中间偃麦草的GISH分析

以染色体组为E^eE^e的二倍体长穗偃麦草（Thinopyrum elongatum,2n=2x=14)、染色体组为E^bE^b的二倍体比萨偃麦草（Th.bessarabicum,2n=2x=14）、染色体组为StStStSt的四倍体拟鹅冠草（Pseudoroegneiria strigosa,2n=4x=28)的总基因组DNA为探针,对中间偃麦草（Th.intermedium)进行GISH分析。结果表明,中间偃麦草是由2个亲缘关系较近的染色体组、1个亲缘关系较远的染色体组构成;中间偃麦草所含的亲缘关系较近的染色体组分别与二倍长穗偃麦草染色体组E^e、比萨偃麦草染色体组E^b、以及1个亲缘关系较远的染色体组与拟鹅冠草染色体组St基本相似,但不完全一样,因此,中间偃麦草的染色体组用E^etE^etE^btStSt表示。     

偃麦草属三个种的染色体组研究

本文综合应用体细胞染色体分带、杂交一代 PMc MI 染色体构型统计和同工酶分析,研究了偃麦草属三个种的染色体组构成。结果表明:中间偃麦草不含与小麦同源的 B 组,有两个组部分同源,其染色体组可用 xE_1E_2表示。长穗偃麦草(10x)也不含 B 组,有四个组两两部分同源,是部分同源异源十倍体,染色体组公式为 xE_1E_1F_1F_2。四倍体偃麦草的两个染色体组间缺少同源性。     

抗条锈病小偃麦双体异附加系山农87074-519的鉴定

综合利用抗性接种鉴定、细胞学分析、SSR分子标记和基因组原位杂交(GISH)技术相结合的方法,对从长穗偃麦草与小麦复合杂交后代中选育的抗条锈病种质系山农87074-519进行了鉴定。结果表明,山农87074-519的根尖细胞染色体数目2n=44,花粉母细胞减数分裂中期I(PMCMI)绝大多数细胞内可观察到22个二价体,平均染色体构型2n=44=21.82Ⅱ 0.36Ⅰ,它与普通小麦中国春杂种F1的多数花粉母细胞内染色体构型为2n=21Ⅱ 1Ⅰ,因此它是1个附加了1对长穗偃麦草染色体的双体异附加系;以假鹅冠草St基因组总DNA作探针进行原位杂交发现山农87074-519的44条染色体中有2条出现黄绿色杂交信号,且杂交信号遍布整条染色体,证明其附加的长穗偃麦草染色体为St基组;利用SSR分子标记技术,在170对SSR引物中筛选出特异引物BARC165,它能稳定地在山农87074-519中扩增出长穗偃麦草特异标记BARC165268;将长穗偃麦草中BARC165的特异扩增片段克隆测序后制备成探针进行原位杂交,可在山农87074-519的间期染色体和有丝分裂中期染色体检测到杂交信号。山农87074-519综合农艺性状较好,对条锈病免疫,其抗性基因为显性,且位于附加的长穗偃麦草St基组染色体上,暂将其表示为YrSt。该种质系在小麦的遗传改良中具有重要利用价值。     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。     

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。     

St基因组中的CRW同源序列在偃麦草中的FISH分析

为了确定十倍体长穗偃麦草(Thinopyrum ponticum, Liu & Wang)和六倍体中间偃麦草(Th. intermedium, [Host] Barkworth & Dewey )的基因组组成, 根据野生一粒小麦(Triticum boeoticum)着丝粒自主型反转录转座子(CRW)序列设计特异引物, 以二倍体拟鹅观草(Pseudoroegneria spicata, Á Löve )基因组 DNA为模板进行PCR扩增, 筛选到一条St基因组着丝粒区相对特异反转录转座子的部分序列pStC1, 长度为1.755 kb (GenBank登录号: FJ952565), 其中有800 bp与小麦着丝粒反转录转座子(CRW)的LTR区高度同源, 另有小部分片段与其外壳蛋白编码基因(gag)部分同源, 并且包含一段富含AGCAAC碱基的重复序列。以pStC1为探针, 对十倍体长穗偃麦草的FISH检测结果显示其基因组组成为两个St组3个E组(St₁St₂E^eE^bE^x); pStC1与中间偃麦草杂交时, 不仅St基因组上有强烈的荧光信号, 而且E基因组一些染色体的近着丝粒区域也有杂交信号, 说明偃麦草属异源多倍体物种在其形成及进化过程中St与E基因组之间在着丝粒及近着丝粒相关区域可能存在协同进化。     

硬粒小麦、提莫菲维小麦与四倍体长穗偃麦草属间杂种的形态和细胞遗传学研究

首次获得硬粒小麦（Triticum durum Desf),提莫菲维小麦（T.timopheevi Zhuk)与四倍体长穗偃麦草（tetraploid Elytrigia elongata)的属间杂种,杂交当代结实率分别为5．29％和1．41％,杂种均表现为多年生,具很强的生活力,形态上呈双亲中间类型,杂种F1自交不育,用普通小麦,硬粒小麦回交,以硬粒小麦为母本的F1均获得交种子,杂种F1花粉母细胞减数分裂中期I染色体配以构型分别为：13．78I＋6．87II＋0．147III,9．10I＋9．11 II＋0．20III,F1形成的二价体主要是四倍体长穗偃麦草染色体之间配对所致,并推测四倍体长穗偃麦草具促进小麦部分同源染色体配对或抑制小麦ph基因作用的特殊遗传系统。     

拟鹅观草St基因组ISSR特异标记的建立

以拟鹅观草(Pseudoroegneria spicata)、中间偃麦草(Thinopyrum intermedium)、长穗偃麦草(Th.elonga-tum)、二倍体簇毛麦(Dasypyrum villosum)、澳冰草(Australopyrum retrofractum)等10份小麦族物种为材料,对100条ISSR引物进行分析,结果显示引物811可以在拟鹅观草(GenBank登录号为EU368859)和中间偃麦草中扩增出一条长441 bp的特异DNA片段命名为St441,而其它供试物种均未扩出.经序列比对、软件分析结合原位杂交结果认为St441为一类新的低拷贝重复序列.利用ISSR引物811对10份不同居群的中间偃麦草、20份披碱草属物种、4份小麦-偃麦草部分双二倍体、6份小麦-茸毛偃麦草后代和12份小麦对照进行扩增,结果发现除对照小麦外均能扩增出St441;进而对小麦-中间偃麦草两套附加系进行扩增,将St441初步定位于包括第四同源群在内的8条St染色体上.同时,发现只含有整条St染色体和St染色体片段的材料能扩增出St441,而仅有Js染色体的材料未扩增出St441.因此,该标记St441可以作为检测不同背景下St染色质的分子标记.     

应用原位杂交及RAPD技术标记抗黄矮病小麦—中间偃麦草染色体异附加系

以生物素标记中间偃麦草基因组ＤＮＡ为探针,与抗黄矮病小麦－中间偃麦草染色体异附加系Ｚ６进行原位杂交,鉴定出附加的１对中间偃麦草染色体。对异附加系Ｚ６和Ｌ１及它们的小麦亲本进行了ＲＡＰＤ分析,从１２０个随机引物中,筛选出２个引物可以扩增出附加染色体的特异ＤＮＡ片段,可作为鉴定导入小麦的中间偃麦草染色质的分子标记。     

中间偃麦草的染色体组构成研究

本文通过系统的细胞遗传学研究、体细胞Ｎ带研究分析了中间偃麦草的染色体组构成,结果表明：中间偃麦草不含小麦的Ｂ组染色体,而含有两组称为远同源（Ｄｉｓｔａｎｔｈｏｍｏｌｏｇｏｕｓ）的染色体组,这两组远同源的染色体不是来自二倍体长穗偃麦草的Ｅ染色体组。     

幼年和成年小白鼠LDH与SOD动态变化的电泳分析

采用聚丙烯酰胺凝胶圆盘电泳分析方法,研究了幼年鼠和成年鼠睾丸乳酸脱氢酶（ＬＤＨ）同工酶与超氧化物歧化酶（ＳＯＤ）的酶带变化。观察结果表明,成年鼠睾丸的凝胶电泳胶条,多数显现六种ＬＤＨ同功酶带,其中ＬＤＨ５和ＬＤＨ４占优势,ＬＤＨ１、ＬＤＨ２、ＬＤＨ３显带较弱,在正极端ＬＤＨ的下面是第６条酶带,相当于Ｃ亚单位（ＬＤＨｃ）的Ｃ４。幼年鼠睾丸的凝胶电泳胶条,只显现ＬＤＨ１和ＬＤＨ２两条同功酶带,以ＬＤＨ     

RAPD and ISSR-assisted identification and development of three new SCAR markers specific for the Thinopyrum elongatum E (Poaceae) genome

Diploid Thinopyrum elongatum, a wild relative of wheat, contains many agronomically desirable traits and has potential for increasing genetic variability and introducing desirable characters in this crop. Few molecular markers are available for rapid screening of T. elongatum genome segments in the wheat genetic background. We used 36 RAPD primers and 33 ISSR primers to screen for polymorphisms in the common wheat variety Chinese Spring and in T. elongatum. Two RAPD markers and one ISSR marker, designated OPF03(1407), LW10(1487) and UBC841(701), were identified and were specific for the T. elongatum E genome. Three pairs of primers flanking these specific sequences were designed to produce SCAR markers. All three SCAR markers were T. elongatum E genome-specific. Two of these SCAR markers, SCAR(807) and SCAR(577), were present in all seven T. elongatum chromosomes, while SCAR(839) was specific for T. elongatum chromosomes 2E and 3E. These newly developed SCAR markers should be useful for detecting alien genome chromatin or chromosome segments in the genetic background of common wheat.     

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.     

异源四倍体鲫鲤及其原始亲本遗传变异的微卫星标记分析

采用从鲤中分离出来的32对微卫星DNA标记,对异源四倍体鲫鲤、红鲫和野鲤的基因组DNA进行了研究。在筛选出的15对微卫星引物中,随引物不同,各等位基因数为1～8个,大小在100～420bp之间。从3个不同群体内部的遗传相似系数来看,异源四倍体鲫鲤个体之间的遗传相似系数最大,说明异源四倍体鲫鲤群体内部的遗传变异程度最低,已经形成了一个遗传性状稳定的群体。从3个不同群体之间的遗传相似系数来看,异源四倍体鲫鲤和红鲫遗传相似系数为0．5625,和野鲤的遗传相似系数为0．5125,说明异源四倍体鲫鲤接受原始母本的遗传物质比原始父本野鲤要多一些。微卫星标记与以前报道的RAPD标记的检测结果是相似的,然而由微卫星标记获得的种群内和种群间的遗传距离均大于RAPD,说明微卫星标记比RAPD标记显示出更高的个体多态性。     

Molecular analysis of leaf-rust resistant introgression lines obtained by crossing hexaploid wheat Triticum aestivum with tetraploid wheat Triticum timopheevii

Twenty-four Triticum eastivum x T. timopheevii hybrid lines developed on the basis of five varieties of common wheat and resistant to leaf rust were analyzed by the use of microsatellite markers specific for hexaploid common wheat T. aestivum. Investigation of intervarietal polymorphism of the markers showed that the number of alleles per locus ranged from 1 to 4, depending on the marker (2.5 on average). In T. timopheevii, amplification fragments are produced by 80, 55, and 30% of primers specific to the A, B, and D common wheat genomes, respectively. Microsatellite analysis revealed two major areas of introgression of the T. timopheevii genome: chromosomes of homoeological groups 2 and 5. Translocations were detected in the 2A and 2B chromosomes simultaneously in 11 lines of 24. The length of the translocated fragment in the 2B chromosome was virtually identical in all hybrid lines and did not depend on the parental wheat variety. In 15 lines developed on the basis of the Saratovskaya 29, Irtyshanka, and Tselinnaya 20, changes occurred in the telomeric region of the long arm of the 5A chromosome. Analysis with markers specific to the D genome suggested that introgressions of the T. timopheevii genome occurred in chromosomes of the D genome. However, the location of these markers on T. timopheevii chromosomes is unknown. Our data suggest that the genes for leaf-rust resistance transferred from T. timopheevii to T. aestivum are located chromosomes of homoeological group 2.     

应用5SrDNA间隔序列分析鉴定体细胞杂种

近年来,由于分子生物学的飞速发展,极大地推动了生命科学各分支学科在ＤＮＡ水平上的研究力度。就体细胞杂种的鉴定而言,仅形态、细胞学、生化方面的证据还远远不足,直接从ＤＮＡ水平上鉴定其杂种性质已成为必然。目前,从分子水平上鉴定体细胞杂种的方法有多种,如ＲＦＬＰ图谱分析、ＲＡＰＤ分析等。由于ＲＦＬＰ分析需要特异探针,ＲＡＰＤ分析的重复性较差,在一定程度上限制了两种方法在体细胞杂种鉴定方面的应用。据报道［１］,５ＳｒＤＮＡ（即５ＳｒＲＮＡ基因）在至今研究的所有真核生物中是以串联重复单位组成的,与卫星ＤＮ…     

RAPD method for the identification of intergeneric asymmetric somatic hybrid plants of wheat]

Randomly amplified polymorphic DNA (RAPD) method was used to identify the hybrid nature of three kinds of intergeneric asymmetric somatic hybrid plants of wheat: wheat (Triticum aestivum) + Haynaldia villosa, Wheat + Leymus chinensis and wheat + Agropyron elongatum. It was shown from the electrophoresis profiles that the genome of somatic hybrid plants contained specific section genome of both parents after DNA amplification with arbitrary primers. A specific RAPD product (DNA fragment of 0.77 kbp) of A. elongatum generated with primer OPJ-12 was isolated, purified, labeled and used as a probe. Southern blot from OPJ-12 primer-generated specific section genome of the hybrid (T. aestivum + A. elongatum) hybridized to this probe (0.77 kbp) proved that they are homologous in nature. This paper also discussed the advantage of RAPD method in identification of hybrid plants, especially asymmetric somatic hybrids.     

异源四倍体鲫鲤雌核发育后代的RAPD分析

在优化RAPD(随机扩增多态性DNA)检测条件基础上,从134个随机引物中筛选出53个扩增较好且多态性强的引物,对异源四倍体鲫鲤第1代(G1)、第2代(G2)人工诱导的雌核发育二倍体后代群体的DNA多态性及分子标记进行了分析。结果显示,53个随机引物在G1群体和G2群体中检测到的位点数分别为541、511,其中多态性位点数分别为70、52,多态位点比例分别为12.94%、10.18%。两个群体的平均遗传距离分别为0.0732、0.0464。研究表明,经过连续2代人工雌核发育,G2的遗传多样性明显减少,种质进一步纯化。还从53个随机引物的扩增谱带中找到了2个引物(S50、S223)的特异扩增谱带,可以作为第1、2代雌核发育群体间的分子遗传标记。由计算机软件程序构建的分支系统树清晰地反映了两个雌核发育群体及其个体间的相互关系。       

Genetic relationships between diploid and allotetraploid cherry species (Prunus avium, Prunus x gondouinii and Prunus cerasus)

Prunus avium L. (diploid, AA, 2n=2x=16), Prunus cerasus L. (allotetraploid, AAFF, 2n=4x=32) species, and their hybrid Prunus x gondouinii Rehd., constitute the most widely cultivated cherry tree species. P. cerasus is supposed to be an hybrid species produced by the union of unreduced P. avium gametes and normal P. fruticosa gametes. A continuum of morphological traits between these three species makes their assignation difficult. The aim of this paper is to study the genetic relationships between tetraploid and diploid cherry species. In all, 114 genotypes belonging to these species were analyzed using 75 AFLP markers. The coordinates of these genotypes on the first axis of a correspondence analysis allowed us to clearly distinguish each species, to identify misclassifications and to assign unknown genotypes to one species. We showed that there are specific alleles in P. cerasus, which are not present in the A genome of P. avium and which probably come from the F genome of P. cerasus. The frequencies of each marker in the A and the F genomes were estimated in order to identify A and F specific markers. We discuss the utility of these specific markers for finding the origin of the A and F genomes in the allopolyploid species.