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.

Beyond pathways: Accelerated flavonoids candidate identification and novel exploration of enzymatic properties using combined mapping populations of wheat.

Although forward-genetics-metabolomics methods such as mGWAS and mQTL have proven effective in providing myriad loci affecting metabolite contents, they are somehow constrained by their respective constitutional flaws such as the hidden population structure for GWAS and insufficient recombinant rate for QTL. Here, the combination of mGWAS and mQTL was performed, conveying an improved statistical power to investigate the flavonoid pathways in common wheat. A total of 941 and 289 loci were, respectively, generated from mGWAS and mQTL, within which 13 of them were co-mapped using both approaches. Subsequently, the mGWAS or mQTL outputs alone and their combination were, respectively, utilized to delineate the metabolic routes. Using this approach, we identified two MYB transcription factor encoding genes and five structural genes, and the flavonoid pathway in wheat was accordingly updated. Moreover, we have discovered some rare-activity-exhibiting flavonoid glycosyl- and methyl-transferases, which may possess unique biological significance, and harnessing these novel catalytic capabilities provides potentially new breeding directions. Collectively, we propose our survey illustrates that the forward-genetics-metabolomics approaches including multiple populations with high density markers could be more frequently applied for delineating metabolic pathways in common wheat, which will ultimately contribute to metabolomics-assisted wheat crop improvement.

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.

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.

A high-resolution genotype-phenotype map identifies the TaSPL17 controlling grain number and size in wheat.

BACKGROUND: Large-scale genotype-phenotype association studies of crop germplasm are important for identifying alleles associated with favorable traits. The limited number of single-nucleotide polymorphisms (SNPs) in most wheat genome-wide association studies (GWASs) restricts their power to detect marker-trait associations. Additionally, only a few genes regulating grain number per spikelet have been reported due to sensitivity of this trait to variable environments. RESULTS: We perform a large-scale GWAS using approximately 40 million filtered SNPs for 27 spike morphology traits. We detect 132,086 significant marker-trait associations and the associated SNP markers are located within 590 associated peaks. We detect additional and stronger peaks by dividing spike morphology into sub-traits relative to GWAS results of spike morphology traits. We propose that the genetic dissection of spike morphology is a powerful strategy to detect signals for grain yield traits in wheat. The GWAS results reveal that TaSPL17 positively controls grain size and number by regulating spikelet and floret meristem development, which in turn leads to enhanced grain yield per plant. The haplotypes at TaSPL17 indicate geographical differentiation, domestication effects, and breeding selection. CONCLUSION: Our study provides valuable resources for genetic improvement of spike morphology and a fast-forward genetic solution for candidate gene detection and cloning in wheat.

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.

Genetic basis of geographical differentiation and breeding selection for wheat plant architecture traits.

BACKGROUND: Plant architecture associated with increased grain yield and adaptation to the local environments is selected during wheat (Triticum aestivum) breeding. The internode length of individual stems and tiller length of individual plants are important for the determination of plant architecture. However, few studies have explored the genetic basis of these traits. RESULTS: Here, we conduct a genome-wide association study (GWAS) to dissect the genetic basis of geographical differentiation of these traits in 306 worldwide wheat accessions including both landraces and traditional varieties. We determine the changes of haplotypes for the associated genomic regions in frequency in 831 wheat accessions that are either introduced from other countries or developed in China from last two decades. We identify 83 loci that are associated with one trait, while the remaining 247 loci are pleiotropic. We also find 163 associated loci are under strong selective sweep. GWAS results demonstrate independent regulation of internode length of individual stems and consistent regulation of tiller length of individual plants. This makes it possible to obtain ideal haplotype combinations of the length of four internodes. We also find that the geographical distribution of the haplotypes explains the observed differences in internode length among the worldwide wheat accessions. CONCLUSION: This study provides insights into the genetic basis of plant architecture. It will facilitate gene functional analysis and molecular design of plant architecture for breeding.

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.

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.

QTL Mapping for Wheat Seed Dormancy in a Yangmai16/Zhongmai895 Double Haploid Population.

Pre-harvest sprouting (PHS) of wheat reduces grain yield and quality, and it is strongly affected by seed dormancy. Therefore, identification of quantitative trait loci (QTL) for seed dormancy is essential for PHS resistance breeding. A doubled haploid (DH) population, consisting of 174 lines from the cross between Yangmai16 (YM16) and Zhongmai895 (ZM895) was used to detect QTLs for seed dormancy and grain color. For seed dormancy, a total of seven QTLs were detected on chromosomes 2A, 3A, 3D, 4D, 5B and 5D over four environments, among which Qdor.hzau-3A, Qdor.hzau-3D.1 and Qdor.hzau-3D.2 were stably detected in more than two environments. For grain color, only two QTLs, Qgc.hzau-3A and Qgc.hzau-3D were detected on chromosomes 3A and 3D, which physically overlapped with Qdor.hzau-3A and Qdor.hzau-3D.1, respectively. Qdor.hzau-3D.2 has never been reported elsewhere and is probably a novel locus with allelic effect of seed dormancy contributed by weakly dormant parent ZM895, and a KASP marker was developed and validated in a wheat natural population. This study provides new information on the genetic dissection of seed dormancy, which may aid in further improvement for marker-assisted wheat breeding for PHS resistance.

Variations in phenological, physiological, plant architectural and yield-related traits, their associations with grain yield and genetic basis.

BACKGROUND AND AIMS: Physiological and morphological traits play essential roles in wheat (Triticum aestivum) growth and development. In particular, photosynthesis is a limitation to yield. Increasing photosynthesis in wheat has been identified as an important strategy to increase yield. However, the genotypic variations and the genomic regions governing morphological, architectural and photosynthesis traits remain unexplored. METHODS: Here, we conducted a large-scale investigation of the phenological, physiological, plant architectural and yield-related traits, involving 32 traits for 166 wheat lines during 2018-2020 in four environments, and performed a genome-wide association study with wheat 90K and 660K single nucleotide polymorphism (SNP) arrays. KEY RESULTS: These traits exhibited considerable genotypic variations in the wheat diversity panel. Higher yield was associated with higher net photosynthetic rate (r = 0.41, P < 0.01), thousand-grain weight (r = 0.36, P < 0.01) and truncated and lanceolate shape, but shorter plant height (r = -0.63, P < 0.01), flag leaf angle (r = -0.49, P < 0.01) and spike number per square metre (r = -0.22, P < 0.01). Genome-wide association mapping discovered 1236 significant stable loci detected in the four environments among the 32 traits using SNP markers. Trait values have a cumulative effect as the number of the favourable alleles increases, and significant progress has been made in determining phenotypic values and favourable alleles over the years. Eleven elite cultivars and 14 traits associated with grain yield per plot (GY) were identified as potential parental lines and as target traits to develop high-yielding cultivars. CONCLUSIONS: This study provides new insights into the phenotypic and genetic elucidation of physiological and morphological traits in wheat and their associations with GY, paving the way for discovering their underlying gene control and for developing enhanced ideotypes in wheat breeding.

Linking genetic markers with an eco-physiological model to pyramid favourable alleles and design wheat ideotypes.

Genetic markers can be linked with eco-physiological crop models to accurately predict genotype performance and individual markers' contributions in target environments, exploring interactions between genotype and environment. Here, wheat (Triticum aestivum L.) yield was dissected into seven traits corresponding to cultivar genetic coefficients in an eco-physiological model. Loci for these traits were discovered through the genome-wide association studies (GWAS). The cultivar genetic coefficients were derived from the loci using multiple linear regression or random forest, building a marker-based eco-physiological model. It is then applied to simulate wheat yields and design virtual ideotypes. The results indicated that the loci identified through GWAS explained 46%-75% variations in cultivar genetic coefficients. Using the marker-based model, the normalized root mean square error (nRMSE) between the simulated yield and observed yield was 13.95% by multiple linear regression and 13.62% by random forest. The nRMSE between the simulated and observed maturity dates was 1.24% by multiple linear regression and 1.11% by random forest, respectively. Structural equation modelling indicated that variations in grain yield could be well explained by cultivar genetic coefficients and phenological data. In addition, 24 pleiotropic loci in this study were detected on 15 chromosomes. More significant loci were detected by the model-based dissection method than considering yield per se. Ideotypes were identified by higher yield and more favourable alleles of cultivar genetic traits. This study proposes a genotype-to-phenotype approach and demonstrates novel ideas and tools to support the effective breeding of new cultivars with high yield through pyramiding favourable alleles and designing crop ideotypes.

QTL mapping and KASP marker development for seed vigor related traits in common wheat.

Seed vigor is an important parameter of seed quality, and identification of seed vigor related genes can provide an important basis for highly efficient molecular breeding in wheat. In the present study, a doubled haploid (DH) population with 174 lines derived from a cross between Yangmai16 and Zhongmai 895 was used to evaluate 10 seed vigor related traits in Luoyang during the 2018-2019 cropping season and in Mengjin and Luoning Counties during 2019-2020 cropping season for three environments. Quantitative trait locus (QTL) mapping of 10 seed vigor related traits in the DH population resulted in the discovery/identification of 28 QTLs on chromosomes 2B, 3D, 4B, 4D, 5A, 5B, 6A, 6B, 6D, 7A and 7D, explaining 3.6-23.7% of the phenotypic variances. Among them, one QTL cluster for shoot length, root length and vigor index was mapped between AX-89421921 and Rht-D1_SNP on chromosome 4D in the physical intervals of 18.78-19.29 Mb (0.51 Mb), explaining 9.2-20.5% of the phenotypic variances. Another QTL for these traits was identified at the physical position 185.74 Mb on chromosome 5B, which was flanked by AX-111465230 and AX-109519938 and accounted for 8.0-13.3% of the phenotypic variances. Two QTLs for shoot length, shoot fresh weight and shoot dry weight were identified in the marker intervals of AX-109384026-AX-111120402 and AX-111651800-AX-94443918 on chromosomes 6A and 6B, explaining 8.2-11.7% and 3.6-10.3% of the phenotypic variance, respectively; both alleles for increasing phenotypic values were derived from Yangmai 16. We also developed the KASP markers for the QTL cluster QVI.haust-4D.1/QSL.haust-4D/QRL.haust-4D, and validated in an international panel of 135 wheat accessions. The germplasm, genes and KASP markers were developed for breeders to improve wheat varieties with seed vigor related traits.

Wheat traits and the associated loci conferring radiation use efficiency.

Wheat (Triticum aestivum L.) radiation use efficiency (RUE) must be raised through crop breeding to further increase the yield potential, as the harvest index is now close to its theoretical limit. Field experiments including 209 wheat cultivars which have been widely cultivated in China since the 1940s were conducted in two growing seasons (2018-2019 and 2019-2020) to evaluate the variations of phenological, physiological, plant architectural, and yield-related traits and their contributions to RUE and to identify limiting factors for wheat yield potential. The average annual genetic gain in grain yield was 0.60% (or 45.32 kg ha-1 year-1 ; R2 = 0.44, P < 0.01), mainly attributed to the gain in RUE (r = 0.85, P < 0.01). The net photosynthetic rates were positively and closely correlated with grain RUE and grain yield, suggesting source as a limiting factor to future yield gains. Thirty-four cultivars were identified, exhibiting not only high RUE, but also traits contributing to high RUE and 11 other critical traits - of known genetic basis - as potential parents for breeding to improve yield and RUE. Our findings reveal wheat traits and the associated loci conferring RUE, which are valuable for facilitating marker-assisted breeding to improve wheat RUE and yield potential.

High Resolution Genome Wide Association Studies Reveal Rich Genetic Architectures of Grain Zinc and Iron in Common Wheat (Triticum aestivum L.).

Biofortification is a sustainable strategy to alleviate micronutrient deficiency in humans. It is necessary to improve grain zinc (GZnC) and iron concentrations (GFeC) in wheat based on genetic knowledge. However, the precise dissection of the genetic architecture underlying GZnC and GFeC remains challenging. In this study, high-resolution genome-wide association studies were conducted for GZnC and GFeC by three different models using 166 wheat cultivars and 373,106 polymorphic markers from the wheat 660K and 90K single nucleotide polymorphism (SNP) arrays. Totally, 25 and 16 stable loci were detected for GZnC and GFeC, respectively. Among them, 17 loci for GZnC and 8 for GFeC are likely to be new quantitative trait locus/loci (QTL). Based on gene annotations and expression profiles, 28 promising candidate genes were identified for Zn/Fe uptake (8), transport (11), storage (3), and regulations (6). Of them, 11 genes were putative wheat orthologs of known Arabidopsis and rice genes related to Zn/Fe homeostasis. A brief model, such as genes related to Zn/Fe homeostasis from root uptake, xylem transport to the final seed storage was proposed in wheat. Kompetitive allele-specific PCR (KASP) markers were successfully developed for two major QTL of GZnC on chromosome arms 3AL and 7AL, respectively, which were independent of thousand kernel weight and plant height. The 3AL QTL was further validated in a bi-parental population under multi-environments. A wheat multidrug and toxic compound extrusion (MATE) transporter TraesCS3A01G499300, the ortholog of rice gene OsPEZ2, was identified as a potential candidate gene. This study has advanced our knowledge of the genetic basis underlying GZnC and GFeC in wheat and provides valuable markers and candidate genes for wheat biofortification.

Increased precipitation and nitrogen addition accelerate the temporal increase in soil respiration during 8-year old-field grassland succession.

Ecological succession after disturbance plays a vital role in influencing ecosystem structure and functioning. However, how global change factors regulate ecosystem carbon (C) cycling in successional plant communities remains largely elusive. As part of an 8-year (2012-2019) manipulative experiment, this study was designed to examine the responses of soil respiration and its heterotrophic component to simulated increases in precipitation and atmospheric nitrogen (N) deposition in an old-field grassland undergoing secondary succession. Over the 8-year experimental period, increased precipitation stimulated soil respiration by 11.6%, but did not affect soil heterotrophic respiration. Nitrogen addition increased both soil respiration (5.1%) and heterotrophic respiration (6.2%). Soil respiration and heterotrophic respiration linearly increased with time in the control plots, resulting from changes in soil moisture and shifts of plant community composition from grass-forb codominance to grass dominance in this old-field grassland. Compared to the control, increased precipitation significantly strengthened the temporal increase in soil respiration through stimulating belowground net primary productivity. By contrast, N addition accelerated temporal increases in both soil respiration and its heterotrophic component by driving plant community shifts and thus stimulating soil organic C. Our findings indicate that increases in water and N availabilities may accelerate soil C release during old-field grassland succession and reduce their potential positive impacts on soil C accumulation under future climate change scenarios.

Fine mapping of QPm.caas-3BS, a stable QTL for adult-plant resistance to powdery mildew in wheat (Triticum aestivum L.).

KEY MESSAGE: A stable QTL QPm.caas-3BS for adult-plant resistance to powdery mildew was mapped in an interval of 431 kb, and candidate genes were predicted based on gene sequences and expression profiles. Powdery mildew is a devastating foliar disease occurring in most wheat-growing areas. Characterization and fine mapping of genes for powdery mildew resistance can benefit marker-assisted breeding. We previously identified a stable quantitative trait locus (QTL) QPm.caas-3BS for adult-plant resistance to powdery mildew in a recombinant inbred line population of Zhou8425B/Chinese Spring by phenotyping across four environments. Using 11 heterozygous recombinants and high-density molecular markers, QPm.caas-3BS was delimited in a physical interval of approximately 3.91 Mb. Based on re-sequenced data and expression profiles, three genes TraesCS3B02G014800, TraesCS3B02G016800 and TraesCS3B02G019900 were associated with the powdery mildew resistance locus. Three gene-specific kompetitive allele-specific PCR (KASP) markers were developed from these genes and validated in the Zhou8425B derivatives and Zhou8425B/Chinese Spring population in which the resistance gene was mapped to a 0.3 cM interval flanked by KASP14800 and snp_50465, corresponding to a 431 kb region at the distal end of chromosome 3BS. Within the interval, TraesCS3B02G014800 was the most likely candidate gene for QPm.caas-3BS, but TraesCS3B02G016300 and TraesCS3B02G016400 were less likely candidates based on gene annotations and sequence variation between the parents. These results not only offer high-throughput KASP markers for improvement of powdery mildew resistance but also pave the way to map-based cloning of the resistance gene.

Rht24b, an ancient variation of TaGA2ox-A9, reduces plant height without yield penalty in wheat.

Rht-B1b and Rht-D1b, the 'Green Revolution' (GR) genes, greatly improved yield potential of wheat under nitrogen fertilizer application, but reduced coleoptile length, seedling vigor and grain weight. Thus, mining alternative reduced plant height genes without adverse effects is urgently needed. We isolated the causal gene of Rht24 through map-based cloning and characterized its function using transgenic, physiobiochemical and transcriptome assays. We confirmed genetic effects of the dwarfing allele Rht24b with an association analysis and also traced its origin and distribution. Rht24 encodes a gibberellin (GA) 2-oxidase, TaGA2ox-A9. Rht24b conferred higher expression of TaGA2ox-A9 in stems, leading to a reduction of bioactive GA in stems but an elevation in leaves at the jointing stage. Strikingly, Rht24b reduced plant height, but had no yield penalty; it significantly increased nitrogen use efficiency, photosynthetic rate and the expression of related genes. Evolutionary analysis demonstrated that Rht24b first appeared in wild emmer and was detected in more than half of wild emmer and wheat accessions, suggesting that it underwent both natural and artificial selection. These findings uncover an important genetic resource for wheat breeding and also provide clues for dissecting the regulatory mechanisms underlying GA-mediated morphogenesis and yield formation.