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Key Message: We developed and validated 56 gene-specific semi-thermal asymmetric reverse PCR (STARP) markers for 46 genes of important wheat quality, biotic and abiotic stress resistance, grain yield, and adaptation-related traits for marker-assisted selection in wheat breeding. Development of high-throughput, low-cost, gene-specific molecular markers is important for marker-assisted selection in wheat breeding. In this study, we developed 56 gene-specific semi-thermal asymmetric reverse PCR (STARP) markers for wheat quality, tolerance to biotic and abiotic stresses, grain yield, and adaptation-related traits. The STARP assays were validated by (1) comparison of the assays with corresponding diagnostic STS/CAPS markers on 40 diverse wheat cultivars and (2) characterization of allelic effects based on the phenotypic and genotypic data of three segregating populations and 305 diverse wheat accessions from China and 13 other countries. The STARP assays showed the advantages of high-throughput, accuracy, flexibility, simple assay design, low operational costs, and platform compatibility. The state-of-the-art assays of this study provide a robust and reliable molecular marker toolkit for wheat breeding programs.

Key Message: Genetic dissection uncovered a major QTL QTKW.caas-4BS corresponding with a 483 kb deletion that included genes ZnF, EamA and Rht-B1. This deletion was associated with increased grain weight and semi-dwarf phenotype. Previous studies identified quantitative trait loci (QTL) for thousand kernel weight (TKW) in the region spanning the Rht-B1 locus in wheat (Triticum aestivum L.). We recently mapped a major QTL QTKW.caas-4BS for TKW spanning the Rht-B1 locus in a recombinant inbred line (RIL) population derived from Doumai/Shi 4185 using the wheat 90K array. The allele from Doumai at QTKW.caas-4BS significantly increased TKW and kernel number per spike, and conferred semi-dwarf trait, which was beneficial to improve grain yield without a penalty to lodging. To further dissect QTKW.caas-4BS, we firstly re-investigated the genotypes and phenotypes of the RILs and confirmed the QTL using cleaved amplified polymorphic sequence (CAPS) markers developed from flanking SNP markers IWA102 and IWB54814. The target sequences of the CAPS markers were used as queries to BLAST the wheat reference genome RefSeq v1.0 and hit an approximate 10.4 Mb genomic region. Based on genomic mining and SNP loci from the wheat 660K SNP array in the above genomic region, we developed eight new markers and narrowed QTKW.caas-4BS to a genetic interval of 1.5 cM. A 483 kb deletion in Doumai corresponded with QTKW.caas-4BS genetically, including three genes ZnF, EamA and Rht-B1. The other 15 genes with either differential expressions and/or sequence variations between parents were also potential candidate genes for QTKW.caas-4BS. The findings not only provide a toolkit for marker-assisted selection of QTKW.caas-4BS but also defined candidate genes for further functional analysis.

Key Message: Genetic diversity, population structure, LD decay, and selective sweeps in 687 wheat accessions were analyzed, providing relevant guidelines to facilitate the use of the germplasm in wheat breeding. Common wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Landraces were subjected to strong human-mediated selection in developing high-yielding, good quality, and widely adapted cultivars. To investigate the genome-wide patterns of allelic variation, population structure and patterns of selective sweeps during modern wheat breeding, we tested 687 wheat accessions, including landraces (148) and cultivars (539) mainly from China and Pakistan in a wheat 90 K single nucleotide polymorphism array. Population structure analysis revealed that cultivars and landraces from China and Pakistan comprised three relatively independent genetic clusters. Cultivars displayed lower nucleotide diversity and a wider average LD decay across whole genome, indicating allelic erosion and a diversity bottleneck due to the modern breeding. Analysis of genetic differentiation between landraces and cultivars from China and Pakistan identified allelic variants subjected to selection during modern breeding. In total, 477 unique genome regions showed signatures of selection, where 109 were identified in both China and Pakistan germplasm. The majority of genomic regions were located in the B genome (225), followed by the A genome (175), and only 77 regions were located in the D genome. EigenGWAS was further used to identify key selection loci in modern wheat cultivars from China and Pakistan by comparing with global winter wheat and spring wheat diversity panels, respectively. A few known functional genes or loci found within these genome regions corresponded to known phenotypes for disease resistance, vernalization, quality, adaptability and yield-related traits. This study uncovered molecular footprints of modern wheat breeding and explained the genetic basis of polygenic adaptation in wheat. The results will be useful for understanding targets of modern wheat breeding, and in devising future breeding strategies to target beneficial alleles currently not pursued.

Background: Identification of loci for grain yield (GY) and related traits, and dissection of the genetic architecture are important for yield improvement through marker-assisted selection (MAS). Two genome-wide association study (GWAS) methods were used on a diverse panel of 166 elite wheat varieties from the Yellow and Huai River Valleys Wheat Zone (YHRVWD) of China to detect stable loci and analyze relationships among GY and related traits., Results: A total of 326,570 single nucleotide polymorphism (SNP) markers from the wheat 90 K and 660 K SNP arrays were chosen for GWAS of GY and related traits, generating a physical distance of 14,064.8 Mb. One hundred and twenty common loci were detected using SNP-GWAS and Haplotype-GWAS, among which two were potentially functional genes underpinning kernel weight and plant height (PH), eight were at similar locations to the quantitative trait loci (QTL) identified in recombinant inbred line (RIL) populations in a previous study, and 78 were potentially new. Twelve pleiotropic loci were detected on eight chromosomes; among these the interval 714.4-725.8 Mb on chromosome 3A was significantly associated with GY, kernel number per spike (KNS), kernel width (KW), spike dry weight (SDW), PH, uppermost internode length (UIL), and flag leaf length (FLL). GY shared five loci with thousand kernel weight (TKW) and PH, indicating significantly affected by two traits. Compared with the total number of loci for each trait in the diverse panel, the average number of alleles for increasing phenotypic values of GY, TKW, kernel length (KL), KW, and flag leaf width (FLW) were higher, whereas the numbers for PH, UIL and FLL were lower. There were significant additive effects for each trait when favorable alleles were combined. UIL and FLL can be directly used for selecting high-yielding varieties, whereas FLW can be used to select spike number per unit area (SN) and KNS., Conclusions: The loci and significant SNP markers identified in the present study can be used for pyramiding favorable alleles in developing high-yielding varieties. Our study proved that both GWAS methods and high-density genetic markers are reliable means of identifying loci for GY and related traits, and provided new insight to the genetic architecture of GY.

Key Message: We identified 21 new and stable QTL, and 11 QTL clusters for yield-related traits in three bread wheat populations using the wheat 90 K SNP assay. Identification of quantitative trait loci (QTL) for yield-related traits and closely linked molecular markers is important in order to identify gene/QTL for marker-assisted selection (MAS) in wheat breeding. The objectives of the present study were to identify QTL for yield-related traits and dissect the relationships among different traits in three wheat recombinant inbred line (RIL) populations derived from crosses Doumai × Shi 4185 (D × S), Gaocheng 8901 × Zhoumai 16 (G × Z) and Linmai 2 × Zhong 892 (L × Z). Using the available high-density linkage maps previously constructed with the wheat 90 K iSelect single nucleotide polymorphism (SNP) array, 65, 46 and 53 QTL for 12 traits were identified in the three RIL populations, respectively. Among them, 34, 23 and 27 were likely to be new QTL. Eighteen common QTL were detected across two or three populations. Eleven QTL clusters harboring multiple QTL were detected in different populations, and the interval 15.5-32.3 cM around the Rht-B1 locus on chromosome 4BS harboring 20 QTL is an important region determining grain yield (GY). Thousand-kernel weight (TKW) is significantly affected by kernel width and plant height (PH), whereas flag leaf width can be used to select lines with large kernel number per spike. Eleven candidate genes were identified, including eight cloned genes for kernel, heading date (HD) and PH-related traits as well as predicted genes for TKW, spike length and HD. The closest SNP markers of stable QTL or QTL clusters can be used for MAS in wheat breeding using kompetitive allele-specific PCR or semi-thermal asymmetric reverse PCR assays for improvement of GY.

Background: Black point is a serious threat to wheat production and can be managed by host resistance. Marker-assisted selection (MAS) has the potential to accelerate genetic improvement of black point resistance in wheat breeding. We performed a genome-wide association study (GWAS) using the high-density wheat 90 K and 660 K single nucleotide polymorphism (SNP) assays to better understand the genetic basis of black point resistance and identify associated molecular markers., Results: Black point reactions were evaluated in 166 elite wheat cultivars in five environments. Twenty-five unique loci were identified on chromosomes 2A, 2B, 3A, 3B (2), 3D, 4B (2), 5A (3), 5B (3), 6A, 6B, 6D, 7A (5), 7B and 7D (2), respectively, explaining phenotypic variation ranging from 7.9 to 18.0%. The highest number of loci was detected in the A genome (11), followed by the B (10) and D (4) genomes. Among these, 13 were identified in two or more environments. Seven loci coincided with known genes or quantitative trait locus (QTL), whereas the other 18 were potentially novel loci. Linear regression showed a clear dependence of black point scores on the number of favorable alleles, suggesting that QTL pyramiding will be an effective approach to increase resistance. In silico analysis of sequences of resistance-associated SNPs identified 6 genes possibly involved in oxidase, signal transduction and stress resistance as candidate genes involved in black point reaction., Conclusion: SNP markers significantly associated with black point resistance and accessions with a larger number of resistance alleles can be used to further enhance black point resistance in breeding. This study provides new insights into the genetic architecture of black point reaction.

Water soluble carbohydrates (WSC) in stems play an important role in buffering grain yield in wheat against biotic and abiotic stresses; however, knowledge of genes controlling WSC is very limited. We conducted a genome-wide association study (GWAS) using a high-density 90K SNP array to better understand the genetic basis underlying WSC, and to explore marker-based breeding approaches. WSC was evaluated in an association panel comprising 166 Chinese bread wheat cultivars planted in four environments. Fifty two marker-trait associations (MTAs) distributed across 23 loci were identified for phenotypic best linear unbiased estimates (BLUEs), and 11 MTAs were identified in two or more environments. Liner regression showed a clear dependence of WSC BLUE scores on numbers of favorable (increasing WSC content) and unfavorable alleles (decreasing WSC), indicating that genotypes with higher numbers of favorable or lower numbers of unfavorable alleles had higher WSC content. In silico analysis of flanking sequences of trait-associated SNPs revealed eight candidate genes related to WSC content grouped into two categories based on the type of encoding proteins, namely, defense response proteins and proteins triggered by environmental stresses. The identified SNPs and candidate genes related to WSC provide opportunities for breeding higher WSC wheat cultivars., Competing Interests: The authors have declared that no competing interests exist.

Key Message: Nine QTL for black point resistance in wheat were identified using a RIL population derived from a Linmai 2/Zhong 892 cross and 90K SNP assay. Black point, discoloration of the embryo end of the grain, downgrades wheat grain quality leading to significant economic losses to the wheat industry. The availability of molecular markers will accelerate improvement of black point resistance in wheat breeding. The aims of this study were to identify quantitative trait loci (QTL) for black point resistance and tightly linked molecular markers, and to search for candidate genes using a high-density genetic linkage map of wheat. A recombinant inbred line (RIL) population derived from the cross Linmai 2/Zhong 892 was evaluated for black point reaction during the 2011-2012, 2012-2013 and 2013-2014 cropping seasons, providing data for seven environments. A high-density linkage map was constructed by genotyping the RILs with the wheat 90K single nucleotide polymorphism (SNP) chip. Composite interval mapping detected nine QTL on chromosomes 2AL, 2BL, 3AL, 3BL, 5AS, 6A, 7AL (2) and 7BS, designated as QBp.caas-2AL, QBp.caas-2BL, QBp.caas-3AL, QBp.caas-3BL, QBp.caas-5AS, QBp.caas-6A, QBp.caas-7AL.1, QBp.caas-7AL.2 and QBp.caas-7BS, respectively. All resistance alleles, except for QBp.caas-7AL.1 from Linmai 2, were contributed by Zhong 892. QBp.caas-3BL, QBp.caas-5AS, QBp.caas-7AL.1, QBp.caas-7AL.2 and QBp.caas-7BS probably represent new loci for black point resistance. Sequences of tightly linked SNPs were used to survey wheat and related cereal genomes identifying three candidate genes for black point resistance. The tightly linked SNP markers can be used in marker-assisted breeding in combination with the kompetitive allele specific PCR technique to improve black point resistance.

Key Message: We developed and validated a robust marker toolkit for high-throughput and cost-effective screening of a large number of functional genes in wheat. Functional markers (FMs) are the most valuable markers for crop breeding programs, and high-throughput genotyping for FMs could provide an excellent opportunity to effectively practice marker-assisted selection while breeding cultivars. Here we developed and validated kompetitive allele-specific PCR (KASP) assays for genes that underpin economically important traits in bread wheat including adaptability, grain yield, quality, and biotic and abiotic stress resistances. In total, 70 KASP assays either developed in this study or obtained from public databases were validated for reliability in application. The validation of KASP assays were conducted by (a) comparing the assays with available gel-based PCR markers on 23 diverse wheat accessions, (b) validation of the derived allelic information using phenotypes of a panel comprised of 300 diverse cultivars from China and 13 other countries, and (c) additional testing, where possible, of the assays in four segregating populations. All KASP assays being reported were significantly associated with the relevant phenotypes in the cultivars panel and bi-parental populations, thus revealing potential application in wheat breeding programs. The results revealed 45 times superiority of the KASP assays in speed than gel-based PCR markers. KASP has recently emerged as single-plex high-throughput genotyping technology; this is the first report on high-throughput screening of a large number of functional genes in a major crop. Such assays could greatly accelerate the characterization of crossing parents and advanced lines for marker-assisted selection and can complement the inflexible, high-density SNP arrays. Our results offer a robust and reliable molecular marker toolkit that can contribute towards maximizing genetic gains in wheat breeding programs.

Key Message: We cloned TaSST genes, developed a gene-specific marker for TaSST-D1, and identified three QTL in the Doumai/Shi 4185 RIL population. TaSST-D1 is within one of the three QTL. Sucrose:sucrose-1-fructosyltransferase (1-SST), a critical enzyme in the fructan biosynthetic pathway, is significantly and positively associated with water soluble carbohydrate (WSC) content in bread wheat stems. In the present study, wheat 1-SST genes (TaSST) were isolated and located on chromosomes 4A, 7A and 7D. Sequence analysis of TaSST-D1 revealed 15 single nucleotide polymorphisms (SNP) in the third exon between cultivars with higher and lower WSC content. A cleaved amplified polymorphism sequence (CAPS) marker, WSC7D, based on the polymorphism at position 1216 (C-G) was developed to discriminate the two alleles. WSC7D was located on chromosome 7DS using a recombinant inbred line (RIL) population from a Doumai/Shi 4185 cross, and a set of Chinese Spring nullisomic-tetrasomic lines. TaSST-D1 co-segregated with the CAPS marker WSC7D and was linked to SNP marker BS00108793&#95;51 on chromosome 7DS at a genetic distance of 6.1 cM. It explained 8.8, 10.9, and 11.3% of the phenotypic variances in trials at Beijing and Shijiazhuang as well as the averaged data from those environments, respectively. Two additional QTL (QWSC.caas-4BS and QWSC.caas-7AS) besides TaSST-D1 were mapped in the RIL population. One hundred and forty-nine Chinese wheat cultivars and advanced lines tested in four environments were used to validate a highly significant (P < 0.01) association between WSC7D and WSC content in wheat stems. WSC7D can be used as a gene-specific marker for improvement of stem WSC content in wheat breeding programs.

Key Message: Fifty-six QTL for flour color-related traits and polyphenol oxidase activity were identified using a genome-wide linkage mapping of data from a RIL population derived from a Gaocheng 8901/Zhoumai 16 cross., Abstract: Flour color-related traits, including L*, a*, b*, yellow pigment content (YPC), and polyphenol oxidase (PPO) activity are important parameters influencing the quality of wheat end-use products. Mapping quantitative trait loci (QTL) for these traits and characterization of candidate genes are important for improving wheat quality. The aims of this study were to identify QTL for flour color-related traits and PPO activity and to characterize candidate genes using a high-density genetic linkage map in a common wheat recombinant inbred line (RIL) population derived from a cross between Gaocheng 8901 and Zhoumai 16. A linkage map was constructed by genotyping the RILs with the wheat 90 K iSelect array. Fifty-six QTL were mapped on 35 chromosome regions on homoeologous groups 1, 2, 5 and 7 chromosomes, and chromosomes 3B, 4A, 4B and 6B. Four QTL were for PPO activity, and the others were for flour color-related traits. Compared with previous studies, five QTL for a*, two for b*, one for L*, one for YPC and one for PPO activity were new. The new QTL on chromosome 2DL was involved in both a* and YPC, and another on chromosome 7DS affected both a* and L*. The scan for SNP sequences tightly linked to QTL for flour color-related traits against the wheat and/or related cereals genomes identified six candidate genes significantly related to these traits, and five of them were associated with the terpenoid backbone biosynthesis pathway. The high-density genetic linkage map of Gaocheng 8901/Zhoumai 16 represents a useful tool to identify QTL for important quality traits and candidate genes.

Stripe rust is one of the most devastating diseases of wheat (Triticum aestivum) worldwide. Adult-plant resistance (APR) is an efficient approach to provide long-term protection of wheat from the disease. The Chinese winter wheat cultivar Zhong 892 has a moderate level of APR to stripe rust in the field. To determine the inheritance of the APR resistance in this cultivar, 273 F6 recombinant inbred lines (RILs) were developed from a cross between Linmai 2 and Zhong 892. The RILs were evaluated for maximum disease severity (MDS) in two sites during the 2011-2012, 2012-2013 and 2013-2014 cropping seasons, providing data for five environments. Illumina 90k SNP (single nucleotide polymorphism) chips were used to genotype the RILs and their parents. Composite interval mapping (CIM) detected eight QTL, namely QYr.caas-2AL, QYr.caas-2BL.3, QYr.caas-3AS, QYr.caas-3BS, QYr.caas-5DL, QYr.caas-6AL, QYr.caas-7AL and QYr.caas-7DS.1, respectively. All except QYr.caas-2BL.3 resistance alleles were contributed by Zhong 892. QYr.caas-3AS and QYr.caas-3BS conferred stable resistance to stripe rust in all environments, explaining 6.2-17.4% and 5.0-11.5% of the phenotypic variances, respectively. The genome scan of SNP sequences tightly linked to QTL for APR against annotated proteins in wheat and related cereals genomes identified two candidate genes (autophagy-related gene and disease resistance gene RGA1), significantly associated with stripe rust resistance. These QTL and their closely linked SNP markers, in combination with kompetitive allele specific PCR (KASP) technology, are potentially useful for improving stripe rust resistances in wheat breeding.

Key Message: Three novel QTL for peroxidase activity were mapped, and gene-specific markers for TaPod-A1 were developed and validated using RILs derived from the Doumai/Shi 4185 cross and 281 wheat cultivars. TaPod-A1 is within one of the three QTL. Peroxidase (POD) activity in grain is an important factor determining the color of flour and end-use products of wheat, such as noodles and steamed bread. Mapping QTL for POD activity, characterization of POD genes and development of gene-specific markers are important for molecular marker-assisted selection in wheat breeding. Quantitative trait loci (QTL) for POD activity in common wheat were mapped using a recombinant inbred line (RIL) population derived from a Doumai/Shi 4185 cross grown in four environments and genotyped using the wheat 90 K iSelect assay. Three novel QTL for POD activity, QPod.caas-3AL, QPod.caas-4BS and QPod.caas-5AS, were identified on chromosomes 3AL, 4BS and 5AS, explaining 5.3-21.2% of phenotypic variance across environments. The full-length genomic DNA (gDNA) sequence of a POD gene, designated TaPod-A1, on chromosome 3A was characterized by homolog cloning and PCR verification. Two complementary dominant sequence-tagged site (STS) markers, POD-3A1 and POD-3A2, were developed based on single nucleotide polymorphisms (SNPs) between two alleles at the TaPod-A1 locus, amplifying 291- and 766-bp fragments in cultivars with lower and higher POD activities, respectively. The two gene-specific markers were mapped on chromosome 3AL using a set of Chinese Spring (CS) nulli-tetrasomic lines, and ditelosomic lines 3AL and 3AS. QTL analysis indicated that QPod.caas-3AL co-segregated with the gene-specific markers POD-3A1 and POD-3A2. POD-3A1 and POD-3A2 were verified on 281 wheat cultivars and advanced lines, and showed significant (P < 0.05) associations with POD activities. POD-3A1 and POD-3A2 may be useful as markers for improving color attributes in wheat breeding programs.