俄罗斯小麦籽粒硬度及puroindoline基因等位变异的分布

小麦籽粒硬度是重要的品质性状之一,对小麦加工品质具有重要影响.为了合理引进种质资源,培育高品质小麦品种,本研究利用小麦硬度指数测定仪JYDB100×40对引进俄罗斯小麦品种的籽粒硬度进行检测,利用STS标记和变温PCR对控制籽粒硬度基因puroindoline的主要等位变异进行分析.结果显示:208个俄罗斯引进小麦品种硬度指数范围为45.6 ～79.2,硬质麦品种为190个(91.35％),混合麦品种为18个(8.65％).Pina基因有Pina-D1a、Pina-D1b和杂合Pina-D1a/ Pina-D1b共3种类型,所占比例分别为87.02％、10.10％和2.88％.Pinb基因有Pinb-D1a、Pina-D1b、Pinb-D1c和双位点突变Pinb-D1bc 4种类型,所占比例分别为37.02％、55.77％、3.85％和3.37％.统计结果显示:携带Pina-D1b+Pinb-D1a基因小麦籽粒硬度指数显著大于Pina-D1a+Pinb-D1b,Pina-D1a+Pinb-D1a和Pina-D1a+Pinb-D1bc.其他puroindoline等位变异之间没有显著差异.俄罗斯硬质麦种质资源丰富,是改善我国小麦籽粒硬度和品质的重要遗传材料.     

81份国外小麦品种(系)Puroindoline基因分子鉴定与分析

为更好地利用国外小麦Puroindoline优异变异类型,以从乌克兰、俄罗斯、西班牙等4个国家搜集的81份小麦品种(系)为试验材料,采用单籽粒谷物特性测定仪(SKCS)、分子标记技术及DNA测序技术,对其硬度表型、Puroindoline不同变异类型进行鉴定与分析。结果表明,试验材料中硬质麦比例较高,为92.6%,软质麦比例较低,仅为7.4%,且没有发现混合型小麦,SKCS硬度指数范围较宽,为25~86。硬质麦的Puroindoline基因型检测中,共检测到Pina-D1b、Pinb-D1b、Pinb-D1c和Pinb-D1d 4种类型,其中Pinb-D1b所占比例较高,占58.7%,Pina-D1b和Pinb-D1c则分别为28.0%和12.0%,而PinbD1d类型所占比例极低,仅为1.3%。Puroindoline不同等位变异类型的籽粒硬度大小也存在差异,其中Pina-D1b突变型的硬度值最高,野生型最低,且Pina-D1b与Pinb-D1c两种硬质类型的籽粒硬度呈显著性差异,而Pinb-D1b、Pinb-D1c与Pinb-D1d之间的籽粒硬度差异不显著。     

基于173份小麦评价糯蛋白亚基基因(Wx)的分子标记和构建其多重PCR体系

糯蛋白亚基基因(Wx)组成与小麦淀粉品质特性密切相关,筛选和建立其高效的鉴定方法和体系,对小麦品质育种改良和种质资源快速评价具有重要意义。本研究基于SDS-PAGE方法检测173份小麦(Triticum aestivum L.)品种(系)糯蛋白亚基组成的结果,评价序列标签位点(sequence tagged sites,STS)标记的有效性,利用有效的STS标记构建Wx基因分子检测的多重PCR体系。SDS-PAGE方法检测结果表明,20份小麦品种(系)缺失Wx-B1蛋白亚基,1份3个Wx蛋白亚基全部缺失,依次占11.6%和0.6%。4个共显性和2个显性Wx基因STS标记检测供试小麦的结果,与SDS-PAGE结果完全一致。构建了两套分子标记检测的多重PCR体系,其中多重PCR体系Ⅰ能够同时检测Wx-A1和Wx-B1位点的4种等位变异,体系Ⅱ可同时检测Wx-B1和Wx-D1位点的4种等位变异,两套多重PCR体系检测的结果与SDS-PAGE和单一PCR的检测结果也完全一致。筛选的6个STS标记和构建的两套PCR检测体系,用于小麦Wx基因的分子标记辅助选择,将有助于提高小麦品种淀粉品质评价和选育的效率。     

六倍体小黑麦品种资源Glu-1位点的多态性

利用SDS-PAGE技术分析了我国新疆101份和波兰11份六倍体小黑麦品种资源高分子量谷蛋白亚基(HMW-GS)组成,共检测到17种高分子量谷蛋白亚基,其中Glu-A1位点编码的HMW-GS有3种变异类型,即1(a)、2*(b)和Null(c),Glu-B1位点编码的HMW-GS有8种变异类型,即7(a)、7+8(b)、7+9(c)、6+8(d)、20(e)、13+19(g)、7+18(r)和6.8+20y(s),Glu-R1位点编码的HMW-GS有6种变异类型,即1r+4r(a)、2r+6.5r(b)、6r+13r(c)、2r+9r(d)、6.5r(e)和0.8r+6r(f)。这些小黑麦品种的HMW-GS组成以Null(c)、7+18(r)和6r+13r(c)为主,分别占58.93%、67.90%和58.00%。在112份供试材料中共检测到30种HMW-GS组合变异类型,其中[Null,7+18,6r+13r(c,r,c)]和[2*,7+18,6r+13r(b,r,c)]出现频率较高,分别为16.91%和16.02%,其他类型组合出现频率较低,个别材料具有少见的特殊亚基组合,如[2*,7+18,2r+9r(b,r,d)]、[2*,6.8+20y,2r+6.5r(b,s,b)]等类型。分析2个地域品种的遗传多样性,发现新疆品种的遗传变异范围小于波兰品种,波兰品种在Glu-1位点上的遗传变异更丰富。结果还显示,在六倍体小黑麦的人工进化过程中Glu-B1位点发生了很大变异,产生了小黑麦特有的7+18(r)和6.8+20y(s)亚基,而且频率很高,为小麦品质改良提供了丰富的基因资源。     

2019—2020山东小麦区试品系55个基因的等位基因分布

为分析2019—2020年山东省小麦区试参试品系携带优异等位基因情况,利用产量、品质、抗性、开花期等55个基因的64个竞争性等位基因特异性聚合酶链式反应（KASP）标记对126份参试品系进行分子标记检测。结果表明,90%以上KASP标记具有较好的分型结果,可高效地进行基因型鉴定。20个基因的优异等位基因频率高于80%,包括Vrn-A1、Vrn-B1、Vrn-D1、Ppd-A1、Ppd-B1、Ppd-D1、Psy-D1、Glu-A3g、Rht-D1、Pinb-D1、TaCwi-A1-1、TaGW2-6B、TaSus1-7B、TaGASR7-A1、1-feh-w3、TaDreb-B1、PRA1、PRR-B1、TaFT3-B1和TaMOT1-D1;26个基因的优异等位基因频率低于30%,包括Vp1-B1、Ppo-D1、TaPds-B1、Zds-A1、TEF-7A、Lr46、Glu-B3g、TaGS5-A1、TaCwi-4A、1B/1R、Rht-B1、Lr68、TaELF3-B1、Pina-D1、Sbwm1、TaPHS1、Pm21、COMT-3B、TaCKX-D1、TaSdr-B1、Pch1、...     

中国小麦品种醇溶蛋白Gli-1和Gli-2编码位点等位基因组成分析

利用酸性聚丙烯酰胺凝胶电泳(A-PAGE)方法,鉴定分析了我国部分重要小麦品种或种质醇溶蛋白Gli-1和Gli-2编码位点等位基因组成特点.36个品种的研究结果表明,我国小麦品种在醇溶蛋白几个主要位点上均存在较大的变异度,Nei氏遗传变异系数(H)达0.775.6个位点一共检测到59个不同的醇溶蛋白等位基因,其中出现频率较高的有8个等位基因,即Gli-B2g(55.56 %)、Gli-D1k(50 %)、Gli-A1a(33.33 %)、Gli-A2f(30.56 %)、Gli-B1b(22.22 %)、Gli-D1f(22.22 %)、Gli-B2b(22.22 %)和Gli-D2g(22.22 %).国外品种中很少的4个等位基因(Gli-D1f、Gli-A2f、Gli-B2g和Gli-D2g)在我国具有较高的频率.可作为1BL/1RS易位标记的Gli-B1l等位基因在中国品种中的频率较高.另外,还发现一些优质醇溶蛋白等位基因(如Gli-B1b、Gli-B2c、Gli-A2b等)在我国品种中出现的频率很低,这可能是中国小麦品种品质普遍较差的一个原因.     

小麦微核心种质的Viviparous-B1基因多态性及其籽粒休眠性的检测和鉴定

根据GenBank中小麦Viviparous(Vp1)-B1基因序列设计特异引物,检测中国小麦(Triticum aestivum)微核心种质,结果表明,在中国小麦微核心种质中Vp1-B1存在较为丰富的等位变异,共检测出4种等位类型,分别为Vp1-B1a(35.3%)、Vp1-B1b(18.5%)、Vp1-B1c(40.7%)和Vp1-B1e(5.5%).其中,具有Vp1-B1a基因的小麦品种其籽粒平均萌芽指数较高(49.8%);Vp1-B1b、Vp1-B1c和Vp1-B1e大都分布在萌芽指数较低的品种中,特别是具有Vp1-B1b等位基因的品种平均萌芽指数最低,仅为18.9%,具有Vp1-B1c和Vp1-B1e等位基因的品种平均萌芽指数分别为25.7%和25.6%.经改良的变性PAGE凝胶电泳检测及测序分析表明,Vp1-B1e为新的等位类型,和Vp1-B1c基因的相似性最高,达99%;和后者相比,其在第3内含子区域发生ATAT 4个碱基的插入以及2个SNP.     

利用甲基磺酸乙酯(EMS)诱导小麦-华山新麦草染色体易位的研究

小麦(Triticum aestivum)种质24-3-1是通过染色体工程手段获得的一个高抗条锈病的小麦-华山新麦草二体异附加系。为了提高其遗传稳定性,促进华山新麦草(Psathyrostachys huashanica)优异基因的有效利用,对24-3-1进行甲基磺酸乙酯(ethylmethylsulfone,EMS)化学处理来诱导易位系。本研究利用细胞学和基因组原位杂交(genomic in situ hybridization,GISH)对其M2进行鉴定和易位系的筛选,并分析了不同EMS浓度对小麦-华山新麦草染色体易位的诱导效应。结果显示,在930个M2单株中共检测出了61个含有小麦-华山新麦草易位染色体的植株,易位频率为6.56%。其中7个单株检测出含有1条易位染色体,5个单株检测出含有1条易位染色体+1条华山新麦草染色体,20个单株检测出含有2条易位染色体,3个单株检测出含有3条易位染色体,26个单株检测出含有4条易位染色体。根尖细胞学观察和基因组原位杂交证明,含有2条易位染色体的单株为小麦-华山新麦草易位系;含有4条易位染色体的单株为小麦-华山新麦草易位-易位附加系。1.0%EMS为诱导小麦-华山新麦草染色体易位的最适浓度。本研究不仅为小麦特殊遗传材料的建立提供了重要的基因资源,同时也为小麦育种提供了新的种质。     

抗白粉病糯性小麦的多重PCR分子鉴定技术

为同时鉴定小麦糯性和白粉病抗性,本研究利用已知3对引物建立多重PCR体系,分别扩增WxA1、Wx-B1、Wx-D1和Pm21基因的分子标记,其目的片段大小分别为:230/265、854、204和1400 bp。经过对供试品种和部分F2分离群体的Wx蛋白进行SDS-PAGE电泳检测,其结果与多重PCR结果一致,证明该多重PCR体系准确可靠,可提高小麦糯性兼抗白粉病的选育效率。     

新疆小麦地方品种资源HMW-GS的遗传多样性组成分析

为了明确新疆小麦地方品种高分子量麦谷蛋白亚基的遗传多样性,并为小麦品质改良提供基础材料,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE) 技术,分析了源自新疆地区的282份小麦地方品种的高分子量麦谷蛋白亚基组成。结果表明,在Glu-A1、Glu-B1和Glu-D1位点上的等位变异分别为3、6和 5种,三个位点上的优势亚基依次为null、7+8和2+12,其频率分别是75.5%、90.8%和72.0%。在Glu-1位点共检测到20种亚基组合,其中(null, 7+8, 2+12)组合的频率最高,为52.8%, 其次是(null, 7+8, 2.6+12)和(2*, 7+8, 2+12)组合,其频率分别为14.1%和11.0%,其它亚基组合的频率均低于10%。另外,在Glu-D1位点上还检测到一个新的亚基2.6+12。在供试的282份新疆地方品种中发现了两份具有优质亚基组合的材料,它们的亚基组成为(2*, 7+9, 5+10)和(1, 7+9, 5+10),这些地方品种可作为改良小麦品质性状的重要遗传资源。     

Development of SNP assays for genotyping the puroindoline b gene for grain hardness in wheat using pyrosequencing

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.) and has been reported to result from either a failure to express puroindoline a (Pina) or single-nucleotide mutations in puroindoline b (Pinb). Up to now, seven alleles from Pinb-D1a to Pinb-D1g were identified in bread wheat. Compared to the DNA coding region of Pinb-D1a (allele for softness), six single-nucleotide polymorphisms (SNPs) were detected in six alleles for Pinb-D1. In this study, we used pyrosequencing technology to develop two SNP assays for identification of the seven Pinb alleles and characterized SNP variations in the Pinb of 493 European wheat varieties. Of the three hardness alleles Pinb-D1b, Pinb-D1c, and Pinb-D1d detected in this study, Pinb-D1b was the most predominant hardness allele in European hard wheats. The hardness genotypes of partial German wheat varieties available confirmed the reliability and validation of the SNP assays developed for the Pinb locus. Therefore, pyrosequencing technology offers an efficient, precise, and reliable concept for high-throughout genotyping to assist selection of grain hardness genes in wheat quality breeding programs.     

我国不同时期小麦品种高分子量麦谷蛋白亚基组成比较

用SDS-PAGE方法测定了我国10个小麦主产省份171份小麦品种和高代品系的高分子量麦谷蛋白亚基(HMW-GS)组成。鉴定出18种HMW-GS,40种HMW-GS组成形式,其中20种亚基其组成形式只在一个品种(系)中出现。Glu-A1位点亚基1和Null出现最多,Glu-B1位点7 8和7 9亚基对占绝对优势,Glu-D1位点2 12亚基对出现频率最高。Null、7 9、2 12、Null,7 8,2 12,1、7 8,2 12,1、7 9、2 12等亚基组成形成出现频率最高,占分析品种的49.71%。与前人研究相比,新育成品种HMW-GS亚基组成发生了明显变化,面包优质亚基(对)1、5 10出现的频率显著升高,亚基多态性增加,组成形式明显改善,这些对于品质改良和品种选育是非常有利的,新育成品种Glu-1品质评分已超过7。尽管个别品种亚基组成好,品质优良,但总体上看,我国小麦品种与其它国家相比品质还存在一定差距,提高5 10、17 18等优质亚基的频率是改善我国小麦面包品质的重要措施。     

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.     

(英文)

对来源于美、中、俄及埃塞阿比亚等22个国家的142份硬粒小麦材料的种子贮藏蛋白位点及遗传变异进行了研究。供试的硬粒小麦(Triticum durum Desf )材料共检测出37条醇溶蛋白条带，无1条带纹为所有材料共有，多态性达到100％，说明硬粒小麦具有丰富的醇溶蛋白等位变异。聚类分析将142份供试材料分为3个大类，材料间遗传差异大小在不同的国家有所不同，表明醇溶蛋白带型与地理来源有一定关系。高分子量谷蛋白电泳共分离出14种亚基和15种亚基组合，但是优质亚基所占比例不高，这可能是因为硬粒小麦加工用途的特殊性，使得多年的育种并未太多改变硬粒小麦高分子量谷蛋白亚基等位变异的频率，促成优质亚基的累计。     

(英文)

用SDS-PAGE方法测定了我国10个小麦主产省份171份小麦品种和高代品系的高分子量麦谷蛋白亚基（HMW-GS）组成。鉴定出18种HMW-GS，40种HMW-GS组成形式，其中20种亚基其组成形式只在一个品种（系）中出现。Glu-A1位点亚基1和Null出现最多，Glu-B1位点7＋8和7＋9亚基对占绝对优势，Glu-D1位点2＋12亚基对出现频率最高。Null、7＋9、2＋12、Null，7＋8，2＋12，1、7＋8，2＋12，1、7＋9、2＋12等亚基组成形成出现频率最高，占分析品种的49.71％。与前人研究相比，新育成品种HMW-GS亚基组成发生了明显变化，面包优质亚基（对）1、5＋10出现的频率显著升高，亚基多态性增加，组成形式明显改善，这些对于品质改良和品种选育是非常有利的，新育成品种Glu-1品质评分已超过7。尽管个别品种亚基组成好，品质优良，但总体上看，我国小麦品种与其它国家相比品质还存在一定差距，提高5＋10、17＋18等优质亚基的频率是改善我国小麦面包品质的重要措施。     

Influence of Puroindolines A and B Individually and in Combination on Wheat Milling and Bread Traits

Endosperm texture in wheat (Triticum aestivum L.) is determined by the Pina and Pinb genes located within the Hardness (Ha) locus on chromosome 5D. We have previously shown that Pina and Pinb can act alone to produce intermediate-textured grain or act together to produce soft grain. The objective here was to isolate the role of PINA and PINB individually and in combination on milling and bread traits by analyzing F₃ recombinant lines created by crosses between PINA and PINB null cultivars with Pina-D1a and Pinb-D1a overexpressing transgenic lines. Homozygous lines that contained either the Pina-D1b/Pinb-D1a (Pina null) or Pina-D1a/Pinb-D1e (Pinb null) Ha locus with or lacking transgenically added Pina or Pinb were analyzed for milling and bread traits. Addition of Pina-D1a to Pina-D1b/Pinb-D1a and addition of Pinb-D1a to Pina-D1a/Pinb-D1e Ha locus genotypes gave soft grain with lower flour yield, flour ash, and a higher proportion of small flour particles. Addition of Pinb-D1a produced greater negative effects on loaf volume than addition of Pina-D1a. Grain hardness, flour protein, flour ash, and mixograph water absorption were positively correlated, which is indicative of the complex phenotype conditioned by PINs. The results demonstrate that PIN overexpression leads to a reduction in grain hardness and reduced flour yield, flour ash, and flour particle size. PIN expression also results in reduced loaf volume and flour water absorption.     

Cloning and phylogenetic analysis of polyphenol oxidase genes in common wheat and related species

Cloning and phylogenetic analysis of polyphenol oxidase (PPO) genes in common wheat and its relatives would greatly advance the understanding of molecular mechanisms of grain PPO activity. In the present study, six wheat relative species, including T. urartu, T. boeoticum, T. monococcum, T. dicoccoides, T. durum and Ae. tauschii, were sampled to isolate new alleles at Ppo-A1 and Ppo-D1 loci corresponding to common wheat PPO genes, and seven new alleles were identified from these species, which were designated as Ppo-A1c (from T. urartu), Ppo-A1d (T. boeoticum), Ppo-A1e (T. monococcum and T. durum), Ppo-A1f (T. dicoccoides), Ppo-A1g (T. durum), Ppo-D1c (Ae. tauschii) and Ppo-D1d (Ae. tauschii), respectively. Five out of the seven alleles detected in the wheat relatives contained an open reading frame (ORF) of 1,731 bp, encoding a polypeptide of 577 residues, which is the same as those of Ppo-A1 and Ppo-D1 genes in common wheat, whereas, the full-length ORF of the allele Ppo-A1g from T. durum was not obtained, and a 73-bp deletion occurred in the third exon of Ppo-D1d, an allele from Ae. tauschii, resulting in a shorter polypeptide of 466 amino acids. The 191-bp insertion in the first intron reported previously in common wheat was also found in T. dicoccoides lines, implying that more than one tetraploid wheat lines may be involved in the origination of common wheat. Phylogenetic trees were constructed using the genomic DNA sequences of the seven alleles, together with four from common wheat and four partial PPO gene sequences deposited in GenBank. The genome tribe A was divided into two clusters, one of which contained Ppo-A1d and Ppo-A1e, and the other included the remaining five alleles at Ppo-A1 locus. The alleles from different clusters showed high sequence divergences, indicated by dozens of SNPs and five to six InDels. The genome tribe D comprised the alleles Ppo-D1a, Ppo-D1c, Ppo-D1d and Ppo-D1b, and the former three were clustered together, showing significant sequence divergence from Ppo-D1b. In addition, the relationships between these allelic variants and grain PPO activities were also discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic diversity of landraces of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from hilly areas of Uttaranchal,India

Seed samples of 27 landraces of wheat were collected from farmers’ fields of hilly areas of Himalaya in Uttaranchal state of India during April 2004. Genetic diversity among 41 genotypes (cultivars and landraces of wheat) was studied using morphological traits, microsatellite markers and SDS-PAGE of HMW-GS. The dendrogram and PCA (Principal Component Analysis) based on morphological data clearly separated landraces of wheat from cultivars. In the dendrogram based on microsatellite markers data all the wheat cultivars released after the introduction of high yielding dwarf wheat varieties from CIMMYT, used in this study, were grouped separately with the exception of NP4. The pre-green revolution indigenous varieties grouped with landraces suggesting that the same had been probably developed through selection among landraces in India. The landraces had higher diversity for HMW-glutenin subunits coded by Glu-B1, with distinct subunit combinations 6 + 8, 7 + 9, 13 + 16, than within the wheat cultivars analyzed. Most of the landraces except IITR10 and IITR14 are clearly distinct from the indigenous and modern wheat cultivars released in India in the 20th century. More than half of the landraces were heterogeneous mixture of plants with different glume color, awnness, grain color and HMW-GS profile and hence need purification through single plant selection. Some of the landraces with resistance to yellow rust and powdery mildew and distinct HMW-GS subunits can be used in appropriate breeding programs. It will be desirable to conserve and protect the landraces as geographical indications of Uttaranchal.