离子束介导玉米DNA的水稻变异后代AFLP分析

利用离子束介导法将玉米（郑单14）DNA片段转入水稻豫粳6号,经过5年的筛选得到了基本稳定遗传的变异株系。本研究通过对64对AFLP选扩引物的筛选,优选出了18对选扩引物,它们能同时在2个变异株系中扩增出大量的DNA指纹条带,并且同时能扩增出差异带以及“目的带”。用AFLP分子标记初步分析了2个变异株系与对照间的差异,变异株系中出现了新带、缺失带和“目的带”等几种情况,这些DNA水平上的差异,表明离子束介导玉米DNA育成的水稻材料可能已经插入了玉米的DNA。     

香蕉离体试管芽诱变育种的研究 Ⅴ.漳蕉8号株系基因组变异检测

应用 2 0条Sangon的随机引物 ,对6 0 Coγ射线辐照诱变所获新株系漳蕉 8号(原漳农 8号 )及其对照品种台湾北蕉 (MusAAAGrandCavendish)的总DNA进行RAPD ,结果发现有 1 2条引物扩增出 68条谱带 ,其中同源带 53条 ,占总带数的77 9% ,差异带 1 5条 ,占总带数的 2 2 1 % ;漳蕉 8号与对照多态性差异高达 2 4 6% ,且显示出稳定性遗传。其中S10 和S19的 3条差异带 ,可作为漳蕉 8号株系鉴定和防退化复壮的筛选标记。     

猪基因组AFLP指纹图谱的构建

摘要：对猪（Sus scrofa）基因组AFLP指纹图谱构建的各项条件进行了优化,包括双酶切消化、接头连接、预扩增和选择性扩增、凝胶电泳银染技术及荧光标记检测法等,建立了稳定的猪基因组DNA的AFLP指纹图谱构建方法,采用E32/T32引物组合在45个猪种基因组混合DNA中检测到28个多态标记。     

水稻空间诱变的遗传变异及突变体的AFLP分子标记

搭载“神舟三号”航天飞船的水稻纯合种子在返回地面后SP3代种子出现颖壳、茎杆和叶片的主脉变异为金黄色的突变体,其他性状无明显变异,经遗传分析,颜色变异属一对隐性基因控制的性状变异。同时利用AFLP分子标记的方法,对航天诱变的水稻突变体进行基因组对比分析,通过聚丙烯酰胺电泳寻找突变体(黄金1号)多态性DNA片段,经过一系列引物筛选,找到了5条多态性DNA条带,对开展突变体基因功能研究和杂交水稻聚合育种有十分重要的意义。     

利用RAPD和AFLP标记分析烟草种质资源的遗传多样性

从200条10 bp的RAPD引物中筛选获得28条多态性引物，对48份烟草材料的基因组DNA进行扩增。共获得184条DNA扩增片断带，其中多态性带86条，平均多态检出率为46.7%。用4对AFLP选择性扩增引物对48份烟草材料进行选择性扩增，共获得321条扩增带，其中174条具有多态性，平均多态检出率为54.2%。根据RAPD和AFLP标记的结果，采用UPGMA法进行聚类分析，两种方法获得了相似但不完全相同的结果，两者都可以将48份烟草资源分为两大类群，即黄花烟草(Nicotiana rustica)群和普通烟草(N.tobacum)群，普通烟草可进一步分为4组；48份材料的遗传距离变幅在1.4～11.0之间，其聚类结果与理论上所期望的结果基本一致，AFLP标记技术比RAPD标记技术能更好地从分子水平上揭示烟草种质资源的遗传背景、亲缘关系及演化规律。     

超低温小麦种子和幼苗保存及遗传变异分析

 以小麦（Triticum aestivum L.）品种周98165和开18的种子与幼苗为材料,对它们超低温（液氨中）保存前后的发芽和成活率及田间生长指标进行了研究,并采用AFLP和MSAP技术对其遗传稳定性作了初步探讨。结果表明,小麦种子经超低温保存后,其发芽率和成活率与对照相比有一定的差异,但田间生长,除分蘖数稍低于对照外,其他生长指标与对照相比没有明显区别。而超低温保存后的幼苗,各项生长指标与对照均有显著差异。AFLP技术分析了超低温保存前后的样品,同一品种不同处理间的AFLP带型一致,统计的1500多条带中未见明显差异带型,表明在超低温保存前后无论是种子还是幼苗,其基因组DNA序列均没有发生变异。进一步用MSAP技术分析了超低温保存后的样品的甲基化水平,13对引物共扩增出590多条清晰的带,与对照相比,超低温保存后的材料均产生了不同程度的甲基化变化。其中变异带最多的达83条,变异率高达13.67%,且脱甲基化变化是主要趋势。     

黄瓜果皮品质性状AFLP分析条件的优化

以果皮性状差异明显的两个黄瓜自交系0349-5和0345-2为试材,提取高质量的DNA,利用AFLP技术和64对引物对两个亲本DNA进行了分析,共得到79条差异带,每对引物平均产生1.23条差异带,多态性带的比例为1.4%,其中有8对引物表现的差异带多于6条。     

利用RAPD标记鉴定山葡萄种质

采用修改后的CTAB法获得了高质量的基因组DNA.利用随机扩增多态性即RAPD标记对山葡萄7份种质进行鉴定,用4个引物(从30个引物中筛选)对试材进行PCR扩增,共扩增出30条谱带,平均每条引物产生7.5条谱带,其中21条谱带为多态性谱带,占总谱带数的70%.不同引物扩增的谱带数不同,范围在6～9条之间.利用4个引物扩增出的多态性谱带可以将7份山葡萄种质区分.     

玫瑰黄链霉菌Men-myco-93-63与其突变株间的DNA多态性分析和序列特征性扩增区域(SCAR)标记

玫瑰黄链霉菌(Streptomyces roseoflavus )Men-myco-93-63是分离自马铃薯疮痂病自然衰退土壤中的一株拮抗菌。前期研究表明,该菌株及其发酵液对棉花黄萎病菌（Verticillium dahliae）等多种重要的植物病原真菌具有较强的抑制作用。利用AFLP对Men-myco-93-63及其8株抗生素生物合成阻断突变株进行了基因组DNA多态性分析。结果表明,38对AFLP引物共产生3 142条谱带,其中2 362条为多态性谱带,占总带数的75.2 %。从96对引物中筛选得到了3对引物：E-CA/M-GA,E-GA/M-AC和E-GA/M-AG,分别扩增出254、312和209 bp 3条仅在Men-myco-93-63中存在,命名为EM254、EM312和EM209。将这3个片段回收、克隆和测序,成功地将其转化为SCAR标记,可以更加方便地用于对突变株的分子检测。     

转OsbHLH1和Bar基因水稻及相关特性分析

OsbHLH1基因编码bHLH(basic helix-loop-helix,碱性螺旋-环-螺旋)类转录因子,与水稻耐低温相关。草铵膦是一种高效低毒灭生性除草剂,Bar基因表达产物能解除草铵膦的毒性。本实验对通过农杆菌(Agrobacterium tumefaciens)浸染法获得的以粳型水稻(Oryza sativaL.subsp.japonica Kato)恢复系淮C17为受体的转OsbHLH1和Bar基因水稻进行研究,鉴定其除草剂抗性和耐低温能力,考查其农艺性状。草铵膦筛选发现Bar基因正常表达;对草铵膦筛选获得的转基因植株进行PCR、Southern、RT-PCR和实时荧光定量PCR检测,发现OsbHLH1基因已经整合到水稻基因组中并且在水稻中表达。转基因T3代株系芽期(萌发芽长0.5mm)在2℃条件下处理6d,第6号转基因株系死苗率为17.8%,对照死苗率为61.1%。T3代苗期(一叶一心)在8～10℃下处理7d,第5、6、9、11号株系的根长显著长于对照,第3、5、11号株系的根数显著多于对照,第3号株系的平均鲜重显著重于对照。芽期、苗期耐冷性试验表明,OsbHLH1基因在水稻中过量表达显著提高了水稻芽期、苗期的耐低温能力。对转基因T2代的农艺性状进行考查,株高、千粒重、结实率、理论产量等农艺性状与对照相比较均出现了显著的变化,外源基因的导入对水稻的农艺性状有明显影响。研究结果将为选育抗除草剂、耐低温杂交粳稻新组合提供新种质。     

利用SRAP分子标记评价小麦三雌蕊近等基因系的遗传背景

普通小麦三雌蕊突变体(TP)具有明显的穗粒数优势,为评估该突变体在育种中的利用价值,进行了近等基因系培育。以三雌蕊突变体(TP)为供体,单雌蕊中国春、川麦28、绵阳29、内麦9号为轮回亲本,经7代回交和4代自交,初步培育出三雌蕊近等基因系CSTP、CM28TP、MY29TP和NM9TP。利用128对SRAP引物对培育的近等基因系及轮回亲本进行遗传分析,结果表明:(1)128对引物共扩增出978条谱带。其中有120对引物的扩增产物具有多态性,占所用引物的93.8%。这120对引物共扩增出638个差异谱带,占总谱带数的65.2%;(2)利用128对SRAP引物计算9个材料之间的遗传相似系数。其中中国春与CSTP的相似系数为0.9346,绵阳29与MY29TP的遗传相似系数为0.9070,川麦28与CM28TP的遗传相似系数为0.9397,内麦9号与NM9TP的遗传相似系数为0.8732;(3)通过聚类分析筛选出2对遗传相似性大于0.93的近等基因系,即CM28TP与川麦28、CSTP与中国春。     

浙江省南部地方梨品种的SSR和AFLP遗传多样性研究

为了解浙江省南部地方梨品种的遗传多样性,采用10对苹果和3对梨上的SSR引物以及6对AFLP引物对来自温岭和丽水的18个地方梨品种或类型进行了鉴定分析。结果表明10对苹果引物在梨上扩增出62条条带。13对SSR引物的观察杂和度(He)为0.056～0.944,平均值为0.57,而香农指数(I)的范围0.340～2.013,平均值为1.22。6对AFLP引物组合共扩增出364条带,平均每对引物扩增60.67条,平均多态性比例为73.35%。采用UPGMA聚类分析法构建的SSR和AFLP系统树中,云和梨、云和粗花雪梨与云和细花雪梨在2个系统树中聚在一起,虽然相互间有较高的遗传相似度,但属于不同的品种;3个同名为老雪梨的品种(老雪梨1、老雪梨2和老雪梨3)分散在不同的组中,可能为异物同名。SSR和AFLP聚类结果基本吻合,但AFLP的结果更符合品种的地域分布情况。     

尼里——拉菲奶水牛的RAPD分析

以尼里—拉菲奶水牛基因池为模板,从22条10 bp的随机引物中筛选出17条具有清晰多态条带的引物,从中选取5条,对尼里—拉菲奶水牛基因组DNA进行RAPD扩增,结果得到53条带,其中12条表现多态,多态频率23.85%;根据N e i氏公式计算品种内的遗传相似指数F的平均值为0.9500,遗传距离指数D的平均值为0.0500。     

卫星搭载后玉米诱变系的SRAP分析

为探索空间诱变对玉米种子后代的影响,本研究用16对SRAP引物对玉米自交系＂968＂及其诱变系进行PCR检测,共检测到154个等位基因变异,每对引物检测出5～18个等位基因,平均为9.6个;94份材料间的遗传相似系数表明诱变系与对照间的遗传相似系数变化幅度为0.481～1.000,平均为0.903,其中诱变系37号与对...     

Genetic diversity and differentiation in Ethiopian populations of Phytolacca dodecandra as revealed by AFLP and RAPD analyses

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analyses were performed on six populations (a total of 89 individuals) of Phytolacca dodecandra (endod) collected in Ethiopia. The populations were selected based on our previous investigation to represent two altitude groups: lowland/central-highland (1600–2500 m) and highland (2501–3000 m). A total of 197 AFLP and 68 RAPD markers were scored from 5 primer pairs and 12 random primers, respectively. The overall patterns obtained for AFLPs and RAPDs from diversity, cluster and principal component analyses were very comparable. However, the moderate correlation (r = 0.56) between AFLP and RAPD similarity matrices as well as the discrepancies in diversity estimates between the two techniques in some populations and in the lowland/central-highland plants could be due to differences in sensitivity of reaction conditions, bias in scoring of bands, number of markers used for analyses, and/or parts of the genome surveyed. For both AFLP and RAPD, the lowland/central-highland populations showed higher polymorphism and Shannon information measure (H) than the highlands. Cluster and principal component analyses performed for both marker types have also clearly demonstrated the differentiation of all the lowland/central highland plants from those of the highlands, in agreement with our previous conclusion. Markers scored from any of the five AFLP primer pairs were sufficient to clearly distinguish the two altitude groups; with RAPD, selection of about 8 informative markers produced by seven random primers was needed for the same purpose.     

Genetic diversity analysis of hulless barley from Shangri-la region revealed by SSR and AFLP markers

For adding the hulless barley resources of Shangri-la region to the global barley resource library, a basic work was done by us to assess their genetic diversity of this region. The genetic diversity of 60 hulless barley samples collected from three counties in Shangri-la region of Yunnan Province, were studied using SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) markers. A total of 70 alleles were detected for 19 pairs of SSR primers, and 525 band containing 464 polymorphic bands were revealed for 5 pairs of AFLP primers. The value of polymorphism information content (PIC) ranged from 0.03 to 0.86 for SSR primers. The total numbers of alleles were 51, 55, 43 in three populations and the polymorphic bands were 188, 205 and 141. The genetic distances and genetic identity among the three populations showed their close relationship. The gene diversity among populations relative to the total population diversity (Gst) was 0.13 for SSR markers and 0.02 for AFLP markers and indicated that just 13 and 2% variations were among populations, respectively. The UPGMA cluster analysis revealed that all of the samples grouped randomly rather than clustered into distinct groups corresponding to their populations, row types and spring/fall types. We concluded that there was high genetic diversity in the population of Shangri-la region and the formation of diversity was related to complex environment and inhabitants’ traditional practices.     

基于RAPD标记的福建省稻曲病菌遗传多样性分析

为了解福建省稻曲病菌群体的遗传多样性和遗传组成,应用随机扩增多态性RAPD (random amplified polymorphic DNA)技术分析了来自福建省不同水稻种植区的102个稻曲病菌(Ustilaginoidea virens)的遗传多样性.从100条随机引物中筛选出10条扩增带清晰、重复性好的引物进行稻曲病菌多态性扩增,共扩增出157条带,多态性条带比率为82.17％,遗传距离变化范围为0.02～0.67.遗传多样性分析表明,福建省稻曲病菌具有丰富的遗传多样性,相对于其它地区而言,福建闽西地区分离的菌株遗传多样性水平最高PPB=76.43,H=0.2212,I=0.3383),晚稻分离的菌株群体遗传多样性(PPB=91.08,H=0.2402,I=0.3655)高于早稻群体(PPB=63.06,H=0.1892,I=0.2870).聚类分析显示,在遗传距离0.349水平上,供试的所有菌株可被划分成7个遗传聚类组(R1～R7),聚类组R1为优势聚类组,包含有80个菌株,其内又存有一些亚组.这些菌株的聚类与菌株的地理来源及水稻品种无明显相关性.但是在遗传距离0.330水平上,来自宁化的10个菌株可被明显划分成早稻群体和晚稻群体.初步分析认为菌株的地理来源、水稻品种及其生长季节是影响福建省稻曲病菌遗传多样性的主要因素,在稻曲病菌的遗传变异以及该病的发生和流行中可能起重要的作用.     

Analysis of genetic diversity in a sweet potato (Ipomoea batatas L.) germplasm collection from Tanzania as revealed by AFLP

Sweet potato (Ipomoea batatas L.) is the fifth most important crop in the developing countries after rice, wheat, maize and cassava. The amplified fragment length polymorphism (AFLP) method was used to study the genetic diversity and relationships of sweet potato accessions in the germplasm collection of Sokoine University of Agriculture, Morogoro and Sugarcane Research Institute, Kibaha, Tanzania. AFLP analysis of 97 sweet potato accessions using ten primer combinations gave a total of 202 clear polymorphic bands. Each one of the 97 sweet potato accessions could be distinguished based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the un-weight pair-group method using arithmetic average. AFLP-based genetic similarity varied from 0.388 to 0.941, with a mean of 0.709. Cluster analysis using genetic similarity divided the accessions into two main groups suggesting that there are genetic relationships among the accessions. Principal Coordinate analysis confirmed the pattern of the cluster analysis. Analysis of molecular variance revealed greater variation within regions (96.19%) than among regions (3.81%). The results from the AFLP analysis revealed a relatively low genetic diversity among the germplasm accessions and the genetic distances between regions were low. A maximally diverse subset of 13 accessions capturing 97% of the molecular markers diversity was identified. We were able to detect duplicates accessions in the germplasm collection using the highly polymorphic markers obtained by AFLP, which were found to be an efficient tool to characterize the genetic diversity and relationships of sweet potato accessions in the germplasm collection in Tanzania.