Development and Characterizations of EST-SSR Markers in Rubber Tree(Hevea brasiliensis)

To further develop EST-SSR marker in rubber tree, we assembled the sequences downloaded from NCBI and Malaysia EST databases of rubber tree. By analyzing the assembled 3 733 unigenes, we identified 566 potential SSR sites in this study. That is to say, there was one EST-SSR in every 3.96 kb. The di-nu-cleotide repeat was the most abundant type, fol owed by tri-, hexa-, tetra- and pen-ta-nucleotide repeat. The most common number of repeat units was 5, fol owed by more than 12, 6 and 7. Of 51 SSR motifs identified in this study, di-, tri-, tetra-, penta- and hexa-nucleotide repeats were 6, 26, 5, 3 and 11 types, respectively. The GA/CT di-nucleotide repeat was the most abundant motif, fol owed by TC/AG, AT/TA, CTT/GAA, TTC/AAG and TCT/AGA. In total, 158 new EST-SSRs were developed and amplified with the DNA of RRIM600 as a template. The results showed that the PCR products of 99 EST-SSRs generated clear amplifying bands. The EST-SSR markers developed in this study further enrich the number of molecular marker in rubber tree, and they wil be widely applied in DNA fingerprinting, genetic diversity, marker-assisted selection and genetic mapping, etc.

Haplotyping of Rice Genotypes Using Simple Sequence Repeat Markers Associated with Salt Tolerance

Salt stress is a major problem in most of the rice growing areas in the world. A major QTLSaltol associated with salt tolerance at the seedling stage has been mapped on chromosome 1 in rice.This study aimed to characterize the haplotype diversity at Saltol and additional QTLs associated withsalt tolerance. Salt tolerance at the seedling stage was assessed in 54 rice genotypes in the scale of 1to 9 score at EC = 10 dSm^-1 under controlled environmental conditions. Seven new breeding linesincluding three KMR3/O. rufipogon introgression lines showed similar salt tolerant ability as FL478 andcan be good sources of new genes/alleles for salt tolerance. Simple sequence repeat （SSR） markerRM289 showed only two alleles and RM8094 showed seven alleles. Polymorphic information contentvalue varied from 0.55 for RM289 to 0.99 for RM8094 and RM493. Based on 14 SSR markers, the 54lines were clearly separated into two major clusters. Fourteen haplotypes were identified based onSaltol linked markers with FL478 as the reference. Alleles of RM8094 and RM3412 can discriminatebetween the salt tolerant and susceptible genotypes clearly and hence can be useful in marker-assistedselection at the seedling stage. Other markers RM10720 on chromosome 1 and RM149 and RM264 onchromosome 8 can also distinguish tolerant and susceptible lines but with lesser stringency.

Genetic Variation in Triticum turgidum L. ssp. turgidum Landraces from China Assessed by EST-SSR Markers

It was helpful for the wheat improvement to evaluate the genetic resources of Triticum turgidum L. ssp. turgidum landraces. In this study, 68 turgidum landraces accessions, belonging to four geographic populations in China, were investigated by using EST-SSR markers. A total of 63 alleles were detected on 22 EST-SSR loci, and the number of alleles on each locus ranged from 1 to 5, with an average of 2.9. The results of the analysis of molecular variance （AMOVA） indicated that 92.5% of the total variations was attributed to the genetic variations within population, whereas only 7.5% variations among populations. Although the four populations had similar genetic diversity parameters, Sichuan population was yet distinguished from other populations when comparing the population samples in pairs. Significant correlations were detected by the statistic analysis among six genetic diversity parameters among each other. The selection difference between heterozygosty and homozygosty was also observed among different EST-SSR locus. The genetic similarity （GS） ranged from 0.18 to 0.98, with the mean of 0.72, and all accessions could be clustered into 7 groups. The dendrogram suggested that the genetic relationships among turgidum accessions evaluated by EST-SSR markers were unrelated to their geographic distributions. These results implied that turgidum landraces from China had the unique characters of genetic diversity.

Transcriptome analysis of the endangered Notopterygium incisum: Coldtolerance gene discovery and identification of EST-SSR and SNP markers

Notopterygium incisum C. C. Ting ex H. T. Chang(Apiaceae) is an endangered perennial herb in China. The lack of transcriptomic and genomic resources for N. incisum greatly hinders studies of its population genetics and conservation. In this study, we employed RNA-seq technology to characterize transcriptomes for the flowers, leaves, and stems of this endangered herb. A total of 56 million clean reads were assembled into 120,716 unigenes with an N50 length of 850 bp. Among these unigenes, 70,245(58.19%) were successfully annotated and 65,965(54.64%) were identified as coding sequences based on their similarities with sequences in public databases. We identified 21 unigenes that had significant relationships with cold tolerance in N. incisum according to gene ontology(GO) annotation analysis. In addition, 13,149 simple sequence repeats(SSRs) and 85,681 single nucleotide polymorphisms were detected as potential molecular genetic markers. Ninety-six primer pairs of SSRs were randomly selected to validate their amplification efficiency and polymorphism. Nineteen SSR loci exhibited polymorphism in three natural populations of N. incisum. These results provide valuable resources to facilitate future functional genomics and conservation genetics studies of N. incisum.

Effective Procedure for Development of EST-SSR Markers Using cDNA Library

The present study was conducted to develop EST-SSR markers using the cDNA library from rice plant. Total RNA extracted from the leaves of brown plant hopper resistance gene originated from a rice cultivar “Cheongcheong” and sensitive rice cultivar “Nakdong” were used to synthesize a cDNA library. As a result of analyzing the cDNA library, the 17 EST-SSR primer sets were developed. This study enables to provide effective marker assisted selection (MAS) methods on the selection of white-backed planthopper resistance gene originated from a rice plant more simply, quickly and precisely. Furthermore, using this marker’s advantage of deriving from cDNA, it is possible to identify the white-backed planthopper resistance gene. In addition, this study introduces a technique for construction of a cDNA library safely without using radioactivity.

Confirmation of Pearl Millet-Napiergrass Hybrids Using EST-Derived Simple Sequence Repeat (SSR) Markers

Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids (“PMN”;Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both “Seeded-yet-Sterile” biofuel PMN feedstocks and additional PMN cultivar-and parental species-specific markers.

Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus

Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.

Analysis of Simple Sequence Repeats Information from Floral Expressed Sequence Tags Resources of Papaya (<i>Carica papaya</i>L.)

Papaya (Carica papaya L.) is one of the most economically, medicinally and nutritionally important tropical fruit crops. Expressed sequence tags (ESTs) derived simple sequence repeat (SSR) markers are more valuable as they are derived from conserved genic portion. Development of EST-SSRs markers through in silico approach is cheaper, less time consuming and labour-intensive. In this study, we aimed to mine SSRs and developed EST-SSR primers from papaya floral ESTs. A total of 75,846 papaya floral ESTs were downloaded from public database National Centre for Biotechnology Information (NCBI). A total of 26,039 floral unigenes (7961 contigs and 18,078 singletons) were generated after assembly of these ESTs. From these floral unigenes, 433,782 perfect SSRs, 204,968 compound SSRs and 6061 imperfect SSRs were mined, respectively. In perfect SSRs, mononucleotide repeats were most abundant (94.7%) followed by tri- (3.1%) and di-nucleotide repeats (1.7%). The frequencies of tetra-, hexa- and penta-nucleotide repeats accounted for only (0.17%), (0.04%) and (0.03%), respectively. In mononucleotide repeats, the most abundant motif was A/T (69.3%) and in di- and tri-nucleotide repeats were AG/CT (61%) and AAG/CTT (31%), respectively. In imperfect SSRs, mononucleotide repeats (56.5%) were most abundant. 176 different types of motifs were identified. A total of 3807 primer pairs for floral papaya ESTs were successfully designed. These developed EST-SSR primers are being used for the genetic improvement of papaya such as study of cross-transferability across genera/species, evaluation of genetic diversity, and identification of sex-specific markers. These EST derived SSRs can also be used in filling gaps in existing linkage maps in papaya.

“过”的信息突显性及其解释力

以往研究用终结性或可重复性来解读经历体标记“过”的语法意义,但各有反例。文章提出,“过”应该满足信息突显的语用条件,终结性与可重复性则源自信息突显的两个实现条件。因为信息突显性的制约,肯定和否定语境下的“V过”包含截然相反的信息量。信息突显依赖“图形”和“背景”的配置。图形是“过”标记的事件,通过终结性实现突显,是终结义的来源;背景则是事件集合,通过“数”概念起到衬托的作用,是可重复义的来源。文章引入突显概念,完善终结义和可重复义的内涵,将两者看作突显性的一体两面,实现了两个语法意义的融合。

基于转录组数据的马氏珠母贝EST-SSR位点的信息分析及其多态性检测

为获得稳定可靠的马氏珠母贝SSR分子标记,本文利用MISA软件对转录组测序数据进行了大规模的EST-SSR标记发掘,并分析了位点信息及其多态性。结果表明,马氏珠母贝转录组测序所获得的74007条Unigenes中检测出9872个EST-SSR位点,位点出现频率为13.34%,平均每5102 bp含有1个SSR位点。在转录组SSR中共有132种重复基元类型,其中单核苷酸重复基元为主要类型,占SSR总数的81.46%;单核苷酸重复以A/T基序为主,占SSR总数的71.27%。基于筛选的SSR序列,应用Primer3软件进行引物的批量设计,共为5922条EST-SSR序列成功设计出17766对引物。随机选择80对引物对EST-SSR多态性进行检测,共有62对引物成功扩增出稳定条带,占引物总数的77.5%;其中,17对EST-SSR引物表现出个体多态性,多态性比率为27.42%。以上研究为马氏珠母贝遗传多样性、遗传图谱构建及分子辅助育种研究提供了有效工具,对于马氏珠母贝种质资源保护、优良品种培育和珍珠养殖业的健康发展均具有重要意义。

基于SSR标记与农艺性状对三七集团选择群体的评价

三七为人参属多年生草本植物,生长和繁育周期长,群体间的遗传多样性低,但群体内单株间表型变异大,人参皂苷含量差异尤为明显。本研究根据三七中5种人参皂苷(三七皂苷R1、人参皂苷Rg1、人参皂苷Rb1、人参皂苷Re、人参皂苷Rd)含量构建了11份集团群体(SL1~SL11),对当代和第一代集团群体人参皂苷含量进行了比较分析。同时,基于三七基因组,使用MISA软件进行全基因组水平SSR标记开发,鉴定到255239个SSR标记,筛选出17对多态性SSR标记。使用SSR标记对第一代群体进行遗传多样性评价。结果表明,11份第一代集团群体观察杂合度Ho较高(0.4583~0.6042),遗传分化程度低(Fst=0.0447),且具有较高基因流(Nm=11.6189);第一代群体间总皂苷含量无显著性差异而三七皂苷R1变异系数高于总皂苷和其他单体皂苷,且群体SL8三七皂苷R1含量显著高于其他群体。综上,本研究以皂苷含量为目标性状构建了高皂苷含量的集团群体,并利用SSR标记评价了群体遗传多样性,构建的三七皂苷R1高含量群体可作为后续育种材料。

中国虎纹蛙多碱基重复微卫星位点的多态性

[目的]筛选出中国虎纹蛙高质量多态性的微卫星标记,用于该物种的鉴定以及遗传多样性、种群结构、遗传连锁图谱分析。[方法]利用新一代测序技术,从中国虎纹蛙部分基因组中筛选出33个三核苷酸重复、42个四核苷酸重复和1个五核苷酸重复微卫星位点,共76个多碱基重复微卫星位点。所有位点对浙江丽水学院两栖爬行动物实验室人工饲养的种群(n=30)进行了扩增和基因型分析。[结果]每个位点观察到的等位基因数和多态信息含量(PIC)的平均值分别为4个(2~7个)和0.514(0.164~0.822),观测杂合度(H_(O))和期望杂合度(H_(E))分别为0.00~0.97和0.18~0.82。有2个位点(THC141和THC377)显著偏离哈迪温伯格平衡,在11个位点中发现了显著的连锁不平衡。[结论]这些新筛选的多态性微卫星标记有助于进一步研究中国虎纹蛙的遗传多样性和开展遗传育种,为制定有效的保护策略和开展分子辅助选择提供依据。

水芹SSR分子标记开发与遗传多样性分析

水芹是伞形科水芹属多年生草本植物,是一种重要的药食两用蔬菜作物。在中国,水芹的种植区域十分广泛,然而目前对其种质资源的鉴定、培育及遗传信息的研究较少。本研究利用溧阳白芹基因组开发水芹简单重复序列(SSR)分子标记,分析55份水芹的遗传多样性并用非加权组平均法(UPGMA)构建系统进化树,同时用SSR扩增条带数据构建DNA指纹图谱。结果显示,共鉴定到325699个SSR位点,其中单核苷酸SSR重复单元、二核苷酸SSR重复单元、三核苷酸SSR重复单元、四核苷酸SSR重复单元、五核苷酸SSR重复单元、六核苷酸SSR重复单元的出现频率分别为33.94%、54.62%、9.31%、1.66%、0.17%、0.29%,其中二核苷酸SSR重复单元数最多,有177887个,且A/T(占比为29.98%)和AT/AT(占比为35.70%)是较丰富的重复类型。UPGMA分析结果表明,33对高多态性引物[多态信息含量(PIC)>0.25]可将55份水芹材料分为4组。利用筛选出的4对引物(Oj-084、Oj-110、Oj-112、Oj-156)可以将55份水芹材料完全区分开,并且可构建指纹图谱。研究结果可为水芹种质资源鉴定、保护及分子遗传育种提供有力依据。

大顶苦瓜种质资源的遗传多样性分析与指纹图谱构建

为实现对大顶苦瓜种质的快速精准鉴定,以包含主栽品种在内的26份大顶苦瓜种质为材料,通过筛选高多态性简单重复序列(simple sequence repeat,SSR)核心标记,对其进行聚类分析和指纹图谱构建。结果表明:在苦瓜基因组6种SSR基序单元类型中,二核苷酸SSR标记的多态性最好,适用于进一步从中筛选苦瓜SSR核心标记。在遗传相似系数0.80处,40个SSR标记将26份大顶苦瓜种质分为3个类群,第一类群包含18份种质,果形以短圆锥形为主;第二类群包含5份种质,果形以长圆锥形为主;第三类群包含3份种质,其果形与大顶苦瓜相似,但含有非大顶苦瓜种质背景。利用MC05_69594、MC04_50530、MC06_87314和MC10_146038这4个SSR核心标记可对26份大顶苦瓜种质进行有效鉴定,并为每份大顶苦瓜种质建立独特的QR编码指纹图谱,其中,1号大顶和3号大顶疑为相似材料。构建的大顶苦瓜指纹图谱可以为大顶苦瓜种质鉴定与育种利用提供科学依据。

兰属植物EST-SSR标记开发及应用

本研究利用兰属植物杂交种转录组测序数据开发EST-SSR标记,分析标记的多态性及兰属种质的遗传多样性,为兰属植物种质资源的创新和分类研究提供参考。结果表明,转录组测序分析获得113780条Unigene,从中共识别到23709个SSR位点,SSR位点出现频率为20.84%。从设计的200对引物中筛选出20对具有多态性,并且扩增条带大小与预期相符的EST-SSR标记引物,对48份兰属种质材料进行PCR扩增,平均多态位点数为3.0,共检测到81.00个等位基因,平均每对引物检测到4.05个等位基因。观测杂合度平均值为0.3208,期望杂合度平均值为0.5070;Shannon’s信息指数的变化范围为0.2338~1.4724,平均值为0.9321,多态信息含量为0.1103~0.6622。对4个参试兰属植物种群进行遗传多样性分析,春兰和大花蕙兰的F1杂交种群与大花蕙兰种群亲缘关系较近,与春兰种群的亲缘关系较远。聚类分析结果表明,遗传相似系数为0.72时,48份种质聚成7类,春兰和大花蕙兰的F1代杂交种群大多聚入第I类,春兰种群聚入第V类,第II类、第III类、第IV类、第VII类均为大花蕙兰种群。

香菇EST-SSR标记的开发及应用

从NCBI中下载了12 184条香菇（Lentinula edodes）表达序列标签（expressed sequence tag,EST）序列,处理后得到全长3 681 177 bp的4 684个Unigene。在其中共发掘出142个简单重复序列（simple sequencerepeat,SSR）,分布于120条EST中,分布频率为2.99%（平均分布距离：25.924 kb）。其中,单、二、三核苷酸重复是主要的重复类型,A/T,AG/CT,ACG/CTG是单、二、三核苷酸的主要重复基序,分别占所有EST-SSR的23.23%、21.83%和10.56%。利用引物设计软件PrimerPremier5.0设计了40对引物,并利用6%变性聚丙烯酰胺凝胶在18个香菇品种中进行检测。其中,22对引物为多态性引物,多态率为55%。应用NTSYS软件的聚类方法分析表明,18份材料遗传相似系数范围为0.375-0.984,与前人研究结果相符。

SSR分子标记在玉米研究中的应用

SSR简单重复序列也称微卫星DNA,是由特异性的引物进行PCR扩增分析的一种分子标记技术。简要介绍了SSR分子标记的原理,分析了SSR分子标记在玉米的种质资源、品种纯度鉴定、真伪鉴定、遗传多样性、种质性状等方面的应用。通过研究表明:能够利用SSR分子标记技术对玉米的品种纯度鉴定、真伪性鉴定、遗传结构、亲缘关系、优劣群体的划分、种质性状等方面进行分析,同时也能为以后研究玉米的遗传连锁图谱构建、分子标记辅助育种、基因定位和种质资源等方面提供参考。

基于表型性状及SSR标记的天麻种质资源遗传多样性分析

目的:研究天麻的遗传多样性,分析杂交、自交天麻种质资源间的差异。方法:以26个天麻种质资源为材料,基于形态学标记对各种质的株高、蒴果数、花序长度、蒴果长度、蒴果宽度、块茎长度、块茎宽度、单重、有效成分含量等表型性状进行方差分析、聚类分析及相关性分析;结合SSR分子标记进行遗传多样性研究,并构建SSR指纹图谱。结果:天麻表型性状中,各性状的变幅不同,变幅较大的为蒴果重、单重、块茎长、鲜品产量、干品产量、天麻素含量、对羟基苯甲醇含量;变幅较小的为蒴果宽、茎秆粗、肚脐眼直径、横纹环数、折干率。相关性分析结果显示,部分性状间相关性显著;表型性状聚类结果显示,在阈值为11.32时,将天麻资源分为2大类群。从合成的7对引物中筛选出3对SSR特异性引物,PCR扩增获得较清晰的SSR指纹图谱。UPGMA聚类可明显区分天麻自交系及杂交系,与表型聚类分析结果较为一致。结论:利用形态学标记及SSR分子标记相结合的方法可有效区分天麻杂交组合及自交组合。

胃癌干细胞表面标志物的研究进展

胃癌干细胞(GCSC)在胃癌的发生、发展、侵袭、转移中发挥了重要的作用,因此探索灵敏度高、特异性强的GCSC表面标志物极其重要。本文阐述了常用及最新的GCSC标志物,其中CD44^(+)GCSC是最早发现具有启动肿瘤生长、维持肿瘤自我更新能力的独特亚群,是目前研究较为成熟的标志物之一。重复含G蛋白偶联受体5同样具有明确的细胞干性,但其作为GCSC标志物不是特异的,而CD133^(+)细胞是否具有细胞干性,还需进一步探索其机制。SOX2、醛脱氢酶及其同工酶、NME/NM23核苷二磷酸激酶2具有干细胞潜能,为GCSC表面标志物提供了更多的可能及选择。

利用EST-SSR、IT-ISJ分子标记研究四倍体与八倍体柳枝稷的遗传多样性

柳枝稷是重要的能源植物之一,为研究四倍体Alamo与通过秋水仙素加倍后的八倍体之间的遗传差异,本研究以65份四倍体Alamo与其诱变的八倍体为研究材料,通过EST-SSR与IT-ISJ两种分子标记进行遗传分析。结果表明,EST-SSR引物共扩增出245条带,多态性比率为99.59%,观测等位基因Na为1.9878,有效等位基因Ne为1.1718,基因多样性指数H为0.1308,Shannon信息指数I为0.2388,其中四倍体Alamo的各遗传参数(226条,88.93%,1.8816,1.1823,0.1349,0.2398)多高于八倍体Alamo(217条,92.24%,1.9184,1.1652,0.1228,0.2234)。IT-ISJ引物共扩增出165条条带,多态性比率为96.36%,观测等位基因Na为1.9878,有效等位基因Ne为1.5777,基因多样性指数H为0.3354,Shannon信息指数I为0.4999,四倍体Alamo各项指数(145条,94.55%,1.9333,1.6366,0.3602,0.5280)均高于八倍体Alamo(139条,84.24%,1.7515,1.4377,0.2573,0.3864)。通过UPGMA分析表明,65个柳枝稷材料中四倍体与八倍体没有明显区分开,表明其没有产生明显的遗传分化。通过AMOVA分析表明,EST-SSR和IT-ISJ的遗传变异分别有94.78%和80.76%发生在倍性内,有5.22%和19.24%发生在倍性间,表明柳枝稷倍性间没有明显的差异。