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.

Development and validation of KASP assays for genes underpinning key economic traits in bread wheat.

Drought stress at the reproductive stage causes pollen sterility and grain loss in wheat (Triticum aestivum). Drought stress induces abscisic acid (ABA) biosynthesis genes in anthers and ABA accumulation in spikes of drought-sensitive wheat varieties. In contrast, drought-tolerant wheat accumulates lower ABA levels, which correlates with lower ABA biosynthesis and higher ABA catabolic gene expression (ABA 8'-hydroxylase). Wheat TaABA8'OH1 deletion lines accumulate higher spike ABA levels and are more drought sensitive. ABA treatment of the spike mimics the effect of drought, causing high levels of sterility. ABA treatment represses the anther cell wall invertase gene TaIVR1, and drought-tolerant lines appeared to be more sensitive to the effect of ABA. Drought-induced sterility shows similarity to cold-induced sterility in rice (Oryza sativa). In cold-stressed rice, the rate of ABA accumulation was similar in cold-sensitive and cold-tolerant lines during the first 8 h of cold treatment, but in the tolerant line, ABA catabolism reduced ABA levels between 8 and 16 h of cold treatment. The ABA biosynthesis gene encoding 9-cis-epoxycarotenoid dioxygenase in anthers is mainly expressed in parenchyma cells surrounding the vascular bundle of the anther. Transgenic rice lines expressing the wheat TaABA8'OH1 gene under the control of the OsG6B tapetum-specific promoter resulted in reduced anther ABA levels under cold conditions. The transgenic lines showed that anther sink strength (OsINV4) was maintained under cold conditions and that this correlated with improved cold stress tolerance. Our data indicate that ABA and ABA 8'-hydroxylase play an important role in controlling anther ABA homeostasis and reproductive stage abiotic stress tolerance in cereals.

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Control of Abscisic Acid Catabolism and Abscisic Acid Homeostasis Is Important for Reproductive Stage Stress Tolerance in Cereals

Functional markers (FM) are developed from sequence polymorphisms present in allelic variants of a functional gene at a locus. FMs accurately discriminate alleles of a targeted gene, and are ideal molecular markers for marker-assisted selection in wheat breeding. In this paper, we summarize FMs developed and used in common wheat. To date, more than 30 wheat loci associated with processing quality, agronomic traits, and disease resistance, have been cloned, and 97 FMs were developed to identify 93 alleles based on the sequences of those genes. A general approach is described for isolation of wheat genes and development of FMs based on in silico cloning and comparative genomics. The divergence of DNA sequences of different alleles that affect gene function is summarized. In addition, 14 molecular markers specific for alien genes introduced from common wheat relatives were also described. This paper provides updated information on all FMs and gene-specific STS markers developed so far in wheat and should facilitate their application in wheat breeding programs.

Functional markers in wheat: current status and future prospects

Identification of genes for yield components, plant height and yield-related physiological traits and tightly linked molecular markers is of great importance in marker-assisted selection (MAS) in wheat breeding. In the present study, 246 F8 RILs derived from the cross of Zhou 8425B/Chinese Spring were genotyped using the high-density Illumina iSelect 90K single nucleotide polymorphism (SNP) assay. Field trials were conducted at Zhengzhou and Zhoukou of Henan Province, during the 2012–2013 and 2013–2014 cropping season sunder irrigated conditions, providing data for four environments. Analysis of variance (ANOVA) of agronomic and physiological traits revealed significant differences (P<0.01) among RILs, environments, and RIL × environment interactions. Broad-sense heritabilities of all traits including thousand kernel weight (TKW), plant height (PH), spike length (SL), kernel number per spike (KNS), spike number/m2 (SN), normalized difference in vegetation index at anthesis (NDVI-A) and at 10 days post-anthesis (NDVI-10), SPAD value of chlorophyll content at anthesis (Chl-A) and at 10 days post-anthesis (Chl-10) ranged between 0.65 and 0.94. A linkage map spanning 3,609.4 cM was constructed using 5,636 polymorphic SNP markers, with an average chromosome length of 171.9 cM and marker density of 0.64 cM/marker. A total of 866 SNP markers were newly mapped to the hexaploid wheat linkage map. Eighty-six QTL for yield components, plant height and yield-related physiological traits were detected on 18 chromosomes except 1D, 5D and 6D, explaining 2.3 to 33.2% of the phenotypic variance. Ten stable QTL were identified across four environments, viz. QTKW.caas-6A.1, QTKW.caas-7AL, QKNS.caas-4AL, QSN.caas-1AL.1, QPH.caas-4BS.2, QPH.caas-4DS.1, QSL.caas-4AS, QSL.caas-4AL.1, QChl-A.caas-5AL and QChl-10.caas-5BL. Meanwhile, 10 QTL-rich regions were found on chromosome 1BS, 2AL (2), 3AL, 4AL (2), 4BS, 4DS, 5BL and 7AL exhibiting pleiotropic effects. These QTL or QTL clusters are tightly linked to SNP markers, with genetic distances to the closest SNPs ranging from 0 to 1.5 cM, and could serve as target regions for fine mapping, candidate gene discovery, and marker-assisted selection in wheat breeding.

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Genome-Wide Linkage Mapping of QTL for Yield Components, Plant Height and Yield-Related Physiological Traits in the Chinese Wheat Cross Zhou 8425B/Chinese Spring.

Incorporation of genetic diversity into elite wheat (Triticum aestivum L., 2n1⁄4 6x1⁄4 42, AABBDD) cultivars has long been recognized as a means of improving wheat productivity and securing global wheat supply. Synthetic hexaploid wheat (SHW) genotypes recreated from its two progenitor species, the tetraploid, Triticum turgidum (2n1⁄4 4x1⁄4 28, AABB) and its diploid wild relative, Aegilops tauschii (2n1⁄4 2x1⁄4 14, DD) are a useful resource of new genes for hexaploidwheat improvement. These include many productivity traits such as abiotic (drought, heat, salinity/sodicity, andwaterlogging) and biotic (rusts, septoria, barley yellow dwarfvirus (BYDV), crownrot, tan spot, spot blotch, nematodes,powderymildew, and fusarium head blight) stress resistance/tolerances as well as novel grain quality traits. Numerous SHWs have been produced globally by various institutions including CIMMYT-Mexico, ICARDA-Syria, Department of Primary Industries (DPI), Victoria-Australia, IPK-Germany, Kyoto University-Japan, and USDAARS. This review examines the varied aspects in the utilization of synthetics for wheat improvement including the traits and genes identified, mapped, and transferred to common wheat. It has also been demonstrated that synthetic backcross-derived lines (SBLs, i.e., when SHW is crossed to adapted local bread varieties) show significant yield increases and thus, enhanced yield performance across a diverse range of environments, demonstrating their potential for improving wheat productivity worldwide. This is particularly evident in moisturelimited environments. The use of SBLs, advanced backcross QTL analysis, chromosome introgression lines, and whole genome association mapping is contributing to the elucidation of the genetic architecture of some of the traits. The contribution of transgressive segregation to enhanced phenotypes and the mechanisms including its genetic and physiological basis are yet to be elucidated. 36 FRANCIS C. OGBONNAYA ET AL.

Synthetic Hexaploids: Harnessing Species of the Primary Gene Pool for Wheat Improvement

Improvement of end-use quality in bread wheat depends on a thorough understanding of current wheat quality and the influences of genotype (G), environment (E), and genotype by environment interaction (G x E) on quality traits. Thirty-nine spring-sown spring wheat (SSSW) cultivars and advanced lines from China were grown in four agro-ecological zones comprising seven locations during the 1998 and 1999 cropping seasons. Data on 12 major bread-making quality traits were used to investigate the effect of G, E, and G x E on these traits. Wide range variability for protein quantity and quality, starch quality parameters and milling quality in Chinese SSSW was observed. Genotype and environment were found to significantly influence all quality parameters as major effects. Kernel hardness, flour yield, Zeleny sedimentation value and mixograph properties were mainly influenced by the genetic variance components, while thousand kernel weight, test weight, and falling number were mostly influenced by the environmental variance components. Genotype, environment, and their interaction had important effects on test weight, mixing development time and RVA parameters. Cultivars originating from Zone VI (northeast) generally expressed high kernel hardness, good starch quality, but poor milling and medium to weak mixograph performance; those from Zone VII (north) medium to good gluten and starch quality, but low milling quality; those from Zone VIII (central northwest) medium milling and starch quality, and medium to strong mixograph performance; those from Zone IX (western/southwestern Qinghai-Tibetan Plateau) medium milling quality, but poor gluten strength and starch parameters; and those from Zone X (northwest) high milling quality, strong mixograph properties, but low protein content. Samples from Harbin are characterized by good gluten and starch quality, but medium to poor milling quality; those from Hongxinglong by strong mixograph properties, medium to high milling quality, but medium to poor starch quality and medium to low protein content; those from Hohhot by good gluten but poor milling quality; those from Linhe by weak gluten quality, medium to poor milling quality; those from Lanzhou by poor bread-making and starch quality; those from Yongning by acceptable bread-making and starch quality and good milling quality; and those from Urumqi by good milling quality, medium gluten quality and good starch pasting parameters. Our findings suggest that Chinese SSSW quality could be greatly enhanced through genetic improvement for targeted well-characterized production environments.

Effect of environment and genotype on bread-making quality of spring-sown spring wheat cultivars in China

Progress of wheat breeding in China and the future perspective

Stripe rust,caused by Puccinia striiformis f.sp.tritici,and powdery mildew,caused by Blumeria graminis f.sp.tritici,are the devastating diseases in common wheat(Triticum aestivum L.) worldwide.Use of adult-plant resistance(APR) genes is an important method for the development of durable resistant cultivars.A total of 72 quantitative trait loci(QTLs) for APR to stripe rust and 82 QTLs for APR to powdery mildew were integrated into a linkage map based on the information of DNA markers linked to individual QTL.Eight gene clusters(≥5 QTLs) conferred resistance to both stripe rust and powdery powdery,among them,Yr18/Lr34/Pm38,Yr29/Lr46/Pm39,and Yr46/Lr67 showed resistance to stripe rust,leaf rust and powdery mildew.Yr18/Lr34/Pm38 and Yr36 have been cloned.Xiannong 4 and Xiaoyan 6 were very important resistant germplasm for APR to stripe rust and powdery midew.The application of APR to wheat breeding in China was summarized.The use of APR genes will be a major method for improving stripe rust and powdery mildew resistance in wheat breeding.The strategies for APR on wheat breeding were also discussed.

Progress and perspective in research of adult-plant resistance to stripe rust and powdery mildew in wheat

Improved lodging resistance is important for achieving high yield in irrigated environments. This study was conducted to determine genotypic variation in lodging resistance and related morphological traits among winter wheat cultivars planted at two densities, and to identify key traits associated with lodging resistance. Lodging performance of 28 genotypes, including 24 released cultivars and four advanced lines, was evaluated at 250 plants per square meter and 500 plants per square meter in Shandong province during the 2008-2009 and 2009-2010 crop seasons. At the higher density, the average grain yield was 2.6% higher, even though lodging score rose by as much as 136%. The higher planting density increased lodging through increased leaf area index (LAI), plant height, center of gravity and length of basal internodes, and reduced grain weight per spike and diameter of the lower two stem internodes. LAI, center of gravity and diameter of first internodes, as the important indicators for lodging resistance, were significantly correlated with lodging score, with R= 0.62, 0.59 and -0.52 (P<0.01), respectively. Plant pushing resistance was significantly associated with diameter and length of the first internodes (R = 0.71-0.77, P<0.01), indicating it could be used to assess the strength of the lower stem. Higher planting density could be used to select genotypes with lodging resistance in irrigated environments. Cultivars carrying high plant density tolerance and high yield potential, such as Jimai 22 and Liangxing 66, were recommended as leading cultivars for production as well as elite crossing parents for further increasing yield potential in the Yellow and Huai Valleys Winter Wheat Zone in China.

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He Zhonghu

Papers

Effect of environment and genotype on bread-making quality of spring-sown spring wheat cultivars in China

Progress of wheat breeding in China and the future perspective

Progress and perspective in research of adult-plant resistance to stripe rust and powdery mildew in wheat

Lodging resistance and yield potential of winter wheat: effect of planting density and genotype

Allelic variation at the TaZds-A1 locus on wheat chromosome 2A and development of a functional marker in common wheat