用磁珠富集法分离亚麻基因组微卫星分子标记

应用Dynal磁珠-生物素标记的微卫星探针(CT)₁₅与亚麻基因组DNA酶切片段杂交,捕获300~1 500 bp含有微卫星序列的DNA片段,连接到pMD18-T载体中,构建富集微卫星序列的小片段插入文库。利用接头引物和根据微卫星核心序列设计的引物VRV(CT)₁₅使用PCR方法直接对文库筛选,从422个转化子中获得了104个阳性克隆,对其进行测序分析,获得了97个微卫星序列,微卫星序列的富集效率达到22.99%,PCR扩增筛选效率93.27%。对97个微卫星序列进行比对分析,其中51个重复序列的两端序列高度相似,据其设计的特异引物对阳性克隆进行2次筛选,能淘汰相似度高的同类序列,提高筛选亚麻微卫星标记的效率。     

黄花柳基因组微卫星分离及多态性位点检测

以生物素标记的(CT)15和(GT)15为探针进行杂交,借助磁珠筛选出含微卫星的Sau3A I酶切片段,构建了黄花柳微卫星富集文库,获得960个阳性克隆.随机挑选360个阳性克隆进行测序,发现含有微卫星的克隆比例达45%,最后获得的44条非同源性克隆中共含有53个微卫星位点,其中(TC/AG)n,(GA/CT)n,(CA/GT)n比例最高,占74%.随机选取其中的18个微卫星位点设计引物,用5个种个体混合DNA组成模板池,检测引物种间的多态性信息,初步筛选出5对引物在柳属种间有很好的通用性.说明微卫星富集法开发SSR标记不失为一种有效的方法.     

磁珠富集法筛选马尾松微卫星标记

将马尾松核基因组DNA用Sau3A Ⅰ酶切后,电泳回收300～1 000 bp片段.在回收的片段上连接接头PCR后与用生物素标记的微卫星探针(AC)15、(AG)15杂交运用磁珠富集含有微卫星序列的DNA片段.将获得的序列通过PCR扩增后,连接pGEM-T载体,转化入感受态大肠杆菌,得到微卫星序列文库.然后用PCR法直接对文库进行扩增,获得58个阳性克隆,经测序分析,获微卫星序列33个,并成功设计出马尾松微卫星引物19对.     

地黄微卫星富集文库构建及特性分析

应用Dynal磁珠-生物素标记的微卫星探针(AC)8,(AG)8和(ATG)12与地黄基因组DNA酶切片段杂交,捕获含有微卫星序列的DNA片段,连接到pMD 18-T栽体上,转入感受态细胞Trans 5 α,构建地黄富集微卫星文库.利用M13F和M13R载体序列引物筛选文库,对插入片段长度为400～ 800 bp的克隆进行测序.共获得96条序列,48条(50％)含有微卫星位点,其中完美型占66％,非完美型22％,混合型12％.微卫星重复基元中,二核苷酸(AG)n和三核苷酸(CAT)n最为常见.     

利用FIASCO技术进行波纹巴非蛤微卫星

为揭示波纹巴非蛤种质遗传特性、开发种质库,利用FIASCO(Fast Isolation by AFLP of Sequences Containing Repeats)技术开展了其基因组微卫星标记的分离与筛选研究。基因组DNA经限制性内切酶Mse I 酶切后与接头连接,用生物素标记的(CA)15或(AAG)7探针与其杂交,然后用磁珠富集、洗脱获得单链目的片段,经PCR扩增后形成双链,最后进行克隆转化,构建微卫星富集文库。挑选克隆用探针引物(CA)15或(AAG)7和载体引物进行PCR筛选,测序得到含有微卫星DNA的序列,根据序列设计和合成微卫星引物,进行引物适用性分析,并分析了湛江群体的遗传结构。结果表明,8对微卫星引物在湛江群体共检测到108个等位基因,每个位点等位基因数为5~19,期望杂合度为0.666~0.926,观测杂合度为0.400~0.882,4个位点(Pun4,Pun5,Pun6,Pun7)显著偏离哈迪-温伯格平衡(P<0.00625);PIC介于0.62~0.92,所有位点均属于高度多态位点(PIC>0.5)。说明FIASCO技术适合于波纹巴非蛤微卫星标记的分离与筛选,筛选得到的8个微卫星位点能用于波纹巴非蛤遗传多样性分析及野生群体与养殖群体的群体结构分析。     

扁吻鱼微卫星分子标记的筛选及特征分析

通过磁珠富集法筛选扁吻鱼的微卫星分子标记。利用限制性内切酶Sau3AI对扁吻鱼的基因组DNA进行酶切,并选取片段大小在250～750 bp间的片段,利用PCR技术进行全基因组扩增,采用生物素标记的(CA)8探针对微卫星片段进行富集。富集后的片段与T载体连接后利用DH5α大肠杆菌进行转化,然后以Sau3AI为引物对菌落进行PCR扩增,扩增后选片段大小符合条件的菌落挑取至测序板,进行测序。结果表明:PCR筛选共获得563个阳性克隆,其中测序192个阳性克隆后发现有53个微卫星序列;序列分析表明,完美型占67.92%,非完美型为22.64%;混合性标记占9.43%。根据测序结果设计微卫星引物24对,经PCR扩增筛选,琼脂糖凝胶电泳结果显示,其中22对引物可扩增出清晰的条带,其中具有多态性的13对。利用13对多态性引物对扁吻鱼野生群体进行扩增,PCR扩增结果显示,等位基因数为3～6,多态信息含量(PIC)为0.625 3,12个位点处于高度多态水平(PIC>0.5)。     

基于ISSR和AFLP标记开发甜菜 SSR 引物的研究

本研究以ISSR-PCR和AFLP标记原理为基础,介绍一种新的分离甜菜基因组微卫星引物的方法。首先对甜菜基因组DNA进行酶切并连接已知序列的接头,构建基因组DNA酶切文库,同时用一个或两个ISSR引物,扩增文库中两端含微卫星序列片段并进行克隆测序,根据测序结果设计微卫星序列间的IP1引物和IP1与微卫星序列间IP2 引物;再根据侧翼序列克隆原理,采用巢式PCR进行基因组步移,扩增IP2引物下游序列,根据巢式PCR产物测序结果,设计微卫星序列另一侧的引物IP3 ,IP2和IP3即为SSR标记引物,对获得的SSR引物进行PCR验证,结果表明SSR引物产率为16%,本研究获得的SSR引物具有较高的多态性,对于后续的遗传多样性检测和遗传连锁图构建具有重要意义。     

香椿(Toona sinensis)微卫星富集文库构建及特性分析

采用Dynal磁珠富集法构建了香椿微卫星富集文库,通过测序结果对文库的特性进行了分析。实验使用改良CTAB法提取香椿基因组DNA,用(AC)8、(AG)8和(ATG)123种带有生物素标记的探针与香椿基因组DNA的酶切片段进行杂交,将磁珠捕获的含有微卫星序列的DNA片段插入p MD 18-T载体,并转入感受态细胞Trans 5α构建克隆,经筛选后得到含356个克隆的香椿基因组微卫星富集文库。从富集文库中挑选插入片段长度为400~800 bp的128个克隆进行测序,其中含有SSR的序列77条,得率达60.16%,其中完美型占82.69%,非完美型8.65%,混合型8.65%。上述结果为SSR位点的进一步开发奠定了基础。     

ISSR分子标记及其在植物研究中的应用

ISSR(inter simple sequence repeat)分子标记是一种以微卫星序列为引物,进行多位点PCR扩增的技术.ISSR技术简易、快捷,同时兼备AFLP(扩增酶切片段多态性)、SSR(简单序列重复)和RAPD(DNA随机扩增多态性)等分子标记方法的优点.引物设计无需预知基因组序列,只要是目标区域的长度在可扩增范围内,就能扩增出微卫星重复序列间的DNA片段.ISSR标记以其高多态性,已被广泛应用于种质收集、品种鉴定、遗传多样性、系截系、遗传作图、基因定位、标记辅助选择、预测基因组SSR基序的丰度以及SSR引物开发等研究.本文对ISSR技术及其在植物研究中的应用进行全面的概述.     

基于绵羊皮肤组织ESTs开发新型微卫星标记

利用生物信息学方法,在从国际公共生物数据库检索获得绵羊皮肤组织相关的3295条EST序列中筛选微卫星标记。结果表明,从绵羊皮肤组织相关的EST序列中搜索到微卫星209个,含有微卫星的序列196 个,占整个EST 序列数据库的6.3% , 其中双碱基重复150个,三碱基重复19个,四碱基重复12个,五碱基重复17个,六碱基重复11个。在这些微卫星序列中,AC/TG重复在双碱基类型中最丰富,占双碱基微卫星序列总数的71.8%。根据筛选到的微卫星序列设计并合成引物22对 ,其中18 对引物有扩增产物,且条带清晰, 6对引物在中国美利奴羊群内多态性丰富,平均PIC达0.7001,为后期与毛品质进行相关性分析,寻找毛用性状新型分子标记奠定了基础。     

Identification and characterization of microsatellites in eggplant

The potential of microsatellite markers for use in genetic studies in eggplant, Solanum melongena, has been evaluated. A genomic library of eggplant was screened for GA and GT repeat motifs to isolate microsatellite clones. The frequency of each repeat motif in the eggplant genome was found to be every 3200 kb for GA repeats and every 820 kb for GT repeats. Sixty‐one per cent of GT repeats were found to directly flank AT repeats. A total of 37 polymerase chain reaction (PCR) primer pairs were designed, 23 of which amplified a single product or several products. The level of microsatellite polymorphism was evaluated by using S. melongena lines and related Solanum species. Two to six alleles per primer pair were displayed in the S. melongena lines and two to 13 alleles were displayed in the Solanum relatives. Seven microsatellites showed polymorphism between parental lines of the mapping population and segregated in a codominant Mendelian manner. These microsatellite loci were distributed throughout the linkage map.     

Development and utilization of novel SSRs in foxtail millet [Setaria italica (L.) P. Beauv.]

Although the foxtail millet [Setaria italica (L.) P. Beauv.] is recently regarded as a model crop for studying functional genomics of biofuel grasses, its genetic improvement to some extent was limited due to the non‐availability of molecular markers, particularly the microsatellite markers and the saturated genetic linkage map. Considering this, we attempted to generate a significant number of microsatellite markers in cultivar ‘Prasad’. Two hundred and fifty‐six clones were sequenced to generate 41.82‐kb high‐quality sequences retrieved from genomic library enriched with dinucleotide repeat motifs. Microsatellites were identified in 194 (76%) of the 256 positive clones, and 64 primer pairs (pp) were successfully designed from 95 (49%) unique SSR‐containing clones. The 67.4% primer designing ability, 100% PCR amplification efficiency and 45.3% polymorphic potential in the parents of F₂ mapping population established the efficacy of genomic microsatellites. All the 64 microsatellite markers displayed high level of cross‐species amplification (~67%) in 10 millets and non‐millets species. These experimental findings suggest the utility and efficacy of SSRs in diverse genotyping applications, resolving QTLs, phylogenetic relationships and transferability in several important grass species.     

Development and variability analysis of microsatellite markers in peach

A genomic DNA library enriched with AG/CT repeats has been developed from the peach cultivar ‘Merrill O'Henry’. The enrichment method was efficient, with 61% of the clones obtained carrying a microsatellite sequence and a yield of one polymorphic microsatellite every 2.17 sequenced clones. From 35 microsatellites detected, 24 were polymorphic in a set of 25 cultivars including 14 peaches and 11 nectarines. A total of 82 alleles were found with the polymorphic microsatellites, with an average of a 37% of observed heterozygosity. Microsatellites with a high number of repeats were generally those having the largest number of alleles. All cultivars except two (‘Spring Lady’ and ‘Queencrest’) could be individually distinguished with the markers used. Just three selected microsatellites were enough for the discrimination of 24 out of the 25 possible genotypes. Cluster analysis grouped all nectarines in a single cluster. Peaches, with 75 of the 82 alleles found, were more variable than nectarines, with only 64. Microsatellites appear to be powerful and suitable markers for application in peach genetics and breeding.     

Abundance, polymorphism and genetic mapping of microsatellites in oilseed rape (Brassica napus L.)

The objective of the present study was to estimate the abundance and degree of polymorphism of simple sequence repeat (SSR) markers in rapeseed. By screening about 45000 clones of a small inserts library of rapeseed total DNA the abundances of GA/TC and CA/TG simple sequence repeats in the rapeseed genome were estimated to be approximately one repeat every 100 kb and 400 kb, respectively. After sequencing 13 positive clones, primer pairs could be designed for 11 microsatellite loci. Seven of these primer pairs produced reproducible amplification products in a set of 31 rapeseed genotypes, with one pair amplifying two independent products, giving a total of eight amplified loci. The different microsatellite loci displayed between one and three visible alleles. At four loci, additional null alleles were observed. With up to four alleles, polymorphic microsatellite markers show significantly higher allele numbers in rapeseed than restriction fragment length polymorphism (RFLP) markers. Four of the eight microsatellite markers could be mapped on four different linkage groups of an RFLP map of the rapeseed genome.     

Development of simple sequence repeat (SSR) markers in Eucalyptus from amplified inter-simple sequence repeats (ISSR)

Eucalyptus spp. are widely used in exotic plantations. Since many of these trees are derived from vegetative propagation, the routine identification of clones has become increasingly important. The most widely used molecular based method for fingerprinting these clones is by random amplified polymorphic DNAs (RAPDs). Although this technique is useful, its results are not very repeatable, especially between laboratories. The aim of this study was to develop microsatellite markers that are highly repeatable, and to investigate their value in Eucalyptus fingerprinting. Typically, this process involves the expensive procedure of constructing an enriched genomic library. However, we used an intersimple sequence repeat (ISSR) polymerase chain reaction (PCR)‐based enrichment technique for microsatellite‐rich regions. With this relatively inexpensive method, microsatellite‐rich regions were amplified directly from genomic DNA, after which PCR products were cloned and sequenced. From these microsatellite‐rich sequences, primer sets were constructed to amplify mono‐, di‐, tri‐, hexa‐and nona‐nucleotide repeats. These markers were all inherited in a Mendelian fashion in the progeny of a test cross between two Eucalyptus grandis trees. The primer sets developed were also able to amplify the corresponding microsatellite loci from five different Eucalyptus spp., namely E. grandis, E. nitens, E. globulus, E. camaldulensis and E. urophylla.     

Occurrence of microsatellites in spinach sequences from computer databases and development of polymorphic SSR markers

Microsatellites are valuable tools as molecular markers in plant breeding. To establish genetic linkage maps or for population studies, information about the occurrence and usability of microsatellite markers in different species is necessary. Sequences of spinach Spinacia oleracea from computer databases were therefore searched for the presence of microsatellites. Sixty simple sequence repeats were found in 237 spinach sequences with a total of 349.4 kb DNA. After removing duplicated sequences, 50 different microsatellites with various motifs remained. Differences between nuclear and chloroplast DNA were not in the number of microsatellites but in their type and length. Chloroplast sequences from spinach contain only short strings of A and AT repeats, whereas nuclear sequences show a wider variety of motifs. Flanking primers for polymerase chain reaction (PCR) analysis were designed for 13 of these microsatellites and tested with two different varieties of spinach. Twelve primer pairs gave amplification products and seven of these showed polymorphisms in the variety ‘Wiremona’ but only one in the variety ‘Monatol’. These markers may be used for linkage analysis or population studies in spinach.     

DNA typing of hops (Humulus lupulus) through application of RAPD and microsatellite marker sequences converted to sequence tagged sites (STS)

Summary Both random amplified polymorphic DNA and microsatellite repeat sequences were investigated as DNA markers for distinguishing hop cultivars. Microsatellite sequences converted to STS markers proved to be most successful. The relative abundance of microsatellite repeat sequences in the hop genome varied depending on the sequence class. Of the repeat types investigated the dinucleotide repeats (GA)_n and (GT)_n are the most highly represented in the hop genome. Microsatellite repeat sequences in hops have been shown to be highly polymorphic and are very informatives as STS molecular markers. A DNA typing system using sequence-tagged microsatellite site markers has been developed which will not only be useful for hop cultivar identification but also marker assisted breeding and quality control purposes.     

Isolation of 1,796 SSR clones from SSR-enriched DNA libraries of bunching onion (Allium fistulosum)

Bunching onion (Allium fistulosum L.) is one of the most important vegetables in Japan. To establish a genetic basis for molecular breeding of bunching onion, we isolated 1,796 simple sequence repeat (SSR) clones by large-scale sequencing of SSR-enriched genomic DNA libraries. Of these, 1,331 (74.1%) contained (GT) _n repeats (n > 5), while 314 (17.5%) were (GA) _n -containing clones. The average number of SSR repeats was 10.5 and 10.4 in the (GT) _n - and (GA) _n -containing clones, respectively. In a sample of five bunching onion inbred lines, an average of 3.2 alleles were detected in the 100 SSR loci investigated, with the polymorphic information content averaging 0.55. These results indicate that bunching onion SSRs are very rich sources of highly informative genetic markers.