葎草雄性连锁的ISSR标记克隆及SCAR标记的建立

以雌雄异株植物薇草(Humulus scandens L.)为材料,通过优化扩增体系中的退火温度、Mg2+浓度及模板浓度等主要影响因素,利用简单重复序列间扩增标记对其雌雄株性别的基因组差异进行研究.结果表明,62条ISSR引物中有40条引物能扩增出稳定的条带,共产生302条带.引物164扩增出1条雄性连锁标记,经克隆测序,该片段长度为553 bp,AT含量为64.3%,根据测序结果设计特异引物,将该标记转化为稳定性更好的SCAR标记.     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA（random amplified polymorphic DNA,RAPD)技术获得与大麻性别连锁的分子标记,将10株雄性大麻或10株雌性麻的单个DNA样品等量混合分别组成雄性或雌性DNA池（DNApool),以提供具有相同遗传背景的雄,雄性DNA样品。每个随机引物分别用三个不同的循环程序进行PCR扩增,在30个随机引物中,用引物S401扩增得到一条约2．5kb雄性多态性片段,对该片段进行了克隆和序列分析 ,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR（Sequence characterized amplified regions)分子标记。     

菠菜性别相关的ISSR标记

菠菜为雌雄异株植物,用CTAB法提取其雌、雄株成株幼嫩叶片DNA,分别构建雌、雄株DNA池,以之为模板,用已优化的ISSR体系扩增,在74条ISSR引物中,I62扩增出一条约1 200 bp雌性连锁标记,回收纯化该特异扩增片段,将其连接于pUCm-T载体,转化进大肠杆菌JM109菌株,并检测及测序。回收克隆和测序后发现该片段全长1 176 bp,富含AT,AT占57.0%。根据测序结果设计1对25 bp的特异引物将这个雌性连锁的ISSR标记转化为稳定性和特异性更好的SCAR标记。该特异引物对随机选取的雌雄菠菜单株进行PCR扩增,在雌株中均有1 176 bp的特异条带,而雄株中均无。此特异条带的获得为菠菜性别相关基因的克隆奠定基础。     

马铃薯瓢虫与茄二十八星瓢虫SCAR标记的建立及应用

为了探索快速鉴定马铃薯瓢虫Henosepilachna vigintioctomaculata(Motschulsky)和茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)的分子生物学方法,本研究在随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)的基础上,分别设计了可以鉴别两个物种的序列特征扩增区域(sequence characterized amplified regions,SCAR)标记。从随机合成的60条引物中筛选出来2条特异性引物(分别为OPI-6和OPJ-15),引物OPI-6在马铃薯瓢虫中扩增出约750 bp的特异性条带,引物OPJ-15在茄二十八星中扩增出约750 bp的特异性条带,根据测序结果设计了两对SCAR引物对筛选结果进行验证,发现根据OPI-6的测序结果所设计的SCAR引物(OPI-6 test)仅能在马铃薯瓢虫中扩增出645 bp的条带,而根据OPJ-15的测序结果所设计的SCAR引物(OPJ-15 test)仅能在茄二十八星瓢虫中扩增出436 bp的条带。这两对SCAR引物能够准确、稳定且快速地区分马铃薯瓢虫与茄二十八星瓢虫,对这两种害虫的精准防控具有重要意义。     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)技术获得与大麻性别连锁的分子标记.将10株雄性大麻或10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNApool),以提供具有相同遗传背景的雌、雄性DNA样品.每个随机引物分别用三个不同的循环程序进行PCR扩增.在30个随机引物中,用引物401扩增得到一条约2.5kb雄性多态性片段.对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)分子标记.     

与苹果Co基因紧密连锁的RAPD标记的筛选及其SCAR标记转换

以短枝富士(Spur Fuji)X舞姿(Telamon)的105株F1群体为试材,利用RAPD技术,结合集群分类分析法(BSA)进行了苹果柱型基因(Co)分子标记的研究。通过对300条随机引物的筛选,获得一个与Co基因紧密连锁的RAPD标记S1142682,连锁距离为2．86cM。对该标记片段进行序列测定,然后根据序列特点设计了4条特异引物(其中正向引物与反向引物各两条)。PCR结果显示,这4条引物的4种组合都可以扩增出柱型性状的特征带。选其中之一进行群体上的分析,结果表明该SCAR标记特征带与柱型性状的共分离行为与原RAPD标记表现一致。可见,此组合的引物可以作为该SCAR标记的特异引物。通过对S1142682标记片段序列分析发现,在 45～ 251区域含有一个可编码68个氨基酸残基的ORF。     

与棱果沙棘性别相关的RAPD标记

应用RAPD技术筛选与棱果沙棘性别相关的分子标记,对棱果沙棘雌雄株的基因组DNA进行混合分组分析（BSA）,在194条随机引物中有50条引物能够在雌雄DNA反应池间形成多态性条带,应用这50条引物分别对棱果沙棘雌雄个体（雌雄个体各选取5个）进行RAPD分析,其中引物S10扩增得到1个约为1030 bp的与雌性相关的RAPD标记。该标记的获得进一步表明棱果沙棘雌雄株间存在基因水平的差异,为棱果沙棘的性别研究提供分子依据。进一步利用该雌性特异位点设计出更加稳定的SCAR标记,可望用于棱果沙棘的早期性别的准确鉴别。     

杉木种子随体染色体SCAR标记的建立

回收、纯化由引物OPB07(5’-GGTGACGCAG-3’) OPB18(5’-CCACAGCAGT-3’)扩增而得的杉木(Cunninghamia lanceolata(Lamb.)Hook)种子随体染色体特异性RAPD(随机扩增的DNA多态性分析)片段OPB07-18907,将该片段克隆至pUCm-T载体,转化受体菌后筛选出阳性克隆,并对插入片段进行测序,根据序列特点设计两对SCAR(序列特异性扩增区)引物,PCR结果显示,这两对引物的4种组合都可以扩增出属于随体染色体的特征带,适宜退火温度为57℃。成功将特异RAPD标记OPB07-18907转化为稳定的SCAR标记。开发随体染色体SCAR标记目的是:一方面能在分子水平上鉴定微分离的杉木随体染色体,另一方面,也可以将杉木已构建的遗传图谱中连锁群与染色体进行对应。探讨了该SCAR标记对杉木核型分析的作用。     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphicDNA)分子标记技术,寻找谭清苏铁(Cycas tanqingii)中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465(CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域(SCAR)分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

SCAR标记在茶树种质资源鉴定中的应用

SCAR标记是一种在RAPD技术的基础上发展起来的新型分子标记技术,提高了分子标记辅助选择育种的效率,在茶树种质资源的合理开发与利用中具有广阔的应用前景.运用优化后的RAPD反应体系对10个茶树品种的基因组DNA进行遗传差异分析,随机引物S89、S4分别在白毫早和福云6号中扩增得到长度为498 bp、1 622 bp的差异片段,命名为BHZ498、FY1622.根据它们的测序结果分别设计了一对特异引物,BHZ498的特异引物为SB1/SB2;FY1622的特异引物为SC1/SC2,用这两对特异引物对10个茶树品种的基因组DNA进行扩增.引物SB1/SB2和SC1/SC2分别在白毫早和福云6号中扩增出唯一的一条扩增带,而这两对引物在其他供试茶树材料中均无相应的扩增带,结果表明已将BHZ498、FY1622标记成功转化成SCAR标记.     

银杏雌雄基因组DNA间的差异性分析

本研究应用RAPD技术,应用300个10bp随机单引物及200对随机双引物组合,检测了雌雄异株银杏基因组DNA的多态性。结果表明:雌雄基因组间具有极高的相似性,在检测到的3450个标记中,仅获得1个与银杏雄性基因组相关的RAPD标记。以该标记为探针,与雌雄银杏基因组DNA的Southern杂交分析,其杂交信号在两性之间表现为限制性片段长度多态性,该结果为寻找银杏早期性别鉴定的探针以及在细胞和分子水平进一步研究其性别问题奠定了基础。     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

WHBE 兔、日本大耳白兔和新西兰兔遗传多样性的 RAPD 分析

目的：应用随机引物扩增多态性DNA技术（ random amplified polymorphic DNA , RAPD）对大耳白黑眼兔（ white hair black eyes rabbit , WHBE rabbit ）、日本大耳白兔（ Japanese white rabbit , JW rabbit ）和新西兰兔（New Zealand white rabbit, NZW rabbit）3个实验兔品系进行遗传分析。方法选用90只实验兔的皮肤组织样品提取基因组DNA,用60个随机引物对实验兔基因组DNA进行PCR扩增,根据电泳结果筛选出多态性较高的引物进行RAPD-PCR分析,再利用Popgene 3．2统计软件对3个品系的扩增条带进行遗传分析,获得实验数据。结果分析结果表明：（1）60个随机引物中筛选出25个多态性较高的引物,3个品系实验兔共检测到493个扩增片段,长度在100～1800 bp之间,筛选的25个引物中,其中16个引物既可扩增出3个品系共同的DNA条带,也可扩增出WHBE兔特有的特征条带;（2） WHBE兔位点数为234个,其中多态位点数166个,多态位点比为70．94％,JW兔位点数为228个,其中多态位点数122个,多态位点比为53．51％,NZW兔位点数为231个,其中多态位点数94个,多态位点比为40．69％;（3）三个群体的Shannon多样性指数分别为0．3385,0．2222和0．1905;（4） JW兔和NZW兔的遗传相似系数最高,为0．8443,其次为WHBE兔和JW兔的遗传相似系数,为0．8204,WHBE兔和NZW兔的遗传相似系数最低,为0．7862。结论结果表明WHBE兔与JW兔和NZW兔之间有遗传的相似性,也存在着遗传差异,应用RAPD技术可以很好地检测实验兔不同品系之间以及同一品系不同个体之间的亲缘关系。     

银杏性别相关分子标记

利用RAPD技术寻找银杏(Ginkgo biloba L.)中与性别相关的分子标记。筛选了1200个10bp的随机引物,产生了8372个RAPD条带。只有S1478产生一条大小为682bp、雄性特异的分子标记,该分子标记被命名为S1478—682,出现在所有雄性植株中,而所有雌性植株都不具有该分子标记。通过在北京和沈阳种植的银杏植株的RAPD推广验证,说明该分子标记可以用来检测银杏植株的性别。     

应用RAPD标记技术对树鼩遗传多样性的分析

目的建立树鼩RAPD（random amplified polymorphic DNA）遗传标记分析方法,了解中缅树鼩种群的遗传多样性。方法采用PCR技术对40条随机引物进行优化,筛选出能有效用于树鼩群体遗传分析的RAPD位点,对48只树鼩个体的基因组DNA进行PCR扩增,并应用Popgene 1.32与RAPDistance Package Version 1.04等软件进行数据处理,分析树鼩的群体遗传特性。结果20个RAPD引物共检测到113个位点,平均每个引物可扩增出5.65个位点,其中多态位点数为69个（占61.1%）。个体间的遗传相似系数平均为0.8307,个体间遗传距离在0.09-0.27之间,平均遗传距离为0.1693。雄性群体的遗传多态度（H0）（0.1864）略高于雌性群体（0.1470）,平均遗传多态度（Hpop）为0.1667;树鼩遗传多态度所占的比例在群体内为48.29%,而雌、雄群体间为51.71%。NJ法进行聚类分析得出T15、T33和T47号树鼩个体聚类成一大类,其余45只树鼩个体聚成另一大类,雌、雄个体呈相互交叉现象。结论实验所筛选的随机引物可有效用于中缅树鼩种群的遗传结构分析。本树鼩群体具有较好的遗传多样性。     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Integration genetic linkage map construction and several potential QTLs mapping of Chinese shrimp (Fenneropenaeus chinensis) based on three types of molecular markers

In this study, totally 54 selected polymorphic SSR loci of Chinese shrimp (Fenneropenaeus chinensis), in addition with the previous linkage map of AFLP and RAPD markers, were used in consolidated linkage maps that composed of SSR, AFLP and RAPD markers of female and male construction, respectively. The female linkage map contained 236 segregating markers, which were linked in 44 linkage groups, and the genome coverage was 63.98%. The male linkage map contained 255 segregating markers, which were linked in 50 linkage groups, covering 63.40% of F. chinensis genome. There were nine economically important traits and phenotype characters of F. chinensis were involved in QTL mapping using multiple-QTL mapping strategy. Five potential QTLs associated with standard length (q-standardl-01), with cephalothorax length (q-cephal-01), with cephaloghorax width (q-cephaw-01), with the first segment length (q-firsel-01) and with anti-WSSV (q-antiWSSV-01) were detected on female LG1 and male LG44 respectively with LOD> 2.5. The QTL q-firsel-01 was at 73.603 cM of female LG1. Q-antiWSSV-01 was at 0 cM of male LG44. The variance explained of these five QTLs was from 19.7-33.5% and additive value was from -15.9175 to 7.3675. The closest markers to these QTL were all SSR, which suggested SSR marker was superior to AFLP and RAPD in the QTL mapping.     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphic DNA)分子标记技术,寻找谭清苏铁（Cycas tanqingii）中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465 (CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域（SCAR）分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。