Effect of genotyping errors on linkage map construction based on repeated chip analysis of two recombinant inbred line populations in wheat (Triticum aestivum L.).

Linkage maps are essential for genetic mapping of phenotypic traits, gene map-based cloning, and marker-assisted selection in breeding applications. Construction of a high-quality saturated map requires high-quality genotypic data on a large number of molecular markers. Errors in genotyping cannot be completely avoided, no matter what platform is used. When genotyping error reaches a threshold level, it will seriously affect the accuracy of the constructed map and the reliability of consequent genetic studies. In this study, repeated genotyping of two recombinant inbred line (RIL) populations derived from crosses Yangxiaomai × Zhongyou 9507 and Jingshuang 16 × Bainong 64 was used to investigate the effect of genotyping errors on linkage map construction. Inconsistent data points between the two replications were regarded as genotyping errors, which were classified into three types. Genotyping errors were treated as missing values, and therefore the non-erroneous data set was generated. Firstly, linkage maps were constructed using the two replicates as well as the non-erroneous data set. Secondly, error correction methods implemented in software packages QTL IciMapping (EC) and Genotype-Corrector (GC) were applied to the two replicates. Linkage maps were therefore constructed based on the corrected genotypes and then compared with those from the non-erroneous data set. Simulation study was performed by considering different levels of genotyping errors to investigate the impact of errors and the accuracy of error correction methods. Results indicated that map length and marker order differed among the two replicates and the non-erroneous data sets in both RIL populations. For both actual and simulated populations, map length was expanded as the increase in error rate, and the correlation coefficient between linkage and physical maps became lower. Map quality can be improved by repeated genotyping and error correction algorithm. When it is impossible to genotype the whole mapping population repeatedly, 30% would be recommended in repeated genotyping. The EC method had a much lower false positive rate than did the GC method under different error rates. This study systematically expounded the impact of genotyping errors on linkage analysis, providing potential guidelines for improving the accuracy of linkage maps in the presence of genotyping errors.

Orchestrating seed storage protein and starch accumulation toward overcoming yield-quality trade-off in cereal crops.

Achieving high yield and good quality in crops is essential for human food security and health. However, there is usually disharmony between yield and quality. Seed storage protein (SSP) and starch, the predominant components in cereal grains, determine yield and quality, and their coupled synthesis causes a yield-quality trade-off. Therefore, dissection of the underlying regulatory mechanism facilitates simultaneous improvement of yield and quality. Here, we summarize current findings about the synergistic molecular machinery underpinning SSP and starch synthesis in the leading staple cereal crops, including maize, rice and wheat. We further evaluate the functional conservation and differentiation of key regulators and specify feasible research approaches to identify additional regulators and expand insights. We also present major strategies to leverage resultant information for simultaneous improvement of yield and quality by molecular breeding. Finally, future perspectives on major challenges are proposed.

Dynamic metabolite QTL analyses provide novel biochemical insights into kernel development and nutritional quality improvement in common wheat.

Despite recent advances in crop metabolomics, the genetic control and molecular basis of the wheat kernel metabolome at different developmental stages remain largely unknown. Here, we performed widely targeted metabolite profiling of kernels from three developmental stages (grain-filling kernels [FKs], mature kernels [MKs], and germinating kernels [GKs]) using a population of 159 recombinant inbred lines. We detected 625 annotated metabolites and mapped 3173, 3143, and 2644 metabolite quantitative trait loci (mQTLs) in FKs, MKs, and GKs, respectively. Only 52 mQTLs were mapped at all three stages, indicating the high stage specificity of the wheat kernel metabolome. Four candidate genes were functionally validated by in vitro enzymatic reactions and/or transgenic approaches in wheat, three of which mediated the tricin metabolic pathway. Metabolite flux efficiencies within the tricin pathway were evaluated, and superior candidate haplotypes were identified, comprehensively delineating the tricin metabolism pathway in wheat. Finally, additional wheat metabolic pathways were re-constructed by updating them to incorporate the 177 candidate genes identified in this study. Our work provides new information on variations in the wheat kernel metabolome and important molecular resources for improvement of wheat nutritional quality.

The TaSOC1-TaVRN1 module integrates photoperiod and vernalization signals to regulate wheat flowering.

Wheat needs different durations of vernalization, which accelerates flowering by exposure to cold temperature, to ensure reproductive development at the optimum time, as that is critical for adaptability and high yield. TaVRN1 is the central flowering regulator in the vernalization pathway and encodes a MADS-box transcription factor (TF) that usually works by forming hetero- or homo-dimers. We previously identified that TaVRN1 bound to an MADS-box TF TaSOC1 whose orthologues are flowering activators in other plants. The specific function of TaSOC1 and the biological implication of its interaction with TaVRN1 remained unknown. Here, we demonstrated that TaSOC1 was a flowering repressor in the vernalization and photoperiod pathways by overexpression and knockout assays. We confirmed the physical interaction between TaSOC1 and TaVRN1 in wheat protoplasts and in planta, and further validated their genetic interplay. A Flowering Promoting Factor 1-like gene TaFPF1-2B was identified as a common downstream target of TaSOC1 and TaVRN1 through transcriptome and chromatin immunoprecipitation analyses. TaSOC1 competed with TaVRT2, another MADS-box flowering regulator, to bind to TaVRN1; their coding genes synergistically control TaFPF1-2B expression and flowering initiation in response to photoperiod and low temperature. We identified major haplotypes of TaSOC1 and found that TaSOC1-Hap1 conferred earlier flowering than TaSOC1-Hap2 and had been subjected to positive selection in wheat breeding. We also revealed that wheat SOC1 family members were important domestication loci and expanded by tandem and segmental duplication events. These findings offer new insights into the regulatory mechanism underlying flowering control along with useful genetic resources for wheat improvement.

Rht12b, a widely used ancient allele of TaGA2oxA13, reduces plant height and enhances yield potential in wheat.

KEY MESSAGE: We identified a new wheat dwarfing allele Rht12b conferring reduced height and higher grain yield, pinpointed its causal variations, developed a breeding-applicable marker, and traced its origin and worldwide distribution. Plant height control is essential to optimize lodging resistance and yield gain in crops. RHT12 is a reduced height (Rht) locus that is identified in a mutationally induced dwarfing mutant and encodes a gibberellin 2-oxidase TaGA2oxA13. However, the artificial dwarfing allele is not used in wheat breeding due to excessive height reduction. Here, we confirmed a stable Rht locus, overlapping with RHT12, in a panel of wheat cultivars and its dwarfing allele reduced plant height by 5.4-8.2 cm, equivalent to Rht12b, a new allele of RHT12. We validated the effect of Rht12b on plant height in a bi-parent mapping population. Importantly, wheat cultivars carrying Rht12b had higher grain yield than those with the contrasting Rht12a allele. Rht12b conferred higher expression level of TaGA2oxA13. Transient activation assays defined SNP-390(C/A) in the promoter of TaGA2oxA13 as the causal variation. An efficient kompetitive allele-specific PCR marker was developed to diagnose Rht12b. Conjoint analysis showed that Rht12b plus the widely used Rht-D1b, Rht8 and Rht24b was the predominant Rht combination and conferred a moderate plant height in tested wheat cultivars. Evolutionary tracking uncovered that RHT12 locus arose from a tandem duplication event with Rht12b firstly appearing in wild emmer. The frequency of Rht12b was approximately 70% (700/1005) in a worldwide wheat panel and comparable to or higher than those of other widely used Rht genes, suggesting it had been subjected to positive selection. These findings not only identify a valuable Rht gene for wheat improvement but also develop a functionally diagnostic tool for marker-assisted breeding.

Molecular characterization of stable QTL and putative candidate genes for grain zinc and iron concentrations in two related wheat populations.

KEY MESSAGE: Major QTL for grain zinc and iron concentrations were identified on the long arm of chromosomes 2D and 6D. Gene-based KASP markers were developed for putative candidate genes TaIPK1-2D and TaNAS10-6D. Micronutrient malnutrition is one of the most common public health problems in the world. Biofortification, the most attractive and sustainable solution to surmount malnutrition requires the development of micronutrient enriched new crop cultivars. In this study, two recombinant inbred line (RIL) populations, ZM175/XY60 and ZM175/LX987, were used to identify QTL for grain zinc concentration (GZnC), grain iron concentration (GFeC) and thousand grain weight (TGW). Eight QTL for GZnC, six QTL for GFeC and five QTL for TGW were detected. Three QTL on chromosomes 2DL and 4BS and chromosome 6A showed pleiotropic effects on all three traits. The 4BS and 6A QTL also increased plant height and might be Rht-B1a and Rht25a, respectively. The 2DL locus within a suppressed recombination region was identified in both RIL populations and the favorable allele simultaneously increasing GZnC, GFeC and TGW was contributed by XY60 and LX987. A QTL on chromosome 6DL associated only with GZnC was detected in ZM175/XY60 and was validated in JD8/AK58 RILs using kompetitive allele-specific PCR (KASP) marker K_AX-110119937. Both the 2DL and 6DL QTL were new loci for GZnC. Based on gene annotations, sequence variations and expression profiles, the phytic acid biosynthesis gene TaIPK1-2D and nicotianamine synthase gene TaNAS10-6D were predicted as candidate genes. Their gene-based KASP markers were developed and validated in a cultivar panel of 343 wheat accessions. This study investigated the genetic basis of GZnC and GFeC and provided valuable candidate genes and markers for breeding Zn- and Fe-enriched wheat.

Molecular identification and validation of four stable QTL for adult-plant resistance to powdery mildew in Chinese wheat cultivar Bainong 64.

KEY MESSAGE: Four stable QTL for adult-plant resistance (APR) to powdery mildew were identified on chromosome arms 1DL, 2BS, 2DL, and 6BL in the widely grown Chinese wheat cultivar Bainong 64. These QTL had no effect on response to stripe rust or leaf rust. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating fungal disease. Seedlings of Chinese wheat Bainong 64 are susceptible to Bgt, but adult plants have maintained resistance since it was released in 1996. A population of 171 recombinant inbred lines (RILs) developed from cross Jingshuang 16/Bainong 64 (JS16/BN64) was used to dissect genetic components of powdery mildew resistance. A genetic map comprising 5383 polymorphic markers was constructed using the 15 K SNP chip and kompetitive allele-specific PCR (KASP) markers. Composite interval mapping identified four stable QTL with favorable alleles all from BN64 on chromosome arms 1DL, 2BS, 2DL, and 6BL in at least four environments. They accounted for 8.3%, 13.8%, 14.4%, and 9.0% of the total phenotypic variation explained (PVE) in maximum, respectively. QPmjbr.caas-1DL, situated about 22 Mb from centromere, is probably a new QTL. QPmjbr.caas-2DL located near the end of arm 2DL and explained the largest PVE. Using genetic maps populated with KASP markers, QPmjbr.caas-2BS and QPmjbr.caas-6BL were fine mapped to a 1.8 cM genetic intervals spanning 13.6 Mb (76.0-89.6 Mb) and 1.7 cM and 4.9 Mb (659.9-664.8 Mb), respectively. The four QTL independent of stripe rust and leaf rust resistance were validated for powdery mildew resistance in another RIL population related to BN64 and a cultivar panel using representative KASP markers. Since BN64 has been a leading cultivar and an important breeding parent in China, the QTL and markers reported in this study will be useful for marker-assisted selection of APR.

Functional analysis of polyphenol oxidase 1 gene in common wheat.

Polyphenol oxidase (PPO) activity is a major cause of the undesirable brown color of wheat-based products. Ppo1, a major gene for PPO activity, was cloned based on sequence homology in previous studies; however, its function and regulation mechanism remain unclear. In this study, the function and genetic regulation of Ppo1 were analyzed using RNA interference (RNAi) and Targeting Induced Local Lesions IN Genomes (TILLING) technology, and superior mutants were identified. Compared with the control, the level of Ppo1 transcript in RNAi transgenic lines was drastically decreased by 15.5%-60.9% during grain development, and PPO activity was significantly reduced by 12.9%-20.4%, confirming the role of Ppo1 in PPO activity. Thirty-two Ppo1 mutants were identified in the ethyl methanesulfonate (EMS)-mutagenized population, including eight missense mutations, 16 synonymous mutations, and eight intron mutations. The expression of Ppo1 was reduced significantly by 6.7%-37.1% and 10.1%-54.4% in mutants M092141 (G311S) and M091098 (G299R), respectively, in which PPO activity was decreased by 29.7% and 28.8%, respectively, indicating that mutation sites of two mutants have important effects on PPO1 function. Sequence and structure analysis revealed that the two sites were highly conserved among 74 plant species, where the frequency of glycine was 94.6% and 100%, respectively, and adjacent to the entrance of the hydrophobic pocket of the active site. The M092141 and M091098 mutants can be used as important germplasms to develop wheat cultivars with low grain PPO activity. This study provided important insights into the molecular mechanism of Ppo1 and the genetic improvement of wheat PPO activity.

Fine mapping and characterization of a major QTL for plant height on chromosome 5A in wheat.

KEY MESSAGE: We precisely mapped QPH.caas-5AL for plant height in wheat, predicted candidate genes and confirmed genetic effects in a panel of wheat cultivars. Plant height is an important agronomic trait, and appropriately reduced height can improve yield potential and stability in wheat, usually combined with sufficient water and fertilizer. We previously detected a stable major-effect quantitative trait locus QPH.caas-5AL for plant height on chromosome 5A in a recombinant inbred line population of the cross 'Doumai × Shi 4185' using the wheat 90 K SNP assay. Here , QPH.caas-5AL was confirmed using new phenotypic data in additional environment and new-developed markers. We identified nine heterozygous recombinant plants for fine mapping of QPH.caas-5AL and developed 14 breeder-friendly kompetitive allele-specific PCR markers in the region of QPH.caas-5AL based on the genome re-sequencing data of parents. Phenotyping and genotyping analyses of secondary populations derived from the self-pollinated heterozygous recombinant plants delimited QPH.caas-5AL into an approximate 3.0 Mb physical region (521.0-524.0 Mb) according to the Chinese Spring reference genome. This region contains 45 annotated genes, and six of them were predicted as the candidates of QPH.caas-5AL based on genome and transcriptome sequencing analyses. We further validated that QPH.caas-5AL has significant effects on plant height but not yield component traits in a diverse panel of wheat cultivars; its dwarfing allele is frequently used in modern wheat cultivars. These findings lay a solid foundation for the map-based cloning of QPH.caas-5AL and also provide a breeding-applicable tool for its marker-assisted selection. Keymessage We precisely mapped QPH.caas-5AL for plant height in wheat, predicted candidate genes and confirmed genetic effects in a panel of wheat cultivars.

Genetic dissection of grain morphology and yield components in a wheat line with defective grain filling.

KEY MESSAGE: We identified stable QTL for grain morphology and yield component traits in a wheat defective grain filling line and validated genetic effects in a panel of cultivars using breeding-relevant markers. Grain filling capacity is essential for grain yield and appearance quality in cereal crops. Identification of genetic loci for grain filling is important for wheat improvement. However, there are few genetic studies on grain filling in wheat. Here, a defective grain filling (DGF) line wdgf1 characterized by shrunken grains was identified in a population derived from multi-round crosses involving nine parents and a recombinant inbreed line (RIL) population was generated from the cross between wdgf1 and a sister line with normal grains. We constructed a genetic map of the RIL population using the wheat 15K single nucleotide polymorphism chip and detected 25 stable quantitative trait loci (QTL) for grain morphology and yield components, including three for DGF, eleven for grain size, six for thousand grain weight, three for grain number per spike and two for spike number per m2. Among them, QDGF.caas-7A is co-located with QTGW.caas-7A and can explain 39.4-64.6% of the phenotypic variances, indicating that this QTL is a major locus controlling DGF. Sequencing and linkage mapping showed that TaSus2-2B and Rht-B1 were candidate genes for QTGW.caas-2B and the QTL cluster (QTGW.caas-4B, QGNS.caas-4B, and QSN.caas-4B), respectively. We developed kompetitive allele-specific PCR markers tightly linked to the stable QTL without corresponding to known yield-related genes, and validated their genetic effects in a diverse panel of wheat cultivars. These findings not only lay a solid foundation for genetic dissection underlying grain filling and yield formation, but also provide useful tools for marker-assisted breeding.

Efficient proteome-wide identification of transcription factors targeting Glu-1: A case study for functional validation of TaB3-2A1 in wheat.

High-molecular-weight glutenin subunits (HMW-GS), a major component of seed storage proteins (SSP) in wheat, largely determine processing quality. HMW-GS encoded by GLU-1 loci are mainly controlled at the transcriptional level by interactions between cis-elements and transcription factors (TFs). We previously identified a conserved cis-regulatory module CCRM1-1 as the most essential cis-element for Glu-1 endosperm-specific high expression. However, the TFs targeting CCRM1-1 remained unknown. Here, we built the first DNA pull-down plus liquid chromatography-mass spectrometry platform in wheat and identified 31 TFs interacting with CCRM1-1. TaB3-2A1 as proof of concept was confirmed to bind to CCRM1-1 by yeast one hybrid and electrophoretic mobility shift assays. Transactivation experiments demonstrated that TaB3-2A1 repressed CCRM1-1-driven transcription activity. TaB3-2A1 overexpression significantly reduced HMW-GS and other SSP, but enhanced starch content. Transcriptome analyses confirmed that enhanced expression of TaB3-2A1 down-regulated SSP genes and up-regulated starch synthesis-related genes, such as TaAGPL3, TaAGPS2, TaGBSSI, TaSUS1 and TaSUS5, suggesting that it is an integrator modulating the balance of carbon and nitrogen metabolism. TaB3-2A1 also had significant effects on agronomic traits, including heading date, plant height and grain weight. We identified two major haplotypes of TaB3-2A1 and found that TaB3-2A1-Hap1 conferred lower seed protein content, but higher starch content, plant height and grain weight than TaB3-2A1-Hap2 and was subjected to positive selection in a panel of elite wheat cultivars. These findings provide a high-efficiency tool to detect TFs binding to targeted promoters, considerable gene resources for dissecting regulatory mechanisms underlying Glu-1 expression, and a useful gene for wheat improvement.

Molecular characterization and validation of adult-plant stripe rust resistance gene Yr86 in Chinese wheat cultivar Zhongmai 895.

KEY MESSAGE: Adult-plant stripe rust resistance gene Yr86 in Chinese wheat cultivar Zhongmai 895 was mapped to the physical interval 710.2-713.2 Mb on the long arm of chromosome 2A. Adult-plant resistance to stripe rust is generally more durable than all-stage resistance. Chinese wheat cultivar Zhongmai 895 showed stable stripe rust resistance at the adult-plant stage. To map the genetic loci underlying its resistance, 171 doubled haploid (DH) lines from a Yangmai 16/Zhongmai 895 cross were genotyped with the wheat 660 K SNP chip. Disease severities of the DH population and parents were assessed in four environments. A major QTL designated QYryz.caas-2AL was mapped to interval 703.7-715.3 Mb on the long arm of chromosome 2A using both chip-based and KASP (kompetitive allele-specific PCR) marker-based methods, explaining 31.5 to 54.1% of the phenotypic variances. The QTL was further validated in an F2 population of cross Emai 580/Zhongmai 895 with 459 plants and a panel of 240 wheat cultivars using KASP markers. Three reliable KASP markers predicted a low frequency (7.2-10.5%) of QYryz.caas-2AL in the test panel and remapped the gene to the physical interval 710.2-713.2 Mb. Based on different physical positions or genetic effects from known genes or QTL on chromosome arm 2AL, the gene was predicted to be a new one for adult-plant stripe rust resistance and was named Yr86. Twenty KASP markers linked to Yr86 were developed in this study based on wheat 660 K SNP array and genome re-sequencing. Three of them are significantly associated with stripe rust resistance in natural population. These markers should be useful for marker-assisted selection and also provide a starting point for fine mapping and map-based cloning of the new resistance gene.

Dissecting pleiotropic functions of the wheat Green Revolution gene Rht-B1b in plant morphogenesis and yield formation.

The utilization of reduced plant height genes Rht-B1b and Rht-D1b, encoding homeologous DELLA proteins, led to the wheat Green Revolution (GR). However, the specific functions of GR genes in yield determination and the underlying regulatory mechanisms remained unknown. Here, we validated that Rht-B1b, as a representative of GR genes, affects plant architecture and yield component traits. Upregulation of Rht-B1b reduced plant height, leaf size and grain weight, but increased tiller number, tiller angle, spike number per unit area, and grain number per spike. Dynamic investigations showed that Rht-B1b increased spike number by improving tillering initiation rather than outgrowth, and enhanced grain number by promoting floret fertility. Rht-B1b reduced plant height by reducing cell size in the internodes, and reduced grain size or weight by decreasing cell number in the pericarp. Transcriptome analyses uncovered that Rht-B1b regulates many homologs of previously reported key genes for given traits and several putative integrators for different traits. These findings specify the pleiotropic functions of Rht-B1b in improving yield and provide new insights into the regulatory mechanisms underlying plant morphogenesis and yield formation.

QTL Mapping Reveals Both All-Stage and Adult-Plant Resistance to Powdery Mildew in Chinese Elite Wheat Cultivars.

Powdery mildew caused by Blumeria graminis f. sp. tritici is a threat to wheat production in China. Mapping quantitative trait loci (QTL) for resistance to powdery mildew and developing breeder-friendly markers are important initial steps in breeding resistant cultivars. An all-stage resistance gene and several QTL were identified using a population of 254 recombinant inbred lines developed from a Jingdong 8/Aikang 58 cross. The population was evaluated for powdery mildew resistance across six field environments over three consecutive growing seasons utilizing two different mixtures of B. graminis f. sp. tritici isolates, named #Bgt-HB and #Bgt-BJ. Using genotypic data obtained from the Wheat TraitBreed 50K single-nucleotide polymorphism array, seven stable QTL were identified on chromosome arms 1DL, 2AL, 2DS, 4DL, 5AL, 6BL.1, and 6BL.2. The QTL on 2AL conferred all-stage resistance to B. graminis f. sp. tritici race E20 in greenhouse tests and explained up to 52% of the phenotypic variance in field trials but was resistant only against #Bgt-HB. The gene involved in this QTL was predicted to be Pm4a based on genome location and gene sequence. QPmja.caas-1DL, QPmja.caas-4DL, and QPmja.caas-6BL.1 were identified as potentially new QTL for powdery mildew resistance. QPmja.caas-2DS and QPmja.caas-6BL.1 were effective against both B. graminis f. sp. tritici mixtures, indicating their probable broad-spectrum resistance. A Kompetitive allele-specific PCR marker closely linked to QPmja.caas-2DS was developed and validated in a panel of 286 wheat cultivars. Because both Jingdong 8 and Aikang 58 have been leading cultivars and breeding parents, the QTL and marker reported represent valuable resources for wheat researchers and breeders.

Impact of "Green Revolution" gene Rht-B1b on coleoptile length of wheat.

Wheat coleoptile is a sheath-like structure that helps to deliver the first leaf from embryo to the soil surface. Here, a RIL population consisting of 245 lines derived from Zhou 8425B × Chinese Spring cross was genotyped by the high-density Illumina iSelect 90K assay for coleoptile length (CL) QTL mapping. Three QTL for CL were mapped on chromosomes 2BL, 4BS and 4DS. Of them, two major QTL QCL.qau-4BS and QCL.qau-4DS were detected, which could explain 9.1%-22.2% of the phenotypic variances across environments on Rht-B1 and Rht-D1 loci, respectively. Several studies have reported that Rht-B1b may reduce the length of wheat CL but no study has been carried out at molecular level. In order to verify that the Rht-B1 gene is the functional gene for the 4B QTL, an overexpression line Rht-B1b-OE and a CRISPR/SpCas9 line Rht-B1b-KO were studied. The results showed that Rht-B1b overexpression could reduce the CL, while loss-of-function of Rht-B1b would increase the CL relative to that of the null transgenic plants (TNL). To dissect the underlying regulatory mechanism of Rht-B1b on CL, comparative RNA-Seq was conducted between Rht-B1b-OE and TNL. Transcriptome profiles revealed a few key pathways involving the function of Rht-B1b in coleoptile development, including phytohormones, circadian rhythm and starch and sucrose metabolism. Our findings may facilitate wheat breeding for longer coleoptiles to improve seedling early vigor for better penetration through the soil crust in arid regions.

Fine mapping of reduced height locus RHT26 in common wheat.

KEY MESSAGE: We fine mapped RHT26 for plant height in wheat, confirmed its genetic effects in a panel of wheat cultivars and predicted candidate genes. Development of wheat cultivars with appropriate plant height (PH) is an important goal in breeding. Utilization of semi-dwarfing genes Rht-B1b and Rht-D1b triggered wheat Green Resolution in the 1960s. Since these genes also bring unfavorable features, such as reduced coleoptile length and grain weight, it is necessary to identify alternative reduced height genes without yield penalty. Here we constructed a high-density genetic map of a recombinant inbred line population derived from the cross of Zhongmai175 and Lunxuan987 and detected a stable genetic locus for PH, designated RHT26, on chromosome arm 3DL in all of six environments, accounting for 6.8-14.0% of the phenotypic variances. RHT26 was delimited to an approximate 1.4 Mb physical interval (517.1-518.5 Mb) using secondary mapping populations derived from 22 heterozygous recombinant plants and 24 kompetitive allele-specific PCR markers. Eleven high-confidence genes were annotated in the physical interval according to the Chinese Spring reference genome, and four of them were predicted as candidates for RHT26 based on genome and transcriptome sequencing analyses. We also confirmed that RHT26 had significant effects on PH, but not grain yield in a panel of wheat cultivars; its dwarfing allele has been frequently used in wheat breeding. These findings lay a sound foundation for map-based cloning of RHT26 and provide a breeding-applicable tool for marker-assisted selection.

Fine Mapping of Stripe-Rust-Resistance Gene YrJ22 in Common Wheat by BSR-Seq and MutMap-Based Sequencing.

Identification and accurate mapping of new resistance genes are essential for gene pyramiding in wheat breeding. The YrJ22 gene is a dominant stripe-rust-resistance gene located at the distal end of chromosome 2AL, which was identified in a leading Chinese-wheat variety, Jimai 22, showing high resistance to CYR32, a prevalent race of Puccinia striiformis tritici (Pst) in China. In the current study, 15 F1 and 2273 F2 plants derived from the cross of Jimai 22/Avocet S were used for the fine-mapping of YrJ22. The RNA-Seq of resistant and susceptible bulks of F2 plants (designated BSR-Seq) identified 10 single-nucleotide polymorphisms (SNP) in a 12.09 Mb physical interval on chromosome 2AL. A total of 1022 EMS-induced M3 lines of Jimai 22 were screened, to identify susceptible mutants for MutMap analysis. Four CAPS markers were developed from SNPs identified using BSR-Seq and MutMap. A linkage map for YrJ22 was constructed with 11 CAPS/STS and three SSR markers. YrJ22 was located at a 0.9 cM genetic interval flanked by markers H736 and H400, corresponding to a 340.46 kb physical region (768.7-769.0 Mb), including 13 high-confidence genes based on the Chinese Spring reference genome. TraesCS2A01G573200 is a potential candidate-gene, according to linkage and quantitative real-time PCR (qPCR) analyses. The CAPS marker H732 designed from an SNP in TraesCS2A01G573200 co-segregated with YrJ22. These results provide a useful stripe-rust-resistance gene and molecular markers for marker-assisted selection in wheat breeding and for further cloning of the gene.

Fine mapping of a stripe rust resistance gene YrZM175 in bread wheat.

KEY MESSAGE: A stripe rust resistance gene YrZM175 in Chinese wheat cultivar Zhongmai 175 was mapped to a genomic interval of 636.4 kb on chromosome arm 2AL, and a candidate gene was predicted. Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is a worldwide wheat disease that causes large losses in production. Fine mapping and cloning of resistance genes are important for accurate marker-assisted breeding. Here, we report the fine mapping and candidate gene analysis of stripe rust resistance gene YrZM175 in a Chinese wheat cultivar Zhongmai 175. Fifteen F1, 7,325 F2 plants and 117 F2:3 lines derived from cross Avocet S/Zhongmai 175 were inoculated with PST race CYR32 at the seedling stage in a greenhouse, and F2:3 lines were also evaluated for stripe rust reaction in the field using mixed PST races. Bulked segregant RNA-seq (BSR-seq) analyses revealed 13 SNPs in the region 762.50-768.52 Mb on chromosome arm 2AL. By genome mining, we identified SNPs and InDels between the parents and contrasting bulks and mapped YrZM175 to a 0.72-cM, 636.4-kb interval spanned by YrZM175-InD1 and YrZM175-InD2 (763,452,916-764,089,317 bp) including two putative disease resistance genes based on IWGSC RefSeq v1.0. Collinearity analysis indicated similar target genomic intervals in Chinese Spring, Aegilops tauschii (2D: 647.7-650.5 Mb), Triticum urartu (2A: 750.7-752.3 Mb), Triticum dicoccoides (2A: 771.0-774.5 Mb), Triticum turgidum (2B: 784.7-788.2 Mb), and Triticum aestivum cv. Aikang 58 (2A: 776.3-778.9 Mb) and Jagger (2A: 789.3-791.7 Mb). Through collinearity analysis, sequence alignments of resistant and susceptible parents and gene expression level analysis, we predicted TRITD2Bv1G264480 from Triticum turgidum to be a candidate gene for map-based cloning of YrZM175. A gene-specific marker for TRITD2Bv1G264480 co-segregated with the resistance gene. Molecular marker analysis and stripe rust response data revealed that YrZM175 was different from genes Yr1, Yr17, Yr32, and YrJ22 located on chromosome 2A. Fine mapping of YrZM175 lays a solid foundation for functional gene analysis and marker-assisted selection for improved stripe rust resistance in wheat.

Genetic networks underlying salinity tolerance in wheat uncovered with genome-wide analyses and selective sweeps.

KEY MESSAGE: A genetic framework underpinning salinity tolerance at reproductive stage was revealed by genome-wide SNP markers and major adaptability genes in synthetic-derived wheats, and trait-associated loci were used to predict phenotypes. Using wild relatives of crops to identify genes related to improved productivity and resilience to climate extremes is a prioritized area of crop genetic improvement. High salinity is a widespread crop production constraint, and development of salt-tolerant cultivars is a sustainable solution. We evaluated a panel of 294 wheat accessions comprising synthetic-derived wheat lines (SYN-DERs) and modern bread wheat advanced lines under control and high salinity conditions at two locations. The GWAS analysis revealed a quantitative genetic framework of more than 200 loci with minor effect underlying salinity tolerance at reproductive stage. The significant trait-associated SNPs were used to predict phenotypes using a GBLUP model, and the prediction accuracy (r2) ranged between 0.57 and 0.74. The r2 values for flag leaf weight, days to flowering, biomass, and number of spikes per plant were all above 0.70, validating the phenotypic effects of the loci discovered in this study. Furthermore, the germplasm sets were compared to identify selection sweeps associated with salt tolerance loci in SYN-DERs. Six loci associated with salinity tolerance were found to be differentially selected in the SYN-DERs (12.4 Mb on chromosome (chr)1B, 7.1 Mb on chr2A, 11.2 Mb on chr2D, 200 Mb on chr3D, 600 Mb on chr6B, and 700.9 Mb on chr7B). A total of 228 reported markers and genes, including 17 well-characterized genes, were uncovered using GWAS and EigenGWAS. A linkage disequilibrium (LD) block on chr5A, including the Vrn-A1 gene at 575 Mb and its homeologs on chr5D, were strongly associated with multiple yield-related traits and flowering time under salinity stress conditions. The diversity panel was screened with more than 68 kompetitive allele-specific PCR (KASP) markers of functional genes in wheat, and the pleiotropic effects of superior alleles of Rht-1, TaGASR-A1, and TaCwi-A1 were revealed under salinity stress. To effectively utilize the extensive genetic information obtained from the GWAS analysis, a genetic interaction network was constructed to reveal correlations among the investigated traits. The genetic network data combined with GWAS, selective sweeps, and the functional gene survey provided a quantitative genetic framework for identifying differentially retained loci associated with salinity tolerance in wheat.

Fine mapping and characterization of a major QTL for grain weight on wheat chromosome arm 5DL.

KEY MESSAGE: We fine mapped QTL QTKW.caas-5DL for thousand kernel weight in wheat, predicted candidate genes and developed a breeding-applicable marker. Thousand kernel weight (TKW) is an important yield component trait in wheat, and identification of the underlying genetic loci is helpful for yield improvement. We previously identified a stable quantitative trait locus (QTL) QTKW.caas-5DL for TKW in a Doumai/Shi4185 recombinant inbred line (RIL) population. Here we performed fine mapping of QTKW.caas-5DL using secondary populations derived from 15 heterozygous recombinants and delimited the QTL to an approximate 3.9 Mb physical interval from 409.9 to 413.8 Mb according to the Chinese Spring (CS) reference genome. Analysis of genomic synteny showed that annotated genes in the physical interval had high collinearity among CS and eight other wheat genomes. Seven genes with sequence variation and/or differential expression between parents were predicted as candidates for QTKW.caas-5DL based on whole-genome resequencing and transcriptome assays. A kompetitive allele-specific PCR (KASP) marker for QTKW.caas-5DL was developed, and genotyping confirmed a significant association with TKW but not with other yield component traits in a panel of elite wheat cultivars. The superior allele of QTKW.caas-5DL was frequent in a panel of cultivars, suggesting that it had undergone positive selection. These findings not only lay a foundation for map-based cloning of QTKW.caas-5DL but also provide an efficient tool for marker-assisted selection.