by classical molecular-genetic diversity analysis. Mean heterozygosity of various loci was estimated to be 0.15. Linkage disequilibrium (LD) was used to identify 17 LD blocks across various chromosomes with sizes from 0.154 Kb to 126.28 Mb. CAMS-142 of chromosome 1 was significantly associated with both capsaicin (CA) and dihydrocapsaicin (DCA) levels. Further, CAMS-142 was located in an LD block of 98.18 Mb. CAMS-142 amplified bands of 244, 268, 283 and 326 bp. Alleles 268 and 283 bp had positive effects on both CA and DCA levels, with an average R ² of 12.15 % (CA) and 12.3 % (DCA). Eight markers from seven different chromosomes were significantly associated with fruit weight, contributing an average effect of 15 %. CAMS-199, HpmsE082 and CAMS-190 are the three major quantitative trait loci located on chromosomes 8, 9, and 10, respectively, and were associated with fruit weight in samples from both years of the study. This research demonstrates the effectiveness of using genome-wide SSR-based markers to assess features of LD and genetic diversity within C. annuum.     

QTL mapping and confirmation for tolerance of anaerobic conditions during germination derived from the rice landrace Ma-Zhan Red

Wide adoption of direct-seeded rice practices has been hindered by poorly leveled fields, heavy rainfall and poor drainage, which cause accumulation of water in the fields shortly after sowing, leading to poor crop establishment. This is due to the inability of most rice varieties to germinate and reach the water surface under complete submergence. Hence, tolerance of anaerobic conditions during germination is an essential trait for direct-seeded rice cultivation in both rainfed and irrigated ecosystems. A QTL study was conducted to unravel the genetic basis of tolerance of anaerobic conditions during germination using a population derived from a cross between IR42, a susceptible variety, and Ma-Zhan Red, a tolerant landrace from China. Phenotypic data was collected based on the survival rates of the seedlings at 21 days after sowing of dry seeds under 10 cm of water. QTL analysis of the mapping population consisting of 175 F_2:3 families genotyped with 118 SSR markers identified six significant QTLs on chromosomes 2, 5, 6, and 7, and in all cases the tolerant alleles were contributed by Ma-Zhan Red. The largest QTL on chromosome 7, having a LOD score of 14.5 and an R ² of 31.7 %, was confirmed using a BC₂F₃ population. The QTLs detected in this study provide promising targets for further genetic characterization and for use in marker-assisted selection to rapidly develop varieties with improved tolerance to anaerobic condition during germination. Ultimately, this trait can be combined with other abiotic stress tolerance QTLs to provide resilient varieties for direct-seeded systems.     

Identification of simple sequence repeat markers linked to lipoxygenase-1 gene in soybean

Off-flavour generated in soy products is ascribed to soybean seed lipoxygenase-1, lipoxygenase-2 and lipoxygenase-3, controlled by single dominant genes Lox1, Lox2 and Lox3, respectively. Lox2 locus has already been mapped and reported to be tightly linked with Lox1 locus. The objective of the present study was to map Lox1 locus by investigating the SSR markers reported to be linked with Lox2 locus and the neighbouring SSR markers in two mapping populations of 116 and 91 plants developed from LSb1 × PI408251 and JS335 × PI408251, respectively. Parental polymorphism was surveyed using SSR markers Sat_074, Satt522 reported to be linked with Lox2 locus and the SSR markers in its proximity. F_2:3 seeds were used for assaying lipoxygenase-1 to identify the genotype of the F₂ individuals. SSR marker Satt656 was found to be tightly linked with Lox1 locus at distance of 3.6 and 4.8 cM in the mapping population of LSb1 × PI408251 and JS335 × PI408251, respectively. SSR marker Satt656 can be useful for marker assisted selection for transferring recessive allele of lipoxygenase-1 in the background of high yielding soybean genotypes.     

Patterns of genetic diversity and linkage disequilibrium in a highly structured Hordeum vulgare association-mapping population for the Mediterranean basin

Population structure and genome-wide linkage disequilibrium (LD) were investigated in 192 Hordeum vulgare accessions providing a comprehensive coverage of past and present barley breeding in the Mediterranean basin, using 50 nuclear microsatellite and 1,130 DArT^® markers. Both clustering and principal coordinate analyses clearly sub-divided the sample into five distinct groups centred on key ancestors and regions of origin of the germplasm. For given genetic distances, large variation in LD values was observed, ranging from closely linked markers completely at equilibrium to marker pairs at 50 cM separation still showing significant LD. Mean LD values across the whole population sample decayed below r ² of 0.15 after 3.2 cM. By assaying 1,130 genome-wide DArT^® markers, we demonstrated that, after accounting for population substructure, current genome coverage of 1 marker per 1.5 cM except for chromosome 4H with 1 marker per 3.62 cM is sufficient for whole genome association scans. We show, by identifying associations with powdery mildew that map in genomic regions known to have resistance loci, that associations can be detected in strongly stratified samples provided population structure is effectively controlled in the analysis. The population we describe is, therefore, shown to be a valuable resource, which can be used in basic and applied research in barley.     

Genome-wide association studies of agronomic and quality traits in a set of German winter barley (Hordeum vulgare L.) cultivars using Diversity Arrays Technology (DArT)

A set of about 100 winter barley (Hordeum vulgare L.) cultivars, comprising diverse and economically important German barley elite germplasm released during the last six decades, was previously genotypically characterized by single nucleotide polymorphism (SNP) markers using the Illumina GoldenGate BeadArray Technology to detect associations with phenotypic data estimated in three-year field trials at 12 locations. In order to identify further associations and to obtain information on whether the marker type influences the outcome of association genetics studies, the set of winter barley cultivars was re-analyzed using Diversity Arrays Technology (DArT) markers. As with the analysis of the SNPs, only polymorphic markers present at an allele frequency >5 % were included to detect associations in a mixed linear model (MLM) approach using the TASSEL software (P?≤?0.001). The population structure and kinship matrix were estimated on 72 simple sequence repeats (SSRs) covering the whole barley genome. The respective average linkage disequilibrium (LD) analyzed with DArT markers was estimated at 5.73 cM. A total of 52 markers gave significant associations with at least one of the traits estimated which, therefore, may be suitable for marker-assisted breeding. In addition, by comparing the results to those generated using the Illumina GoldenGate BeadArray Technology, it turned out that a different number of associations for respective traits is detected, depending on the marker system. However, as only a few of the respective DArT and Illumina markers are present in a common map, no comprehensive comparison of the detected associations was feasible, but some were probably detected in the same chromosomal regions. Because of the identification of additional marker–trait associations, it may be recommended to use both marker techniques in genome-wide association studies.     

A first insight into population structure and linkage disequilibrium in the US peanut minicore collection

Knowledge of genetic diversity, population structure, and degree of linkage disequilibrium (LD) in target association mapping populations is of great importance and is a prerequisite for LD-based mapping. In the present study, 96 genotypes comprising 92 accessions of the US peanut minicore collection, a component line of the tetraploid variety Florunner, diploid progenitors A. duranensis (AA) and A. ipaënsis (BB), and synthetic amphidiploid accession TxAG-6 were investigated with 392 simple sequence repeat (SSR) marker bands amplified using 32 highly-polymorphic SSR primer pairs. Both distance- and model-based (Bayesian) cluster analysis revealed the presence of structured diversity. In general, the wild-species accessions and the synthetic amphidiploid grouped separately from most minicore accessions except for COC155, and were eliminated from most subsequent analyses. UPGMA analysis divided the population into four subgroups, two major subgroups representing subspecies fastigiata and hypogaea, a third group containing individuals from each subspecies or possibly of mixed ancestry, and a fourth group, either consisting of COC155 alone if wild species were excluded, or of COC155, the diploid species, and the synthetic amphidiploid. Model-based clustering identified four subgroups- one each for fastigiata and hypogaea subspecies, a third consisting of individuals of both subspecies or of mixed ancestry predominantly from Africa or Asia, and a fourth group, consisting of individuals predominantly of var fastigiata, peruviana, and aequatoriana accessions from South America, including COC155. Analysis of molecular variance (AMOVA) revealed statistically-significant (P < 0.0001) genetic variance of 16.87% among subgroups. A total of 4.85% of SSR marker pairs revealed significant LD (at r² ≥ 0.1). Of the syntenic marker pairs separated by distances < 10 cM, 11–20 cM, 21–50 cM, and > 50 cM, 19.33, 5.19, 6.25 and 5.29% of marker pairs were found in strong LD (P ≤ 0.01), in accord with LD extending to great distances in self pollinated crops. A threshold value of r² > 0.035 was found to distinguish mean r² values of linkage distance groups statistically from the mean r² values of unlinked markers; LD was found to extend to 10 cM over the entire minicore collection by this criterion. However, there were large differences in r² values among marker pairs even among tightly-linked markers. The implications of these findings with regard to the possibility of using association mapping for detection of genome-wide SSR marker-phenotype association are discussed.     

Association studies of dormancy and cooking quality traits in direct-seeded indica rice

Association analysis was applied to a panel of accessions of Assam rice (indica) using 98 SSR markers for dormancy-related traits and cooking quality. Analysis of population structure revealed 10 subgroups in the population. The mean r ² and D ^′ value for all intrachromosomal loci pairs was 0.24 and 0.51, respectively. Linkage disequilibrium between linked markers decreased with distance. Marker-trait associations were investigated using the unified mixed-model approach, considering both population structure (Q) and kinship (K). Genome-wide scanning, detected a total of seven significant marker-trait associations (P < 0.01), with the R ² values ranging from 12.0 to 18.0%. The significant marker associations were for grain dormancy (RM27 on chromosome 2), α-amylase activity (RM27 and RM234 on chromosomes 2 and 7, respectively), germination (RM27 and RM106 on chromosome 2), amylose (RM282 on chromosome 3) and grain length elongation ratio (RM142 on chromosome 4). The present study revealed the association of marker RM27 with traits like dormancy, α-amylase activity and germination. Simple correlation analysis of these traits revealed that these traits were positively correlated with each other and this marker may be useful for simultaneous improvement of these traits. The study indicates the presence of novel QTLs for a few traits under consideration. The study reveals association of traits like dormancy, α-amylase activity, germination, amylose content, grain length elongation ratio with SSR markers indicating the feasibility of undertaking association analysis in conjunction with germplasm characterization.     

Validation of SSR markers linked to null kunitz tryspin inhibitor allele in Indian soybean [Glycine max (L.) Merr.] population

Kunitz trypsin inhibitor, a proteinaceous antinutritional factor present in soybean seeds, is responsible for inferior nutritional quality of raw soybean and incompletely processed soy products. The objective of the present investigation was to validate the SSR markers (Satt228 and Satt409) reported to be linked to Ti locus in an Indian soybean population generated from the cross between soybean cultivar LSb1 (TiTi) and PI542044 (titi). Parental polymorphism was surveyed using Satt409, Satt228 and 5 SSR markers in the neighbouring genomic region of Ti locus. A portion of the cotyledon of F₂ seeds was used for analyzing the presence or absence of kunitz trypsin inhibitor polypeptide electrophoretically while the remaining portion containing the embryo was used for raising the F₂ plants (104) for the development of mapping population. The SSR marker Satt228 reported to be tightly linked with Ti locus was not found to be polymorphic for the parents used in our study. Satt409 was found to be linked with Ti locus at 4.7 cM. Besides, a new marker Satt538 was found to be linked with Ti locus at a distance of 17.8 cM. Thus, the SSR marker Satt409 can be useful for Marker Assisted Selection for transferring titi allele in the background of Indian soybean genotypes.     

Identification of germplasm with stacked QTL underlying seed traits in an inbred soybean population from cultivars Essex and Forrest

Soybean (Glycine max (L.) Merr.) seed provides valuable oil (~200 g/kg) and protein (~400 g/kg) co-products. Seed composition variations result from several quantitative trait loci (QTL) that act through development. The objectives here were to identify loci underlying seed traits in the Essex × Forrest (EF94)-derived recombinant inbred line (RIL) population which has low frequencies of marker polymorphisms. Seed weight, protein, and oil were measured over 3 years: 2001, 2003, and 2005. Essex’s seeds were larger (141 mg/seed), higher in protein (406 g/kg), and lower in oil (190 g/kg) than Forrest’s (115 mg/seed, 395 g protein/kg, and 203 g oil/kg). Marker analysis included 413 markers for trait associations used for ANOVA, interval mapping, and composite interval mapping. Eleven QTL in nine genomic regions were associated (LOD >2; P < 0.0077) with seed traits. Two QTL, for mean protein and seed size, were clustered on linkage group (LG) E (chromosome Gm16). QTL for protein content alone were found on LG C2 (Gm6), LG D1b (Gm2), LG H (Gm12), and LG I (Gm20). The alleles from Essex, the high-protein parent, underlay higher protein (4–10 g/kg) at four of five loci. A QTL for mean oil was found on LG A2 (Gm18) and on LG I (Gm 20). The alleles from Forrest underlay higher oil (3–4 g/kg). Five separate QTL for mean seed weight were found on LG A1 (Gm05), LG N (Gm15), two on LG B1 (Gm11) and one on LG N (Gm3). The alleles from Essex underlay greater seed weight (0.4–0.66 g/100 seeds). The QTL positions were consistent with reported loci. Germplasm that contained all five beneficial alleles at the QTL underlying protein was significantly higher in protein and yield than Essex (409.7–412.3 g/kg) and included RILs 49 and 62. The germplasm identified can be useful for further breeding of the many traits and QTL measured in each line.     

Verification of marker–trait associations in biparental winter barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) DH populations

In previous genome-wide association studies, marker–trait associations for grain yield and additional traits of agronomic importance were identified in the German winter barley (Hordeum vulgare L.) breeding gene pool. In the present study, seven doubled haploid populations segregating for the relevant alleles at the associated loci were used to get information whether these marker–trait associations can be verified in biparental populations and reliably used in applied barley breeding. The doubled haploid populations were phenotyped in field trials at two to five locations each in 1 year and genotyped by 40 trait-associated single nucleotide polymorphisms using an Illumina VeraCode GoldenGate assay. Large phenotypic variation was observed for all traits within at least one doubled haploid population. For 19 out of 58 marker–trait associations tested, the phenotypic means of both marker classes were significantly (p ≤ 0.005) different, thus confirming the association of the respective marker and the quantitative trait locus detected. For example, doubled haploid lines derived from a cross of ‘Malta’ × ‘Goldmine’ carrying different marker alleles differed by 0.41 t/ha in mean grain yield. The 19 (out of 58) marker–trait associations verified correspond to 10 (out of 27) genomic regions. Markers that were verified to be associated with a quantitative trait locus can be implemented directly in winter barley breeding for the selection of parental lines and marker-assisted pedigree selection.     

A Genome Scan for Quantitative Trait Loci Associated with Vibrio anguillarum Infection Resistance in Japanese Flounder (Paralichthys olivaceus) by Bulked Segregant Analysis

A recent genetic linkage map was employed to detect quantitative trait loci (QTLs) associated with Vibrio anguillarum resistance in Japanese flounder. An F1 family established and challenged with V. anguillarum in 2009 was used for QTL mapping. Of the 221 simple sequence repeat (SSR) markers used to detect polymorphisms in the parents of F1, 170 were confirmed to be polymorphic. The average distance between the markers was 10.6 cM. Equal amounts of genomic DNA from 15 fry that died early and from 15 survivors were pooled separately to constitute susceptible bulk and resistance bulk DNA. Bulked segregant analysis and QTL mapping were combined to detect candidate SSR markers and regions associated with the disease. A genome scan identified four polymorphic SSR markers, two of which were significantly different between susceptible and resistance bulk (P?=?0.008). These two markers were located in linkage group (LG) 7; therefore, all the SSR markers in LG7 were genotyped in all the challenged fry by single marker analysis. Using two different models, 11–17 SSR markers were detected with different levels of significance. To confirm the associations of these markers with the disease, composite interval mapping was employed to genotype all the challenged individuals. One and three QTLs, which explained more than 60 % of the phenotypic variance, were detected by the two models. Two of the QTLs were located at 48.6 cM. The common QTL may therefore be a major candidate region for disease resistance against V. anguillarum infection.