麻竹EST-SSR标记开发及其对慈竹变异类型的分析研究

利用麻竹(Dendrocalamus latiflorus)已有的EST序列,开发了一批EST-SSR分子标记,并用于慈竹(Bambusa emeiensis)栽培变异类型的多态性研究。结果表明,在麻竹9574个EST中找到了331个EST,含有381个SSR位点,SSR出现的频率为3.98%。EST-SSR的重复类型共有59个,其中2个核苷酸的重复最多,占63.8%,其次是3个核苷酸的重复序列。根据含有SSR的EST序列设计出了44对引物,对慈竹及其变异类型进行了扩增效率、多态性及通用性检测,其中37对引物能够扩增出稳定且清晰的条带,且扩增具有多态性,扩增片段长度主要集中在100～1000 bp,引物有效率达84.1%。遗传相似性分析结果显示,所检测样品间遗传距离为0.263～0.840,平均为0.552,其中‘黑笋慈竹’与‘琴丝慈竹’‘、蛇头慈竹’与‘龙头慈竹’之间的遗传距离较近,而‘罗汉慈竹’与其他样品的遗传距离最远。     

龙眼顶芽转录组简单重复序列(SSR)标记信息分析及分子标记开发

随着新一代测序技术的发展,大量的转录组数据和表达序列标签(EST)成为开发简单重复序列(SSR)标记的可利用资源。本研究利用MISA软件筛选龙眼(Dimocarpus longan)顶芽转录组数据库序列,从114 445条龙眼转录组unigene序列中发现11 546个SSR位点,SSR出现频率为10.09%。其中1 975条unigene含有两个或两个以上EST-SSR位点,占所有SSR位点的比例为17.10%,SSR出现的平均距离为7.52 kb。从龙眼转录组SSR核苷酸基序类型来看,二核苷酸(52.11%)和三核苷酸(46.15%)出现频率最高,占所有核苷酸出现频率的99.26%。在龙眼转录组SSR中二核苷酸重复基元出现频率最高的是AG/CT(4 250个,占36.81%),三核苷酸重复基元出现频率最高的是AAG/CTT(1 109个,占9.61%)。对含SSR位点的9 571条unigene序列进行引物设计,共设计出了8 347对SSR位点特异引物。随机挑选合成50对EST-SSR引物,以‘石硖’、‘储良’、‘古山2号’、‘立冬本’等四份龙眼材料的基因组DNA为模板对这批引物进行PCR扩增、筛选,结果表明,其中21对引物能产生理想的PCR产物,有效扩增率为42%;16对引物扩增条带具有多态性,占有效引物的76.2%;16对多态性引物共扩增获得50个条带,其中多态性片段21个,每对引物平均产生1.31个多态性片段。     

华仁杏EST-SSR标记引物设计与开发

随着生物科技的进步,ESTs（表达序列标签）已经成为开发SSR（简单重复序列）标记的重要资源。本文利用NCBI公共数据库下载蔷薇科EST序列22 458条,使用SSRHunter1.3软件进行了SSR搜索,从中获得22 527条SSR,应用Primer5.0软件设计并经由Oligo7.0软件检测,共得到61对EST-SSR引物。利用这些引物对8个华仁杏品种进行了PCR扩增及检测,得到10对能产生清晰多态性条带的EST-SSR标记,标明了10对引物的序列,为进一步开展华仁杏SSR分子标记辅助育种研究奠定了基础。     

中国96个荔枝种质资源的EST-SSR遗传多样性分析

根据本实验室已获得的荔枝果皮cDNA文库EST序列,通过SSRIT在线检索,从3391条EST序列中,发现305条含有SSR,占整个文库EST的8.99%。利用SSR-ESTs序列共设计100对EST-SSR引物,其中62对在荔枝上有扩增产物,50对有扩增多态性,即具有一定的通用性。接着从96份荔枝种质中选取12个品种的基因组DNA,开展核心引物筛选,共筛选出多态性较好的EST-SSR分子标记30个;这30个EST-SSR分子标记在96份资源共扩出284条带,不同引物的扩增条带在3～18条之间,平均9.47条,其中有282条为多态性带,多态率高达99.30%,每对引物的Nei's基因多样度范围为0.186～0.396,香农信息指数范围为0.318～0.558;此外,系统聚类分析结果表明,在相似系数0.5525处,可将96份荔枝种质资源分成了8大类群,该8大类群基本与其生态类型和植物学性状特征相符。在此基础上,还对荔枝的主栽品种和特殊种质进行鉴别,结果表明,该30个EST-SSR分子标记在不同品种间可产生较清晰可辨的多态性差异,为荔枝品种以及种质资源鉴别和鉴定的分子指纹的构建奠定了良好基础。     

亚麻EST-SSR信息分析与标记开发

与基因组SSR相比,以EST为基础的EST-SSR分子标记具有自身的优点。本研究从11240条亚麻（Linum sitatissmum L.）EST序列中检索出877条含有SSR的序列,其出现频率为7.8%。其中以三核苷酸重复出现的频率最高,占总SSR序列的60.1%;其次是二核苷酸重复,占21.9%;四、五和六核苷酸重复占18%。根据这些含SSR的EST序列共设计了73对SSR引物,在8份亚麻材料间通过PCR扩增检测,有63对引物扩增出清晰条带,引物可用率86.3%;有17对引物在8份亚麻材料间显现出多态性,占可扩增引物的26.3%。     

15,665个黄瓜EST-SSR位点的引物设计及多态性检测

目前,国际公共数据库中黄瓜EST序列数量的迅速增加为SSR标记的开发提供了极为便利的资源。本研究从葫芦科基因组数据库下载513,801条黄瓜EST,经EST-trimmer软件和CD-HIT程序预处理,共获得381,022条非冗余EST。利用Perl程序MISA搜索到SSR位点15,665个,检出率为4.11%。利用Primer3软件成功设计了9,145对黄瓜EST-SSR引物,随机抽取10对引物对5个黄瓜品种进行多态性分析发现,其中仅2对引物能检测到多态性。该数据为下一步开发新的黄瓜EST-SSR标记奠定了一定的基础。     

漆树EST-SSR引物开发

利用NCBI数据库进行漆树EST-SSR引物开发,从NCBI数据库中共下载漆树EST序列87 856条。利用MISA软件进行序列处理、拼接及聚类后,从87 856条漆树EST序列中拼接组装成3 979条非冗余序列,含SSR位点的EST序列出现频率占EST序列总数的4.5%,从3 979条非冗余序列中检测到487个SSRs微卫星位点,出现频率为12.2%。这些SSR位点中,三核苷酸和二核苷酸重复基元所占比例较高。采用Primer5.0软件,共成功设计50对EST-SSR引物,50对EST-SSR引物在25个漆树个体上均能扩增出清晰的电泳条带,其中18对引物检测出了多态性条带,扩增率达36%。     

家蚕第12连锁群EST-SSR标记的筛选和分析

为了探究家蚕Bombyx mori EST-SSR标记的多态性, 对检索获得的家蚕第12连锁群的4 465条EST序列进行了分析, 整理和拼接后得到581条非冗余EST序列, 总长度约为480 kb。其中, 有122条序列中共检测到154个EST-SSR, 占所研究的EST序列的2.73%, 平均每3.12 kb 含有一个EST-SSR。在所检测的EST-SSR中, 三核苷酸和四核苷酸重复是主导类型, 分别占总数的36.36%和28.57%,大部分表现为Perfect形式; 核苷酸重复平均长度约为16.2 bp, 最长为30 bp。进一步进行同源性分析, 发现有26条序列可以在NCBI中检索到同源序列, 在这些序列中一共含有40个SSR, 其中14个(35.0%)位于5′-UTR, 11个(27.5%)位于3′-UTR, 15个(37.5%)位于CDS区。根据筛选到的微卫星序列设计11对引物, 其中8对引物有扩增产物, 且条带清晰; 应用引物ES1204对8个家蚕品种进行PCR扩增都呈现多态性。结果说明通过家蚕EST数据库发掘SSR标记是一条可行的途径。     

柑桔BES SSR标记开发及连锁图延伸和加密

为了系统性地开发和拓展柑桔SSR标记,通过对公布的柑桔BAC文库末端序列(BAC-End sequence,BES)进行SSR分析,选择1500个SSR位点设计合成并检测323对引物。结果表明:(1)从总长度为28.1 Mb的46 339条序列中共检测出22 403个SSR位点,约每2条序列就会出现一个SSR位点,发生频率为48%,相当于平均1.25kb的序列中就会出现1个SSR,频率约为柑桔EST的2倍,且不同核心重复序列的SSR发生特点与EST也不同。(2)所合成的323对引物中,有效扩增316对,扩增率约98%,173对表现多态性,总多态性比率约55%,多态性引物中单核苷酸重复类型15对,双核苷酸重复类型100对,三核苷酸及以上重复类型58对,表明柑桔BES中具有较为丰富的多态性SSR标记。(3)结合已发表的遗传作图数据,对总计349个多态性位点进行遗传连锁分析,获得的新遗传连锁图谱共含有9个连锁群、334个SSR标记、总长844.2cM、平均图距2.53cM,延长和加密了先前的图谱。该研究开发的新SSR标记为开展柑桔遗传鉴定分析和遗传图谱构建提供了新的标记来源,加密的遗传图谱为柑桔的基因定位、图位克隆和标记辅助育种等奠定了基础,SSR分析结果也为其他物种SSR标记的开发提供了参考。     

利用SSR与RAPD分子标记评估甘蔗品种的遗传多样性

利用SSR与RAPD两种分子标记对美国、中国台湾以及中国大陆不同甘蔗育种单位选育的甘蔗品种或亲本材料的遗传多样性进行评估。其中19对SSR引物共扩增出87条带,多态性带为84条,多态性比例为96.55%,扩增出的条带数范围为2~8条,平均每对引物扩增出4.58条带,引物的PIC值范围为0.34~0.93,平均0.64。21条RAPD引物共扩增出184条带,扩增条带数范围为3~16,平均每条引物扩增8.76条带,其中多态性带为184,多态性比例为100%,引物PIC范围为0.53~0.97,平均0.86。结果表明,两种分子标记都能较好的评估甘蔗品种的遗传多样性。     

Genetic dissection of scent metabolic profiles in diploid rose populations

The scent of flowers is a very important trait in ornamental roses in terms of both quantity and quality. In cut roses, scented varieties are a rare exception. Although metabolic profiling has identified more than 500 scent volatiles from rose flowers so far, nothing is known about the inheritance of scent in roses. Therefore, we analysed scent volatiles and molecular markers in diploid segregating populations. We resolved the patterns of inheritance of three volatiles (nerol, neryl acetate and geranyl acetate) into single Mendelian traits, and we mapped these as single or oligogenic traits in the rose genome. Three other volatiles (geraniol, β-citronellol and 2-phenylethanol) displayed quantitative variation in the progeny, and we mapped a total of six QTLs influencing the amounts of these volatiles onto the rose marker map. Because we included known scent related genes and newly generated ESTs for scent volatiles as markers, we were able to link scent related QTLs with putative candidate genes. Our results serve as a starting point for both more detailed analyses of complex scent biosynthetic pathways and the development of markers for marker-assisted breeding of scented rose varieties.     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。     

Development of EST-SSRs by an Efficient FIASCO-Based Strategy: A Case Study in Rare Minnow (Gobiocyrpis Rarus)

Type I markers are useful for comparative mapping and other genetic analyses, but relatively difficult to develop. In the present study a microsatellite (SSR)-enriched cDNA library was constructed for the first time using the fast isolation by AFLP of sequences containing repeats (FIASCO) method in a small fish, Chinese rare minnow (Gobiocypris rarus). A total of 97.4% of the expressed sequence tags (ESTs) contained targeted CA-repeats, in which 29 unique EST-SSRs were identified. Ten out of the 28 loci for which primer pairs were designed were polymorphic with alleles ranging from three to seven (mean 4.50). Some of these EST-SSRs can be amplified in other species. These results proved that cDNA-FIASCO is an efficient way to isolate novel EST-SSRs in a fish.     

Development of EST-SSRs by an efficient FIASCO-based strategy: a case study in rare minnow (Gobiocyrpis rarus)

Type I markers are useful for comparative mapping and other genetic analyses, but relatively difficult to develop. In the present study a microsatellite (SSR)-enriched cDNA library was constructed for the first time using the fast isolation by AFLP of sequences containing repeats (FIASCO) method in a small fish, Chinese rare minnow (Gobiocypris rarus). A total of 97.4% of the expressed sequence tags (ESTs) contained targeted CA-repeats, in which 29 unique EST-SSRs were identified. Ten out of the 28 loci for which primer pairs were designed were polymorphic with alleles ranging from three to seven (mean 4.50). Some of these EST-SSRs can be amplified in other species. These results proved that cDNA-FIASCO is an efficient way to isolate novel EST-SSRs in a fish.     

Development and analysis of EST-SSRs for flax (Linum usitatissimum L.)

A set of 146,611 expressed sequence tags (ESTs) were generated from 10 flax cDNA libraries. After assembly, a total of 11,166 contigs and 11,896 singletons were mined for the presence of putative simple sequence repeats (SSRs) and yielded 806 (3.5%) non-redundant sequences which contained 851 putative SSRs. This is equivalent to one EST-SSR per 16.5 kb of sequence. Trinucleotide motifs were the most abundant (76.9%), followed by dinucleotides (13.9%). Tetra-, penta- and hexanucleotide motifs represented <10% of the SSRs identified. A total of 83 SSR motifs were identified. Motif (TTC/GAA)n was the most abundant (10.2%) followed by (CTT/AAG)n (8.7%), (TCT/AGA)n (8.6%), (CT/AG)n (6.7%) and (TC/GA)n (5.3%). A total of 662 primer pairs were designed, of which 610 primer pairs yielded amplicons in a set of 23 flax accessions. Polymorphism between the accessions was found for 248 primer pairs which detected a total of 275 EST-SSR loci. Two to seven alleles were detected per marker. The polymorphism information content value for these markers ranged from 0.08 to 0.82 and averaged 0.35. The 635 alleles detected by the 275 polymorphic EST-SSRs were used to study the genetic relationship of 23 flax accessions. Four major clusters and two singletons were observed. Sub-clusters within the main clusters correlated with the pedigree relationships amongst accessions. The EST-SSRs developed herein represent the first large-scale development of SSR markers in flax. They have potential to be used for the development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping and fingerprinting cultivars for example. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

普通小麦SSR和EST-SSR引物对冰草通用性的比较分析

选用定位于普通小麦7个部分同源群的534对SSR引物和351对EST-SSR引物分别对普通小麦品种‘Fukuho’和四倍体冰草‘Z559’的基因组DNA进行扩增,结果显示:有475对(89.0%)SSR引物和314对(89.5%)EST-SSR引物对‘Fukuho’能有效扩增,226对(42.3%)SSR和258对(73.5%)EST-SSR引物对‘Z559’能有效扩增,表明小麦EST-SSR对冰草的通用性明显高于SSR;扩增强带比率SSR和EST-SSR引物分别为76.1%、84.1%,说明小麦EST-SSR在冰草上扩增带的质量亦优于SSR。选择上述在‘Fukuho’和‘Z559’基因组DNA之间有多态性扩增且带谱清晰的SSR和EST-SSR引物各60对,对‘Fukuho’、‘中国春’、‘北京8号’和二、四、六倍体冰草‘Z804’、‘Z559’、‘Z1075’的基因组DNA再行PCR扩增,结果显示,40对(66.7%)SSR和22对(36.7%)EST-SSR引物在‘Fukuho’、‘中国春’和‘北京8号’间扩增产物表现多态性,且前者高于后者;50对(83.3%)SSR和52对(86.7%)EST-SSR引物在冰草‘Z804’、‘Z559’和‘Z1075’间扩增产物表现多态性,两者相当。通用性、多态性和扩增强带比率综合比较表明,普通小麦EST-SSR和SSR经筛选虽都能转用于冰草,但两者相比EST-SSR更优。     

Development of microsatellite markers in potato and their transferability in some members of Solanaceae

We have developed thirty new microsatellite markers in potato by screening genomic libraries and ESTs. Genomic libraries of potato cultivar Kufri Bahar were screened for sequences containing microsatellite motifs GA, GT, ACA, ATC, GAA, TAA and GATA. Using flanking sequences, PCR primers were designed for microsatellites identified from genomic libraries and ESTs. Sixteen new primer pairs from genomic libraries and fourteen from ESTs along with seven previously published primer pairs amplified PCR products in the selected genotypes comprising of 65 Solanum tuberosum lines and 14 other species of the potato gene pool. Neighbor-joining tree based on genetic distance matrix developed using microsatellite markers successfully distinguished all these genotypes in the expected size range. Seventeen microsatellites could also be cross-amplified in at least one of the five members of solanaceae, namely tomato, eggplant, pepper, petunia and tobacco. The new microsatellite markers obtained in this study will be useful in various genetic and taxonomic studies in potato and related genomes.