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。     

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。     

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.     

长江中下游麦区小麦品种籽粒硬度及puroindoline基因等位变异的分子检测

为了明确长江中下游麦区小麦籽粒硬度及puroindoline基因型的分布,以该麦区105份小麦育成品种为材料,利用单籽粒硬度仪(SKCS)测定其籽粒硬度,利用分子标记检测和基因序列分析鉴定puroindoline基因的等位变异。结果表明,在长江中下游麦区历年育成的小麦品种中软质麦比例较高,占52.4%,硬质麦和混合麦分别占38.1%和9.5%;硬质麦和混合麦中存在Pinb-D1b、Pina-D1b和Pinb-D1p三种变异类型,突变频率分别为29.5%、10.5%和3.8%。     

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.     

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。     

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／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。     

软质小麦品质的辅助选择

为建立软质小麦品质的筛选和评价体系,对黄淮麦区和长江下游麦区20个不同硬度小麦品种的Puroindoline基因型以及全麦粉和面粉的部分相关品质性状进行了检测.结果表明,20个品种中,2个品种为Pina-D1b硬质突变型,6个品种为Pinb-D1b硬质突变型,其余12个品种为软质小麦;硬、软质小麦的全麦粉硬度和SDS沉淀值的差异均达到极显著水平,但碱水保持力差异不显著;硬、软质小麦面粉的碱水保持力和溶剂保持力均达到极显著差异.性状间的相关分析表明,全麦粉硬度与SDS沉淀值、面粉的碱水保持力、水溶剂保持力、碳酸钠溶剂保持力、蔗糖溶剂保持力均呈极显著相关,相关系数分别为0.64、0.94、0.94、0.91和0.81,SDS沉淀值与乳酸溶剂保持力相关也极显著,相关系数0.80.这些结果说明Puroindoline基因型检测、全麦粉硬度和SDS沉淀值检测可以作为软质小麦的早代筛选方法.     

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。     

Genetic and environmental factors affecting grain texture in common wheat

Thirteen wheat cultivars grown in six locations were compared for kernel weight, protein content and grain texture, as determined by the Single Kernel Characterization System (SKCS). Moreover, puroindolines a (Pin-A) and b (Pin-B) bound to starch were quantified by densitometric scanning of A-PAGE fractionations. All cultivars shared allele Pina-D1a coding for wild-type Pin-A, and differed from each other in allele composition at Pinb-D1 coding for Pin-B. Cultivars with Pinb-D1a exhibited soft grain and high amounts of Pin-A and Pin-B compared to cultivars with Pinb-D1b or Pinb-D1d. Significant genetic variation for grain hardness and Pin-A level was detected in soft cultivars. The ratio between Pin-A and Pin-B levels in soft cultivars was approximately 6:5, whereas it varied between 9:5 and 10:1 in hard cultivars. Protein content was significantly correlated with Pin-B content (r=0.34) and SKCS value (r=0.36) in soft wheats. Significant correlations (0.68 and 0.73 for soft and hard wheats, respectively) were observed between Pin-A and Pin-B levels. Grain hardness was not correlated with puroindoline levels and Pin-A/Pin-B ratio in both textural classes. By contrast, kernel weight was found to act as a major environmental factor affecting grain texture in both soft and hard wheats.     

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。     

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.     

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.     

Identification of new puroindoline genotypes and their relationship to flour texture among wheat cultivars

Puroindoline genotypes (Pina and Pinb) and their encoded proteins related to grain hardness were studied in various common wheat cultivars from Australia, China, Japan, Korea and North America. Most of the hard wheats had the Pinb-D1b genotype with a glycine to serine mutation at position 46. In addition to the known Pina and Pinb genotypes, cultivars were found with Pina and Pinb double-null mutations (Pina-D1b/Pinb-D1h (t)) and a new Pinb frameshift mutation (designated Pinb-D1i (t)) within the region encoding a tryptophan-rich domain. This new Pinb frameshift mutation was found only in Chinese cultivars. Endosperm proteins encoded by Pina and Pinb in these cultivars were analysed by 2D-gel electrophoresis (IPG×SDS-PAGE). Cultivars with Pina and Pinb double-null mutations showed no PIN-a or PIN-b protein, and cultivars with Pinb-D1i (t) had no PIN-b protein. Surprisingly, cultivars with Pinb-D1b had severely reduced amounts of PIN-b and cultivars with Pinb-D1c showed no PIN-b proteins. Grain hardness among cultivars having mutated Pinb may be explained by the amount of PIN-b protein and not by the type of amino acid substitutions.