利用小麦微卫星引物建立簇毛麦染色体组特异性标记

选位于普通小麦1A-7A、1B-7B、1D-7D染色体上的102对微卫星引物对多年生簇毛麦、二倍体簇毛麦、小麦-簇毛麦双二倍体与后代和普通小麦中国春、R25、R111、MY11进行了PCR扩增, 发现引物对Xgwm301可以在含簇毛麦染色体的材料中扩出一条长415 bp的特异片段(命名为Xgwm301/415), 而所有供试小麦均未扩出此片段。进而用一套中国春-二倍体簇毛麦附加系来进行扩增, 发现1V-7V染色体均可以扩出该片段, 说明该片段为簇毛麦1V-7V染色体所共有。因此, Xgwm301/415是簇毛麦染色体组上的一个特异片段, 可以用来快速跟踪检测导入到普通小麦背景中的簇毛麦染色体。     

簇毛麦染色体组特异性RAPD标记的筛选、定位和应用

以普通小麦中国春、中国春－簇毛麦二体附加系以及不同来源的簇毛麦为材料,用100个10碱基随机引物进行RAPD扩增。引物OPF02能在不同来源的簇毛麦及所有中国春－簇毛麦二体附加系中扩增出一条长约750bp的片段OPF02 750。普通小麦和硬粒小麦不能扩增出该片段。因此,OPF02 750为分布于簇毛麦所有染色体上的一个簇毛麦染色体组特异片段。用引物OPF02对普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体以及几个普通小麦的簇毛麦二体代换系、二体附加系进行检测,发现NAU302已经丢失了其所附加的簇毛麦3V染色体。     

簇毛麦基因组特异性PCR标记的建立和应用

以普通小麦中国春、簇毛麦、中国春－簇毛麦二体附加系和代换系为材料进行RAPD分析,筛选出一个簇毛麦基因组特异性RAPD片段OPFO2757,该片段分布于簇毛麦所有染色体上。在对OPFO2757进行克隆、测序的基础上,设计一对PCR引物,建立了簇毛麦基因组特异性PCR标记。用这对PCR引物对不同普通小麦品种、不同硬粒小麦品种、不同居群的簇毛麦、中国春－簇毛麦二体附加系、中国春－簇毛麦二体代换系、普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体等材料进行扩增,凡具有簇毛麦染色体的材料都能扩增出一条长为677bp的DNA片段,而不具簇毛麦染色体的材料包括大麦、黑麦、长穗偃麦草、中间偃麦草等不能扩增出该片段。所以,该特异性PCR标记可用于快速跟踪检测小麦背景中的簇毛麦染色体。     

利用AABBDDDD八倍体培育小麦-簇毛麦二体附加系的研究

对普通小麦（Triticumaestivum）-节节麦（Aegilopssquarrosa）八倍体（2n=8x=56,AABBDDDD）与硬粒小麦（Triticumdurum）-簇毛麦（Haynaldiavillosa）六倍体（2n=6x＝42,AABBVV）杂交后,将所得七倍体杂种（AABBDDV）进行连续自交,在F4代中利用C-分带鉴定出可能的簇毛麦6V二体附加系95－7和2V二体附加系26－7,其花粉母细胞染色体在减数分裂中期I的配对构型分别为0.14I＋20．42＋1．5和0．10I＋20．07＋1.82;进一步将95－7和26－7的基因组DNA用EcoRI酶切,分别用小麦族第6部分同源群短臂探针Psr113和第2部分同源群长臂探针BCD240进行Southern杂交,结果显示具有簇毛麦的特异杂交带,进一步确证了95－7和26－7分别是普通小麦-簇毛麦6V和2V二体附加系。     

利用离果山羊草3C染色体诱导簇毛麦4V染色体结构变异

通过普通小麦农林26-离果山羊草3C异附加系与普通小麦-簇毛麦4V（4D）代换系杂交,杂交F1代与普通小麦回交,综合运用染色体构型分析、C-分带和荧光原位杂交等技术从BC1F2、BC1F3代中鉴定出涉及簇毛麦4V染色体的易位系、端体、等臂染色体系等变异植株,表明离果山羊草3C染色体可有效诱发簇毛麦4V染色体结构变异,是创造小麦-簇毛麦4V易位系的一种有效途径。     

簇毛麦NBS类R基因相关序列的分子标记开发及其染色体定位

小麦近缘种簇毛麦携带许多尚未克隆的抗病(R)基因。NBS-LRR类型的R基因占已克隆植物R基因的绝大多数,因此,本研究根据NBS-LRR类型R基因的保守序列设计引物,从簇毛麦基因组DNA和cDNA中扩增获得23条相关序列。基于其中5条抗病基因同源序列(RGAs)H-56/d6、H-66/b2和CDS40设计引物,对小麦、簇毛麦、硬-簇双二倍体及其杂种以及已知携带个别簇毛麦染色体或染色体臂的小麦材料进一步进行PCR扩增,结果表明:3对引物均可对簇毛麦、硬-簇双二倍体进行特异扩增;同时,源于序列H-66/b2的引物可对1VL和6VL染色体臂进行特异扩增;源于序列CDS40的引物可在同时携带1VL和2VS或同时携带2VS和4V的小麦材料以及具有6VL的小麦材料中特异扩增,而H-56/d6的引物在携带1VL、2VS、4V和6V染色体臂或染色体的小麦背景中都不能获得目的片段的扩增。这些结果不仅为外源染色体臂在小麦背景中的追踪与鉴定提供了新的分子标记,而且这些标记还与外源染色体或染色体臂上的抗病基因或抗病基因同源物紧密连锁或共分离。     

用分子原位杂交（GISH）鉴定小麦－簇毛麦双倍体、附加系、代换系和易位系

用生物素标记的簇毛麦（Ｈａｙｎａｌｄｉａｖｉｌｌｏｓａ）染色体组ＤＮＡ（ｔｏｔａｌｇｅｎｏｍｉｃＤＮＡ）作探针,以普通小麦染色体组ＤＮＡ作遮盖（用量１：２００左右）,进行有丝分裂中期和减数分裂中期Ｉ染色体的分子原位杂交（ＧＩＳＨ）,经抗生物素蛋白－辣根过氧化物酶复合物（ｂｉｏ－ｓｔｒｅｐｔａｖｉｄｉｎ－ｈｏｒｓｅｒａｄｉｓｈｐｅｒｏｘｉｄａｓｅ）和联苯胺四盐酸（ＤＡＢ）检测显色后,小麦－簇毛麦双倍体、附加系、代换系和易位系中的簇毛麦染色体及染色体片段显棕色,与显浅蓝色的小麦染色体可明显区分。用ＧＩＳＨ不仅可以检测导入小麦中的簇毛麦染色质,而且可以清楚地显示出易位染色体断裂点的确切位置。将ＧＩＳＨ用于减数分裂期染色体配对分析,还可以清晰形象地显示出同源和非同源染色体之间的配对和分离情况。     

大麦6H染色体特异性标记的筛选和鉴定

从大麦、小麦和小麦－大麦６Ｈ染色体附加系ＲＡＰＤ分析筛选出对６Ｈ染色体特异的２个ＲＡＰＤ标记,转换为特异性ＰＣＲ标记,利用标记对不同植物材料进行ＰＣＲ扩增鉴定。表明凡含有大麦６Ｈ染色体的材料（Ｂｅｔｚｅｓ、Ｉｇｒｉ、ＣＳ６Ｈ附加系）均能扩增出特异带;而不含６Ｈ染色体的材料,包括小科、黑麦、长穗偃麦草、中间偃麦草、簇毛麦以及含有其他大麦染色体的小麦附加系均不主增出特异带。可见,２对ＰＣＲ引物具有大麦     

小麦-簇毛麦种质系''山农030713''的细胞学和SSR鉴定

利用形态学、细胞学以及SSR标记技术对从硬簇麦和Am 3的杂种后代中选育的种质系‘山农030713’进行了鉴定,结果表明:种质系‘山农030713’大田生长整齐一致,农艺性状较好,且对白粉病免疫;其根尖细胞染色体数目为2n=42,花粉母细胞减数分裂中期Ⅰ(PM C MⅠ)染色体构型为2n=21Ⅱ;它与普通小麦的杂种F1PM C MⅠ多数细胞中形成21个二价体,且常有四价体出现,可能伴有染色体的结构变异;SSR分析证明‘山农030713’基本染色体组成为AABBDD,引物X gwm 99-1A在‘山农030713’中扩增出簇毛麦的特异带,表明‘山农030713’中有来自于簇毛麦的遗传物质,此特异带可作为识别‘山农030713’的SSR标记.综合形态学、细胞学和SSR分析结果推测,‘山农030713’可能是一个小麦-簇毛麦易位系.     

中间偃麦草染色体2Ai-2特异PCR新标记的建立和St基因组特异序列的克隆

中间偃麦草麦、小麦和小麦－中间偃麦草2Ai－2附加系Z1、Z2、X6,代换系ZD28等进行RAPD分析,从320个RAPD引物中,鉴定出2Ai－2染色体特异的2个RAPD标记OPO05650和OPMO414000。利用这2个特异OPO05和OPM04,PCR扩增普通小麦CS（ABD）及其近缘植物中间偃麦草（E1E2St）、拟鹅冠草（St）,长穗偃麦草（E）、簇毛麦（V）、黑麦（R）、大麦（H）粗山羊草（D）等基因组DNA。结果表明,OPO05650和OPO41400均是2Ai－2染色体上St基因组区域的特异标记。将上棕2个特异片段分离回收、克隆、测序,根据测序结果重新设计、合成特异引物,成功地转换RAPD标记为SCAR（sequence characterizked amplifed region）标记SC－05和SC－M4。利用SCAR标记对不同材料进行分析的结果表明,凡含有2Ai－2染色体的抗黄矮病材料及拟鹅冠草均产生一条扩增带,不含2Ai－2染色体的材料,包括小麦、长穗麦草、簇毛麦、黑麦、在麦、粗山羊草以有含有其他他中间偃麦草染色休的附加系,均没有扩增产物,说明上棕2个SCAR标记是中间偃麦草2Ai－2染色体的特异性PCR标记,且是2Ai－2染色体上St基因组区域的特异性标记。克隆与鉴定中间偃麦草的2个SCAR扩增片段TiSCO5和TiSCM4。结果表明,克隆的中间偃麦草TiSCO5和TiSCM4特异片段,分别是St基因组特异性的寡拷贝序列有多拷贝重复序列,为St基因组遗传研究的新探针。     

Identification, mapping, and application of polymorphic DNA associated with resistance gene Pm21 of wheat.

A new powdery mildew resistance gene designated Pm21, from Haynaldia villosa, a relative of wheat, has been identified and incorporated into wheat through an alien translocation line. Cytogenetic and biochemical analyses showed that chromosome arms 6VS and 6AL were involved in this translocation. Random amplified polymorphic DNA (RAPD) analysis was performed on recipient wheat cultivar Yangmai 5, the translocation line, and H. villosa with 180 random primers. Eight of the 180 primers amplified polymorphic DNA in the translocation line, and the same results were obtained in four replications. Furthermore, RAPD analysis was reported for substitution line 6V, seven addition lines (1V-7V), and the F1, as well as F2 plants of (translocation line x 'Yangmai 5'), using two of the eight random primers. One RAPD marker, specific to chromosome arm 6VS, OPH17-1900, could be used as a molecular marker for the detection of gene Pm21 in breeding materials with powdery mildew resistance introduced from H. villosa. Key words : RAPD analysis, 6VS-specific marker, Pm21, Erysiphe graminis f.sp. tritici, Triticum aestivum - Haynaldia villosa translocation.     

Microsatellites confirm the authenticity of inter-varietal chromosome substitution lines of wheat (Triticum aestivum L.)

Ninety-five wheat microsatellite markers (WMS) were used to verify the authenticity of the set of Saratovskaya 29/Yanetzkis Probat inter-varietal wheat chromosome substitution lines developed using Saratovskaya 29 as the recipient variety. Polymorphic markers were available for all chromosome arms except 4DS, 6DS and 7DS. Each chromosome substitution line was tested by 2–8 microsatellite markers. The results demonstrate that most of the lines are correct. Out of 21 lines tested 17 showed the expected microsatellite pattern of the donor variety. Two entire chromosomes, 1B and 7A, and two chromosome arms, 3AL and 6DS, were not substituted with Yanetzkis Probat in their respective lines. Three microsatellite markers located in the distal regions of chromosome arms 4AL, 3BS and 5BL in the corresponding substitution lines did not reveal the expected microsatellite pattern of the recipient variety. The possible causes of the incorrect substitution line development and the appearance of incorrect distal microsatellite markers are discussed. The data confirm the idea that microsatellite markers provide ideal tools for testing the authenticity of genetic stocks of wheat. Received: 27 August 1999 / Accepted: 8 October 1999     

Marker Assisted Selection of Fusarium Head Blight Resistance Genes in Two Doubled­ Haploid Populations of Wheat

Fusarium head blight, caused primarily by Fusarium graminearum, is the most important wheat disease in Canada causing both grain yield and quality losses. Selection for resistance to Fusarium head blight in breeding programs has been difficult because of the complex inheritance of resistance and the environmental effect on disease development and expression. The present study was conducted to examine microsatellite markers associated with resistance to Fusarium head blight and evaluate the effectiveness of these microsatellite markers in selecting for resistance to Fusarium head blight in two doubled-haploid populations segregating for Sumai 3-derived resistance genes. Both doubled-haploid populations were evaluated for resistance to Fusarium head blight by inoculation with F. graminearum in the greenhouse. Eight microsatellite markers from chromosomes 3BS, 6B and 5AL were applied to both doubled-haploid populations. The most significant microsatellite markers were found on the short arm of chromosome 3B, explaining 12% and 36% of phenotypic variation for resistance in the DH181/AC Foremost and AC Foremost/93FHB 21 doubled-haploid populations, respectively. Another important microsatellite marker, gwm644 on 6B, explained 21 % of the phenotypic variation for resistance to Fusarium head blight in the DH181/AC Foremost doubled-haploid population. There was a general lack of marker polymorphism on 5AL for the parents used in this study. Microsatellite markers on chromosome 3BS in addition to microsatellite markers on 6B have the potential for accelerating the development of wheat cultivars with improved Fusarium head blight resistance through the use of marker-assisted selection.     

Chromosomal locations of the genes for histones and a histone gene-binding protein family HBP-1 in common wheat

The chromosomal locations of the genes in common wheat that encode the five histones and five members of the HBP (histone gene-binding protein)-1 family were determined by hybridizing their cloned DNAs to genomic DNAs of nullitetrasomic and telosomic lines of common wheat, Triticum aestivum cv. Chinese Spring. The H1 and H2a genes are located on different sets of homoeologous chromosomes or chromosome arms, namely, 5A, 5B and 5D, and 2AS, 2BS and 2DS, respectively. Genes for the other histones, H2b, H3 and H4, are found in high copy number and are dispersed among a large number of chromosomes. The genes for all members of the HBP-1 family are present in small copy numbers. Those for HBP-1a(1) are located on six chromosome arms, 3BL, 5AL, 5DL, 6AL, 6BS and 7DL, whereas those for each HBP-1a(c14), 1a(17), 1b(c1), and 1b(c38) are on a single set of homoeologous chromosome arms; 4AS, 4BL, 4DL; 6AS, 6BS, 6DS; 3AL, 3BL, 3DL; and 3AS, 3BS, 3DS, respectively. The genes for histones H1 and H2a, and for all members of the HBP-1 family except HBP-1a(1) are assumed to have different phylogenetic origins. The genes for histone 2a and HBP-1a(17) are located in the RFLP maps of chromosomes 2B and 6A, respectively. Gene symbols are proposed for all genes whose chromosomal locations have been determined.     

Identification of chromosome arms influencing expression of the HMW glutenins in wheat

The high-molecular-weight (HMW) glutenin genes, located on the group 1L chromosome arms, are a major determinant for baking quality in wheat ( Triticum aestivum L.). In addition, the HMW glutenin genes provide a valuable model system for studying the evolution and regulation of orthologous and paralogous genes in polyploid species. The goal of this study was to identify loci that modify the expression of the HMW glutenins, and to map them to specific chromosome arms. Comparisons were made between endosperms with zero versus three (or three versus six) doses for each of the 42 chromosome arms of wheat. SDS-PAGE and scanning densitometry were used to quantify the protein expression levels of the four HMW glutenin genes in cv. Chinese Spring, for each of the dosage comparisons. Fifteen chromosome arms were found to have significant effects on Glu-B1-1, excluding the structural gene dosage effect: eight positive effects on 1AL, 2AS, 2BL, 2DS, 5DS, 6AL, 6DL, and 7AL and seven negative effects on 1BS, 1DS, 1DL, 4DL, 6BS, 6DS, and 7AS. Nineteen chromosome arms had significant effects on Glu-B1-2, excluding the structural gene dosage effect: eight positive effects on 1AL, 2AS, 2BS, 3AL, 4BL, 6DS, 7BL and 7DS and 11 negative effects on 1AS, 1BS, 1DS, 1DL, 2AL, 2BL, 3DS, 4BS, 4DL, 5BL, and 6BS. Twenty chromosome arms had significant effects on Glu-D1-1, excluding the structural gene dosage effect: 11 positive effects on 1AL, 1BL, 2BS, 2DS, 5BS, 5DS, 6AL, 6DS, 6DL, 7AL, and 7BL and nine negative effects on 1AS, 1BS, 1DS, 2BL, 4DL, 5BL, 5DL, 6BL, and 7DS. Twenty-five chromosome arms had significant effects on Glu-D1-2, excluding the structural gene dosage effect: 17 positive effects on 1BL, 2AS, 2BS, 2DS, 2DL, 3AS, 3AL, 3BS, 5AS, 5BS, 5DL, 6AL, 6DL, 7AL, 7BS, 7BL, and 7DL and eight negative effects on 1DS, 4DL, 5AL, 5BL, 6BS, 6BL, 6DS and 7DS. Of the 164 gene-chromosome arm tests performed, about 52% (85/164) showed no significant effects, and 48% (79/164) showed significant effects, excluding the structural gene dosage effects. Of the significant effects, 56% (44/79) were positive effects, and 44% (35/79) were negative effects. Comparisons of dosage effects on orthologous loci (both x-type or both y-type HMW glutenins) showed that orthologous HMW glutenin genes are largely influenced by the same regulatory systems. Less correlation was found for comparisons between paralogous genes, although considerable conservation was observed at this level as well. These observations suggest that after polyploidization, many of the duplicated orthologous regulatory loci were inactivated by mutation, thus consolidating control over the HMW glutenin genes. Possible candidates for orthologous regulatory genes were identified in maize and barley. This study represents the first comprehensive search of the wheat genome for regulators of the HMW glutenins.     

普通小麦—簇毛麦异附加系和异代换系的C—分带鉴定

用改良的C-分带技术鉴定南京农业大学细胞遗传研究室获得的普通小麦的簇毛麦V_2、V_3、V_4、V_6、V_7染色体异附加系和V_2、V_5异代换系,得到与N-分带和染色体配对分析一致的结果,并且由于C-分带可同时鉴别小麦全部21对染色体,鉴定出V_2异代换系中被代换掉的小麦染色体为1A。