小麦糯性基因的多重PCR分子鉴定

采用多重 PCR 的方法, 对其反应条件进行优化, 以获得用于小麦糯性(Wx)基因分析的稳定PCR体系。应用两对引物, 分别扩增小麦 Wx-A1、Wx-B1、Wx-D1 基因, 目的片段大小分别为: 230 bp/265 bp、854 bp和 204 bp。经反复验证, 结果准确可靠, 重复性好, 成本低, 可以在同一PCR反应体系中对 3 个Wx 基因进行同时筛选鉴定。该体系可用于 Wx 蛋白基因的分子标记辅助选择, 可以提高小麦淀粉品质评价和糯麦选育的效率。     

47份外引小麦种质资源Waxy和HMW-GS分子检测与品质性状分析

为有效利用外引小麦种质资源,本研究对收集的47份外引小麦种质材料进行Waxy和HMW-GS等位基因的分子检测,并分析了其直链淀粉、支链淀粉、湿面筋等品质参数。结果表明,在Wx-A1位点存在3种类型:Wx-A1a、Wx-A1g和WxA1b,39份材料(82.98%)为Wx-A1a类型;Wx-B1位点3种类型:Wx-B1a、Wx-B1e和Wx-B1b,37份材料(78.72%)为Wx-B1a类型;Wx-D1位点2种类型:Wx-D1a和Wx-D1b,46份材料具有Wx-D1a类型;共鉴定出8种Wx-1位点等位基因组合,31份材料(65.96%)为Wx-A1a/B1a/D1a。在Glu-A1位点,含有等位基因Ax2^*、Null和Ax1类型的材料分别为18份、18份和11份;在Glu-D1位点,含有等位基因Dx2和Dx5类型的材料分别为23份(48.94%)和21份(44.68%),含有等位基因Dy12和Dy10类型的材料分别为22份(46.81%)和20份(42.55%),具有Dy10+Dy12类型材料2份;共鉴定出19种Glu-A1/D1等位基因组合,7份材料含有Null/Dx5+Dy12。含有Wx-A1a/B1a/D1a材料的直链淀粉含量相对较高,支链淀粉含量相对较低;含有优质等位基因Ax1或Ax2^*兼Dx5+Dy10材料的湿面筋含量相对较高。总体上这些外引种质资源Waxy和HMW-GS等位基因类型丰富,可为种质资源合理利用和现代普通小麦品质改良提供参考依据。     

黄淮冬麦区不同时期主推品种淀粉合成酶基因分子标记鉴定

利用普通小麦直链淀粉合成酶GBSS基因及支链淀粉关键合成酶SSⅡ基因的特异分子标记鉴定了黄淮冬麦区自20世纪50年代以来253份主推品种,发现8份品种缺失Wx-B1基因,其中5份材料（小偃168、秦麦1号、83S502、山东935031、山东928802）是新发现的缺失Wx-B1基因的小麦品种,未发现Wx-A1、Wx-D1基因缺失类型品种;所鉴定253份品种的SSⅡ基因均为野生型,未发现缺失突变类型。通过对8份缺失Wx—B1基因品种直链淀粉含量分析,发现这些品种的直链淀粉含量差异较大．变异范围为19．9％-33．0％,其中豫麦47、83S502和中育5号3个品种的直链淀粉含量较低,分别为19．9％、21．3％和26．4％,可以用于优质面条小麦品质改良。     

高分子量麦谷蛋白1Bx14亚基AS-PCR标记

根据已发表的1Bx14亚基的基因序列在不同位点设计了10对特异引物,从中筛选出1对引物,对HMW-GS在Glu-1Bx位点已知的10个小麦品种进行了PCR扩增.结果表明,具有1Bx14亚基的4个品种都能扩增出1条1 256 bp左右的特异带.用这一特异标记对山东省种植面积较大的40个品种进行PCR扩增(即等位专一PCR,AS-PCR),发现仅有5个品种携带1Bx14亚基.该AS-PCR标记可用于检测小麦品种在该位点的亚基组成,与SDS-PAGE相比,可显著提高检测的准确性和效率,可为种质鉴定和育种工作提供参考.     

利用回交结合Wx基因分子标记培育部分糯性小麦

颗粒结合型淀粉合成酶Ⅰ(GBSSI,Waxy蛋白)是小麦胚乳中直链淀粉合成的关键酶。小麦基因组中存在3个Waxy基因(Wx-A1、Wx-B1、Wx-D1)。在白火麦中Wx-D1位点的突变(Wx-D1b)引起Wx-D蛋白的缺失,导致直链淀粉含量下降,其面粉表现出部分糯性。与目前生产上推广品种相比,白火麦的农艺性状较差,产量非常低。为了培育农艺性状优良的部分糯性小麦,我们将白火麦与具有优良农艺性状的小麦品种PH85-16、济南17和烟农15(轮回亲本)分别进行杂交,后代分别与相应的三个轮回亲本回交五代,在每代中均选择农艺性状与轮回亲本相近并含有Wx-D1b的后代。在第六代自交后选择具有Wx-D1b的纯合体,选出的单株连续自交三代。获得了六个农艺性状与轮回亲本相似的品系,它们均携带纯合的Wx-D1b位点。研究表明采用回交的方法并结合基于Wx基因序列的分子标记技术,是培育优良部分糯性小麦的一种非常有效的方法。本研究培育出的部分糯性小麦品系可以直接用于大田生产。     

利用回交法与Wx基因分子标记辅助选择培育糯性小麦

以中国春糯性位点全套近等基因系为研究材料,对小麦Wx基因的6个STS标记和1个CAPS标记进行了筛选。改良PCR扩增条件以及产物检测方式后,从这些标记中筛选出3个标记,包括鉴定Wx-A1、Wx-D1位点的2个共显性STS标记和Wx-B1位点的1个显性STS标记,用于本研究中糯性小麦的分子标记辅助育种。在育种过程中,首先配制全糯材料“98Y1441”与推广品种“川育12”的杂交组合,采用籽粒碘染法从其F2种子中选择全糯基因型个体与回交亲本川育12杂交,如此反复自交、回交,历经数代异地加代繁殖得到BC5F2代回交改良群体。利用上述3个分子标记从该群体中筛选出了8种Wx基因型,经卡方检验,其分离比符合3对基因的分离比例,其中基因型为aabbdd的植株有2株,直链淀粉含量分别为1.81%和0.82%,为全糯小麦;基因型为AAbbdd, aabbDD的部分糯性植株各有1株,直链淀粉含量分别为15.24%和17.57%。研究中获得的BC5 F2代群体的农艺性状接近回交亲本,并明显优于全糯材料“98Y1441”,表明采用回交法与Wx基因分子标记辅助选择相结合,有助于培育高产、优质的全糯和部分糯小麦。     

中国糯小麦研究进展

由于糯小麦缺失(Wx-A1、Wx-B1和Wx-D1)3对蜡质基因,其直链淀粉含量低于1%,因此在食品工业和非食品工业中有着巨大的应用价值.自20世纪90年代以来,糯小麦研究已成为国际热点.虽然我国开展糯小麦研究起步较迟,但在糯小麦Wx基因资源的筛选、糯小麦的遗传和育种、糯小麦淀粉品质评价等方面取得了较快的进展.本文就中国在上述领域的研究进展作一综述,并就糯小麦研究提出几点建议.     

中国小麦微核心种质资源Psy基因的等位变异

根据已发表的麦族植物体Psy基因序列的保守区设计引物PsyO2,克隆小麦Psy基因（片段）。结果表明,PsyO2引物的扩增产物出现2种带型：196bp和233bp,序列分析表明两条特异条带涵盖了小麦Psy基因第2外显子全部序列,相差的37bp为Psy基因第2内含子中的一段插入序列,可反映不同黄色素含量（YPC）,属小麦风,,基因的等位变异。验证试验表明,248份小麦微核心种质中有153份材料（占样品数的65．7％）扩增出196bp条带,群体内YPC均值7．314mg kg^-1,属高YPC范畴;另有95份材料（占样品总数的38．3％）扩增出233bp条带,群体内YPE均值为5．207mg kg^-1,属低YPC范畴,方差分析表明二者YPC差异达1％极显著水平差异,说明上述37bp的插入序列是导致小麦品种间YPC产生差异的原因之一,因此该引物扩增的Psy基因对小麦YPC具有显著影响,引物PsyO2是对小麦YPC进行分子鉴定的重要标记。     

小麦Glu-B3位点LMW-GS基因的克隆及序列分析

以小麦特殊遗传材料———六倍体普通小麦阿勃二体、1A缺体、1B缺体和1D缺体,四倍体硬粒小麦墨西粒卡以及二倍体节节麦的总基因组DNA为模板,对D Ovidio等曾报道的硬粒小麦Glu-B3位点LMW-GS基因特异引物对P1(5-′tcctgagaagtgcatgacatg-3′)和P2(5-′gtaggcaccaactccggtgc-3′)进行了PCR扩增验证.结果表明,该引物对同样能特异扩增普通小麦Glu-B3位点LMW-GS基因.利用这对引物通过AS-PCR方法克隆得到优良小麦品种小偃6号1B染色体1个LMW-GS基因片段.该基因全长为1 089 bp,包含了完整的编码区和其上游318 bp的胚乳特异表达启动子区.该基因被命名为XY-Glu-B3-LMW2(GenBank登录号为DQ630442).XY-Glu-B3-LMW2的推测蛋白含256个氨基酸(包括N-端20个氨基酸的信号肽),其成熟蛋白有8个保守的Cys残基,均分布在C-末端区.XY-Glu-B3-LMW2是从小偃6号克隆到的第2个LMW-GS基因.     

改进的多片段重叠延伸PCR制作基因多位点突变

为在研究工作中提高制作目标基因多位点突变体的效率,对常规重叠延伸PCR进行适当改进:对于相距较近的两个突变位点,只需设计一对突变引物各自涵盖其中一个位点即可一次突变;对于相距较远的两个位点,可以采用三片段重叠延伸PCR的办法解决;两者结合则可一次性突变多个位点。以制作RBCT的6位点突变体PSM6为例,利用上述策略,设计两对突变引物;采用OE-PCR法,第一轮PCR扩增出三个片段,第二轮PCR同时利用三片段重叠延伸产物作为模板扩增出目标基因突变体,再按常规分子克隆方法将其连入质粒载体。经测序检测,发现得到了预期的目标基因多位点突变体。因此,采用灵活的引物设计策略,结合多片段重叠延伸PCR即可一次性制作基因的多位点突变体,此方案可解决研究工作中大多数多位点突变问题。     

小麦Wx-B1基因酶切片段长度多态性及其与直链淀粉含量的关系

采用序列标志位点(sequence-tagged sites,STS)引物,对35个小麦品种的Wx-B1基因位点上的近第4个内含子区域的序列进行了扩增,用限制性内切酶BamHI切割.结果表明,扩增片段有的能被酶切,有的不能.酶切片段出现两种长度多态性,其长度与直链淀粉含量(AC)呈负相关.AC在约20%以上的小麦品种扩增的片段能够被BamHI切割,而在约20%以下的不能.以上结果表明,Wx-B1基因在扩增序列区域有多态性,这可以作为一种分子标记在育种中预测不同小麦品种的直链淀粉含量,以改良小麦品质.     

Waxy protein deficiency and chromosomal location of coding genes in common wheat

Deficiency of the wheat waxy (Wx) proteins (Wx-A1, Wx-B1 and Wx-D1) was studied in 1,960 cultivars derived from several countries. Gel electrophoretic analyses revealed that the null allele for the Wx-A1 protein occurred frequently in Korean, Japanese and Turkish wheats but was relatively rare in cultivars from other countries and regions. About 48% of the wheats deficient for the Wx-B1 protein were from Australia and India. One Chinese cultivar lacked the WxD1 protein. While 9 Japanese cultivars were deficient in both the Wx-A1 and Wx-B1 proteins, no cultivars lacked both the Wx-A1 and Wx-D1 proteins, both the Wx-B1 and Wx-D1 proteins or all three Wx proteins. Two-dimensional gel electrophoresis revealed polymorphisms of the three Wx proteins that varied according to isoelectric points or molecular weight. The Wx-A1 gene coding the Wx-A1 protein and the Wx-B1 gene coding the Wx-B1 protein were localized in the distal regions of chromosome arms 7AS and 4AL, respectively, by deletion mapping using the deletion lines developed in the common wheat cultivar Chinese Spring.     

Differential effects of Wx-A1, -B1 and -D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat

Waxy (Wx) protein is a granule-bound starch synthase (GBSS) responsible for amylose production in cereal endosperm. Eight isolines of wheat (Triticum aestivum L.) having different combinations of presence and absence of three Wx proteins, Wx-A1, -B1, and -D1, were produced in order to elucidate the effect of Wx protein deficiencies on the apparent amylose content and starch-pasting properties. An improved SDS gel electrophoresis showed that ’Bai Huo’ (a parental wheat) carried a variant Wx-B1 protein from an allele, Wx-B1e. Thus, wheat lines of types 1, 2, 4, and 6 examined in this study contained a variant Wx-B1 allele and not the standard allele, Wx-B1a. The results from 3 years of experiments using 176 lines derived from two cross-combinations showed that apparent amylose content increased the least in type 8 (waxy) having no Wx proteins and, in ascending order, increased in type 5 (only the Wx-A1 protein is present) <type 7 (Wx-D1) <type 6 (Wx-B1) <type 3 (Wx-A1 and -D1) <type 4 (Wx-A1 and -B1) <type 2 (Wx-B1 and -D1) <type 1 (three Wx proteins). However, Tukey’ s studentized range test did not detect significant differences in some cases. Densitometric analysis suggested that the amylose content was related to the amount of the Wx protein in the eight types. Parameters in the Rapid Visco-Analyzer test and swelling power were correlated to amylose content. Consequently, amylose content and pasting properties of starch were determined to be influenced the most by the lack of the Wx-B1 protein, followed by a lack of Wx-D1, and leastly by the Wx-A1 deficiency, which indicated the presence of differential effects of the three null alleles for the Wx protein. Received: 1 February 1999 / Accepted: 10 April 1999     

Rapid classification of partial waxy wheats using PCR-based markers.

Mutations in the three homeologous waxy loci Wx-A1, Wx-B1, and Wx-D1 of a waxy wheat line have previously been characterized at the molecular level. Using combinations of these mutations, six types of partial waxy wheat plus wild type and waxy wheat (types 1-8) can be produced. Here, we describe primer sets for all three loci that can be used under a single set of conditions, allowing 32 lines to be characterized as types 1-8 in a single PCR run using a 96-well plate. Using multiplex PCR, mutations at the Wx-B1 and Wx-D1 loci can be identified in a single PCR, reducing the number of reactions necessary to identify and select the desired partial waxy wheat line. A single multiplex PCR can be used to detect all three mutations when products are analyzed using capillary electrophoresis on a microchip device. The PCR conditions and primers are effective with a number of cultivars from other countries, indicating that the mutations found at the Wx-A1 and Wx-B1 loci of these cultivars likely have the same origins as the mutations in the corresponding loci of the waxy wheat line used in this study. The PCR selection method described here is an easy and effective alternative to the commonly used SDS-PAGE methods for identification of null alleles.     

Molecular comparison of<Emphasis Type="Italic"> waxy</Emphasis> null alleles in common wheat and identification of a unique null allele

PCR selection markers for the identification of null waxy alleles were used to screen for waxy mutations in 168 common wheat cultivars. In all cultivars where the Wx-B1 protein was absent, the Wx-B1 allele was identical to the previously identified mutation carried by Kanto 107. Although most cultivars missing the Wx-A1 protein also carried the same Wx-A1 mutation as found in Kanto 107, all of the Turkey Wx-A1 mutants produced a different PCR fragment, implying the presence of a different mutation. Sequencing of this fragment indicated the mutation, which consisted of a 173-bp insertion in an exon, was in a different location than the previously identified Wx-A1 mutation. An 8-bp duplication of the Wx-A1 sequence flanked each end of the insertion, and an element with reverse complementary sequences was present at both ends of the insertion. These structures correspond with the features of class II transposable elements. Hence, the Turkey null Wx-A1 mutation was likely caused by the movement of a transposon, and this spontaneous mutation appears to be present in a limited geographical area.Communicated by C. Möllers