松树、杨树及桉树表达基因序列微卫星比对分析

微卫星是生物基因组中变异频率最快的序列,结构基因中微卫星重复数的变化会引起基因的框移突变,导致基因表达完全不同或截短的蛋白。因此在进化过程中,基因区微卫星会受到强烈选择的影响。为研究基因区微卫星在不同树种中的变化情况,在本研究中,利用SPUTNIK程序分析了NCBI数据库中松树（Pinus spp.）、杨树（Populus spp.）及桉树（Eucalyptus spp.）的表达序列标签（express sequence tag,EST）序列各3万条。结果显示,桉树和杨树EST序列含有微卫星的比例比较接近,分别为18.7%和15.3%,而在松树中则发生了较大分化,只有8.2%。研究发现,三碱基重复单元是这3个树种编码序列中微卫星的主要重复类型。除三碱基重复微卫星外,桉树和杨树EST序列中其它类型微卫星的丰度随着重复单元长度的增加而减少,而在松树中则呈相反现象。同时值得注意的是松树EST序列中变异频率快的微卫星（〉20bp）数量明显比桉树及杨树少。研究还发现,3个树种中微卫星获得或丢失重复单元的速率都随着重复单元的增加而降低。本研究首次报道了不同树种基因区微卫星比较研究,发现了一些松树与杨树、桉树相比较EST序列中所含微卫星在丰度及变异频率方面存在的异同。基因所含微卫星序列对基因的功能有重要影响,本研究的结果将为了解不同树种中基因区微卫星的特征提供重要参数,同时也将为利用所研究树种的EST序列开发多态性高的微卫星标记提供有益的生物信息学参考。     

菠菜性别相关EST-SSR标记的开发及应用

为了明确菠菜EST序列中SSR的总体特点,开发菠菜EST-SSR引物;为利用EST-SSR引物进行菠菜性别相关特异序列的克隆奠定基础,本文从NCBI上获得1093条EST,利用在线软件SSRIT检测所含SSR序列,并进行分析。共检索出68条SSR序列,分布于64条EST中,检出率为6.22%,包括22种重复基元。其中二核苷酸重复基元的EST-SSR占主导地位,占总SSR数目的32.3%。利用在线引物设计软件Primer3.0设计了7对EST-SSR引物,在适合的PCR反应体系下,分别以雌、雄菠菜DNA基因组为模板,对设计的EST-SSR引物进行筛选,结果显示以EST序列HS097148设计的一对引物从菠菜雌雄基因组中扩增出一条雄性特异的条带,表明通过菠菜EST-SSR引物获得菠菜性别相关特异序列是可行的。     

大白菜表达序列标签SSR标记分析*

对大白菜（Brassica campestris L．ssppekinensis）13007个EST序列进行拼接共得到7714个片段重叠群（contig），其中10．4％（890个）非冗余EST含有SSR标记（EST-SSR），标记间平均间隔距离为3．9kb，单、双和三核苷酸重复序列大约各占1／3左右。CDS所包含的三核苷酸重复序列最多，而且AAG／CTT重复序列的含量最高。通过同源性比较分析，获得功能已知的SSR-ESTs774个，共计含有846个SSR，其中50．2％位于5＇-UTR，31．4％位于3＇-UTR，18．4％位于CDS。实验选取123个SSR座位设计引物，利用芜菁花叶病毒（Turnip mosaic virus，TuMV）的高抗和高感大白菜F6代自交系材料各2份，分析EST-SSR标记多态性。结果表明，其中37个标记可进行有效扩增，但是没有表现出多态性，23个标记（18．7％）至少在1份材料中具有多态性。对这些EST-SSR标记在大白菜和其它芸薹属植物的系统发育关系分析、遗传作图和基因定位的应用进行了讨论。     

铁皮石斛EST-SSR分子标记的开发

为了开发铁皮石斛EST-SSR分子标记,利用SSRIT软件对铁皮石斛近缘物种-金钗石斛的15 383条EST序列进行SSR位点查找,利用Primer 5和Oligo 7软件进行引物设计和评价,之后经PCR扩增和琼脂糖凝胶电泳对引物进行初步筛选,并通过聚丙烯酰氨凝胶电泳对所筛选的引物进行有效性检测。结果表明,SSRIT软件共查找到370条SSR位点序列,共有379个SSR位点,候选SSR位点出现的频率为2.46%。二核苷酸、三核苷酸和六核苷酸重复是最主要的重复类型,分别占41.42%、26.91%和27.44%,其中二核苷酸重复中AG/CT(20.84%)和GA/TC(18.73%)最丰富;三核苷酸重复中TCT/AGA(4.22%)和GA/TC(3.96%)最丰富;而六核苷酸重复基元较多。EST-SSR平均长度为24 bp,平均每8.5条EST序列包含1个SSR位点。设计的106对EST-SSR引物中有50对能扩增出理想的PCR产物,引物有效扩增率为47.17%,其中有43对能够扩增出多态性条带,占可扩增引物的86%。本研究开发的43对引物为铁皮石斛遗传多样性分析、种质鉴定、遗传图谱构建和功能基因研究等提供了重要的参考价值。     

银杏EST序列中微卫星的分布特征

本文利用从NCBI下载的21590条银杏EST序列,分析了银杏（表达序列标签微卫星）EST-SSR在银杏EST序列的分布和比较了在不同长度EST序列中的SSR特性。在剔除冗余和低质量序列后,得到总长为5708.385kb的无冗余EST序列7961条,发现了405个EST序列（5.09%）含有475个SSR,长度400~1000bp的EST序列含SSR位点数为445个,占SSR总数的93.68%。二核苷酸和三核苷酸基元类型是银杏EST-SSR的主要类型,分别占SSR总数的73.89%和24.00%,最常见的SSR基元是：（AT）n、（AG）n、（AC）n、（AAG）n和（AAT）n。通过对银杏EST序列中SSR位点信息的发掘分析,为有针对性地设计EST-SSR引物,开发银杏EST-SSR分子标记奠定基础。     

结球白菜结球前期基因表达序列标签（EST）分析

以结球白菜(Brassica rapa L.ssp．pekinensis)结球前期心叶为材料，构建了结球前期球叶cDNA文库，随机挑选克隆单向单次测序后，共得到1162条峰图良好和插入片段长度大于150bp的可用序列。BLASTX及BLASTN序列比对分析表明，94．8％(1102／1162)的表达序列标签(EST)可在蛋白质或核苷酸水平上找到同源类似物。同源性最大的蛋白质按物种来源分析后发现，大约77％的功能已知蛋白质来自拟南芥(Arabidopsis thaliana)，另外还有60个EST与已发表的植物基因没有同源性，这一部分EST对于研究结球白菜独特的生理和形态发育途径以及开展基因组分子作图具有重要的意义。同源蛋白质按其功能进行分类表明，参与蛋白质合成过程的酶或蛋白质的EST数量最多，其次是参与能量代谢过程(包括光合作用)的EST序列。在核苷酸水平上，全部EST中只有51％的同源物来自拟南芥。对上述1162条EST进行片段重叠群分析(contig analysis)共获得895个非冗余片段重叠群，其中723个仅由一个EST组成(即singletons)，表明结球白菜结球过程中表达的基因种类繁多。大量EST的获得为进一步了解结球白菜结球机制及获取相关基因提供了重要的序列信息。     

竹类植物EST-SSRs分布特征及应用

本研究利用毛竹已有的EST序列,开发竹类植物EST-SSR标记,旨在对竹子的分子分类进行研究。通过对3087条毛竹(Phyllostachys pubescens)EST序列进行筛选,获得了408条含有SSR的毛竹EST,在所有的EST-SSR中,三核苷酸重复类型(49.75%)最多,其次是二核苷酸重复类型(43.14%)、五核苷酸重复类型(4.41%)、四核苷酸重复类型(1.72%)和六核苷酸重复类型(0.98%)。不同基序类型中,GAG/CTC基序和AG基序分别是三核苷酸重复类型和二核苷酸重复类型中含量最高的重复基序,分别占18.72%和71.02%。当SSR长度等于18bp时,所检测到的SSR数量最多,达140条。随着SSR长度的增加,所检测到的SSR数量呈下降的趋势。根据搜索出的EST序列,共设计出25对引物,对12个竹类植物进行了扩增效率、多态性及通用性检测。其中18对引物能够扩增出稳定且清晰的条带,16对引物扩增具有多态性,扩增片段长度主要集中在100～500bp,引物有效率达64%。依据扩增结果进行遗传相似性分析,12种竹类植物可分为两大类群:丛生竹类群(clump bamboos)和散生竹类群...     

香菇表达序列标签(EST)的SSR分析及EST-SSR引物的筛选

从真菌基因组计划网站(FGP)和NCBI网站数据库下载了总长度为8.1Mb的11150条香菇的EST(包括香菇的10条cDNA)序列，通过SSRhunter 1.3软件结合手工查找，从中发现2.83％即316条EST含有一共469个SSR.，平均每17.3kb出现一个EST-SSR。在所有EST-SSR中，三碱基和六碱基SSR出现最多，分别占EST-SSR总数的38.00％和20.00%，出现较多的基元为(A)n、(T)n、(GA)n、(AG)n、(TGA)n、(GAT)n和(TCTTT)n，占所有EST-SSR的35.39％。利用Oligo 6.0软件设计了51对EST-SSR引物，并选用其它物种的24对引物，对香菇菌株进行了PCR扩增。实验结果表明，设计的51对引物有39对具有明显的扩增产物，选用的其它物种的24对引物均没有清晰可见的特异性扩增产物。部分有扩增产物的EST-SSR引物具有多态性。     

中国野生葡萄抗白粉病基因RAPD标记的克隆及序列分析

对获得的中国野生葡萄抗白粉病基因RAPD标记OPW02-1756、OPO11-964、OPY13- 661、OPB11- 520、OPW05-766、OPV03-1365和OPJ16-759进行了回收、克隆、测序及序列分析。结果表明,7个RAPD标记的实际长度分别是1756bp、964bp、661bp、520bp、766bp、1365bp和759bp。OPW02-1756序列与3条欧洲葡萄cDNA克隆获得的EST序列有94 ~ 98%的同源性。OPO11- 964序列与欧洲葡萄“赤霞珠”的1条EST序列有93%同源性,与“霞多丽”的2条果实不同发育时期获得的EST序列有90%和87%的同源性,与甜橙在多种病原菌侵染后通过cDNA文库获得的1条EST序列有83%的同源性。OPO11-964最大的一个阅读框架包含444个碱基,编码147个氨基酸,该氨基酸序列和21个其它生物的未知功能蛋白序列有部分相似性。OPY13-661序列与欧洲葡萄16条EST序列有同源性,其中与“赤霞珠”cDNA克隆的EST有较高的同源性, 与“霞多丽”的2条叶片非生物胁迫有关的EST序列同源性均为89%。OPB11-520序列与拟南芥基因组4条未知功能DNA序列有94%同源性。OPW05-766序列与来自于酿酒葡萄的GAG-POL 前提有50%的同源性。OPV03-1365序列与水稻基因组6条序列有81 ~ 88%的同源性,与拟南芥基因组3条序列有84 ~ 91%的同源性。OPJ16-759与欧洲葡萄“霞多丽”叶片14条非生物胁迫EST序列有83 ~ 100%的同源性,与欧洲葡萄“Cabsau”浆果3条水分胁迫的EST序列有94 ~ 100%的同源性。     

禾本科植物微卫星序列的特征分析和比较

微卫星或简单序列重复（simple sequence repeats,SSRs）广泛存在于真核生物的基因组中,是目前最有用的分子标记之一。本研究首次利用全基因序列对4个禾本科植物种,包括二穗短柄草（Brachypodium distachyon）、水稻（Oryza sativa）、高粱（Sorghum bicolor）和玉米（Zea mays）的SSR的类型、丰度（数量）、相对丰度、频率、长度和偏好性等进行了分析和比较。结果表明,禾本科植物中SSR的丰度与基因组大小成正比,而相对丰度与基因组大小相关不显著;三核苷酸和六核苷酸基序类型比其它类型更为丰富;不同物种皆呈现出明显的基序偏好性。总的来讲,除个别例外,禾本科植物的基因组中更倾向于富含A/T的基序。本研究结果为研究禾本科植物的基因组进化和SSR标记的开发提供了有价值的信息。     

Genic simple sequence repeat markers for measuring genetic diversity in a native food crop: a case study of Australian <Emphasis Type="Italic">Kunzea pomifera</Emphasis> F.Muell. (muntries)

Kunzea pomifera F.Muell. (muntries) is a native food crop in Australia that produces spicy apple-flavoured berries. Orchards were established in the 1990s using a variety of wild-collected selections. No published records are currently available of formal plant breeding although the Rivoli Bay selection is the most widely planted. DNA markers are needed to reliably assess genetic diversity and validate plant selections, however genomic resources of K. pomifera are not available. Initially, based on the large MYB gene family, eight MYB gene fragments were amplified and cloned using a degenerate PCR primer strategy. Targeted amplicon sequencing was subsequently used to evaluate diversity of 19 K. pomifera plants and this revealed the presence of single nucleotide polymorphisms and simple sequence repeats (SSRs). Three genic SSR markers were developed from K. pomifera MYB gene fragments and another three genic SSRs were successfully transferred from K. pulchella (Lindl.) A. S. George. A high transfer rate (75%) was obtained due to selecting SSRs in genic regions (introns or intergenic regions) and using Eucalyptus grandis W.Hill sequences to identify conserved regions. The six SSRs were highly polymorphic with observed heterozygosity (H_o) (average H_o = 0.63) and polymorphic information content (PIC) (average PIC = 0.54). Using the six genic SSRs, 15 unique genotypes were identified from the K. pomifera collections in two orchards. Importantly, plants in a new diversity panel were genotyped and suspected mis-labeling events confirmed. The genic SSRs and approach developed will benefit marker assisted selection programs by increasing the proportion of genic markers that could be linked to important horticultural traits.     

草莓抗炭疽病遗传图谱及其QTL初分析

为获得与草莓炭疽病密切相关的分子标记,需构建高密度与抗病相关的遗传连锁图,本研究以易感草莓炭疽病品种宝交早生（Hokowase）与高抗草莓炭疽病品种甜查理（SweetCharlie）杂交的210个F2代群体材料为作图群体,构建了包含34个AFLP标记和109个SSR标记的分子遗传图谱,并对抗草莓炭疽病相关因素进行了QTL分析。该图谱共包括7个连锁群和133个遗传标记,平均每个连锁群有19个遗传标记。遗传图谱总覆盖长度为451．8cM,标记间平均距离为3．4cM。经复合区间作图法分析得到3个与草莓炭疽病抗性相关基因座（QTL）,其中2个与Colletotrichumacutatum抗性相关,分布在LG3和LG5连锁群上,可解释表型变异的31．6％;1个与Colletotrichumgloeosporioides抗性相关,分布在LG6连锁群上,可解释表型变异的68．4％。     

Chloroplast SSR genetic diversity indicates a refuge for Corylus avellana in northern Portugal

The genus Corylus, a member of the birch family Betulaceae, includes several species that are widely distributed throughout temperate regions of the Northern Hemisphere. The development of microsatellites or simple sequence repeats (SSRs) for non-coding regions of the chloroplast genome and their higher sequence variation compared with coding regions has provided a higher resolution tool for the study of cultivars and closely related taxa. Chloroplast polymorphisms provide a marker system to evaluate the genetic structure of plant populations. This study investigated genetic diversity in three cultivars and 32 genotypes of Corylus avellana L. from Portugal: 13 wild genotypes and 19 Portuguese landraces. Four of ten cpSSR loci were polymorphic, with diversity indices ranging from 0.111 to 0.244. Eleven chlorotypes were detected, and their relationships were analyzed using a network model. Haplotype A was most frequent in landraces and cultivars. Four chlorotypes (H, I, J and L) were found only in wild hazelnuts. The diversity of chlorotypes in the wild hazels, and the limited number reported in cultivars, suggests that northern Portugal was a refuge for hazel during the last ice age.     

SSR Diversity of Vegetable Soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

Edamame [Glycine max (L.) Merr.] is a type of soybean selected for fresh or frozen vegetable use at an immature stage. Since edamame has a similar protein content, milder flavor, nuttier texture, and is easier to cook when compared to grain soybean, it is being promoted as a new vegetable for global consumption. Global production will require breeding programs for local adaptation; however, limited research has been published on genetic diversity of edamame varieties for the assessment of genetic resources. Simple sequence repeats (SSRs) were used to study the genetic diversity among 130 accessions, including edamame cultivars and landraces from Japan, China and the US, and also the new breeding lines in the US. Although it is assumed that elite edamame cultivars would have narrow genetic diversity, seventeen SSRs detected polymorphism to distinguish 99 of the 130 accessions. The cluster analysis generated nine clusters and 18 outliers. Genetic diversity within Japanese edamame was lower than that within Chinese vegetable soybean accessions (maodou), even though only 10 Chinese maodou were analyzed compared to 107 Japanese edamame. Cluster analysis revealed that the patterns of SSR diversity in edamame can generally distinguish maturity classes and testa color. We concluded that Japanese edamame have a narrow genetic base different from others and that SSRs can describe the patterns of genetic diversity among the elite vegetable soybean.     

Comparison of genetic variation among accessions of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> using AFLP and SSR markers

Genetic diversity of 54 accessions of Aegilops tauschii from five countries was assessed using sequence-tagged microsatellites (or simple sequence repeats, SSRs) and amplified fragment length polymorphisms (AFLPs). In the case of AFLP analysis, a total of 256 amplification products obtained, 234 of them were polymorphic across all the 54 accessions. A total of 224 fragments were obtained from the 24 SSR primers and 219 of fragments were polymorphic across all the genotypes screened. Based on both AFLP and SSR markers, the highest percentage of polymorphisms were obtained in Iranian and accessions of unknown origin. The highest polymorphic information content (PIC) value was observed for SSRs (0.82) while the highest marker index (MI) value was for AFLPs (8.5) reflecting the hyper-variability of the first and the distinctive nature of the second system. Principal co-ordinate analysis (PCO) revealed congruent patterns of genetic relationships for both data sets, but did not group accessions strictly according to their geographical origins. Poor correlation was found between AFLP and SSR marker loci. This low association may be due to low number of AFLP and SSR markers. These results show that molecular markers can help to organize the genetic variability and expose useful diversity for breeding purposes.     

Worldwide genotyping of castor bean germplasm (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.) using AFLPs and SSRs

Worldwide genetic diversity in 200 individuals comprising 41 castor bean accessions was assessed using amplified fragment polymorphisms (AFLPs) and simple sequence repeats (SSRs). We found that, despite surveying five continents and 35 countries, genetic diversity in castor bean germplasm is relatively low (overall H _e = 0.126 for AFLPs and 0.188 for SSRs) compared to estimates of genetic diversity in other plant species. Our data also show no geographic structuring of genotypes across continents or countries within continents. An assessment of the congruence between AFLP and SSRs indicates a low correlation (R ² = 0.19) between the two data sets, but each marker class nonetheless shows similar patterns of low-genetic diversity and a lack of geographic structure. Our data do suggest that SSRs yield a higher percentage of polymorphic loci, higher heterozyosity and a greater range of genetic distances, and are therefore more informative than are AFLPs on a locus-by-locus basis. Based on comparisons with numerous other plant species, we suggest that the lower genetic variation in this worldwide collection may be due to one or more factors including: sampling strategies that have not captured the full extent of genetic variation in the species; artifactual variation due to long-term germplasm storage and seed regeneration; or intense selection followed by domestic cultivation of a limited number of castor bean genotypes, which are widely propagated for their horticultural and agro-economic value.