小麦微卫星引物对多枝赖草基因组DNA扩增的研究

用227对小麦微卫星引物对多枝赖草基因组DNA进行PCR扩增，共有81对引物有扩增产物，占所用引物的35．7％。这81对小麦微卫星引物涉及101个微卫星位点，覆盖了小麦全部7个部分同源群。其中有76对引物可在多枝赖草和中国春及丰抗13问扩增出多态性标记。这说明多枝赖草虽然和普通小麦亲缘关系较远，但小麦SSR引物还是可以在多枝赖草上应用的，这不仅拓宽了小麦微卫星引物的使用范围，更重要的是为使用小麦微卫星引物对普通小麦与多枝赖草远缘杂交种质材料进行深入研究奠定了基础。     

利用SSR鉴定普通小麦-多枝赖草二体异附加系 Line24中外源染色体同源群的归属

用227对小麦微卫星引物进行PCR扩增，76对可在多枝赖草和耐黄矮病的普通小麦-多枝赖草二体异附加系Line24的小麦亲本中国春、丰抗13间检测到多态性。和多枝赖草相同而与Line24其他小麦亲本不同的扩增带。在这76对引物中，发现有4对引物能从Line24中扩增出进一步用Line24和普通小麦杂交得到的7个不同的单体异附加系进行验证，也得到同样的结果，说明这4对微卫星引物扩增出的特异带可以作为Line24中多枝赖草染色体的分子标记。根据这4对引物各自对应的微卫星标记位点在小麦染色体上的位置，说明Line24中附加的一对多枝赖草染色体是第3，5，6和7部分同源群多枝赖草染色体相互易位形成的。     

小麦品种百农AK58与周麦18的遗传差异性分析

为解析百农AK58和周麦18的遗传差异,利用表型数据分布于小麦全基因组的305个SSR标记进行分析,以期为小麦大面积种植品种的培育提供参考。方差分析表明,百农AK58和周麦18在株高、千粒质量和单位面积穗数上存在极显著差异,其中百农AK58在株高和单位面积穗数上明显优于周麦18。分子标记比较发现,305对SSR引物中有130对引物在百农AK58与周麦18之间扩增出差异带,多态引物比例达到42.6%。2个品种在A、B、D基因组存在差异的引物数基本一致,分别有34,37,37个,多态引物比例表现为B基因组(43.9%)A基因组(37.1%)D基因组(33.5%)。百农AK58和周麦18的遗传差异在小麦7个部分同源群和21对染色体上也表现一定的不均衡性。在7个部分同源群之间,二者在第5部分同源群的遗传差异最大,多态引物比例达到49.3%,而在染色体水平上,它们在3B、5B、7B、4A的遗传差异相对较大,多态性引物比例均超过50%。百农AK58和周麦18所不同的SSR位点是遗传研究中需要重点关注的基因组区域。     

人工合成六倍体小麦后代衍生群体遗传多样性检测

通过四倍体二粒小麦和节节麦杂交而获得的人工合成六倍体小麦,含有丰富的普通小麦品种改良有益基因,作为拓宽普通栽培小麦性状和新品种改良的新的种质资源已广泛应用于普通小麦的遗传改良实践中.利用分布于小麦A、B、D基因组21条染色体、28个不同染色体臂上的37对微卫星引物,对人工合成六倍体小麦与四川成都平原普通栽培小麦主栽品种杂交、回交经多代选择而形成的117份人工合成六倍体小麦衍生后代高代群体系(其中川麦38、川麦42、川麦43和川麦47为审定品种)进行了DNA分子水平上的分析,共检测到256个等位变异,平均每个SSR标记位点检测到6.92个等位变异,变幅在1到14之间.A、B、D基因组中,D基因组表现出的多态性信息含量最低,为0.4276,B基因组次之,为0.5346,A基因组最高,达到0.6145(A＞B＞D).辛普森指数比较的结果也反映出相同的变化趋势,A基因组最高,为1.1874,B基因组次之,为1.0810,D基因组最小,为0.8046(A＞B＞D).综合多态性信息含量和辛普森指数的估值,表明这一批人工合成六倍体小麦衍生后代群体接受的遗传基因既来自人工合成六倍体小麦,又来自普通栽培小麦,显示杂合度类型丰富,具有较高的遗传差异.根据SSR位点获得的等位基因变异片断的分布情况进行UPGMA聚类,发现A、B、D基因组基凶型间的遗传相似系数较低,A、B、D三个基因组所得平均遗传相似系数为0.4721,其中A基因组平均遗传相似系数为0.3797,B基因组平均遗传相似系数为0.4627,D基因组上平均遗传相似系数为0.5815,反映人工合成六倍体小麦后代衍生材料的遗传多样性处于较高水平.研究结果证明利用人工合成六倍体小麦所具有的普通小麦野生近缘种中的基因库改良现代小麦,丰富其遗传基础,减少其生物和非生物胁迫的脆弱性,是一条行之有效的途径.     

ISSR分子标记及其在植物研究中的应用

ISSR(inter simple sequence repeat)分子标记是一种以微卫星序列为引物,进行多位点PCR扩增的技术.ISSR技术简易、快捷,同时兼备AFLP(扩增酶切片段多态性)、SSR(简单序列重复)和RAPD(DNA随机扩增多态性)等分子标记方法的优点.引物设计无需预知基因组序列,只要是目标区域的长度在可扩增范围内,就能扩增出微卫星重复序列间的DNA片段.ISSR标记以其高多态性,已被广泛应用于种质收集、品种鉴定、遗传多样性、系截系、遗传作图、基因定位、标记辅助选择、预测基因组SSR基序的丰度以及SSR引物开发等研究.本文对ISSR技术及其在植物研究中的应用进行全面的概述.     

小麦微卫星标记在中间偃麦草中通用性研究

利用分布于普通小麦整个基因组的525对微卫星引物,对其在普通小麦与中间偃麦草(Thinopyrumintermedi-um)之间的通用性及其亲缘关系进行了分析。结果表明:202对引物在小麦和中间偃麦草间多态性扩增,所占比率为38.4%,小麦的A,B,D3个基因组多态性引物所占比率分别为34.6%,36.9%和42.2%;说明普通小麦SSR引物在中间偃麦草之间具有通用性,小麦D基因组与中间偃麦草亲缘关系要远于A,B基因组与中间偃麦草关系,表明小麦的A,B,D基因组之间存在遗传差异性。     

人工六倍体小麦后代衍生群体遗传多样性研究

通过四倍体二粒小麦和节节麦杂交而获得的人工合成六倍体小麦,含有丰富的普通小麦品种改良有益基因,作为拓宽普通栽培小麦性状和新品种改良的新的种质资源已广泛应用于普通小麦的遗传改良实践中.利用分布于小麦A、B、D基因组21条染色体、28个不同染色体臂上的37对微卫星引物,对人工合成六倍体小麦与四川成都平原普通栽培小麦主栽品种杂交、回交经多代选择而形成的117份人工合成六倍体小麦衍生后代高代群体系(其中川麦38、川麦42、川麦43和川麦47为审定品种)进行了DNA分子水平上的分析,共检测到256个等位基因变异,平均每个SSR标记位点检测到6.92个等位变异基因.每个SSR位点等位基因变异数在1～14个,变异幅度较大,表明SSR分子标记在人工合成六倍体小麦中表现出高水平的遗传变异.A,B,D基因组中,D基因组表现出的多态性信息含量最低,为0.427 6,B基因组次之,为0.534 6,A基因组最高,达到 0.614 5(A>B>D).辛普森指数比较的结果也反映出相同的变化趋势,A基因组最高,为1.187 4,B基因组次之,为1.081,D基因组最小,为0.804 6(A>B>D).综合多态性信息含量和辛普森指数的估值,表明这一批人工合成六倍体小麦后代衍生高代群体接受的遗传基因既来自人工合成六倍体小麦,又来自普通栽培小麦,显示杂合度类型丰富,具有较高的遗传差异.根据获得的等位基因变异片断的分布情况进行UPGMA聚类,发现A,B,D基因组基因型间的遗传相似系数较低, A,B,D三基因组37个SSR标记位点所得平均遗传相似系数为0.472 1,A基因组平均遗传相似系数为0.379 7,B基因组平均遗传相似系数为0.462 7,D基因组上平均遗传相似系数为0.581 5,反映出人工合成六倍体小麦后代衍生群体材料的遗传多样性处于较高水平.本研究结果证明利用人工合成六倍体小麦所具有的普通小麦野生近缘种中的基因库改良现代小麦,丰富其遗传基础,减少其生物和非生物胁迫的脆弱性,是一条行之有效的途径.     

苦荞全基因组SSR位点特征分析与分子标记开发

目前苦荞SSR多态性标记数量较少,根据已发表的苦荞基因组测序数据,利用MISA软件对1~6核苷酸重复的SSR位点进行了查找和序列特征分析,批量设计引物并对引物进行了有效性和多态性检测。结果表明,苦荞基因组中共检测到1 640个SSR位点,其中三核苷酸重复型SSR最多,占比63.29%,五核苷酸重复型最少,仅占0.12%。AT/TA、AAG/CTT、ACC/GGT和ATC/GAT为出现频率较高的重复基序。苦荞基因组SSR序列长度变化范围为12~476bp,平均长度23.14bp,长度12~19bp的占比71.71%,长度≥20bp的占比28.29%。根据不同类型SSR位点设计并合成引物479对,选择200对引物对5份苦荞资源和3份甜荞资源进行多态性检测,有56对扩增出多态性条带,17对在苦荞种质中产生多态性条带,48对在甜荞种质中产生多态性条带,9对同时在两种种质中产生多态性条带。利用苦荞全基因组序列可实现SSR标记的批量开发,可鉴定出适用于苦荞和甜荞遗传多样性分析、遗传图谱构建和品种鉴定等研究的SSR引物。     

小麦Glu-D3和Glu-B3位点LMW-GS基因特异引物设计与PCR扩增

用CTAB法提取小麦基因组DNA，根据GenBank中公布的已知LMW-GS基因序列，设计并合成染色体位点特异PCR引物1～7；利用特殊小麦材料——六倍体普通小麦阿勃二体、1A、1B和1D缺体，四倍体小麦及二倍体的一粒小麦和节节麦的基因组DNA为模版，在优化的PCR体系下进行特异性扩增和引物验证。结果表明：引物3和引物4为小麦谷蛋白Glu-D     

普通小麦背景中长穗偃麦草[Lophopyrum elongatum（Host）A．Loeve]E染色体组的RGAP特异标记

利用已知植物抗病基因编码氨基酸保守区域NBS—LRR（核苷酸结合位点-富亮氨酸区域）设计了42个简并引物组合，运用抗病基因类似物多态性（resistance gene analog polymorphism，RGAP）分子标记技术，对中国春、中国春-长穗偃麦草双二倍体及其附加系和代换系基因组DNA进行PCR扩增。结果表明，共有38对引物组合获得扩增产物，其中35对在普通小麦中国春、中国春-长穗偃麦草双二倍体中能扩增出多态性，平均每个引物组合扩增出38．5个片段。在普通小麦背景下，共获得275条长穗偃麦草E基因组多态件谱带，占扩增总谱带数的17．44％，揭示出在普通小麦背景下E基因组和普通小麦A、B、D基因组间的高丰度遗传变异。另外，利用RGAP分子标记技术，构建了一套完整的长穗偃麦草1E～7E染色体的特异RGAP标记。为小麦背景中长穗偃麦草外源遗传物质的快速检测提供了新途径。     

Waxy proteins in diploid, tetraploid and hexaploid wheats

Electrophoretic analyses of waxy proteins, encoded by genes present at the Wx‐1 loci, present in several cultivars and accessions of hexaploid wheat, Triticum aestivum, have permitted the detection of null alleles at the Wx‐B1 and Wx‐D1 loci. Polymorphism at the Wx‐A1 and Wx‐B1 loci was also investigated in several accessions of tetraploid wheats, Triticum durum, Triticum dicoccoides and Triticum timopheevi, and in diploid species, Triticum urartu, Triticum boeoticum and Triticum monococcum. One null allele at the Wx‐A1 locus and three polymorphic alleles at Wx‐B1 locus were detected in T. durum; a new allele at one of the two waxy loci was identified in the tetraploid wheat T. timopheevi; no polymorphism was detected in diploid species. Polymerase chain reaction techniques made possible the detection of further polymorphism existing at the Wx‐1 loci and the reason for the lack of expression of the null genotypes to be investigated. The null forms detected at each locus have been used to produce complete sets of partial and total waxy lines in durum and bread wheat.     

Transfer of the Glu-D1 Gene from Chromosome 1D to Chromosome 1A in Hexaploid Triticale

To complement previously developed recombinant chromosomes 1R.1D, two series of translocations involving the Glu-D1 gene from chromosome ID to chromosome 1A were produced in hexaploid triticale. These series involve seven independent transfers of allele d encoding for high molecular weight glutenin subunits 5+10 and ten independent transfers involving allele a encoding for HMW glutenin subunits 2 + 12. The frequency of homoeologous recombination between chromosomes 1A and 1D was within the range observed for pairs of homologues in wheat, supporting earlier observations that homoeologous recombination in triticale is frequent. Recombined chromosomes 1A.1D can be used to introduce the Glu-D1 gene to durum wheats, and to manipulate the dosage of Glu-D1 in hexaploid triticale and bread wheat.     

Resistance to stripe rust in durum wheats, A-genome diploids, and their amphiploids

Stripe rust (caused by Puccinia striiformis Westend.) is a wheat disease of worldwide importance. Seedlings of 75 accessions of Triticum boeoticum, 12 of T. monococcum, 16 of T. urartu, 230 of durum wheat (T. turgidum L. var. durum), and 128 amphiploids (genome AAAABB) involving the crosses of the three diploid species (AA) with T. turgidum (AABB) were evaluated in the greenhouse for their reaction to P. striiformis race 14E14. Durum wheats and the amphiploids were also evaluated at two field locations in Mexico with the same race for their adult plant response. Resistant seedling reactions (infection types: 0-3 on a 0-9 scale) were seen for 10 (13%) accessions of T. boeticum, 19 (8%) accessions of T. turgidum and 32 (25%) amphiploids. The remaining accessions were either moderately resistant (ITs 4-6) or susceptible (ITs 7-9). The three amphiploids derived from the crosses of seedling resistant T. boeoticum and T. turgidum, were resistant as seedlings. Among the 51 amphiploids involving one resistant parent, 29 were resistant and the remaining 22 displayed intermediate to susceptible reactions. Suppressors for resistance were common in the A and AB genomes and suppression was resistance gene specific. Forty-five (20%) durums showed adequate field resistance (relative AUDPC <10% of the susceptible check ‘Morocco’). These included the 19 seedling resistant durums. Presence of genes involved in adult plant resistance was evident, because 26 of the remaining adult plant resistant durums had displayed intermediate-susceptible seedling reactions. Though the seedling reactions of the amphiploids varied from low to high, all involving the adult plant resistant durums possessed adequate field resistance. The resistant, newly produced, AAAABB amphiploids are useful genetic resources for stripe rust resistance which could be transferred to the cultivated T. turgidum. This revised version was published online in July 2006 with corrections to the Cover Date.     

不同水肥条件下二倍体、四倍体和六倍体小麦养分吸收、利用效率的研究

试验选用了3个二倍体（Triticum boeoticum，AA；Aegilops speltoides，BB和Ae. tauschii，DD）、2个四倍体（T. dicoccoides，AABB和T. dicoccum，AABB）和1个六倍体（T. aestivum，AABBDD）的小麦进化材料，在不同水肥条件下研究小麦进化中养分效率的差异及水肥条件对小麦养分利用的影响。试验表明，随小麦的进化，养分（N、P和K）吸收量和吸收效率显著增加，但是籽粒养分含量却减少。水分胁迫和低肥处理减少小麦籽粒养分含量、（整株）养分吸收量和吸收效率。小麦的生物量养分利用效率和产量养分利用效率都存在显著的种间差异，而且后者的差异更大。随小麦的进化，生物量养分利用效率和产量养分利用效率均显著增加。6个小麦进化材料的生物量氮、磷利用效率（NUTEb和PUTEb）的大小顺序相同，均为：Ae. speltoides > T. dicoccum、T. dicoccoides、Ae. tauschii、T. boeoticum > T. aestivum，而生物量钾利用效率（KUTEb）是T. aestivum、Ae. tauschii、Ae. speltoides > T. dicoccoides > T. dicoccum > T. boeoticum。Ae. tauschii的产量养分利用效率显著高于其他两个二倍体小麦（AA和BB），表明D组染色体上存在有控制高效利用养分的基因。产量氮、磷、钾利用效率（NUTEg、PUTEg、KUTEg）和收获指数（HI）的大小排序均为：T. aestivum > T. dicoccum、T. dicoccoides、Ae. tauschii > T. boeoticum、Ae. speltoides。水分与养分对生物量养分利用效率有显著影响，而对产量养分利用效率的作用却不显著，说明小麦产量养分利用效率是较为稳定的特性，主要由基因型决定。水分胁迫有利于提高小麦生物量养分利用效率，但是增加施肥量却对其有负作用。     

Starch characterisation and variability in GBSS loci of synthetic hexaploid wheats and their durum and <Emphasis Type="Italic">Aegilops tauschii</Emphasis> parents

Greater variability in starch properties is found in lower ploidy wheats than in commercial hexaploid wheats. This paper reports on the starch properties and variability in granule bound starch synthase (GBSS) loci of 17 diploid (Aegilops tauschii) and 12 tetraploid (durums) potential progenitors of wheat, compared with 29 synthetic hexaploid wheats produced from such progenitors. Starch properties examined were granule size distribution, swelling power, amylose content, gelatinisation and amylose-lipid dissociation properties. A PCR screening method was able to detect the presence or absence of each of the three GBSS genes. It also detected polymorphisms in eight diploids and nine hexaploids, all displaying the same 25 bases deletion in the D genome allele of GBSS. Two tetraploids and five hexaploids were null 4A for GBSS. There was little difference in the amylose contents and amylose-lipid dissociation peak temperatures of the synthetic hexaploids and the lower ploidy wheats. The synthetic hexaploids showed intermediate swelling power values with the durums giving the highest swelling powers. The durums also had higher B granule contents than the A. tauschii accessions, but not as high as the synthetics. However, the A. tauschii samples gave the highest gelatinisation peak temperatures. The presence of the null 4A mutation was positively correlated with swelling power, amylose content and DSC measurements. The new smaller D genome allele of GBSS was associated with slightly higher swelling power. These results confirm the value of wheat progenitor lines as sources of new starch properties for hexaploid wheat. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Polymorphism of waxy proteins in Iberian hexaploid wheats

A collection of 130 cultivars of bread wheat, 332 landraces of bread wheat and 144 spelt wheats was analysed for waxy proteins in the grain. The electrophoretic patterns showed very low polymorphism and most of the hexaploid wheats had the Wx-Ala, Wx-D1a and Wx-B1 alleles of ‘Chinese Spring’. Two alleles were detected at Wx-A1 (Wx-A1a, and Wx-A1b (null)), the latter was present in only 5.1% of the bread wheat landraces and 7.6% ofthe spelt wheats. No allelic variation was found at the Wx-D1 locus and all the hexaploid wheats had the Wx-D1a allele. Wx-B1 was the most polymorphic locus, with three alleles detected: Wx-B1a, Wx-B1b (null) and Wx-Blc coding for a Wx-B1 protein with a slightly different mobility from Wx-B1a. The null Wx-B1b allele was found in 10.8% of the bread wheat cultivars, 21.4% of the bread wheat landraces and 12.5% of the spelt wheats. Among the 604 hexaploid wheats analysed, only two bread wheat landraces (0.6%) and two spelt wheats (1.4%) had the null allele at both Wx-A1 and Wx-B1 loci.     

Effect of genome and ploidy on photosynthesis of wheat

We assessed (1) the effects of addition and doses of the D genome from different sources and (2) the addition of either the A genome or the D genome on the photosynthesis of synthesized hexaploid wheats. On average, the increased doses of the D genome reduced photosynthesis, but the depression was dependent on the source of the D genome. Two accessions of Aegilops squarrosa had depressed photosynthetic rates, but not another accession of Ae. squarrosa. The D genome of cv. Thatcher did not contribute to depress photosynthetic rate. Triticum monococcum had considerably higher photosynthetic rates than Ae. squarrosa. However, addition of the A genome from T. monococcum did not increase the photosynthetic rates of hexaploids. Chlorophyll a : b ratio, functional photosystem II and the core complex of photosystem II did not account for the variation in photosynthetic rate among the genotypes studied. In our experiment, photosynthesis of polyploids was not dependent on photosynthesis rates of the donor genomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

小麦盐胁迫相关基因TaMYB32的克隆与分析

在对小麦全长cDNA克隆进行大规模测序及转录因子功能研究中,筛选到一个盐胁迫相关的MYB转录因子基因,将其命名为TaMYB32。TaMYB32的全长cDNA序列为1250 bp,开放阅读框为732 bp,编码一个具有244个氨基酸的R2R3-MYB转录因子。根据该基因的cDNA序列设计引物,分别在小麦二倍体祖先种乌拉尔图小麦UR206、拟斯卑尔脱山羊草Y2006和粗山羊草Y2282以及六倍体普通小麦中国春和茶淀红中克隆了TaMYB32的基因组和cDNA序列。序列分析表明TaMYB32在小麦二倍体祖先种中存在2种序列,在六倍体小麦中存在4种序列,其中1种序列在进化上非常保守,在二倍体和六倍体中完全相同。对TaMYB32的基因组和cDNA序列比较分析表明它是一个没有内含子的基因。电子定位发现TaMYB32在小麦第六同源群上,每个基因组中有2个拷贝,这与测序结果相吻合。同源序列分析发现,TaMYB32与来自水稻和玉米中的MYB蛋白的相似性分别为72.4 %和73.7%。组织表达特性分析表明该基因在小麦根、茎、叶、雌蕊和花药中均有较强的表达。半定量与实时定量RT-PCR结果表明TaMYB32是一个受盐胁迫诱导表达的基因。     

Transfer of the Glu-D1 Gene from Chromosome 1D of Breadwheat to Chromosome 1R in Hexaploid Triticale

The use of hexaploid triticale as a crop for human consumption has been limited by its inferior bread-making quality. To ameliorate this problem, a segment of chromosome ID of breadwheat with the Glu-D1d allele encoding for high molecular weight glutenin subunits 5 7plus; 10 was translocated to chromosome 1R of the hexaploid triticale ‘Rhino’ through a combination of a centric break-fusion translocation followed by 5D(5B)-induced homoeologous pairing. The resulting recombinant chromosome 1R has a small interstitial segment of ID with the Glu-D1d allele. The maximum physical length of the translocated segment is estimated at about 16.5 % of 1DL. Frequency of translocations involving the long arms of homoeologous group-1 chromosomes in the analyzed progeny suggested that homoeologous recombination in triticale was substantially higher than that previously reported in hexaploid wheat.     

Assessment of genetic diversity in synthetic hexaploid wheats and their Triticum dicoccum and Aegilops tauschii parents using AFLPs and agronomic traits

Synthetic hexaploid wheats are of interest to wheat breeding programs, especially for introducing new genes that confer resistance to biotic and abiotic stresses. A group of 54 synthetic hexaploid wheats derived from crosses between emmer wheat(Triticum dicoccum, source of the A and B genomes) and goat grass (Aegilops tauschii, D genome donor) were investigated for genetic diversity. Using the AFLP technique, dendrograms revealed clear grouping according to geographical origin for the T. dicoccum parents but no clear groups for the Ae. tauschii parents. The geographical clustering of the T. dicoccum parents was also reflected in the dendrogram of their derived synthetic hexaploids. Diversity of the T. dicoccum parents and their derived synthetic hexaploids was further evaluated by measuring 18morphological and agronomic traits on the plants. Clustering based on morphological and agronomic data also reflected geographical origin. However, comparison of genetic distances obtained from AFLP and agronomic data showed no correlation between the two diversity measurements. Nevertheless, similarities among major clusters with the two systems could be identified. Based on percentage of polymorphic markers, the synthetic hexaploids had a considerably higher level of AFLP diversity (39%) than normally observed in cultivated hexaploid wheat (12–21%). This suggests that synthetic hexaploid wheats can be used to introduce new genetic diversity into the bread wheat gene pool. This revised version was published online in July 2006 with corrections to the Cover Date.