银杏EST SSR引物开发

为了开发银杏SSR标记，从NCBI的dbEST数据库中共下载21 604条银杏EST序列，经过去冗余处理和SSR位点搜索，共得到2 122条EST SSR序列。比较发现，二核苷酸和三核苷酸重复基元所占比例较高。利用TRA1.5软件共成功设计1 926对引物，随机选取100对引物进行合成和筛选，共选出22对具有多态性扩增条带的引物。22对引物在50个银杏品种上共扩增出78个条带，其中多态性条带34条。     

Genome-wide development of interspecific microsatellite markers for Saccharum officinarum and Saccharum spontaneum

Sugarcane has a large, complex, polyploid genome that has hindered the progress of genomic research and molecular marker-assisted selection. The user-friendly SSR markers have attracted considerable attention owing to their ideal genetic attributes. However, these markers were not characterized and developed at the genome-wide scale due to the previously lacking high-quality chromosome-level assembled sugarcane genomes. In this present study, 744 305and 361 638 candidate SSRs were identified fro...     

五叶草莓与凤梨草莓种间杂交F_1代的形态学及SSR标记鉴定

运用传统杂交方法,以五叶草莓为母本、凤梨草莓品种‘达赛莱克特’为父本进行远缘杂交,获得了11个F1代株系,利用形态学性状与分子标记相结合进行种间杂种鉴定。对父母本、F1代主要的植物学特征、生物学特性的调查分析结果表明,F1代在叶、花、果实等方面的很多性状不同于父母本,其中大部分介于双亲之间;植株长势明显强于父母本,具有明显的杂种优势。采用草莓属46对SSR引物进行分子鉴定,利用3对引物鉴定出种间杂种。形态学与SSR标记综合鉴定结果表明,全部F1代株系均为五叶草莓与凤梨草莓的种间杂种。     

Genetic Diversity Analysis of Faba Bean （Vicia faba L.） Based on EST-SSR Markers

Faba bean (Viciafaba L.), one of the most important legumes in the world, evolved different types of cultivars due to its partial cross-pollination. The development of simple sequence repeat (SSR) markers from expressed sequence tags (EST) provided a useful tool for investigation of its genetic diversity. The purpose of the present study was to investigate the genetic diversity of faba bean from China and Europe using EST-SSR markers. 5 031 faba bean ESTs from the NCBI database were downloaded and assembled into 1148 unigenes. A total of 107 microsatellites in 96 unigenes were identified, indicating that merely 8.36% of sequences contained SSRs. The most abundant SSR within faba bean was tri-nucleotide repeat motif, and among all the tri-nucleotide repeats, the motif AAG/CTT was the most abundant type. Based on these results, 11 EST-SSR markers were used to assess the genetic diversity of 29 faba bean cultivars from China and Europe with two to three alleles per locus. The polymorphism information content value ranged from 0.0644 to 0.4278 with an average of 0.2919. Principal coordinate analysis (PCA) and phylogenetic clustering based on these 11 EST-SSR markers distinguished these cultivars into different groups. The results indicated that faba bean in China had a narrow genetic basis, and the additional sources of genetic cultivars/accessions should be introduced to enhance the genetic variability. The results of this study proved that the EST-SSR marker is very effective in evaluation of faba bean germplasm.     

轮枝镰孢SSR标记开发及在玉米分离群体遗传多样性分析中的应用

【目的】开发轮枝镰孢（Fusarium verticillioides）的SSR标记,为轮枝镰孢遗传多样性研究提供技术支持。【方法】利用Fastpcr在轮枝镰孢基因组中查找符合条件的SSR位点,采用Primer5.0软件设计引物,利用NTSYS及Popgene分析来自玉米的轮枝镰孢单孢分离物群体的PCR扩增结果。【结果】共设计有效扩增SSR引物158对,经筛选,109对（69.0%）可扩增出2条及以上的条带,其中55对引物（34.8%）多态性较好,可扩增出3条及以上的条带。从11条已组装的轮枝镰孢染色体上各选出2对多态性引物,计22对引物,对66株轮枝镰孢玉米分离物进行扩增,共获得125个等位变异,变异范围为2-11个,平均为5.68个。轮枝镰孢玉米分离物之间Nei’s基因多样性指数为0.1139-0.8687,平均为0.6199,表现出较高的遗传多样性。【结论】基于轮枝镰孢基因组序列开发的SSR标记具有很好的多态性,可用于轮枝镰孢的遗传多样性分析。用22对SSR引物扩增,以遗传相似系数0.3进行划分,可将66株轮枝镰孢玉米分离物分为3群;中国轮枝镰孢玉米分离物的遗传多样性与地理分布无相关性。     

辣椒疫霉EST-SSRs标记的发掘

通过对GenBank上发布的55 848条辣椒疫霉EST的鉴定,在328条EST中发现331个SSR.在筛选的EST序列中SSR平均密度为每23.7 kb含有1个SSR.在鉴定的SSR中,三核苷酸重复基元的SSR类型最多,占鉴定总数的59.5%;其次为二核苷酸乖复基元的SSR类型,为39.3%;四核苷酸重复基元的SSR类型最少,为1.2%.设计40对SSR引物对5个辣椒疫霉菌株基因组DNA进行PCR扩增,有27对引物扩增出SSR特征条带,其中有18对引物在5个辣椒疫霉菌株间扩增出多态性条带25条.基于SSR标记进行聚类分析,可将5个检测大豆疫霉菌菌株划分为3类.该研究是辣椒疫霉的SSR标记开发的首次报道,为辣椒疫霉的遗传变异和分子作图等分析提供了一种有效的分子标记系统.     

柑橘栽培品种（系）DNA指纹图谱库的构建

 【目的】构建柑橘栽培品种（系）的DNA指纹图谱数据库,为建立柑橘种苗纯度及真实性鉴定技术体系和技术规范奠定基础。【方法】利用SSR标记和ISSR标记对102个柑橘栽培品种（系）进行DNA指纹分析,筛选适合的特征引物,构建柑橘栽培品种（系）的指纹图谱数据库。【结果】从200对SSR引物中筛选到重复性好、多态性丰富的12对引物作为柑橘品种（系）鉴定的特征引物,12对SSR特征引物组合可鉴别42个品种（系）;在此基础上,对未出现SSR特征指纹的60个品种（系）进行ISSR指纹分析,从40个ISSR引物中筛选到2个可用于品种鉴定的特征引物,结合SSR标记,可快速、准确地鉴别70个柑橘的品种（系）。并利用这12对SSR特征引物和2个ISSR特征引物构建了70个柑橘栽培品种（系）的DNA特征指纹图谱数据库。【结论】SSR和ISSR标记适于构建柑橘栽培品种DNA指纹图谱库;指纹图谱库的建立为柑橘种苗纯度及真实性鉴定奠定了基础。     

Molecular Identification and Cultivar Fingerprints of Prunus persica (L.) Batsch Germplasms

[Objective] The aim was to study the molecular identification and cultivar fingerprints of Prunus persica (L.) Batsch germplasms.[Method] Sixty peach genotypes,representing China common local cultivars and European samples were screened by microsatellites (simple sequence repeats,SSRs) and Inter-Simple Sequence Repeat (ISSR) markers.[Result] 26 reproducible bands were amplified by Nine SSR primers,and 24 of which were polymorphic; 236 bands were amplified by 30 ISSR primers,and 113 of which were polymorphic.31 genotypes were discriminated with 1-3 distinct polymorphic bands generated from the primers ISSR and SSR.Seven cultivar-specific ISSR fragments and two SSR unique alleles obtained from this study were available to be converted into Sequence Characterized Amplified Region (SCAR) markers.The genetic similarity coefficient (GS) estimated from these molecular data averaged were 0.939 (ranged from 0.856 to 0.983) for ISSR and 0.646 (ranged from 0.240 to 1.000) for SSR,respectively.The combined grouping association indicated that most local Chinese peach cultivars and exotic accessions were clustered together.This could be related to the mode of introduction and maintenance of the peach cultivars involving limited foundation germplasm,exchange of cultivars between plantations,and periodic development of new recombinant cultivars following sexual reproduction.[Conclusion] The results obtained in this work would help to improve the conservation,molecular identification and management of peach germplasm in breeding.     

基于RNA-seq数据大规模挖掘蜜蜂球囊菌的SSR分子标记

基于已获得的球囊菌转录组数据预测微卫星标记(SSR),并进行SSR位点的信息分析和SSR引物的挖掘.利用软件MISA对球囊菌转录组中42610条unigenes进行搜索,共预测出7968个SSRs,分布于5233条unigenes中,其中最主要的重复类型为三核苷酸重复(53.15%),其次为二核苷酸重复(32.32%)和四核苷酸重复(8.46%).二核苷酸重复中的基序主要是AG/CT(占总量的15.8%).针对所有的SSR位点,利用Primer Premier 5软件设计出6956对SSR特异性引物,随机选取20对引物对两个不同来源的球囊菌样品进行SSR位点扩增,共有6对引物成功扩增出符合预期的目的片段.研究结果表明,利用球囊菌转录组数据开发SSR引物是可行的.     

荔枝EST资源的SSR信息分析及EST-SSR标记开发

 【目的】明确荔枝EST序列中SSR的总体特点,开发荔枝EST-SSR引物,为利用EST-SSR分子标记进行荔枝种质资源遗传多样性、连锁图谱构建及亲缘关系研究奠定基础。【方法】应用SSRIT软件,按照设定标准从自行构建的一个荔枝果皮发育关键期的cDNA文库中的1 331条Unigene（惟一序列）中搜索SSR位点。利用软件Primer primer5.0设计EST-SSR引物。选用16份表型差异较大的荔枝种质资源检测引物的有效性及多态性,利用聚丙烯酰胺凝胶(PAGE)进行片段分离。【结果】荔枝的1 331条EST序列中共搜索出220个SSR位点,分布于189条EST中,出现频率是16.53%。这些EST-SSR的平均长度为18.43 bp,平均分布频率1/4.42 kb。在1—6 bp的重复基元中,二核苷酸和三核苷酸重复类型占主导地位,共占总SSR的69.09%,二者出现的频率分别为37.27%和31.82%。共观察到72种重复基元,出现频率最高的是GA/TC（16.82%）;其次是AG/CT、A/T、AT/TA及GAA/TTC,频率分别为14.55%、11.82%、5.00%和3.64%。不同核苷酸数目的重复基元的重复次数差异很大。设计、合成150对EST-SSR引物,122对（81.33%）引物在荔枝中获得有效扩增,100对引物具有多态性。【结论】荔枝EST资源中含有高频率的SSR位点,且EST-SSR 标记开发效率较高。本研究为利用EST-SSR标记开展荔枝遗传研究提供了100 对EST-SSR引物,并为进一步开发荔枝EST-SSR标记提供了含SSR位点的候选序列。     

我国75份小麦品种SNP和SSR指纹图谱构建与比较分析

联合利用Illumina 90K SNP芯片和毛细管高通量SSR检测技术,构建75份育成品种384个SNP和42个SSR位点的指纹图谱,比较两种标记在遗传多样性、遗传相似系数和鉴别能力等方面的特征。结果显示,SNP标记揭示的遗传多样性指数明显低于SSR标记,但能较好地反映品种间的遗传多样性;SNP标记揭示的遗传相似系数明显高于SSR标记,但两者呈极显著线性相关;384个SNP位点鉴定近等基因系的能力低于42个SSR位点,但去除近等基因系后,仅需8个SNP或4个SSR位点组合即可区分剩余的74份品种,表明最优位点组合具有较高的鉴定效率,在品种鉴定时可先采用少量标记进行初鉴,对于极近似品种可加大标记密度。首次结合SSR和SNP标记构建指纹图谱,证实了两者之间的一致性,提出了分子身份鉴定技术标记数量的选择思路,为小麦品种DNA身份鉴定技术标准制定提供了重要的参考依据。     

Development and Characterization of Microsatellite Markers in Brassica rapa ssp. chinensis and Transferability Among Related Species

Simple sequence repeat （SSR） or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an intersimple sequence repeat （ISSR）-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage （Brassica rapa）. A total of 190 SSRs were obtained. Among these, AG or CT （54.7%） was the most frequent repeat, followed by AC or GT （31.6%） of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content （PIC） value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.     

苹果部分种质资源分子身份证的构建

【目的】以国家果树种质兴城梨、苹果圃保存的131份苹果地方品种、育成品种及其野生近缘种为试材,利用TP-M13-SSR标记构建苹果种质分子身份证。【方法】基于TP-M13-SSR指纹图谱,筛选可以将苹果种质区分的引物组合,并对其等位基因进行编码建立种质分子身份证。【结果】（1）从131份材料中随机选取两份材料,对第一次PCR条件进行优化和引物筛选,从32对合成引物中筛选出16对稳定性高和重复性好的TP-M13-SSR引物用于131份苹果属植物指纹图谱构建。（2）16对SSR引物在供试种质间共检测出等位基因326个,每对引物平均检测到等位基因数为20.3个。CH05d04对种质扩增的等位基因数最多为49个,位点期望杂合度最高为0.878;其次是CH01f07a为48个。利用PopGen32软件计算引物的多态性信息含量,16对引物的平均多态性信息含量为0.7558。16对SSR引物可区分供试苹果种质资源数量从11份到71份不等,平均每对SSR引物可区分49份苹果种质,区分率为8.09%-52.21%。其中对苹果种质区分率最高的是CH01f07a,最低的为BGT23b。（3）根据引物扩增的多态性信息含量和对苹果种质的区分率,将两者均较高的引物CH05d04、CH01f07a、CH03d07、CH04e03、CH04h02和CH04g07两两组合,CH04h02和CH01f07a引物组合分辨率最高,可以区分120份苹果种质。继续增加组合中引物数量,在增加到3对引物时,即可将全部苹果种质区分开来。（4）把可以将全部供试苹果种质资源材料全部区分的3对核心引物CH04h02、CH05d04和CH01f07a获得等位基因按照从大到小的顺序排列,并用阿拉伯数字从01开始赋值;将每份材料在3个位点获得的等位基因按照赋值数字编码获得每份供试材料独有的字符串,利用条码技术将每对引物的分子身份证转化成可被机器快速扫描的条码分子身份证。【结论】依据引物扩增的多态性信息含量和对苹果种质的区分率,筛选核心引物组合,区分全部供试苹果地方品种、育成品种及其野生近缘种质资源,并基于指纹图谱构建其可被机器快速识别的分子身份证,使每份种质具有可辨的分子身份证,达到利用最少、最特异引物区分最多苹果种质的目的。     

基于荧光检测技术的小麦品种SSR鉴定体系的建立

【目的】建立基于SSR荧光标记的小麦品种DNA指纹鉴定体系,为中国小麦育成品种鉴定提供高通量技术手段。【方法】收集已定位到小麦21条染色体上的SSR标记引物,通过PCR扩增和变性聚丙烯酰胺凝胶电泳检测技术,筛选在中国小麦育成品种中多态性高的标记,对筛选出的引物5′末端利用6-FAM、HEX、ROX和TAMRA 4种荧光染料之一进行标记,利用DNA分析仪对扩增产物的峰型进行评价并检测不同等位变异的扩增片段大小,选择峰型简单易读、多态性高、较均匀分布到21条染色体上的标记,确定不同位点等位变异的大小及相应的参照品种,建立基于荧光SSR标记的高通量小麦品种鉴定体系。【结果】利用2 438对SSR标记对8份植物学性状差异大的小麦育成品种进行初步筛选,共筛选出260对多态性较高、扩增稳定的SSR引物。利用上述260对引物对48份小麦品种继续进行复筛,选出130对扩增稳定、多态性高、带型好的SSR引物。对这些引物的正向5′末端分别标记6-FAM、HEX、ROX和TAMRA荧光后,利用DNA分析仪进行检测评价,最终选择了42个PCR扩增稳定、峰图简单、多态性高、连锁群分布均匀的SSR荧光标记。根据DNA分析仪检测到的每个标记的不同等位变异大小,为相应位点的不同等位变异进行了命名,并为每个等位变异选取了相应参照品种。根据每个引物标记的荧光和扩增的片段大小范围对42对引物进行合理搭配,在同一毛细管内对多个荧光标记进行检测,提高了DNA指纹数据采集效率,降低了检测成本。利用该体系对1 625份小麦育成品种进行DNA指纹数据采集,在42个位点中共检测到434个等位变异,每个位点的等位变异个数在3-23,平均10.3个;每个位点的PIC值范围为0.240-0.829,平均0.610。利用1 625份小麦育成品种DNA指纹数据,构建了中国小麦育成品种的DNA指纹数据库。【结论】筛选出42对SSR标记引物,建立了基于荧光SSR标记的小麦品种鉴定体系,可用于高通量小麦品种DNA指纹鉴定。     

基于SSR荧光标记的MCID快速鉴定13个湖北茶树品种

[目的]通过人工绘制品种鉴别图(Manual cultivar identification diagram,MCID)快速区分鉴别湖北茶树品种,为湖北茶树种质资源评价、品种保护及苗木纯度早期鉴定提供技术支持.[方法]以湖北省13个优良茶树品种为材料,利用30对均匀分布于遗传连锁图谱上的SSR荧光引物进行PCR扩增,并用荧光标记毛细管电泳检测扩增产物以筛选出核心引物,利用MCID法构建CID图谱.[结果]30对SSR引物共扩增出145个等位基因,各引物等位基因数为3~9个,平均每对引物为4.83个;共检测到194个基因型,各引物检测出的基因型为3~12个,平均每对引物6.47个;多态性信息量(PIC)为0.29~0.85,平均为0.58. 13个茶树品种的Nei's遗传距离为0.19~0.44.基于Nei's遗传距离,采用Neighbor-Joining(NJ)法可将13个品种分为三大类(Nei's遗传距离为0.16),结果表明地理来源相同的品种可能因为具有相似的遗传背景而聚在一起,也可能因为亲本来源不同而存在明显的遗传差异.利用筛选出的3对核心引物(TM547、TM552和TM107)可快速鉴别所有参试品种,通过MCID法构建的CID图谱可直观看出各参试茶树品种鉴定所需要的引物及其对应的基因型.[结论]基于SSR荧光标记的茶树品种MCID鉴定方法具有快速、准确、高效的特点,可用于湖北茶树良种知识产权保护、苗期鉴定及品种区分.     

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var. capitata L.)

The dominant genic male sterility (DGMS) gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage (Brassica oleracea var. capitata L.), has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants (CDMs399-3/CDMs399-3) is a laborious and time-consuming process. For marker-assisted selection (MAS) of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats (EST-SSR) and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis (BSA). By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygous male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.     

利用ISSR和SSR标记分析芒荻类植物资源遗传多样性

利用ISSR和荧光SSR技术对22种芒荻类植物的遗传多样性进行分析,筛选出具有多态性的ISSR和SSR引物各12对,其中ISSR引物共扩增出216个多态性位点,多态性比率为96.86%,Neis指数为0.301 8,平均Shannon指数为0.462 8;SSR引物共扩增出111个多态性位点,多态性比率为67.68%,Neis指数为0.111 5,平均Shannon指数为0.193 3。UPGMA聚类分析结果表明,22份芒荻类植物资源中除五节芒外,相同地域内的芒、荻和南荻在遗传学上难以区分;芒荻类植物资源的遗传分化与其种源的地域分布有一定相关性。Mantel检验显示,2种分子标记在22份芒荻类植物资源间相关不显著(r=0.374,P0.05)。     

SSR fingerprinting of 203 sweetpotato (Ipomoea batatas (L.) Lam.) varieties

Simple sequence repeat (SSR) markers have been shown to be a powerful tool for varieties identification in plants. However, SSR fingerprinting of sweetpotato varieties has been a little reported. In this study, a total of 1294 SSR primer pairs, including 1215 genomic-SSR and 79 expressed sequence tag (EST)-SSR primer pairs, were screened with sweetpotato varieties Zhengshu 20 and Luoxushu 8 and their 2 F₁ individuals randomly sampled, and 273 and 38 of them generated polymorphic bands, respectively. Four genomic-SSR and 3 EST-SSR primer pairs, which showed good polymorphism, were selected to amplify 203 sweetpotato varieties and gave a total of 172 bands, 85 (49.42%) of which were polymorphic. All of the 203 sweetpotato varieties showed unique fingerprint patterns, indicating the utility of SSR markers in variety identification of this crop. Polymorphism information content (PIC) ranged from 0.5824 to 0.9322 with an average of 0.8176. SSR-based genetic distances varied from 0.0118 to 0.6353 with an average of 0.3100 among these varieties. Thus, these sweetpotato varieties exhibited high levels of genetic similarity and had distinct fingerprint profiles. The SSR fingerprints of the 203 sweetpotato varieties have been successfully constructed. The highly polymorphic SSR primer pairs developed in this study have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction in sweetpotato and other plants.     

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.     

杨树品种的SSR分析及鉴定

利用SSR-PCR方法对杨树(Populus)的10个品种进行了基因组多态性分析,选用10对引物扩增出122个DNA片段,其中114个片段呈现多态性,占总扩增片段的93.44%。平均每对引物得到12.2个位点。依据扩增结果进行遗传距离分析,构建了分子树状图。研究结果表明:SSR分析中产生了一些品种特有的指纹图谱。利用DNA扩增结果进行聚类分析,把供试杨树的10个品种分为3类,并对基本品种及种下品种群的遗传关系进行了探讨。