Genome-wide association mapping of phenolic acids in tetraploid wheats

Phenolic acids are major components of cell walls in wheat and have important implications on human health as antioxidants with anti-tumor activity. Our objectives were to identify phenolic acid genes in wheat by single nucleotide polymorphisms (SNPs) detected within the coding sequences of candidate genes, and to identify chromosomal regions associated with single phenolic acids and total soluble phenolic compounds. A set of candidate genes involved in the biosynthesis of hydroxycinnamic acid derivatives were identified by comparative genomics. SNPs found in the coding sequences of six genes (PAL1, PAL2, C4H, C3H, COMT1 and COMT2) were used to determine their chromosomal location and accurate map position on two reference consensus linkage maps. The genome-wide association study (GWAS), based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs, detected 22 quantitative trait loci (QTL) distributed on almost all durum wheat chromosomes. Two QTL for p-coumaric acid were coincident with the phenylalanine ammonia-lyase (PAL2) and p-coumarate 3-hydroxylase (C3H) genes on chromosome arms 2AL and 1AL, respectively. The availability of candidate gene-based markers can allow elucidating the mechanism of phenolic acids accumulation in wheat kernels and exploiting the genetic variability of phenolic acids content for the nutritional improvement of wheat end-products.     

紫娟茶树叶片不同发育期花青素积累及合成相关基因的表达

为明确紫娟茶树（Camellia sinensis cv. Zijuan）叶片发育中花青素积累特性及合成途径上相关基因的表达特点,利用液质联用法（HPLC-MS）、转录组测序（RNA-Seq）和数字基因表达谱技术（DGE）,分析了紫娟茶树芽、第二叶、开面叶和成熟叶4个发育期花青素的种类、含量及合成相关基因的表达水平。结果表明,花青素积累量随叶片发育先增加后减少,第二叶含量最大（9.87βmg·g^-1）、成熟叶含量最小（0.11βmg·g^-1）,与叶色表现相吻合。结构基因PAL在芽、第二叶和开面叶高表达,在成熟叶下调表达;C4H、4CL、CHS、CHI、F3H、F3'H、F3'5'H和ANS表达模式相似,表达量均随叶片发育而降低,在芽期高表达,在成熟叶全部下调表达;FLS在第二叶上调表达,在成熟叶下调表达,与花青素积累情况一致;DFR在各发育期均有上调和下调表达。GT和ACT表达模式相似,在第二叶、开面叶和成熟叶上调表达;ANR和LAR表达模式相似,在芽、第二叶和开面叶高表达,在成熟叶下调表达。bHLH、MYB和WDR在各发育期均有上调或下调表达。说明紫娟茶树叶片不同发育阶段结构基因和调控基因的表达水平不同,导致花青素积累存在差异,具有一定的时间表达特异性。     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

Novel allelic variants encoded at the Glu-D3 locus in bread wheat

Low-molecular weight glutenin subunits (LWM-GS) are important components of wheat (Triticum aestivum L.) gluten, with important effects on end-use quality. The LMW-GS are encoded at Glu-3 loci (Glu-A3, Glu-B3 and Glu-D3, on the short arms of chromosomes 1A, 1B and 1D), each of which exhibits extensive allelic variation. Each locus encodes numerous LMW-GS, some of which have similar electrophoretic mobilities, making it difficult to distinguish among Glu-3 loci. Alleles of the Glu-D3 locus of bread wheat are considered the most problematic to assign. To date, six Glu-D3 alleles, designated a, b, c, d, e and f, have been reported. We report five previously undescribed alleles (g, h, i, j and k), and describe a method for characterizing them using a combination of SDS-PAGE and multiplexed PCR-based DNA markers. This method could be used for accurate identification of Glu-D3 alleles, permitting the estimation of the effects of these alleles on end-use quality and the selection of desirable alleles and allelic combinations in wheat breeding.     

Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunits (LMW-GS) play a key role in determining the processing quality of the end-use products of common wheat. The objectives of this study were to identify genes at Glu-A3 locus, develop the STS markers, and establish multiplex PCR with the STS markers for Glu-A3 alleles. Gene-specific PCR primers were designed to amplify six near-isogenic lines (NILs) and Glenlea with different Glu-A3 alleles (a, b, c, d, e, f and g) defined by the protein electrophoretic mobility. Three Glu-A3 genes with complete coding sequence were cloned, designated as GluA3-1, GluA3-2 and GluA3-3, respectively. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs (single nucleotide polymorphisms) among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. Four multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, and Glu-A3b + Glu-A3e. These markers and multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.     

矮秆基因在中国不同麦区小麦品种中的分布及其对赤霉病抗性的影响

为研究矮秆基因在中国不同麦区的分布,以及株高和小穗密度与赤霉病抗性的相关性,本研究通过分析3个矮秆基因 Rht1、 Rht2和 Rht8在211份不同麦区小麦自然群体中的分布,并结合其在不同环境下株高、小穗密度以及赤霉病抗性的调查数据,分析矮秆基因 Rht1、 Rht2和 Rht8对赤霉病抗性的影响。结果表明：（1）不同麦区矮秆基因的分布频率差异较大, Rht1主要分布在长江中下游麦区, Rht2和 Rht8主要分布在黄淮麦区;（2）与野生型品种相比,携带 Rht1、 Rht2和 Rht8的小麦品种,株高均显著或极显著降低;（3）携带 Rht2和 Rht8的小麦品种,赤霉病抗性均极显著低于野生型品种,而携带 Rht1的小麦品种,赤霉病抗性则极显著高于野生型品种;（4）携带 Rht2和 Rht8的小麦品种,小穗密度显著大于野生型品种,而携带 Rht1的小麦品种,小穗密度则显著降低。因此,不同矮秆基因对小穗密度性状的遗传差异可能是导致小麦赤霉病抗性和不同麦区矮秆基因选择利用差异的部分原因。     

黄淮麦区小麦种质资源矮秆基因分布及其与农艺性状的关系

为了进一步阐明多个矮秆基因的分布及其与小麦农艺性状的关系,运用分子标记对来自我国黄淮麦区的246份小麦种质资源中6个矮秆基因位点(Rht1、Rht2、Rht4、Rht8、Rht9及Rht12)分别进行了检测,同时连续3年调查参试材料株高、穗长、穗下节长、小穗数、旗叶长、旗叶宽、穗粒数、粒长、粒宽和千粒重共10个农艺性状,分析了不同矮秆基因位点对小麦农艺性状的影响。结果表明,6个矮秆基因在黄淮麦区小麦中均具有广泛分布,其中含有Rht1和Rht2基因的小麦品种分布最广。分析矮秆基因位点对小麦农艺性状的影响发现,在Rht1位点,Rht1-B1a和Rht1-B1b两种基因型间的株高没有显著差异;在Rht2位点,拥有Rht2-D1b类型的小麦品种所有年份间的株高和穗下节长较低,但千粒重较高,为相对优良的基因型。排除Rht1和Rht2基因效应后,Rht4、Rht8、Rht9和Rht12位点对黄淮麦区小麦品种不同农艺性状均具有重要影响,其中,Rht4基因位点主要对小麦株高和千粒重具有重要影响,且Rht4-B1b类型为相对优良的基因型;Rht8基因位点主要对小麦穗下节长、穗长和千粒重具有重要影响,且Rht8-D1b类型为相对优良的基因型;Rht9基因位点主要对小麦株高和千粒重具有重要影响,且Rht9-A1a类型为相对优良的基因型;Rht12基因位点主要对小麦千粒重和穗长具有重要影响,且Rht12-A1a类型为相对优良的基因型。进一步分析发现,6个位点中对株高影响最大的是Rht2基因,其次是Rht4基因;有4个位点(Rht1、Rht2、Rht8、Rht12)对千粒重有显著影响,其中Rht2基因的影响最大。分析除Rht1外其他5个位点优良基因型在不同时期小麦品种中的分布发现,从早期历史品种、近期历史品种到现代品种,不同位点优良基因型分布比例总体呈现上升趋势,表明优良矮秆基因型在黄淮麦区小麦品种选育中的利用逐渐增加,尤其是82.9%的现代小麦品种已含有Rht2-D1b类型。     

A novel Puroindoline b-2 variant present in Chinese winter wheat cultivar Yunong 202

The recently identified Puroindoline b-2 (Pinb-2) variants on the homoeologous group 7 chromosomes in bread wheat are Puroindoline-like genes that account for minor grain texture modulation and display activity against bacteria and phytopathogenic fungi. In this study, the coding sequence of the Pinb-2 gene in the Chinese winter wheat cultivar Yunong 202 (Triticum aestivum L.) was amplified with the universal primer, and the obtained 452-bp fragment was cloned and 36 plasmids encompassing the targeted fragment were sequenced from two strands. Results indicated that Yunong 202 possessed Pinb-2v1, Pinb-2v2, Pinb-2v3b and Pinb-2v4 alleles. A new Puroindoline-2 variant was discovered in Yunong 202 as well and was designated as Pinb-2v6. Full alignment showed that Pinb-2v6 possessed 74.0%, 95.4%, 94.7%, 92.3%, 98.7% and 98.0% identity at the DNA level with Pinb-D1a, Pinb-2v1, Pinb-2v2, Pinb-2v3a, Pinb-2v4 and Pinb-2v5 alleles, respectively. This study may provide useful information for further understanding the molecular and genetics basis of grain texture and for illustrating gene duplication events in wheat.     

Anthocyanin accumulation,biosynthesis and antioxidant capacity of black sweet corn (Zea mays L.) during kernel development over two growing seasons

In this study, the content changes of anthocyanins levels, antioxidant activities as well as related genes in their biosynthesis pathways of two seasons black sweet corn kernels were determined. Generally, three anthocyanins contents showed similar trend with peak at 10 days after pollination (DAP) in both seasons existing as free forms, and they gradually grew afterwards as kernel matured. Pelargonidin as the leading component was decreased significantly in autumn comparing to contents in summer which may be affected by lower photosynthetically active radiation (PAR) and distinct gene expression levels. CF1, F3H and LAD as the essential genes in flavonoid and anthocyanin biosynthesis had high expression levels indicating higher production of flavonoid and anthocyanin. Black sweet corn (BSC) exhibited good in vitro and cellular antioxidant abilities compared to many fruits and vegetables indicating that BSC could be developed as a substitute food with high anthocyanin profiles and antioxidant capacity.     

Association of Puroindoline b-B2 variants with grain traits,yield components and flag leaf size in bread wheat (Triticum aestivum L.) varieties of the Yellow and Huai Valleys of China

A total of 169 wheat (Triticum aestivum L.) varieties (landraces and cultivars) were used to asses the relationship between Puroindoline D1 alleles and Puroindoline b-B2 variants and grain hardness, other grain traits, yield components, and flag leaf size. Results indicated that the average SKCS hardness of Pinb-B2v3 varieties was significantly greater than that of Pinb-B2v2 varieties within the soft Puroindoline D1 haplotype sub-group. Conversely, no statistically significant difference was obtained for SKCS hardness between varieties with the Pinb-B2v3 vs. Pinb-B2v2 alleles within the two hard Puroindoline D1 haplotypes (Pinb-D1b and Pinb-D1p sub-groups). Therefore, the Puroindoline b-B2 gene may have a bigger impact on soft wheat varieties than hard. Across all varieties, thousand-kernel weight, grain weight per spike, grain diameter, grain number per spike, flag leaf width and area of Pinb-B2v3 varieties were significantly greater than those possessing Pinb-B2v2. These results indicated that the Pinb-B2v3 allele was associated with preferable grain yield traits compared to the Pinb-B2v2 allele in bread wheat. This study provides evocative information for better understanding the molecular and genetic basis of wheat grain yield.     

Cloning and expression analysis of kenaf (Hibiscus cannabinus L.) major lignin and cellulose biosynthesis gene sequences and polymer quantification during plant development

In order to apply state-of-the-art molecular breeding techniques in fibre crop it is necessary to have a good knowledge of major polymer biosynthesis gene sequences and their expression pattern. Polymerase chain reaction was employed to isolate sequences of the major genes for lignin and cellulose biosynthesis in a kenaf cultivar. CeSA, 4cl, c4h, cad, and ccr gene primers were designed according to their conservative regions; partial sequences of lignin and cellulose biosynthesis genes were obtained. One actin II gene sequence was also isolated from the kenaf genome as a housekeeping gene to be employed in qPCR analysis. Expression levels of genes c4h, cad and CeSA in bark and core from plants harvested at three different growth stages were evaluated. Using qPCR analyses it was found that the expression levels of the two biosynthesis lignin genes in bark tissues increased during plant growth, while a negative trend was recorded in core tissues. In both bark and core, the quantity of lignin was positively correlated to plant growth while cellulose content decreased.     

Genetic characterization of kernel polyphenol oxidases in wheat and related species

Polyphenol oxidase (PPO) activity causes undesirable darkening of raw Asian noodles and other wheat products. In this study we investigate the genetic origins and diversity of wheat kernel PPO. PPO was characterized via activity assays, antigenic staining, and Southern blots in Triticum aestivum, Triticum dicoccoides, Triticum durum, Triticum dicoccum, Triticum monococcum, Triticum urartu, Aegilops speltoides, and Aegilops tauschii. Among these species, PPO activity was well-correlated with antigenic staining intensity toward a wheat kernel-type PPO antibody. High PPO activity was observed in all three T. monococcum accessions (A^m genome), one Ae. speltoides accession, one T. durum accession, and two hexaploid wheat cultivars. Southern blots suggested the presence of two or more kernel-type PPO genes in diploid progenitors of the hexaploid A, B, and D genomes. Whole-kernel PPO activity was evaluated in disomic substitution lines derived from three T. dicoccoides accessions in the background of T. durum ‘Langdon’. PPO activity was primarily associated with chromosome 2A and to a much lower degree with chromosome 2B. DNA sequence comparisons showed that the intron associated with the high PPO allele on chromosome 2AL of hexaploid wheat had 94% nucleotide identity with the homeologous intron found in T. monococcum, a species with high kernel PPO activity. This implies that the ancestral PPO allele on the A genome is one of the high activity, and the low PPO allele found in hexaploid wheat represents a relatively recent genetic alteration. Results confirm the presence of multiple kernel-type PPO genes in the diploid and tetraploid progenitors and relatives of hexaploid wheat. However, it is likely that relatively few of the many kernel-type PPO genes present in wheat contribute substantially to kernel PPO activity. A single genetic locus on homeologous group 2 chromosomes may be the primary cause of high PPO activity in wheat kernels.     

Association analysis of Puroindoline-D1 and Puroindoline b-2 loci with 13 quality traits in European winter wheat (Triticum aestivum L.)

Grain hardness, a major determinant influencing end-use quality of common wheat, is mainly controlled by Puroindoline a-D1 (Pina-D1) and Puroindoline b-D1 (Pinb-D1) genes. Recently, additional puroindoline genes, designated Puroindoline b-2 (Pinb-2), were described. This study examined frequencies of Pin-D1 alleles and Pinb-2 variants in 94 West European wheat genotypes and assessed their association with 13 quality traits considering population and family structure. The survey was completed by analyzing the Grain softness protein-1 gene. Results indicated sequence variation only for Pinb-D1 and Pinb-B2 genes. Pinb-D1b was the predominant hard allele. Pinb-B2v3-1 was the most common Pinb-2 variant, followed by a newly discovered variant Pinb-B2v3-5. Association mapping carried out in the whole sample population showed that Pinb-D1 alleles were associated with 11 quality traits, whereas Pinb-B2 variants were only associated with semolina extraction. Considering only the panel of hard wheat genotypes, variation for flour ash content, sedimentation value, gluten index and loaf volume was found to be associated with Pinb-D1 mutations suggesting that different Pinb-D1 mutations might have particular effects on quality traits. Our study indicated that Pinb-D1d was associated with inferior sedimentation value, gluten index and loaf volume, for which reason this mutation should be disregarded in breeding for quality wheat.     

Identification and molecular characterization of novel LMW-m and -s glutenin genes,and a chimeric -m/-i glutenin gene in 1A chromosome of three diploid Triticum species

Low molecular weight glutenin subunits (LMWGs) are part of the gluten network that gives dough its viscoelastic properties. A-genome-containing diploid species related to modern polyploid wheat show great variability in these subunits. The current study characterized the variability of LMW-m and -s genes, being identified fifteen LMWGs genes in the three species evaluated, of which 14 were novel. Ten were pseudogenes, which are common in cereal prolamins. The rest of genes, with intact coding region, corresponded to the LMW-m genes and some single nucleotide polymorphisms and insertion/deletion events were detected, which could alter protein structure and affect dough quality. Two variants of the LMW-s genes were detected in cultivated einkorn and Triticum urartu, the former being novel. The LMW-m and -s genes were related to TuA3-391 and TuA3-400 genes, and to the Tu-460 gene, respectively, of T. urartu. Screening the sequences characterized for reactive epitopes of celiac disease revealed that LMW-m could be less toxic than other subunits for celiac patients. One novel chimeric gene with features from LMW-m and LMW-i genes was detected in T. urartu, and it produces a novel mature protein that may have a distinguishing effect on dough quality. Novel insights into the evolution of LMWGs genes are also reported. These species are a potential source of novel LMWGs variants.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

Analysis of Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm by Ecotilling and identification of a novel Pinb allele

Kernel hardness is mainly conditioned by allelic variations of Pina-D1 and Pinb-D1 genes located on the short arm of chromosome 5D. In this work, the Ecotilling approach was optimized to investigate Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm, and three Pina and eight Pinb alleles were found. Generally, more Pinb alleles were detected in the accessions coming from the regions that grow winter or a mixture of spring and winter wheats. This was particularly evident for the Southwestern winter wheat, Xinjiang winter–spring wheat and Yellow and Huai River Valley winter wheat regions. A novel variant (designated as Pinb-D1x) was discovered in one of the accessions from the Xinjiang winter–spring wheat region. Compared to wild type (WT) allele Pinb-D1a, two nucleotide substitutions occurred in the coding region of Pinb-D1x, one (at nucleotide position 257) resulting in the replacement of a WT cysteine residue by tyrosine and the other (at nucleotide position 382) creating a premature stop codon. The implications of our data to understanding the diversity of Pina and Pinb alleles in wheat and to future molecular breeding of wheat kernel hardness are discussed.     

A transgenic durum wheat line that is free of marker genes and expresses 1Dy10

As currently practiced, genetic engineering of monocots requires the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to favor the multiplication of the initially transformed cells. In the present paper we have used “minimal gene cassettes” and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Two biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments each excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight (HMW) glutenin subunits, which have been associated with superior bread-making quality and which are absent from durum wheats. The positive selectable marker was the Escherichia coli phosphomannose isomerase (pmi) gene, whose product catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate, allowing plant cells to utilize mannose as a carbon source. PCR assays of genomic DNA from regenerated plants identified 15 T₀ plants that contained the pmi marker gene for an overall transformation efficiency of 1.5%, which is similar to biolistic transformation efficiencies of durum wheat with intact circular plasmids. Line TC-52, which initially contained pmi, non-expressed 1Dx5, and expressed 1Dy10 HMW glutenin subunit transgenes, was further investigated. PCR was used to follow inheritance of the pmi marker gene and 1Dx5 from the T₁ to T₃ generations. Transgene expression was monitored by the chlorophenol-red assay for pmi and SDS-PAGE of seed proteins for 1Dy10. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T₃ generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. Homozygotes containing and expressing only the 1Dy10 transgene were identified in the T₄ generation. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits.     

新疆小麦品种春化和光周期主要基因的组成分析

为了明确新疆冬春麦区小麦春化和光周期基因的分布特点,利用STS标记对185份品种(系)的重要春化基因Vrn-A1、Vrn-B1、Vrn-D1、Vrn-B3和光周期基因Ppd-D1位点的等位变异组成进行了检测和分析。结果表明,在新疆小麦品种中,春化和光周期基因位点显性等位变异分布频率不同。含有春化显性等位变异Vrn-A1的品种47个,占供试品种(系)的25.4%;Vrn-B1为43个,占23.3%;Vrn-D1为38个,占20.5%;Vrn-B3位点不存在显性等位变异。春化显性等位变异Vrn-A1、Vrn-B1和Vrn-D1在冬、春性小麦内的分布比例也不同。在春性小麦品种(系)中,显性等位变异Vrn-A1出现的频率较高(55.3%);其次为Vrn-B1,占50.6%;Vrn-D1占44.7%。在冬性小麦中,仅有显性等位变异Vrn-B1出现,占2.0%。在光周期基因Ppd-D1位点,80.0%的品种(系)携带光不敏感显性等位变异Ppd-D1a;其中在春性和冬性小麦品种(系)中,Ppd-D1a出现的频率分别为83.5%和77.0%。新疆小麦品种(系)中,存在11种春化和光周期基因显性等位变异组合。