南瓜耐盐种质的筛选鉴定及耐盐基因的标记

以27份自交系南瓜为材料,选择出苗率和盐害指数为耐盐鉴定指标,从中筛选出了耐盐的南瓜NM083和不耐盐的南瓜XBZM,并以NM083为父本、XBZM为母本构建F2群体,结合集团混合分离分析法(bulked segregation analysis,BSA)筛选耐盐相关的分子标记。结果表明,在600条RAPD和44对SSR引物中,共有18个引物(10条RAPD和8对SSR)在双亲中显示出多态性,其多态性比例分别为0.43%和4.3%。最终筛选到RAPD引物P597,在双亲及基因池间能稳定扩增出片段大小为685 bp的差异条带。通过160个F2代单株耐盐性试验验证,表明耐盐的F2代单株中能扩增出差异条带,而不耐盐的F2代单株中不能扩增出差异条带,从而初步确定此差异性条带是与南瓜耐盐性紧密连锁的RAPD标记,从而为利用分子辅助育种加速耐盐南瓜新品种的选育提供有效标记。     

甘蓝型油菜细胞质雄性不育恢复基因的分子标记及定位

以甘蓝型油菜恢复系CP015和不育系77A构建的F2群体为供试材料,运用RAPD,SSR和AFLP 3种标记技术,对甘蓝型油菜恢复基因进行分子标记和图谱定位。在260条RAPD引物、185对SSR引物、58对AFLP引物中,在两亲本间筛选多态性高的RAPD引物121条,SSR引物128对,AFLP引物组合26对,进一步通过BSA法筛选,获得了与甘蓝型油菜恢复基因Rf连锁的1个RAPD标记S1009-750和1个AFLP标记E5M11-150,标记与基因Rf之间的遗传距离分别为4.9cM和8.6cM。     

利用微卫星标记分析两个吉富罗非鱼群体的遗传差异

[目的]评价不同吉富罗非鱼群体的遗传多样性,筛选出鉴别不同群体的分子标记,为吉富罗非鱼种质资源保护及其新品种选育提供理论依据.[方法]对48尾来自两个不同群体的吉富罗非鱼进行尾静脉采血,抽提其基因组DNA,应用微卫星标记技术判定其等位基因和基因型,然后采用GenAlEx 6.2、Cervus 3.0和Populations软件进行数据分析,计算有效等位基因数(Ne)等群体遗传多样性相关参数,对两个吉富罗非鱼群体进行遗传差异分析.[结果]从50对微卫星引物中筛选出23对扩增产物稳定、条带清晰、特异性好的引物,扩增出的DNA片段大小在110～388 bp.利用23个微卫星位点,从两个吉富罗非鱼群体中共检测到89个等位基因和163种基因型,平均每个位点的等位基因数(Na)3.8个、基因型7.1种.两个吉富罗非鱼群体的平均观察杂合度(Ho)分别为0.6383和0.5957,平均期望杂合度(He)分别为0.5759和0.5559;23个位点在两个群体中平均多态信息含量(PIC)分别为0.5131和0.4942,平均固定系数(FIS)分别为-0.1184和-0.0718;两个群体间的遗传距离(DA)为0.1287,平均遗传分化系数(FST)为0.135.UNH911、GM141、GM287、GM134等4个位点在两个群体中扩增的条带存在明显差异.[结论]两个吉富罗非鱼群体遗传多样性水平较高,均存在杂合子过剩现象,群体间遗传分化程度中等,具有较强的环境适应能力,且选育空间大.UNH911、GM141、GM287、GM134等4个微卫星位点可作为鉴别两个吉富罗非鱼群体的分子标记,也可用于分子标记辅助育种研究.     

应用AFLP标记筛选甘蓝型油菜R4和22B的多态性

应用AFLP分子标记,对甘蓝型油菜恢复系R4和保持系22B进行引物多态性筛选。在414对AFLP引物中,初步筛选到有多态性的引物组合147对;其中重复性好、多态性比率高的引物组合120对;每对引物可扩增出50条左右的条带,其中,多态性条带数为12.4条。AFLP是构建图谱中效率较高的分子标记。     

利用ISSR和AFLP标记分析石斛种质资源的遗传多样性

本研究利用ISSR和AFLP两种分子标记对51个药用石斛材料(包括35个铁皮石斛种质资源)进行遗传多样性研究。利用ISSR标记筛选出6条引物,共扩增出79条带,平均每条引物扩增条带数约为13.17条,多态条带比率都达到100%。利用AFLP标记筛选出5对引物,共扩增207条带,其中206条具多态性,多态性比率高达99.52%。两标记分析获得的群体种系间关系与变异基本一致,且群体内的分化较小,群体间的分化较大,群体间的基因流动较小。所得到的聚类图表明:ISSR和AFLP标记技术能有效地将研究材料分开,且基本反映了它们之间的亲缘关系,说明两种标记均适合用于石斛植物的遗传多样性研究。     

利用SSR和ISSR分子标记分析崇明白扁豆种质资源的遗传多样性

利用大豆SSR和ISSR分子标记对利马豆、白扁豆、大白芸豆共64份材料进行遗传多样性分析。在48对大豆SSR引物中,筛选出带型清晰、多态性较好的引物11对,扩增出84条多态性条带,每对SSR引物扩增出的多态性条带从3条到12条不等,平均7. 6条;从100条ISSR引物中,筛选出扩增稳定、多态性好的引物19条,扩增出151条多态性条带,每条ISSR引物扩增出的多态性条带从5条到13条不等,平均7. 9条。综合两种分子标记的数据,用NTSYSpc V2. 11软件进行聚类分析,可将64份供试材料分为4个类群:材料64独自为第I类群;材料60、62和63为类群II;材料27和61为类群III;其余材料均为崇明白扁豆,为类群IV。结果显示:所收集的崇明白扁豆材料之间的遗传距离很小,与材料61差异不大,说明崇明白扁豆遗传背景比较狭窄,但与外地的白扁豆和大白芸豆能有效区分。     

四川不同地区硬头黄竹RAPD和ISSR分析

以四川不同地区硬头黄竹为研究对象.通过随机扩增多态性DNA标记(RAPD)和简单序列重复区间(ISSR)标记进行扩增,探讨硬头黄竹的遗传多样性.研究表明,6个RAPD引物扩增出了42条条带,多态性高达69.05%;5个ISSR引物扩增出了47条条带,多态性为61.70%.利用Popgen32进行聚类分析,聚类结果可将不同地区的硬头黄竹类型区分开来.其遗传距离分别为0.182 3～0.602 2和0.043 5～0.672 1,说明RAPD和ISSR分子标记为硬头黄竹的遗传改良提供了理论依据.     

鉴别杂草稻及其杂交后代的分子标记研究

采用随机扩增多态性DNA（RAPD）技术,对东北地区杂草稻生态型及其与栽培稻杂交后代进行分子标记研究．所分析的10个随机引物共扩增出了93条DNA片段,其中多态性条带为36条,多态性比例为38．71％．结果表明,RAPD分子标记可以区别东北地区杂草稻、栽培稻及其杂交后代．     

烟草赤星病菌遗传多样性的ISSR和RAPD标记比较分析

采用ISSR和RAPD 2种分子标记方法对来自不同地区的28份烟草赤星病菌进行遗传多样性分析,筛选后选用10个ISSR引物和10个RAPD引物,ISSR扩增出多态性条带112条,多态性条带百分率为86.82%,菌株间相似性系数为0.53～0.97;RAPD引物扩增出多态性条带70条,多态性条带百分率为81.39%,菌株间相似性系数为0.57～0.94;用SPSS17.0软件对2种标记遗传距离进行相关性分析,发现2种分子标记结果呈显著正相关,表明2种分子标记方法都适合于烟草赤星病菌遗传多样性研究,ISSR是一种多态性优于RAPD的标记技术。根据2种标记的结果,利用NTSYS软件按UPGMA方法进行聚类分析,发现烟草赤星病菌遗传多样性与地理差异没有显著相关性。     

SSR和SCoT标记在美洲黑杨×青杨派杂种无性系遗传差异性分析上的比较

【目的】比较SSR和SCoT分子标记在美洲黑杨×青杨派杂种无性系遗传差异性分析上的应用性。【方法】利用SSR和SCoT标记,对9个美洲黑杨×青杨派杂种无性系及3个亲本的遗传差异性进行分析。【结果】筛选出的12对SSR引物对供试杨树材料共扩增出94条清晰条带,其中多态性条带85条,多态性比率达到90%,标记指数为3.19;筛选出的14条SCoT引物对供试杨树材料共扩增出清晰条带127条,其中多态性条带95条,多态性比率达到75%,标记指数为2.72。聚类分析表明,SSR和SCoT分子标记得出12个无性系的遗传相似系数分别为0.44~0.80和0.40~0.87,平均遗传相似系数分别为0.62和0.64;在相似系数平均值处,SSR和SCoT分子标记将12个杨树材料分别分为3大类和5大类。Mantel检测显示,2种分子标记在12个无性系间的遗传相似性显著相关(r=0.501 3,P=0.003)。【结论】这2种分子标记均适合美洲黑杨×青杨派杂种无性系鉴定和遗传多样性研究,但SSR标记的多态性比率和标记效率均高于SCoT标记。     

Assessment of genetic diversity in glandless cotton germplasm resources by using agronomic traits and molecular markers

Seventy-one glandless cotton germplasm resources were firstly evaluated genetically by using nine agronomic traits, 33 simple sequence repeat (SSR) primers and ten amplified fragment length polymorphism (AFLP) primer combinations. Principal component analysis (PCA) of the agronomic traits showed that the first six principal components (PCs) explained a total of 86.352% of the phenotypic variation. A total of 329 alleles were amplified for 33 SSR primers, and 232 polymorphic bands in a total of 389 bands were obtained by using ten AFLP primer combinations. The average polymorphic information content (PIC) value was 0.80 and 0.18 for SSR primers and AFLP primer combinations, respectively. The DIST (average taxonomic distance) and DICE (Nei and Li’s pairwise distance) coefficients ranged from 0.373 to 3.164 and 0.786 to 0.948, respectively, for agronomic traits and SSR&AFLP data based on UPGMA analysis. This suggested that there was a higher diversity in the evaluated population for both agronomic traits and molecular markers. The Mantel’s test showed that the correlation between the dendrograms based on agronomic traits and SSR&AFLP data was non-significant.     

Assessment of genetic diversity among Chinese upland cottons with Fusarium and/or Verticillium wilts resistance by AFLP and SSR markers

Genetic diversity among 95 Chinese upland cottons with Fusarium and/or Verticillium wilts resistance was estimated using Amplified Fragment Length Polymorphism (AFLP) and Simple Sequence Repeat (SSR) markers. Twenty EcoRI-MseI AFLP and 19 SSR primers with polymorphism were selected to perform the fingerprinting. The results showed that 20 AFLP primer pairs produced a total of 1 480 major bands among 95 genotypes, and 214 were polymorphic bands. The number of total bands per primer pair ranged from 47 to 109, with an average of 74.0. The polymorphism information content (PIC) values for the 20 primer pairs varied from 0.01 (E-ACT/M-CAT) to 0.24 (E-ACA/M-CTA), and the average value was 0.09. Nineteen SSR primers generated 89 DNA bands, of which 61 were polymorphic. The total number of alleles per locus varied from 3 to 8, with an average of 4.7. The average PIC value for the SSR amplification products was 0.69. Genetic similarity estimates for the entire data set ranged from 0.978 to 0.998 based on AFLP and SSR bands. It was proved that the close genetic relationship and narrow genetic diversity existed in the tested cultivars. The clustering patterns could not be correlated to the geographic origin, the pedigree and common parentage of the cultivars.     

The power of microsatellite markers and AFLPs in revealing the genetic diversity of Hashemi aromatic rice from Iran

Hashemi, a popular aromatic rice among Iranians, is famous for its fragrance and taste. Such features are major reasons for its higher price compared to non-aromatic varieties available in Iran. Therefore, the knowledge of genetic diversity of this profitable crop is a fundamental ineterst for plant breeders in future breeding programs. In the present research, genetic diversity among 35 genotypes of Hashemi aromatic rice(Oryza sativa L.) from Guilan and Mazandaran provinces of Iran was estimated using simple sequence repeat(SSR) and amplified fragment length polymorphism(AFLP) markers. Out of 21 SSR and 12 Eco RI-Mse I AFLP marker combinations, only 16 SSRs and 10 AFLPs exhibited polymorphic patterns while others were monomorphic. The 10 AFLP primer combinations produced a total of 142 of bands and 20 were polymorphic(14.08%). Moreover, 40 out of 47 bands amplified with 16 SSR markers showed polymorphism(85.1%). The average number of alleles identified by SSR primers was 2.56 alleles per locus with a range of 2 to 4. The average value of polymorphic information content(PIC) was 0.393 and 0.468 for AFLP and SSR markers, respectively. However, the genetic similarity values ranged from 0.26 to 1 for SSRs and 0.21 to 1 for AFLPs. Later, a unweighted pair group method with arithmetic mean(UPGMA) dendrogram was generated and genotypes were clustered into four groups with SSRs at similarity coefficient of 0.55 while AFLPs clustered them into six groups at similarity coefficient of 0.41. Cluster analysis revealed a narrow genetic diversity and low correlation between geographical differentiation and genetic distance within cultivars.     

19个地方鸡种遗传变异的微卫星和AFLP指纹分析

利用20个微卫星标记和6对AFLP引物组合对19个地方鸡种进行了遗传检测,并根据两种标记 分析的遗传距离进行了聚类分析。结果发现,19个地方鸡种在20个微卫星位点上的平均杂合度为0．582 4- 0．743 2,平均多态信息含量为0．523 8-0．702 3;6对AFLP引物组合在19个地方鸡种中共检测到294条多态性 条带,平均每个引物组合产生49条,同时在每个品种群体中还检测到了数量不等的特异性条带;19个地方鸡种两 种标记分析的聚类结果具有较大的一致性,并与所保存地方鸡种的地理分布、现实状况相吻合。这表明同时应用微 卫星和AFLP指纹分析我国地方鸡种的遗传多态性和品种间的亲缘关系,其结果具有更高的可靠性和科学性。     

Genetic diversity of Ustilago hordei in Tibetan areas as revealed by RAPD and SSR

Covered smut, which is caused by Ustilago hordei(Pers.) Lagerh., is one of the most damaging diseases of highland barley(Hordeum vulgare Linn. var. nudum Hook. f) in Tibetan areas of China. To understand the molecular diversity of U. hordei, a total of 27 isolates, which were collected from highland barley plants from Tibet, Sichuan, Qinghai, and Gansu provinces/autonomous region, were analyzed using random amplified polymorphic DNA(RAPD) and simple sequence repeat(SSR) markers. Among the 100 RAPD primers used, 24 primers exhibited polymorphism. A total of 111 fragments were amplified, of which 103 were polymorphic with a polymorphic rate of 92.79%. The average observed number of alleles(Na), effective number of alleles(Ne), Nei's genetic diversity(H), Shannon's information index(I) and polymorphism information content(PIC) value in the RAPD markers were 1.9279, 1.5016, 0.2974, 0.4503 and 0.6428, respectively. For the SSR markers, 40 of the 111 primer pairs exhibited polymorphism and provided a total of 119 bands, of which 109 were polymorphic and accounted for 91.60% of the total bands. The Na, Ne, H, I and PIC values of the SSR markers were 1.9160, 1.4639, 0.2757, 0.4211 and 0.4340, respectively. The similarity coefficients ranged from 0.4957 to 0.9261 with an average of 0.7028 among all the 27 isolates used. The dendrogram, which was developed based on the RAPD and SSR combined marker dataset showed that the 27 U. hordei isolates were divided into 3 clusters at similarity coefficient of 0.7314. We determined that RAPD and SSR markers can be successfully used to assess the genetic variation among U. hordei isolates. The RAPD markers revealed higher levels of genetic polymorphism than did the SSR markers in this study. There existed a moderate genetic difference among isolates. The molecular variation and differentiation was somewhat associated with geographical origin but not for all of the isolates.     

大蒜转录组简单重复序列标记分析与分子标记开发

为探明大蒜转录组简单重复序列标记(SSR)的特征,开发适合大蒜育种的SSR引物,利用MISA软件对大蒜转录组序列进行SSR位点检测与信息分析,并通过PCR扩增检测引物的有效性和多态性。结果表明:大蒜转录组测序获得444 865条unigenes,共鉴定出141 132个SSR位点,出现频率为31.72%,平均每3.45 kb出现1个SSR位点。单核苷酸和二核苷酸为SSR位点中优势类型,分别占总SSR的68.02%和24.12%;SSR位点共有82种重复基序,以A/T和AT/AT数量较多,占总SSR位点的65.02%和12.37%。利用Primer 3.0共设计出125 616对SSR引物,在随机选取的14对引物中有12对引物能够有效扩增,其中6对引物在35份大蒜资源中表现出多态性,扩增共得到26条多态性条带,每对引物平均产生4.33条条带。UPGMA分析显示,在遗传相似系数0.756 9处,35份大蒜资源可被分成5大类群。综上所述,利用大蒜转录组数据进行SSR分子标记开发能够获得较高频率的SSR位点,且开发的SSR标记可用性强。     

AFLP and RAPD Analysis of the Boer and Indigenous Breeds of the Goat in Jiangsu

Blood and tissue samples were collected from 105 goats including 60 Boer goats (30 for eachsex), 30 Xuhuai goats (15 for each sex) and 15 Haimen goats (7 stud and 8 does). DNA was extracted andDNA pools were constructed on the basis of goat breeds. In 36 selective primer combinations, 29 combinationsamplified totally 3 253 markers including 92 polymorphic markers by amplified fragment length polymor-phism (AFLP). On average, 3.17 polymorphic markers were amplified per combination, with a polymorphicfrequency of 2.8%. The primer combinations amplifying more polymorphic markers (showed in brackets)were involved in E00+ACG/M00+CAA (13), E00+ACG/M00+CAG (10), E00+AAC/M00+CAC (8)and E00+AAC/M00+ACT (7). A total of 183 markers including 60 polymorphic markers were amplified byRAPD from the pooled DNA of three breeds using 22 primers with strong polymorphism and high reproduc-ibility selected from 93 RAPD primers. On average, 2.73 polymorphic markers were amplified per primer,with a polymorphic frequency of 32.8%. The results of AFLP and RAPD coincidently suggested that the ge-netic distance is the closest between Xuhuai and Haimen goat, next between Xuhuai and Boer goat, and the far-thest between Haimen and Boer goat. According to the UPGMA method, Haimen and Xuhuai goats can begathered together as a cluster, then Boer goat. Both methods can be used to implicate the genetic difference ofthese three breeds, in particular AFLP has more polymorphic markers.     

毛白杨多态SSR引物库和种质资源指纹图谱库构建

目的针对毛白杨优树种质资源形态学差异小、不易区分等问题,利用多态SSR引物构建优树种质资源指纹图谱,为毛白杨种质资源管理、新品种选育、知识产权保护等提供参考。方法本研究以山东冠县毛白杨种质资源库469个优树无性系为对象,从毛白杨SSR多态引物筛选入手,通过荧光SSR引物PCR扩增和毛细管电泳仪检测筛选SSR引物,并构建指纹图谱。结果在2 317对SSR引物中,共计得到清晰、特异、多态、稳定扩增的SSR引物406对,这些SSR引物在19条染色体上的分布数量介于3 ~ 39对之间,利用BLAST比对分析可将其中389对SSR引物定位到毛果杨基因组,构建了毛白杨优树种质资源多态SSR引物库。应用多态性较高的SSR核心引物,以及特异的SSR辅助引物相结合的技术策略,筛选出25对多态性较高的SSR核心引物,以及13对特异的SSR辅助引物,构建了毛白杨种质资源库内469个优良无性系指纹图谱库,并完成了指纹图谱的QR编码。结论本研究成功构建了毛白杨种质资源多态SSR引物库和优树无性系指纹图谱库,对与毛白杨类似的林木种质资源遗传变异研究以及品种鉴定、系谱分析等具有重要意义。      

用聚合酶链式反应技术鉴定杂交稻种子纯度的初步研究

应用聚合酶链式反应（ＰＣＲ）技术,对３个杂交稻组合及其相应亲本的核ＤＮＡ进行了分析,筛选出９个随机扩增多态性ＤＮＡ（ＲＡＰＤ）随机引物和１对简单重复序列（ＳＳＲ）引物,可对供试材料进行真伪和纯度鉴定,该技术具有快速,简便,准确的特点,每人每天可测定１８０份样品,适用于杂交稻种子的商业化鉴定。