Development of SSR markers from ESTs of gramineous species and their chromosome location on wheat

A total of 407,663 expressed sequence tags （ESTs） of wheat, barley, maize, rice, and sorghum, obtained from GenBank/dbEST, were used to search for simple sequence repeats （SSRs）. A total of 10,253 EST-SSRs, which accounted for 2.52% of all the ESTs, were identified. Using Primer Premier 5.0, 1367 EST-SSR primer pairs were designed, of which 715 with high quality were synthesized. The 715 primer pairs were tested on wheat, rice, maize, cotton, and soybean under the same PCR conditions, and the effective primer pairs in the five crops were 500 （69.93%）, 383 （53.57%）, 452 （63.22%）, 357 （49.93%）, and 388 （56.27%）, respectively. This indicated a high transferability of EST-SSR markers between far-ranging species. In addition, 139 EST-SSR primer pairs with 240 loci were localized on all the 21 wheat chromosomes by using Chinese Spring nulli-tetrasomic lines of wheat.     

杉木EST-SSR与基因组SSR引物开发

利用已经公布的杉木444条EST序列和未公布的杉木基因组文库中1 142条基因序列,进行引物开发效率的比较。去冗余后,利用MISA 搜索SSR 位点,分别得到109个和39个含有SSR的 位点。杉木EST序列中SSR分布密度为964.58个/Mbp,基因组中平均每Mbp出现1 037.24个SSR。在两个独立来源的数据库序列中,六核苷酸重复均为最多的重复类型,且AT-rich的重复类型占较大比例。AGC/CTG是杉木EST序列和基因组库中最多的三碱基重复,通过Primer 3.0分别设计出SSR引物95对和37对。为考察设计引物在杉木不同种源(群体)中的有效性,取12个种源(个体)的优良个体, 利用随机抽取的10个EST-SSR和8个gSSR(基因组SSR)进行引物筛选,结果表明:EST-SSR和gSSR各有4对引物在12个种源(个体)中表现出明显的多态性,多态率分别为40%和50%。8对多态性的SSR引物共扩增出 25 个多态性等位位点,平均每个引物产生 3.125 个多态性等位位点,平均有效的等位位点为2.399 5,PIC平均值为0.519 1; Hot平均为0.307 4。其中gSSR标记在检测群体间存在较大的分化,4个gSSR比4个EST-SSR扩增出更多的等位位点数、平均等位位点数,以及更大的PIC值。     

Mining,characterization, and exploitation of EST-derived microsatellites in <Emphasis Type="Italic">Gossypium barbadense</Emphasis>

Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of ex-pressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were de-termined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. By mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barba-dense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Further-more, trinucleotide was found to be the most abundant repeat type among 2–6-nucleotide repeat types. It accounted for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG)n accounted for the highest proportion. EST-SSR primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G. hirsutum ‘TM-1’ and G. barbadense ‘Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.     

Analysis of simple sequence repeats markers derived from Phytophthora sojae expressed sequence tags

Five thousand and eight hundred publicly available expressed sequence tags (ESTs) of Phytophthora sojae were electronically searched and 415 simple sequence repeats (SSRs) were identified in 369 ESTs. The average density of SSRs was one SSR per 8.9 kb of EST sequence screened. The most frequent repeats were trinucleotide repeats (50.1%) and the least frequent were tetranucleotide repeats (8.2%). Forty primer pairs were designed and tested on 5 strains of P. sojae. Thirty-three primer pairs had successful PCR amplifications. Of the 33 functional primer pairs, 28 primer pairs produced characteristic SSR bands of the expected size, and 15 primer pairs (45.5%) detected polymorphism among 5 tested strains of P. sojae. Based on the polymorphisms detected with 20 EST-SSR markers, the 5 tested strains of P. sojae were clustered into 3 groups. In this study, the SSR markers of P. sojae were developed for the first time. These markers could be useful for identification, genetic variation study, and molecular mapping of P. sojae and its relative species.     

湿地松转录组SSR分析及EST-SSR标记开发

【目的】湿地松是南方地区优先推广的优质产脂树种。但湿地松分子遗传基础薄弱,基因组序列信息匮乏,影响了湿地松基因组学的深入研究。目前,湿地松分子研究所用的SSR标记主要来自其他近缘种或利用公共数据库中有限的基因序列资源开发的SSR标记,其多态性和通用性较差。为解决这一问题, 笔者根据湿地松转录组测序数据开发EST-SSR位点,并揭示其在转录组序列中的分布类型及特征,为湿地松分子标记辅助育种奠定基础。【方法】利用MISA软件对转录组序列进行SSRs查找和分布特征分析。查找标准参数设置为: 单核苷酸重复>10次,二核苷酸重复>6次,三、四、五、六核苷酸重复>5次。根据SSR位点两端的保守区域,利用Primer3.0设计并随机挑选120对SSR引物,通过琼脂糖电泳和毛细管电泳对来自美国和吉安的113份家系个体进行遗传多样性分析,确定引物多态性。【结果】79 574条unigenes序列中搜索到3 818个SSR位点,出现频率为4.80%,平均18.27 kb出现1个SSR位点,3 373个unigenes含有SSR位点,SSR发生频率(含SSR位点的序列数/搜索序列总数)为4.24%,其中2 980条序列含1个SSR位点,含1个以上SSR位点的序列有393条。在检测到的3 818个SSR标记中,单核苷酸分布最多,其次是二核苷酸和三核苷酸,SSR数量分别占总数的63.54%、19.15%和16.27%,而四、五、六核苷酸重复类型所占比例较小,分别为0.52%、0.13%和0.31%。SSR重复单元的重复次数分布在5~22次之间,除单核苷酸重复外的1 391个SSR中,重复5次的数量最多,为498个(35.80%);重复6次和7次的次之,分别为417个(29.98%)和198个(14.23%);重复10次以上的仅有38个(2.73%)。在检测到的731个二核苷酸重复SSR中,最常出现的重复单元为AT/AT,数量为491个(12.86%),AG/CT和AC/GT两种类型的重复单元出现的次数次之,分别为156(4.09%)和81个(2.12%)。在检测到的621个三核苷酸重复中,AAT/ATT是出现频率最高的单元,共139个(3.64%),其次是AAG/CTT,共122个(3.20%)。3 818个SSR中有24.59%的位置未知,其余的SSR则分布在非编码区域(untranslated region,UTR)或者编码区(coding sequence,CDS)上,分布数量表现为3'UTR>5'UTR>CDS。参试的120对SSR引物,有92对扩增成功(76.78%),其中24对呈现多态(20%)。24对引物(13个二核苷酸重复、7个三核苷酸重复和4个四核苷酸重复)共检测出81个等位基因,每个位点的等位基因数为2~9,平均为3.38个。多态性信息含量(polymorphism information content,PIC)为0.103~0.726,平均为0.349。【结论】通过对湿地松转录组数据的挖掘,共获得 3 818个SSR位点,主要重复单元为AT/AT和AAT/ATT,可扩增出多态性位点的引物重复单元以二、三核苷酸重复为主。基于湿地松转录组序列的SSR标记开发是可行的。     

Characterization, development and exploitation of EST-derived microsatellites in Gossypium raimondii Ulbrich

COTTON IS AN IMPORTANT GLOBAL CASH CROP. IN THE RECENTYEARS, MOLECULAR MARKER TECHNOLOGY HAS BEEN WIDELY APPLIED TO SUCH STUDIES ON COTTON AS GENETIC MAP- PING[1―4], VARIETY PURITY DETECTION[5], GENETIC DIVERSITY ANALYSIS[6], MOLECULAR MARKER-ASSISTED BR…     

33个杨柳品种指纹图谱构建

【目的】构建杨树与柳树优良品种的指纹图谱,对不同品种进行准确鉴定。【方法】利用柳树EST-SSR标记对33个杨树与柳树优良品种进行通用性检测及基因分型,通过核心引物间的组合构建品种指纹图谱。同时采用非加权组平均法进行聚类,分析各品种间亲缘关系。【结果】12个EST-SSR标记共扩增出97条等位片段,每个位点等位基因数5~13个不等,平均为8.1个。12个位点的多态信息含量(PIC)变化范围为0.637 2~0.834 8,平均为0.781 7。优选的3对核心引物中,SALeSSR0340与SALeSSR0346组合可完全区分12个杨树品种,而SALeSSR0259、SALeSSR0340与SALeSSR0346的组合可完全区分所有的33个品种。聚类分析显示33个品种间的遗传相似系数为0.68~0.96,并分成杨属与柳属两大类,与传统的杨柳科系统分类学一致。各品种间亲缘关系聚类结果与遗传背景相吻合。【结论】对33个杨柳树优良品种进行指纹图谱构建与亲缘关系分析,表明EST-SSR标记可有效反映品种间的差异,建立的基因分型体系准确、高效,可为杨树与柳树品种鉴定、保护与推广工作提供科学依据。     

春小麦重组自交系及多种SSR标记构建遗传图谱

 以春小麦重组自交系(RIL)宁春4 号×宁春27 号为作图群体,利用SSR 标记构建小麦遗传连锁图谱。结果表明,用1001对SSR 引物选出亲本间表现多态性的引物307 对,多态性频率为30.7%。利用307 对多态性引物对RIL 群体进行分析,共检测到266 个多态性标记位点。通过χ²检测(P<0.05),有147 个SSR 标记表现为偏分离,偏分离率为55.3%,129 个偏向母本宁春4号,其偏分离位点主要分布在B 和D 基因组上。用Mapmaker 3.0 和Mapdraw 2.1 软件将266 个SSR 位点绘制在小麦遗传连锁图上,该图谱覆盖小麦基因组全长2187.79 cM,标记间的平均遗传距离为8.22 cM。     

Screening and Insert Size Analysis of Candidate C-Genome BAC Clones from Brassica napus

Thirty-two C-genome specific candidate bacterial artificial chromosome (BAC) clones were successfully screened from the BAC library by four-dimensional PCR method with the primer pairs of 75 simple sequence repeat (SSR) markers located in the nine C-genome linkage groups of Brassica napus. The screened 32 BAC clones have an average insert size of 114.2 kb with a range of 30-190 kb. They are the first set of C-genome BAC clones screened from B. napus genomic BAC library. The average insert size of this set of BAC clones presented that the constructed BAC library had a high quality. This set of BAC clones can be used as markers to identify individual chromosomes of B. napus C-genome.     

Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

基于EST-SSR标记鉴定猴耳环自由授粉的全同胞子代

【目的】猴耳环是重要的药用树种,具有较好的工业应用价值。利用EST-SSR标记对猴耳环自然群体1株母树的自由授粉子代进行父本鉴定,从而确定全同胞子代,为基于全同胞家系的后续研究提供材料基础。【方法】以自然群体中母树ELS31自由授粉产生的1 489株子代及该群体内挂果的38株候选父本为材料,利用15个EST-SSR标记检测子代多样性和标记多态性,基于最大似然法鉴定各子代的父本,母本父本均相同的子代即构成全同胞家系,并估算群体内花粉传播距离,检验各父本对应的全同胞家系的多样性,通过卡方检验判断标记是否合乎预期的孟德尔分离比。【结果】15个EST-SSR标记的引物(对)共扩增出89个等位片段。子代群体期望杂合度(H_e)为0.525,表明群体多样性为中等水平;基于自然群体18株无亲缘关系的单株估算的标记平均多态性信息量(PIC)为0.736,表明标记多态性高。在1 489株自由授粉的子代中,确定了857株子代(57.6%)的34株父本。子代数最多的10个父本产生的子代数为26~184株,其他24个父本仅共产生子代139株。未发现自交子代,表明猴耳环是异交物种,自交的可能性极低。对子代数量20株以上的10个父本对应的全同胞家系观测杂合度(H_o)为0.502~0.693,H_e为0.417~0.544,各家系均是H_o大于H_e,表明存在一定程度的杂合子过剩。花粉传播的范围为10.0~559.1 m,平均119.2 m,但主要传播距离在150 m以内。716株子代(83.5%)的父本(10株)与母树距离在150 m以内。距离最远的父本ELS01 (559.1 m)和ELS02 (552.2 m)分别仅产生了9和12株子代。15个标记在子代20株以上的部分或全部全同胞家系中均有不同程度的偏分离,平均每家系的偏分离标记数为8.6个;偏分离最严重的是标记ARCeSSR141,在父本ELS30的全同胞家系中卡方(χ²)值为164.55。【结论】基于15个EST-SSR标记鉴定了猴耳环自然群体1株母本的1 489株自由授粉子代的父本,获得了子代20株以上的10个父本的全同胞家系。这为后续遗传测定、遗传图谱构建和数量性状位点定位等研究提供了材料基础。     

Screening microsatellite markers from EST sequences of bay scallop Argopecten irradians

Seventy-five simple sequence repeats （SSRs） were identified by the bioinformatic analysis from 5008 expressed sequence tags （ESTs） of Argopecten irradians. Among the SSRs, the number of repeat nucleotide varied from 2 to 6. Dinucleotide and trinueleotide repeat motifs were dominant in EST-SSRs of bay scallop, with a proportion of 80% over the total screened SSRs. Twenty-nine pairs of primer were designed based on the flank sequences of the selected ESTs using the software of Primet 5, and verified under the given PCR reaction condition. Eighteen of the 29 primer pairs resulted in the expected products, while the remaining either failed to produce any fragments or yielded products over expected size. Thirteen of the 18 SSRs, accounting for 72%, were detected to show polymorphism in the examined scallop samples. A preliminary test in this study indicated that the majority of the identified SSRs were informative in the cultured bay scallops, making them suitable for the population and other genetic analysis. EST-SSR markers have more advantages than the traditional genomic-derived SSRs and there is a wide range of application in comparative mapping, functional gene cloning and marker assisted selection. This research provides a reference to the identification of EST-SSRs with relative bioinformatic analysis from aquaculture species, as well as to those with a large number of ESTs.     

Construction of a genetic linkage map for cotton based on SRAP

DNA markers have been widely used in construction of molecular genetic linkage maps in plants. The first genetic linkage map of cotton was constructed by Reinish in 1994 using RFLP (restriction fragment length polymorphism)[1], which included 705 polymorphic loci on 41 linkage groups with a total length of 4675 cM. Afterwards, several genetic linkage maps were constructed[2—7], but no map is comparable to this one in marker density. A high-density genetic linkage map could be applied effec…     

棉花种质资源多样性的ISSR聚类分析及主成分分析

利用ISSR分子标记对48份棉花种质资源的亲缘关系及遗传多样性进行分析,结果显示,筛选出的10个ISSR引物扩增出的112条带中,多态性条带占101条,为总条带的90.18%.每个引物均可扩增4～21个多态性位点,平均10.10个,引物平均多样性信息指数(PIC)和多态性谱带百分率(PPB)分别为0.76和87.88%.利用NT-SYSpc 2.1统计分析软件中的UPMGA法对48份棉花种质资源构建亲缘关系树状图,在遗传相似性系数(GS)为0.66水平上可将所有参试材料聚成两大类,且与主成分分析(PCA)结果基本吻合.ISSR标记揭示出这48份棉花种质资源遗传多样性较小,遗传基础比较狭窄.     

Development of simple sequence repeats (SSR) markers of ramie and comparison of SSR and inter-SSR marker systems

Ramie (Boehmeria nivea L. ) is an important bast fiber crop. To study genetic background of this species, we isolated and characterized microsatellite markers of ramie. A genomic library containing inserts of rapid amplification of polymorphic DNA (RAPD)fragments was constructed, and screened by PCR amplification using anchored simple sequence repeats as primers. A total of 26 clones were identified as positives, and 13 microsatellite loci were found after sequencing. The polymorphism of these 13 microsatellite loci was examined and the utility of simple sequence repeats (SSR) and inter-SSR (ISSR) marker systems for genetic characterization compared using 19 selected ramie cultivars. Both approaches successfully discriminated the 19 cultivars which differed in the amount of polymorphism detected. The level of polymorphism detected by SSR was 95.0 %, higher than that by ISSR (72.3 % ), but the average polymorphism information content (PIC) of ISSR (0. 651) was higher than that of SSR (0. 441). The higher PIC value of ISSR suggests that ISSR is more efficient for fingerprinting ramie cultivars than SSR markers. However, because the SSR loci are codominant, they are more suitable for determining the homozygosity levels of ramie, constructing linkage map, quantitative trait loci study of complex traits and marker-as-sisted selection.     

Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase

Telomeres define the ends of chromosomes; they consist of short tandemly repeated DNA sequences loosely conserved in eukaryotes (G1-8(T/A)1-4). Telomerase is a ribonucleoprotein which, in vitro, recognizes a single-stranded G-rich telomere primer and adds multiple telomeric repeats to its 3' end by using a template in the RNA moiety. In conjunction with other components, telomerase may balance the loss of telomeric repeats due to DNA replication. Another role of telomerase may be the de novo formation of telomeres. In eukaryotes like Tetrahymena, this process is an integral part of the formation of macronuclear chromosomes. In other eukaryotes this process stabilizes broken chromosomes. A case of human alpha-thalassaemia is caused by a truncation of chromosome 16 that has been healed by the addition of telomeric repeats (TTAGGG)n. Using an in vitro assay, I show here that human telomerase correctly recognizes the chromosome 16 breakpoint sequence and adds (TTAGGG)n repeats. The DNA sequence requirements are minimal and seem to define two modes of DNA recognition by telomerase.     

松属GAs相关EST SSR标记与火炬松×洪都拉斯加勒比松苗高相关性分析

以火炬松×洪都拉斯加勒比松F1代群体为研究对象,从松树PGI（松类基因索引）数据中筛选出13个与赤霉素（GAs）代谢有关的序列。设计了这13条序列的EST SSR引物对,并筛选出4对引物作为F1代检测的较好的标记。4对引物PCR分析显示在2个亲本和39个子代中共扩增出1 014个多态性位点,其中,杂种F1代扩增出的位点数中有50.19 %与父本相同,5217 %与母本相同,这表明母本（火炬松）和父本（加勒比松）杂交能够得到获得双亲遗传物质的新杂种。4个引物检测的26个等位基因位点中有6个与苗龄6个月的苗高有显著或极显著的相关性,有5个位点与苗龄9个月的苗高有显著或极显著相关性。这为早期选择提供了较好的分子标记。     

Construction of an electronic physical map of Magnaporthe oryzae using genomic position-ready SSR markers

Magnaporthe oryzae is a model for plant pathogenic filamentous fungi. We have assembled a simple sequence repeat (SSR)-based physical map of the species, using in silico sequence data. A set of 120 SSR markers was developed from the genomic sequence of the reference isolate 70-15. These markers were readily amplified from the genomic DNA of other isolates, and high levels of allelic variation characterised the parental isolates of the two crosses tested. All the markers were locatable to one of the seven M. oryzae chromosomes. An SSR-based physical in silico map was constructed, and pre-existing SSR and RFLP loci were integrated into the map, along with 23 Avr (avirulence) genes and two other genes of importance to the plant/pathogen interaction. This map provides a platform for population genetics and functional genomics studies in the model pathogen, and even in other evolu- tionally related pathogens.     

检疫性杂草假高粱及其近似种SSR分子标记的遗传多样性

利用SSR标记对8个假高粱及其近似种样品的遗传多样性进行研究,结果发现:(1)在11对SSR引物中,有5对扩增出多态性,这5对引物共检测到25个多态性位点,每对引物平均能检测到5个多态性位点;(2)根据Nei’s遗传距离分析的聚类树显示:这8个高粱属物种共可分为2个类群,7个亚类群;假高粱和黑高粱、拟高粱和明福1号间的亲缘关系极近;(3)SSR结果揭示出所测8个群体的Gst为0.0864,说明本文研究的Sorghum属8个物种的群体遗传分化程度属于中度。     

Detection of quantitative trait loci and heterotic loci for plant height using an immortalized F2 population in maize

A set of recombinant inbred lines (RIL) derived from Yuyu22, an elite hybrid widespread in China, was used to construct an immortalized F2 (IF2) population comprising 441 different crosses. Genetic linkage maps were constructed containing 10 linkages groups with 263 simple sequence repeat (SSR) molecular markers. Twelve and ten quantitative trait loci (QTL) were detected for plant height in the IF2 and RIL populations respectively, using the composite interval mapping method, and six same QTL were identified in the two populations. In addition, ten unique heterotic loci (HL) located on seven different chromosomes were revealed for plant height using the mid-parent heterosis as the input data. These HL explained 1.26%―8.41% of the genotypic variance in plant height heterosis and most expressed overdominant effects. Only three QTL and HL were located in the same chromosomal region, it implied that plant height and its heterosis might be controlled by two types of genetic mechanisms.