新疆小麦品种籽粒硬度及puroindoline基因等位变异的分子检测

为了解新疆小麦品种籽粒硬度概况和puroindoline基因等位变异类型及其分布,以121份新疆冬、春小麦品种(包括51份农家品种和70份育成品种)为材料,采用单粒谷物特性测试仪(SKCS)和分子标记技术,对其SKCS硬度及puroindoline基因型进行了测试和鉴定。结果表明,新疆小麦硬度变化范围较大,以硬质麦为主,占61.2%。冬性农家品种籽粒硬度高于春性农家品种,冬、春小麦育成品种籽粒硬度基本相同(60.6和60.8)。新疆小麦puroindoline基因的类型丰富,共检测到野生型(Pinb-D1 a)、Pina-D1 b、Pinb-D1 b、Pinb-D1 p、Pinb-D1 ab和Pinb-D1 ac6种类型。硬质麦以Pina-D1 b、Pinb-D1 p、Pinb-D1 b3种突变类型为主,其频率分别为33.8%、31.1%和28.4%。冬性农家品种含有3种类型,以Pina-D1 a/Pinb-D1 b类型居多,春性农家品种含有4种类型,以野生型(Pina-D1 a/Pinb-D1 a)为主,冬、春麦育成品种含有4种常见类型,其中冬麦以Pinb-D1 b类型为主,其频率为39.5%,春麦以Pina-D1 b(PINA缺失)类型为主,其频率为59.4%。另外,冬性农家品种中有4个品种属于Pina-D1 a/Pinb-D1 ab类型,春性农家品种中有1个品种属于Pina-D1 a/Pinb-D1 ac类型,均属硬质麦的稀有突变类型。不同puroindoline基因型的籽粒硬度大小也存在差异,其中Pinb-D1 ab突变型的硬度值最高,Pinb-D1 a最低,并且Pina-D1 b、Pinb-D1 b和Pinb-D1 p3种硬质类型的籽粒硬度没有显著性差异。     

青海小麦籽粒硬度等位变异研究

为了解小麦品种籽粒硬度的遗传多样性,利用单粒谷物硬度测定、PCR扩增和核苷酸测序技术,分析了66份青海小麦品种籽粒硬度主效基因的等位变异。结果表明,青海小麦以硬质类型为主,比例达到47.0%,混合麦比例为19.7%,软质麦比例为33.3%。硬度基因有5种组合类型:野生型、Pina-D1a/PinbD1b、Pina-D1a/Pinb-D1c、Pina-D1a/Pinb-D1x和Pina-D1b/Pinb-D1a。野生型小麦类型比例最高,占59.09%,SKCS硬度指数平均为44.12,变化范围为12.75~84.89。突变类型的品种籽粒均为硬质。因此,在青海硬质小麦可以通过突变类型的分子标记进行选育,软质小麦选育需在利用硬度基因分子标记筛选的基础上进一步考察籽粒硬度性状的表现型。     

江苏淮北地区小麦品种资源籽粒硬度基因等位变异的KASP检测

为了解江苏淮北地区小麦品种资源的籽粒硬度概况及硬度基因型分布规律,以74份近年来江苏淮北地区所育品种(系)和38份来自黄淮其他麦区的常用亲本为材料,采用单籽粒谷物硬度测试仪、KASP标记检测技术和基因扩增及测序技术对其SKCS硬度值及硬度基因型进行鉴定。硬度检测结果表明,供试小麦品种(系)硬度变化范围较大,但硬质麦的比例最大,为70.5%。与常用亲本相比,江苏淮北地区育成品种中软质麦比例较高,为34.3%,但在高代品系中软质麦比例下降到20.5%。基因型检测结果表明,在Puroindoline-D1位点,供试品种(系)中共检测到4种基因型,即野生型(Pina-D1a/Pinb-D1a)、Pina-D1b、Pinb-D1b和Pinb-D1p,其频率依次为25.0%、2.7%、67.9%和4.5%。其中,野生型和Pinb-D1p主要分布在江苏淮北地区。不同硬度基因型的硬度值也存在差异,其中以Pina-D1b基因型的硬度值最高,野生型(Pina-D1a/Pinb-D1a)硬度值最低,Pinb-D1b和Pinb-D1p两硬质类型的籽粒硬度没有显著性差异。在Pinb-2位点,供试品种(系)中共检测到25份材料为Pinb-B2b基因型,包含21份硬质麦、2份混合麦和2份软质麦,其平均硬度值为63.8。     

小麦籽粒硬度及其Pinb-D1基因等位变异的STS标记检测

为了明确内蒙古春小麦和部分引进冬小麦的籽粒硬度及其等位变异类型,用SKCS 4100单籽粒谷物硬度仪和STS分子标记对17份冬小麦、87份春小麦品种扣38份春小麦高代品系的籽粒硬度及其Pinb-Dla,Pinb-D1b和Pinb-Dlc等位基因进行了分析.结果表明,在104份冬、春小麦品种中,籽粒硬度指数变幅为1±16～74±21,其中软质、混合和硬质麦频率分别为23.1%、49.0%和27.9%;Pina-D1a/Pinb-D1a、Pina-D1a/Pinb-D1b和Pina-D1a/Pinb-D1c基因型分憋为68份、27份和4份.在38份高代品系中,籽粒硬度指数变幅为O±18～49±17,软质和混合麦频率分别为68.4%和31.6%,Pina-D1a/Pinb-D1a和Pina-D1a/Pinb-D1b基因型分别为20份和18份.     

陕西小麦籽粒硬度及其基因型分析

籽粒硬度与小麦市场分级定价、磨粉品质和食品加工品质密切相关。为给小麦品种选育和品种推广提供参考依据,用单籽粒谷物硬度测试仪测定了169份陕西小麦品种（系）的籽粒硬度,并利用分子标记检测和基因序列分析确定了硬质麦的基因组成。硬度测定结果表明,陕西参试小麦品种（系）存在硬质麦、混合麦和软质麦3种类型,分别为121、11和37份,依次占71.6%、6.5%和21.9%。陕西不同地区3种籽粒硬度类型所占比例明显不同。基因型分析结果表明,陕西硬质麦存在4种基因型,即PinaD1b、PinbD1b、PinbD1d和PinbD1p,分别有14、97、2和8份材料,占硬质麦比例依次为11.6%、80.2%、1.6%和6.6%。总体而言,陕西小麦以硬质麦为主,硬质麦主要由PinbD1b基因型组成。     

CIMMYT普通冬小麦品种的籽粒硬度及Puroindoline基因等位变异

为给中国小麦种质资源引进、利用和品质改良提供信息,以国际玉米小麦改良中心(CIMMYT)土耳其育种站提供的192份普通冬小麦新品系为材料,采用单籽粒谷物特性测试仪、特异引物PCR扩增和改进的SDS-PAGE凝胶电泳方法对其SKCS硬度及Puroindoline基因型进行了鉴定和分析.结果表明,CIMMYT普通冬小麦以硬质类型为主,但SKCS硬度值普遍偏低,平均值仅为60.7.所调查的192份材料中,硬质麦119份,占62.0%;软质麦49份,占25.5%;混合麦24份,占12.5%.硬质小麦共有4种基因型,分别为PinA蛋白缺失类型(Pina-D1b/Pinb-D1a)90份、Pina-D1a/Pinb-D1b类型27份、Pina-D1a/Pinb-D1d类型2份和Pina D1b/Pinb-D1d类型1份,以PinA蛋白缺失类型为主,占总数的75.6%.Pina-D1b/Pinb-D1d为Pina和Pinb基因的双突变类型.CIMMYT普通冬小麦籽粒硬度及其Puroindoline基因变异类型和分布的信息能够为中国冬小麦品质改良提供理论依据.     

部分春小麦品种籽粒硬度及单粒硬度的频率分布

用单籽粒谷物硬度测定仪对55份春小麦品种(系)的籽粒硬度及其单籽粒硬度的频率分布进行研究,结果表明,品种(系)籽粒硬度变幅为11±18~86±16,其中硬质麦34份(1级29份、2级4份、3级1份),混合麦14份(2级4份,3级10份),软质麦7份(4级1份,5级6份),硬质、混合和软质类型比例分别为61.8%、25.5%和12.7%,极硬或极软类型少。东北春麦区、西北春麦区、青藏高原冬春麦区和新疆冬春麦区品种以硬质类型为主,北部春麦区软质和混合麦比例较高。籽粒硬度的分类与不同硬度范围单籽粒频率分布及其硬度均值有关,硬质麦硬度低于46的籽粒占1%~19%,高于47的籽粒比例为77%~99%,硬度均值58±18~86±16;混合麦硬度指数低于46和高于47的籽粒分别为26%~60%和40%~74%,硬度均值45±19~57±20。软质麦硬度指数低于46的籽粒为81%~97%,高于47的籽粒为3%~20%,硬度均值为11±18~33±19;软质或硬质品种籽粒分布集中,一致性较好;混合型品种籽粒分布分散,一致性较差。     

中国小麦历史品种puroindoline基因等位变异检测

小麦籽粒硬度是市场分级和定价的重要依据,由位于5D染色体短臂的一对主效基因和多个微效基因控制。为了解我国早期小麦品种的硬度分布规律,本试验以来自全国10个省市的126份历史品种和高代品系为材料,对其SKCS硬度和基因型进行了研究。结果表明,我国历史品种中软质麦所占比例较高,为42.9%,显著高于先前报道的我国当前主栽冬麦品种中的软质麦比例;硬质麦所占比例为44.4%,显著低于先前报道的我国主栽冬麦品种中的硬质麦比例。各种puroindoline变异类型中, PinaD1b类型和 PinbD1b类型所占比例较大,分别为42.9%和39.3%,其余硬质类型均为 PinbD1p类型,为17.8%。截至目前,所发现的 PinbD1p类型均在中国品种中,由于早期中国与其他国家种质资源交换较少,因此,中国可能是 PinbD1p类型的起源地。在对各种puroindoline类型的硬度比较后,发现除各种puroindoline变异类型与野生型硬度差异达显著水平外, PinaD1b类型硬度值也显著高于 PinbD1b和 PinbD1p类型,而后二者之间硬度没有显著差异。`     

CIMMYT普通冬小麦品种的籽粒硬度及Puroindoline基因等位变异

为给中国小麦种质资源引进、利用和品质改良提供信息,以国际玉米小麦改良中心（CIMMYT）土耳其育种站提供的192份普通冬小麦新品系为材料,采用单籽粒谷物特性测试仪、特异引物PCR扩增和改进的SDS-PAGE凝胶电泳方法对其SKCS硬度及Puroindoline基因型进行了鉴定和分析。结果表明,CIMMYT普通冬小麦以硬质类型为主,但SKCS硬度值普遍偏低,平均值仅为60．7。所调查的192份材料中,硬质麦119份,占62．0％;软质麦49份,占25．5％;混合麦24份,占12．5％。硬质小麦共有4种基因型,分别为PinA蛋白缺失类型（Pina-D1b／P1nb-D1a）90份、Pina-D1a／Pinb—D1b类型27份Pina—D1a／Pinb—D1d类型2份和Pina-D1b/Pinb-D1d类型1份,以PinA蛋白缺失类型为主,占总数的75．6％。Pina—D1b／Pinb-D1d为Pina和Pinb基因的双突变类型。CIMMYT普通冬小麦籽粒硬度及其Puroindoline基因变异类型和分布的信息能够为中国冬小麦品质改良提供理论依据。     

普通小麦籽粒硬度的分子标记研究

籽粒硬度是决定小麦磨粉品质和食品品质的重要性状，蛋白复合体Friabilin的两种主要肽Puroindolinea（PinA）和Puroindolineb（PinB）是决定籽粒硬度的关键。用单籽粒谷物特性仪（SKCS）、PCR技术和改进的SDS-PAGE方法分析了85份小麦品种的籽粒硬度和基因突变形式，结果表明，与软质小麦相比，硬质小麦品种在基因或蛋白质表达水平上发生了变化，有31份野生型的软质小麦Pina-Dla／Pinb-Dla和6份Pina-D1b／Pinb-D1a突变型，42份Pina-D1a／Pinb-D1b和6份Pina—D1a／Pinb-D1p突变型。     

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.     

Pins基因等位变异对磨粉品质和新疆拉面加工品质的影响

为了探讨新疆冬小麦品种Pins基因等位变异对小麦磨粉品质和新疆拉面加工品质的影响，对109份新疆冬小麦品种的籽粒硬度及其Pins基因等位变异、磨粉品质和新疆拉面加工品质进行测定，初步分析了新疆冬小麦品种资源籽粒硬度Pins基因的分布规律以及不同 Pins基因等位变异对籽粒硬度、磨粉品质和新疆拉面加工品质的影响。结果表明，新疆冬小麦品种属硬质麦类型，Pins基因型以 Pina-D1a、 Pinb-D1b和 Pina-D1a/ Pinb-D1b为主， Pins突变类型及Pins突变基因型组合类型小麦的籽粒硬度均显著高于野生型， Pinb-D1a基因型小麦的籽粒硬度最低，L^*值和a^*值最高，b^*值最低； Pinb-D1ab基因型小麦的吸水率最高。不同Pins基因型组合中，野生型小麦的籽粒硬度、b^*值和吸水率最低； Pina-D1a/ Pinb-D1aa的出粉率最高， Pina-D1a/ Pinb-D1ab的灰分含量最低，吸水率最高。Pins基因及其基因型组合对新疆拉面加工品质无直接影响，主要通过对灰分、面粉色泽和吸水率等磨粉品质的作用对新疆拉面产生间接影响。优质新疆拉面品种中，Pinb基因突变对新疆拉面加工品质的影响大于Pina基因突变，育种中应优先选择Pinb 基因突变型材料，其中 Pina-D1a/ Pinb-D1b可以作为重点选择的基因型组合。     

Influence of puroindoline alleles on milling performance and qualities of Chinese noodles,steamed bread and pan bread in spring wheats

Two trials with a total of 75 spring bread wheat cultivars and advanced lines, were used to evaluate single kernel characterization system hardness, puroindoline alleles, milling yield, flour ash content, flour colour, and end-use qualities for Chinese noodles, steamed bread and pan bread. The results indicated that all International Maize and Wheat Improvement Center hard wheat lines surveyed were Pina-D1b genotype, whereas Pinb-D1b was the most common allele in the remaining cultivars. Genotypes with Pinb-D1b possess significantly lower flour ash content and higher milling yield than those of genotypes with Pina-D1b. For steamed bread, mean scores for loaf volume, crumb colour, width, structure and total score of Pinb-D1b genotypes were significantly higher than those of genotypes with Pina-D1b and wild type. For Chinese fresh white noodles, means for noodle a*, colour score, viscoelasticity and total score with Pinb-D1b were significantly higher than those of Pina-D1b and wild type. Means of loaf volume, texture and total score for Pinb-D1b genotypes were significantly higher than those of Pina-D1b genotypes. This study further illustrated the superiority of the Pinb-D1b allele on milling and processing qualities for various end products and provides useful information for wheat quality improvement.     

Puroindoline genotype,starch granule size distribution and milling quality of wheat

Genetically-diverse wheat samples from the Australian Winter Cereals Collection propagated in two environments were sequenced to identify puroindoline genotypes then the relationships between flour yield, genotype, starch granule size distribution and starch-bound puroindoline protein content were investigated. The Pina-D1a, Pinb-D1b genotype resulted in a higher average flour yield than either the Pina-D1b, Pinb-D1a or the Pina-D1a, Pinb-D1a but the ranges of flour yields for the three genotypes showed considerable overlap. For both hard wheat genotypes (Pina-D1a, Pinb-D1b or Pina-D1b, Pinb-D1a), a higher proportion of type A to type C starch granules was associated with higher flour yield and this relationship accounted for between 31% and 33% of the variation in flour yield. This result is consistent with previously reported findings for soft wheat. For the Pina-D1a, Pinb-D1b genotype, increased flour yield was also associated with a decrease in starch granule-bound puroindoline protein, which accounted for 31–35% of the variation in flour yield across the two environments. The combined effect of starch granule type and associated puroindoline content accounted for 68% of the variation in flour yield within the Pina-D1a, Pinb-D1b genotype.     

Milling and Chinese raw white noodle qualities of common wheat near-isogenic lines differing in puroindoline b alleles

Understanding the effects of different alleles at the puroindoline b (Pinb) locus on processing quality will provide crucial information for quality improvement. Seven near-isogenic lines (NILs) planted at two locations in the 2008 cropping season were used to determine the effect of puroindoline b alleles on milling performance and Chinese raw white noodle (CRWN) quality. The Pina-D1b/Pinb-D1a genotype possessed significantly higher values in grain hardness, protein content and starch damage than other genotypes, whereas the Pina-D1a/Pinb-D1d genotype had the lowest grain hardness and starch damage, with higher break flour yield, and less reduction flour yield, higher flour colour L*, and lower flour colour b*, than other genotypes. Farinograph parameters, except for water absorption, were not significantly affected by variation of puroindoline b alleles. Pina-D1a/Pinb-D1e had the highest peak viscosity, whereas the lowest value was observed in a Pina-D1b/Pinb-D1a genotype. For CRWN quality, higher noodle viscoelasticity was obtained in the genotype Pina-D1a/Pinb-D1e and Pina-D1a/Pinb-D1g, whereas Pina-D1a/Pinb-D1d had a lower smoothness score. Genotypes with Pina-D1a/Pinb-D1e and Pina-D1a/Pinb-D1g produced the best total noodle score. It was concluded that genotype Pina-D1a/Pinb-D1d had better milling qualities, whereas Pina-D1a/Pinb-D1e and Pina-D1a/Pinb-D1g had slightly superior CRWN qualities in comparison with other genotypes.     

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.     

Variation in Friabilin Composition as Determined by A-PAGE Fractionation and PCR Amplification, and its Relationship to Grain Hardness in Bread Wheat

A-PAGE fractionation of starch granule proteins from 63 bread wheat cultivars with contrasting grain texture characteristics revealed two prominent polypeptides and three minor ones, approximately 15 kDa in size. These proteins were found to be encoded by genes on the short arm of chromosome 5D. The two major friabilin components were assumed to correspond to puroindolines a and b (pinA and pinB), as suggested by PCR amplification of genes coding for pinA, glycine-type or serine-type pinB. Two electrophoretic patterns for pinA (presence vs absence) and three patterns for pinB were obtained by A-PAGE. In cultivars with pinA (allele Pina-D1a), pinB was found to be encoded by wild-type Pinb-D1a, serine-type Pinb-D1b or by the novel glycine-type b1 allele. Cultivars lacking pinA (allele Pina-D1b) were shown to contain eitherPinb-D1a or the novel b2 allele, both alleles coding for glycine-type pinB. The intensity of pinB in A-PAGE gels was found to be associated with grain hardness as determined by the SKCS method. Cultivars lacking pinA had the highest SKCS values, suggesting that both pinA and pinB may affect grain texture. In the presence of pinA, cultivars with wild-type allelePinb-D1a had soft grain texture, whereas those with alleles Pinb-D1b or b1 showed increased grain hardness. It is suggested that allele b1 affects the interaction of pinB with starch granules because of a sequence mutation different from the glycine-to-serine change.