Genetic diversity and population structure of modern wheat (Triticum aestivum L.) cultivars in Henan Province of China based on SNP markers.

BACKGROUND: Henan is the province with the greatest wheat production in China. Although more than 100 cultivars are used for production, many cultivars are still insufficient in quality, disease resistance, adaptability and yield potential. To overcome these limitations, it is necessary to constantly breed new cultivars to maintain the continuous and stable growth of wheat yield and quality. To improve breeding efficiency, it is important to evaluate the genetic diversity and population genetic structure of its cultivars. However, there are no such reports from Henan Province. Therefore, in this study, single nucleotide polymorphism (SNP) markers were used to study the population genetic structure and genetic diversity of 243 wheat cultivars included in a comparative test of wheat varieties in Henan Province, aiming to provide a reference for the utilization of backbone parents and the selection of hybrid combinations in the genetic improvement of wheat cultivars. RESULTS: In this study, 243 wheat cultivars from Henan Province of China were genotyped by the Affymetrix Axiom Wheat660K SNP chip, and 21 characteristics were investigated. The cultivars were divided into ten subgroups; each subgroup had distinct characteristics and unique utilization value. Furthermore, based on principal component analysis, Zhoumai cultivars were the main hybrid parents, followed by Aikang 58, high-quality cultivars, and Shandong cultivars. Genetic diversity analysis showed that 61.3% of SNPs had a high degree of genetic differentiation, whereas 33.4% showed a moderate degree. The nucleotide diversity of subgenome B was relatively high, with an average π value of 3.91E-5; the nucleotide diversity of subgenome D was the lowest, with an average π value of 2.44E-5. CONCLUSION: The parents used in wheat cross-breeding in Henan Province are similar, with a relatively homogeneous genetic background and low genetic diversity. These results will not only contribute to the objective evaluation and utilization of the tested cultivars but also provide insights into the current conditions and existing challenges of wheat cultivar breeding in Henan Province, thereby facilitating the scientific formulation of breeding objectives and strategies to improve breeding efficiency.

HSP90.2 promotes CO2 assimilation rate, grain weight and yield in wheat.

Wheat fixes CO2 by photosynthesis into kernels to nourish humankind. Improving the photosynthesis rate is a major driving force in assimilating atmospheric CO2 and guaranteeing food supply for human beings. Strategies for achieving the above goal need to be improved. Here, we report the cloning and mechanism of CO2 ASSIMILATION RATE AND KERNEL-ENHANCED 1 (CAKE1) from durum wheat (Triticum turgidum L. var. durum). The cake1 mutant displayed a lower photosynthesis rate with smaller grains. Genetic studies identified CAKE1 as HSP90.2-B, encoding cytosolic molecular chaperone folding nascent preproteins. The disturbance of HSP90.2 decreased leaf photosynthesis rate, kernel weight (KW) and yield. Nevertheless, HSP90.2 over-expression increased KW. HSP90.2 recruited and was essential for the chloroplast localization of nuclear-encoded photosynthesis units, for example PsbO. Actin microfilaments docked on the chloroplast surface interacted with HSP90.2 as a subcellular track towards chloroplasts. A natural variation in the hexaploid wheat HSP90.2-B promoter increased its transcription activity, enhanced photosynthesis rate and improved KW and yield. Our study illustrated an HSP90.2-Actin complex sorting client preproteins towards chloroplasts to promote CO2 assimilation and crop production. The beneficial haplotype of Hsp90.2 is rare in modern varieties and could be an excellent molecular switch promoting photosynthesis rate to increase yield in future elite wheat varieties.

HSP90.2 modulates 2Q2-mediated wheat resistance against powdery mildew.

Wheat (Triticum aestivum L.) is a critical food crop feeding the world, but pathogens threaten its production. Wheat Heat Shock Protein 90.2 (HSP90.2) is a pathogen-inducible molecular chaperone folding nascent preproteins. Here, we used wheat HSP90.2 to isolate clients regulated at the posttranslational level. Tetraploid wheat hsp90.2 knockout mutant was susceptible to powdery mildew, while the HSP90.2 overexpression line was resistant, suggesting that HSP90.2 was essential for wheat resistance against powdery mildew. We next isolated 1500 clients of HSP90.2, which contained a wide variety of clients with different biological classifications. We utilized 2Q2, a nucleotide-binding leucine repeat-rich protein, as a model to investigate the potential of HSP90.2 interactome in fungal resistance. The transgenic line co-suppressing 2Q2 was more susceptible to powdery mildew, suggesting 2Q2 as a novel Pm-resistant gene. The 2Q2 protein resided in chloroplasts, and HSP90.2 played a critical role in the accumulation of 2Q2 in thylakoids. Our data provided over 1500 HSP90.2 clients with a potential regulation at the protein folding process and contributed a nontypical approach to isolate pathogenesis-related proteins.

Helitron and CACTA DNA transposons actively reshape the common wheat - AK58 genome.

Transposable elements (TEs) play a pivotal role in reshaping the plant genome. Helitrons represent a new class of transposable elements recently discovered in animals and plants. Helitrons, DNA transposons that replicate via a rolling-circle replication mechanism, are a major driving force behind genome evolution. Since the recent divergence of the modern cultivars (e.g., AK58) and landraces (e.g., Chinese Spring), Helitrons appear to have contributed greatly to genome variability. We first identified 214,665 Helitrons in AK58 by HelitronScanner software and further detected 18,668 tandem duplicated Helitron regions (TDHRs) from all the Helitrons identified. There are 39% of TDHRs (7289) translocated since the divergence of the AK58 and Chinese Spring genomes. What interested us even more are the 462 TDHRs exclusive to the AK58 genome. We also found 235 TDHRs in the 21 centromeric regions and these TDHRs contributed to centromere plasticity. Another very interesting DNA transposon, CACTA, accounting for 15% of AK58 genome, was also the focus of this study because they often inserted into gene rich regions. We found that CACTAs have inserted into many agronomically important genes, such as seed dormancy gene TaMFT and vernalization gene TaVrn1, indicating the important role of CACTAs in modern wheat adaptation.

Intra-Varietal Diversity and Its Contribution to Wheat Evolution, Domestication, and Improvement in Wheat.

Crop genetic diversity is essential for adaptation and productivity in agriculture. A previous study revealed that poor allele diversity in wheat commercial cultivars is a major barrier to its further improvement. Homologs within a variety, including paralogs and orthologs in polyploid, account for a large part of the total genes of a species. Homolog diversity, intra-varietal diversity (IVD), and their functions have not been elucidated. Common wheat, an important food crop, is a hexaploid species with three subgenomes. This study analyzed the sequence, expression, and functional diversity of homologous genes in common wheat based on high-quality reference genomes of two representative varieties, a modern commercial variety Aikang 58 (AK58) and a landrace Chinese Spring (CS). A total of 85,908 homologous genes, accounting for 71.9% of all wheat genes, including inparalogs (IPs), outparalogs (OPs), and single-copy orthologs (SORs), were identified, suggesting that homologs are an important part of the wheat genome. The levels of sequence, expression, and functional variation in OPs and SORs were higher than that of IPs, which indicates that polyploids have more homologous diversity than diploids. Expansion genes, a specific type of OPs, made a great contribution to crop evolution and adaptation and endowed crop with special characteristics. Almost all agronomically important genes were from OPs and SORs, demonstrating their essential functions for polyploid evolution, domestication, and improvement. Our results suggest that IVD analysis is a novel approach for evaluating intra-genomic variations, and exploitation of IVD might be a new road for plant breeding, especially for polyploid crops, such as wheat.

The MYB family transcription factor TuODORANT1 from Triticum urartu and the homolog TaODORANT1 from Triticum aestivum inhibit seed storage protein synthesis in wheat.

Seed storage proteins (SSPs) are determinants of wheat end-product quality. SSP synthesis is mainly regulated at the transcriptional level. Few transcriptional regulators of SSP synthesis have been identified in wheat and this study aims to identify novel SSP gene regulators. Here, the R2R3 MYB transcription factor TuODORANT1 from Triticum urartu was found to be preferentially expressed in the developing endosperm during grain filling. In common wheat (Triticum aestivum) overexpressing TuODORANT1, the transcription levels of all the SSP genes tested by RNA-Seq analysis were reduced by 49.71% throughout grain filling, which contributed to 13.38%-35.60% declines in the total SSP levels of mature grains. In in vitro assays, TuODORANT1 inhibited both the promoter activities and the transcription of SSP genes by 1- to 13-fold. The electrophoretic mobility shift assay (EMSA) and ChIP-qPCR analysis demonstrated that TuODORANT1 bound to the cis-elements 5'-T/CAACCA-3' and 5'-T/CAACT/AG-3' in SSP gene promoters both in vitro and in vivo. Similarly, the homolog TaODORANT1 in common wheat hindered both the promoter activities and the transcription of SSP genes by 1- to 112-fold in vitro. Knockdown of TaODORANT1 in common wheat led to 14.73%-232.78% increases in the transcription of the tested SSP genes, which contributed to 11.43%-19.35% elevation in the total SSP levels. Our data show that both TuODORANT1 and TaODORANT1 are repressors of SSP synthesis.

A novel NAC family transcription factor SPR suppresses seed storage protein synthesis in wheat.

The synthesis of seed storage protein (SSP) is mainly regulated at the transcriptional level. However, few transcriptional regulators of SSP synthesis have been characterized in common wheat (Triticum aestivum) owing to the complex genome. As the A genome donor of common wheat, Triticum urartu could be an elite model in wheat research considering its simple genome. Here, a novel NAC family transcription factor TuSPR from T. urartu was found preferentially expressed in developing endosperm during grain-filling stages. In common wheat transgenically overexpressing TuSPR, the content of total SSPs was reduced by c. 15.97% attributed to the transcription declines of SSP genes. Both in vitro and in vivo assays showed that TuSPR bound to the cis-element 5'-CANNTG-3' distributed in SSP gene promoters and suppressed the transcription. The homolog in common wheat TaSPR shared a conserved function with TuSPR on SSP synthesis suppression. The knock-down of TaSPR in common wheat resulted in 7.07%-20.34% increases in the total SSPs. Both TuSPR and TaSPR could be superior targets in genetic engineering to manipulate SSP content in wheat, and this work undoubtedly expands our knowledge of SSP gene regulation.

TubZIP28, a novel bZIP family transcription factor from Triticum urartu, and TabZIP28, its homologue from Triticum aestivum, enhance starch synthesis in wheat.

Starch in wheat grain provides humans with carbohydrates and influences the quality of wheaten food. However, no transcriptional regulator of starch synthesis has been identified first in common wheat (Triticum aestivum) due to the complex genome. Here, a novel basic leucine zipper (bZIP) family transcription factor TubZIP28 was found to be preferentially expressed in the endosperm throughout grain-filling stages in Triticum urartu, the A genome donor of common wheat. When TubZIP28 was overexpressed in common wheat, the total starch content increased by c. 4%, which contributed to c. 5% increase in the thousand kernel weight. The grain weight per plant of overexpression wheat was also elevated by c. 9%. Both in vitro and in vivo assays showed that TubZIP28 bound to the promoter of cytosolic AGPase and enhanced both the transcription and activity of the latter. Knockout of the homologue TabZIP28 in common wheat resulted in declines of both the transcription and activity of cytosolic AGPase in developing endosperms and c. 4% reduction of the total starch in mature grains. To the best of our knowledge, TubZIP28 and TabZIP28 are transcriptional activators of starch synthesis first identified in wheat, and they could be superior targets to improve the starch content and yield potential of wheat.

Identification and characterization of CircRNAs involved in the regulation of wheat root length.

BACKGROUND: Recent studies indicate that circular RNAs (circRNAs) may play important roles in the regulation of plant growth and development. Plant roots are the main organs of nutrient and water uptake. However, whether circRNAs involved in the regulation of plant root growth remains to be elucidated. METHODS: LH9, XN979 and YN29 are three Chinese wheat varieties with contrasting root lengths. Here, the root circRNA expression profiles of LH9, XN979 and YN29 were examined by using high-throughput sequencing technology. RESULTS: Thirty-three and twenty-two differentially expressed circRNAs (DECs) were identified in the YN29-LH9 comparison and YN29-XN979 comparison, respectively. Among them, ten DECs coexisted in both comparisons. As the roots of both LH9 and XN979 were significantly larger and deeper than YN29, the ten DECs coexisting in the two comparisons were highly likely to be involved in the regulation of wheat root length. Moreover, three of the ten DECs have potential miRNA binding sites. Real-time PCR analysis showed that the expression levels of the potential binding miRNAs exhibited significant differences between the long root plants and the short root plants. CONCLUSIONS: The expression levels of some circRNAs exhibited significant differences in wheat varieties with contrasting root phenotypes. Ten DECs involved in the regulation of wheat root length were successfully identified in which three of them have potential miRNAs binding sites. The expression levels of putative circRNA-binding miRNAs were correlated with their corresponding circRNAs. Our results provide new clues for studying the potential roles of circRNAs in the regulation of wheat root length.

Identification of Proteins Using iTRAQ and Virus-Induced Gene Silencing Reveals Three Bread Wheat Proteins Involved in the Response to Combined Osmotic-Cold Stress.

Crops are often subjected to a combination of stresses in the field. To date, studies on the physiological and molecular responses of common wheat to a combination of osmotic and cold stresses, however, remain unknown. In this study, wheat seedlings exposed to osmotic-cold stress for 24 h showed inhibited growth, as well as increased lipid peroxidation, relative electrolyte leakage, and soluble sugar contents. iTRAQ-based quantitative proteome method was employed to determine the proteomic profiles of the roots and leaves of wheat seedlings exposed to osmotic-cold stress conditions. A total of 250 and 258 proteins with significantly altered abundance in the roots and leaves were identified, respectively, and the majority of these proteins displayed differential abundance, thereby revealing organ-specific differences in adaptation to osmotic-cold stress. Yeast two hybrid assay examined five pairs of stress/defense-related protein-protein interactions in the predicted protein interaction network. Furthermore, quantitative real-time PCR analysis indicated that abiotic stresses increased the expression of three candidate protein genes, i.e., TaGRP2, CDCP, and Wcor410c in wheat leaves. Virus-induced gene silencing indicated that three genes TaGRP2, CDCP, and Wcor410c were involved in modulating osmotic-cold stress in common wheat. Our study provides useful information for the elucidation of molecular and genetics bases of osmotic-cold combined stress in bread wheat.

Identification of Winter-Responsive Proteins in Bread Wheat Using Proteomics Analysis and Virus-Induced Gene Silencing (VIGS).

Proteomic approaches were applied to identify protein spots involved in cold responses in wheat. By comparing the differentially accumulated proteins from two cultivars (UC1110 and PI 610750) and their derivatives, as well as the F10 recombinant inbred line population differing in cold-tolerance, a total of 20 common protein spots representing 16 unique proteins were successfully identified using 2-DE method. Of these, 14 spots had significantly enhanced abundance in the cold-sensitive parental cultivar UC1110 and its 20 descendant lines when compared with the cold-tolerant parental cultivar PI 610750 and its 20 descendant lines. Six protein spots with reduced abundance were also detected. The identified protein spots are involved in stress/defense, carbohydrate metabolism, protein metabolism, nitrogen metabolism, energy metabolism, and photosynthesis. The 20 differentially expressed protein spots were chosen for quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression changes at the RNA level. The results indicated that the transcriptional expression patterns of 11 genes were consistent with their protein expression models. Among the three unknown proteins, Spot 20 (PAP6-like) showed high sequence similarities with PAP6. qRT-PCR results implied that cold and salt stresses increased the expression of PAP6-like in wheat leaves. Furthermore, VIGS (virus-induced gene silencing)-treated plants generated for PAP6-like were subjected to freezing stress, these plants had more serious droop and wilt, an increased rate of relative electrolyte leakage, reduced relative water content (RWC) and decreased tocopherol levels when compared with viral control plants. However, the plants that were silenced for the other two unknown proteins had no significant differences in comparison to the BSMV0-inoculated plants under freezing conditions. These results indicate that PAP6-like possibly plays an important role in conferring cold tolerance in wheat.

De novo assembly and comparative analysis of the transcriptome of embryogenic callus formation in bread wheat (Triticum aestivum L.).

BACKGROUND: During asexual reproduction the embryogenic callus can differentiate into a new plantlet, offering great potential for fostering in vitro culture efficiency in plants. The immature embryos (IMEs) of wheat (Triticum aestivum L.) are more easily able to generate embryogenic callus than mature embryos (MEs). To understand the molecular process of embryogenic callus formation in wheat, de novo transcriptome sequencing was used to generate transcriptome sequences from calli derived from IMEs and MEs after 3d, 6d, or 15d of culture (DC). RESULTS: In total, 155 million high quality paired-end reads were obtained from the 6 cDNA libraries. Our de novo assembly generated 142,221 unigenes, of which 59,976 (42.17%) were annotated with a significant Blastx against nr, Pfam, Swissprot, KOG, KEGG, GO and COG/KOG databases. Comparative transcriptome analysis indicated that a total of 5194 differentially expressed genes (DEGs) were identified in the comparisons of IME vs. ME at the three stages, including 3181, 2085 and 1468 DEGs at 3, 6 and 15 DC, respectively. Of them, 283 overlapped in all the three comparisons. Furthermore, 4731 DEGs were identified in the comparisons between stages in IMEs and MEs. Functional analysis revealed that 271transcription factor (TF) genes (10 overlapped in all 3 comparisons of IME vs. ME) and 346 somatic embryogenesis related genes (SSEGs; 35 overlapped in all 3 comparisons of IME vs. ME) were differentially expressed in at least one comparison of IME vs. ME. In addition, of the 283 overlapped DEGs in the 3 comparisons of IME vs. ME, excluding the SSEGs and TFs, 39 possessed a higher rate of involvement in biological processes relating to response to stimuli, in multi-organism processes, reproductive processes and reproduction. Furthermore, 7 were simultaneously differentially expressed in the 2 comparisons between the stages in IMEs, but not MEs, suggesting that they may be related to embryogenic callus formation. The expression levels of genes, which were validated by qRT-PCR, showed a high correlation with the RNA-seq value. CONCLUSIONS: This study provides new insights into the role of the transcriptome in embryogenic callus formation in wheat, and will serve as a valuable resource for further studies addressing embryogenic callus formation in plants.

Genome-wide association study for 13 agronomic traits reveals distribution of superior alleles in bread wheat from the Yellow and Huai Valley of China.

Bread wheat is a leading cereal crop worldwide. Limited amount of superior allele loci restricted the progress of molecular improvement in wheat breeding. Here, we revealed new allelic variation distribution for 13 yield-related traits in series of genome-wide association studies (GWAS) using the wheat 90K genotyping assay, characterized in 163 bread wheat cultivars. Agronomic traits were investigated in 14 environments at three locations over 3 years. After filtering SNP data sets, GWAS using 20 689 high-quality SNPs associated 1769 significant loci that explained, on average, ~20% of the phenotypic variation, both detected already reported loci and new promising genomic regions. Of these, repetitive and pleiotropic SNPs on chromosomes 6AS, 6AL, 6BS, 5BL and 7AS were significantly linked to thousand kernel weight, for example BS00021705_51 on 6BS and wsnp_Ex_c32624_41252144 on 6AS, with phenotypic variation explained (PVE) of ~24%, consistently identified in 12 and 13 of the 14 environments, respectively. Kernel length-related SNPs were mainly identified on chromosomes 7BS, 6AS, 5AL and 5BL. Plant height-related SNPs on chromosomes 4DS, 6DL, 2DS and 1BL were, respectively, identified in more than 11 environments, with averaged PVE of ~55%. Four SNPs were confirmed to be important genetic loci in two RIL populations. Based on repetivity and PVE, a total of 41 SNP loci possibly played the key role in modulating yield-related traits of the cultivars surveyed. Distribution of superior alleles at the 41 SNP loci indicated that superior alleles were getting popular with time and modern cultivars had integrated many superior alleles, especially for peduncle length- and plant height-related superior alleles. However, there were still 19 SNP loci showing less than percentages of 50% in modern cultivars, suggesting they should be paid more attention to improve yield-related traits of cultivars in the Yellow and Huai wheat region. This study could provide useful information for dissection of yield-related traits and valuable genetic loci for marker-assisted selection in Chinese wheat breeding programme.

Development of an integrated linkage map of einkorn wheat and its application for QTL mapping and genome sequence anchoring.

KEY MESSAGE: An integrated genetic map was constructed for einkorn wheat A genome and provided valuable information for QTL mapping and genome sequence anchoring. Wheat is one of the most widely grown food grain crops in the world. The construction of a genetic map is a key step to organize biologically or agronomically important traits along the chromosomes. In the present study, an integrated linkage map of einkorn wheat was developed using 109 recombinant inbred lines (RILs) derived from an inter sub-specific cross, KT1-1 (T. monococcum ssp. boeoticum) × KT3-5 (T. monococcum ssp. monococcum). The map contains 926 molecular markers assigned to seven linkage groups, and covers 1,377 cM with an average marker interval of 1.5 cM. A quantitative trait locus (QTL) analysis of five agronomic traits identified 16 stable QTL on all seven chromosomes, except 6A. The total phenotypic variance explained by these stable QTL using multiple regressions varied across environments from 8.8 to 87.1 % for days to heading, 24.4-63.0 % for spike length, 48.2-79.6 % for spikelet number per spike, 13.1-48.1 % for plant architecture, and 12.2-26.5 % for plant height, revealing that much of the RIL phenotypic variation had been genetically dissected. Co-localizations of closely linked QTL for different traits were frequently observed, especially on 3A and 7A. The QTL on 3A, 5A and 7A were closely associated with Eps-A m 3, Vrn1 and Vrn3 loci, respectively. Furthermore, this genetic map facilitated the anchoring of 237 T. urartu scaffolds onto seven chromosomes with a physical length of 26.15 Mb. This map and the QTL data provide valuable genetic information to dissect important agronomic and developmental traits in diploid wheat and contribute to the genetic ordering of the genome assembly.

ITRAQ-Based Proteomic Analysis of the Metabolic Mechanisms Behind Lipid Accumulation and Degradation during Peanut Seed Development and Postgermination.

Peanut seeds have a high oil content making them an important oil crop. During development and germination, seeds undergo complex dynamic and physiological changes. Changes in lipid metabolism and underlying mechanisms during seed development have been studied extensively by DNA and RNA sequencing; however, there are few studies on dynamic changes of proteomics during peanut seed development and germination. In this study, proteomic analyses were carried out 20, 40, 60, and 80 days after pollination and 5, 10, 20, and 30 days after germination using isobaric tags for relative and absolute quantitation (iTRAQ) technology to determine the protein profiles of lipid dynamics during peanut seed development and postgermination. A total of 5712 of 8505 proteins were identified, quantified, and divided into 23 functional groups, the largest of which was metabolism-related. Further analyses of the proteins and their pathways revealed initiation of fatty acid accumulation at early stages after flowering, while lipid degradation occurred largely through the lipoxygenase-dependent pathway. Protein expression patterns related to lipid accumulation and degradation were also verified at transcript levels using quantitative real-time polymerase chain reaction. The proteome profiles determined here will significantly enrich our understanding of the process of lipid accumulation and degradation and the dynamic changes in metabolic networks during peanut development.

Molecular characterization of lipoxygenase genes on chromosome 4BS in Chinese bread wheat (Triticum aestivum L.).

KEY MESSAGE: This study cloned two novel TaLox genes on chromosome of 4BS and developed a co-dominant marker, Lox-B23, in bread wheat that showed highly significant association with lipoxygenase activity. Lipoxygenase (Lox), a critical enzyme in the carotenoid biosynthetic pathway, significantly influences the color and processing quality of wheat-based products. Two novel Lox genes, designated TaLox-B2 and TaLox-B3, were cloned on chromosome 4BS of Chinese bread wheat. The deduced amino acid sequence showed that both TaLox-B2 and TaLox-B3 genes encoded an 861-aa protein and possessed a lipoxygenase superfamily domain at the 170-838 interval. Two different TaLox-B2 alleles, designated TaLox-B2a and TaLox-B2b, were subsequently discovered. A co-dominant marker, Lox-B23, was developed based on sequences of TaLox-B2a, TaLox-B2b, and TaLox-B3 genes to precisely distinguish these three alleles in Chinese bread cultivars. Among five allelic combinations of Lox genes at Lox-B1, Lox-B2, and Lox-B3 loci, wheat cultivars with TaLox-B1a/TaLox-B2a/TaLox-B3a combination exhibited the highest Lox activity, whereas those with TaLox-B1a/TaLox-B2b/TaLox-B3b combination significantly showed the lowest Lox activity. A RIL population was used to evaluate the influence of TaLox-B3a gene on Lox activity. Results showed that TaLox-B3a gene could significantly increase the Lox activity in bread wheat. Physical mapping indicated that both TaLox-B2 and TaLox-B3 genes were located on chromosome 4BS in bread wheat. This study provides useful information to further understand the molecular and genetic bases of Lox activity in bread wheat.

Discovery, distribution and diversity of Puroindoline-D1 genes in bread wheat from five countries (Triticum aestivum L.).

BACKGROUND: Grain texture is one of the most important characteristics in bread wheat (Triticum aestivum L.). Puroindoline-D1 genes play the main role in controlling grain texture and are intimately associated with the milling and processing qualities in bread wheat. RESULTS: A series of diagnostic molecular markers and dCAPS markers were used to characterize Pina-D1 and Pinb-D1 in 493 wheat cultivars from diverse geographic locations. A primer walking strategy was used to characterize PINA-null alleles at the DNA level. Results indicated that Chinese landraces encompassing 12 different Puroindoline-D1 allelic combinations showed the highest diversity, while CIMMYT wheat cultivars containing 3 different Puroindoline-D1 allelic combinations showed the lowest diversity amongst wheat cultivars from the five countries surveyed. Two novel Pina-D1 alleles, designated Pina-D1s with a 4,422-bp deletion and Pina-D1u with a 6,460-bp deletion in the Ha (Hardness) locus, were characterized at the DNA level by a primer walking strategy, and corresponding molecular markers Pina-N3 and Pina-N4 were developed for straightforward identification of the Pina-D1s and Pina-D1u alleles. Analysis of the association of Puroindoline-D1 alleles with grain texture indicated that wheat cultivars with Pina-null/Pinb-null allele, possessing an approximate 33-kb deletion in the Ha locus, have the highest SKCS hardness index amongst the different genotypes used in this study. Moreover, wheat cultivars with the PINA-null allele have significantly higher SKCS hardness index than those of Pinb-D1b and Pinb-D1p alleles. CONCLUSIONS: Molecular characterization of the Puroindoline-D1 allele was investigated in bread wheat cultivars from five geographic regions, resulting in the discovery of two new alleles - Pina-D1s and Pina-D1u. Molecular markers were developed for both alleles. Analysis of the association of the Puroindoline-D1 alleles with grain texture showed that cultivars with PINA-null allele possessed relatively high SKCS hardness index. This study can provide useful information for the improvement of wheat quality, as well as give a deeper understanding of the molecular and genetic processes controlling grain texture in bread wheat.

Molecular characterization of vernalization and response genes in bread wheat from the Yellow and Huai Valley of China.

BACKGROUND: Flowering time greatly influences the adaptation of wheat cultivars to diverse environmental conditions and is mainly controlled by vernalization and photoperiod genes. In wheat cultivars from the Yellow and Huai Valleys, which represent 60%-70% of the total wheat production in China, the large-scale genotyping of wheat germplasms has not yet been performed in terms of vernalization and photoperiod response alleles, limiting the use of Chinese wheat germplasms to a certain extent. RESULTS: In this study, 173 winter wheat cultivars and 51 spring wheat cultivars from China were used to identify allelic variations of vernalization and photoperiod genes as well as copy number variations of Ppd-B1 and Vrn-A1. Two new co-dominant markers were developed in order to more precisely examine Vrn-A1b, Vrn-B1a, and Vrn-B1b. Two novel alleles at the Vrn-B3 locus were discovered and were designated Vrn-B3b and Vrn-B3c. Vrn-B3b had an 890-bp insertion in the promoter region of the recessive vrn-B3 allele, and Vrn-B3c allele had 2 deletions (a 20-bp deletion and a 4-bp deletion) in the promoter region of the dominant Vrn-B3a allele. Cultivar Hemai 26 lacked the Vrn-A1 gene. RT-PCR indicated that the 890-bp insertion in the Vrn-B3b allele significantly reduced the transcription of the Vrn-B3 gene. Cultivars Chadianhong with the Vrn-B3b allele and Hemai 26 with a Vrn-A1-null allele possessed relatively later heading and flowering times compared to those of Yanzhan 4110, which harbored recessive vrn-B3 and vrn-A1 alleles. Through identification of photoperiod genes, 2 new polymorphism combinations were found in 6 winter wheat cultivars and were designated Hapl-VII and Hapl-VIII, respectively. Distribution of the vernalization and photoperiod genes indicated that all recessive alleles at the 4 vernalization response loci, truncated "Chinese Spring" Ppd-B1 allele at Ppd-B1 locus and Hapl-I at the Ppd-D1 locus were predominant in Chinese winter wheat cultivars. CONCLUSION: This study illustrated the distribution of vernalization and photoperiod genes and identified 2 new Vrn-B3 alleles, 1 Vrn-A1-null allele, and two new Ppd-D1 polymorphism combinations, using developed functional markers. Results of this study have the potential to provide useful information for screening relatively superior wheat cultivars for better adaptability and maturity.

Novel insights into the composition, variation, organization, and expression of the low-molecular-weight glutenin subunit gene family in common wheat.

Low-molecular-weight glutenin subunits (LMW-GS), encoded by a complex multigene family, play an important role in the processing quality of wheat flour. Although members of this gene family have been identified in several wheat varieties, the allelic variation and composition of LMW-GS genes in common wheat are not well understood. In the present study, using the LMW-GS gene molecular marker system and the full-length gene cloning method, a comprehensive molecular analysis of LMW-GS genes was conducted in a representative population, the micro-core collections (MCC) of Chinese wheat germplasm. Generally, >15 LMW-GS genes were identified from individual MCC accessions, of which 4-6 were located at the Glu-A3 locus, 3-5 at the Glu-B3 locus, and eight at the Glu-D3 locus. LMW-GS genes at the Glu-A3 locus showed the highest allelic diversity, followed by the Glu-B3 genes, while the Glu-D3 genes were extremely conserved among MCC accessions. Expression and sequence analysis showed that 9-13 active LMW-GS genes were present in each accession. Sequence identity analysis showed that all i-type genes present at the Glu-A3 locus formed a single group, the s-type genes located at Glu-B3 and Glu-D3 loci comprised a unique group, while high-diversity m-type genes were classified into four groups and detected in all Glu-3 loci. These results contribute to the functional analysis of LMW-GS genes and facilitate improvement of bread-making quality by wheat molecular breeding programmes.

Alveograph and Mixolab parameters associated with Puroindoline-D1 genes in Chinese winter wheats.

BACKGROUND: Grain texture is one of the most important characteristics of bread wheat and has a significant influence on end-use qualities. RESULTS: Forty-three Chinese cultivars were tested under three environments and used to characterise kernel hardness, Puroindoline-D1 alleles and Alveograph and Mixolab parameters. The results indicated that SKCS hardness was positively correlated with Alveograph tenacity and P/L and Mixolab protein weakening (C2) and water absorption and negatively correlated with Mixolab starch gelatinisation (C3), amylasic activity (C4) and starch gelling (C5). Variance analysis showed that Puroindoline-D1 had a significant impact on SKCS hardness and most Alveograph and Mixolab parameters. Furthermore, among three Puroindoline-D1 genotypes, PINA-null/Pinb-D1a possessed the highest SKCS hardness, Alveograph tenacity and W and Mixolab stability and water absorption but the lowest Alveograph extensibility and G and Mixolab C3, C4 and C5. Pina-D1a/Pinb-D1a had the lowest SKCS hardness, Alveograph tenacity and W and Mixolab C2, water absorption and stability but the highest Alveograph extensibility and G and Mixolab C3, C4 and C5. Pina-D1a/Pinb-D1b possessed the lowest Mixolab C2 - C1, C3 - C2, C4 - C3 and C5 - C4. CONCLUSION: Pina-D1a/Pinb-D1a was softer and had lower tenacity and water absorption. PINA-null/Pinb-D1a was harder and had higher tenacity and water absorption. Pina-D1a/Pinb-D1b had lower difference values among Mixolab parameters.