High-throughput simple sequence repeat (SSR) markers development for the kelp grouper (<i>Epinephelus bruneus</i>) and cross-species amplifications for Epinephelinae species

The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

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.

The genome of herpes simplex virus type 1 is prone to form short repeat sequences

Herein, we report a very high content of simple sequence repeats (SSRs) covering 66.12% of the herpes simplex virus type 1 (HSV-1) genome when a low threshold is adopted to define SSRs, indicating that repeat sequence is a very important character of the HSV-1 genome. The repeats with two iterations account for 68.33% of the total repeats. In reality, the genome of HSV-1 is prone to form shorter repeat sequences. For mono-, di- and trinucleotide repeats, the repeat numbers decreased with the increase of repeats iterations, implicating that the formation tendency of SSRs might be from low iterations to high iterations. The high iterations SSRs might have subjected to strong selected pressure and survived to perform different functions. The analysis suggested that the repeats formation may be an essential evolutionary driving force for the HSV-1 genome, and the results might be helpful for studying the genome structure, repeats genesis and genome evolution of HSV-1.

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.

Molecular characterization of sweet potato(Ipomoea batatas[L.]Lam)germplasms for desirable traits by using simple sequence repeats markers

Every breeding program that aims to create new and improved cultivars with desired traits mostly relies on information related to genetic diversity.Therefore,molecular characterization of germplasms is important to obtain target cultivars with desirable traits.Sweet potato[Ipomoea batatas(L.)Lam]is widely considered the world’s most important crop,with great diversity in morphological and phenotypic traits.The genetic diversity of 20 sweet potato germplasms originating from Bangladesh,CIP,Philippines,Taiwan,and Malaysia were compared,which was accomplished by genetic diversity analysis by exploring 20 microsatellite DNA markers for germplasm characterization and utilization.This information was effective in differentiating or clustering the sweet potato genotypes.A total of 64 alleles were generated using the 20 primers throughout the 20 germplasm samples,with locus IBS97 having the highest number of alleles(5),whereas locus IbU33 had the fewest alleles(2).The alleles varied in size from 105(IbU31)to 213 base pairs(IBS34).The Polymorphism Information Content(PIC)values for the loci IbL46 and IBS97 varied from 0.445 to 0.730.IBS97 has the highest number of effective alleles(3.704),compared to an average of 2.520.The average Shannon’s diversity index(H)was 1.003,ranging from 0.673 in IbU3 to 1.432 in IBS97.The value of gene flow(Nm)varied between 0.000 and 0.005,with an average of 0.003,whereas genetic differentiation(FST-values)ranged between 0.901 and 1.000.The sweet potato germplasm included in this study had a broad genetic base.SP1 vs.SP9 and SP12 vs.SP18 germplasm pairings had the greatest genetic distance(GD=0.965),while SP1 vs.SP2 germplasm couples had the least genetic diversity(GD=0.093).Twenty genotypes were classified into two groups in the UPGMA dendrogram,with 16 genotypes classified as group“A”and the remaining four genotypes,SP10,SP18,SP19,and SP20,classified as group“B.”According to cluster analysis,the anticipated heterozygosity(gene diversity)of Nei(1973)was 0.591 on average.In summary,SSR markers successfully evaluated the genetic relationships among the sweet potato accessions used and generated a high level of polymorphism.The results of the present study will be useful for the management of germplasm,improvement of the current breeding strategies,and the release of new cultivars as varieties.

基于EST-SSR标记的沙棘品种鉴定及指纹图谱构建

以沙棘(Hippophae rhamnoides)优良品种“实优1号”为材料,对其叶片进行转录组测序,利用微卫星识别软件(microsatellite identification tool,MISA)和Primer 3(version 2.3.4)对获得的序列进行简单重复序列(simple sequence repeat,SSR)位点挖掘和引物设计,以收集的42份沙棘品种为研究材料,开展聚合酶链式反应(PCR)和毛细管电泳检测,旨在开发一套多态性高、稳定性好和通用性强的表达序列标签微卫星(express sequence tags from simple sequence repeat,EST-SSR)引物,构建沙棘指纹图谱,从而实现沙棘品种的快速准确鉴定,并对沙棘品种间亲缘关系进行分析。“实优1号”转录组测序共获得6196个SSR位点,其中,重复基元类型为182种,SSR基序长度主要分布在10~21 bp区间,占全部SSR的81.58%,主要SSR重复类型为单核苷酸重复(48.72%)、二核苷酸重复(22.68%)和三核苷酸重复(18.85%)。利用筛选出的28对引物在42份沙棘品种中共检测出193个等位基因,等位基因数(Na)、有效等位基因数(Ne)、观测杂合度(Ho)、期望杂合度(He)、多态信息含量(PIC)和Shannon信息指数(I)等遗传多样性参数的均值分别为6.964、3.495、0.617、0.671、0.623和1.384。UPGMA聚类分析表明,42份沙棘品种间的遗传相似性系数为0.601~0.990,当遗传相似性系数为0.694时,供试品种可分为2组;当遗传相似性系数约为0.7402时,供试品种可分为3组。优选6对引物构建指纹图谱,可以实现沙棘品种的快速准确鉴定。该研究可为沙棘的良种鉴定、指纹图谱构建以及遗传多样性和亲缘关系分析等提供分子水平的理论基础和数据支撑。

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。

基于SSR标记的MCID法鉴定新疆地方梨品种及其遗传多样性分析

为深入探究新疆地方梨品种的遗传关系,本研究采用简单重复序列(SSR)标记技术对28个新疆地方梨品种进行遗传多样性分析。同时,利用人工绘制品种鉴别示意图(MCID)法建立品种鉴定图(CID)。结果表明,新疆地方梨品种间遗传差异较小,遗传分化为中等程度。通过构建新疆地方梨品种的聚类树状图,发现28个新疆地方梨具有较高的遗传多样性,遗传相似性系数为0.52~0.98。群体结构分析结果表明,最佳群体群组数值(K)=3时新疆地方梨品种被划分为3个组,各品种之间存在普遍的基因交流。采用MCID法构建新疆地方梨品种的CID图谱,并验证了该方法的有效性。本研究结果可为今后梨种质资源评价和利用提供理论依据。

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。

Parent-offspring relationship recognition based on SSR and mtDNA confirmed resource supplement effect of Fenneropenaeus chinensis release

The resource of Fenneropenaeus chinensis has declined sharply due to excessive fishing intensity,ecological changes and diseases.In order to supplement the fishing yield and restore resources of F.chinensis,the relevant authorities have carried out the activities of stock enhancement and releasing.It can increase biomass and recover resources.However,compared with increasing biomass,there were still few reports on its effect on the recovery of resources.Resource recovery is a process related to whether the released individuals can form a reproductive population.Up to now,there has been a lack of evidence whether the released F.chinensis can complete the entire life history,and form reproduction population.In this study,gravid female shrimp after spawning migration were captured from coastal waters of Haiyang,Qingdao,and Yellow Sea.After identifying parentage relationships using simple sequence repeat(SSR)and mtDNA haplotype,it was finally confirmed that there were eight released individuals in the recapture samples.It was confirmed for the first time that at least part of the released F.chinensis can complete overwintering and reproductive migration,and maintain the migration habits as their wild counterparts.Therefore,we infered that the released shrimp can reproduce under natural conditions,these F.chinensis can form reproductive populations theoretically if without human intervention.These results indicated that enhancenment and release activities have a positive effect on resource recovery.

基于果形对草莓品种遗传多样性SSR分析

本研究利用SSR分子标记技术对国家果树种质北京草莓圃的173份草莓品种进行区分,根据果形利用PopGene软件对其做了聚类分析。结果显示,长圆锥形、短圆锥形、阔圆锥形、圆锥形/长楔形、圆锥形/平楔形、长楔形、近圆形、圆球形/卵圆形的观测纯合度都为0.846 2,扁球形、圆球形/半圆球形、短圆锥形/短楔形的观测纯合度都在0.923 1~0.948 7之间,但是短圆形纯合度最低,为0.692 3,由此发现与其他形状的亲缘关系较远,其余形状相互之间的亲缘关系较近。根据果形分析SSR扩增等位基因的多态性发现,173份草莓品种整体纯合体偏多,其中楔形/长圆锥形的无杂合体,而短圆形的纯合体最少,草莓品种整体亲缘关系较近。

三倍体葡萄柚实生后代多倍体的发掘与SSR遗传鉴定

【目的】从三倍体葡萄柚自然授粉的实生后代中发掘多倍体,希望获得一批多倍体新种质,并为探索三倍体遗传规律奠定基础。【方法】秋季采摘三倍体葡萄柚成熟果实,剥取种子,MT+1 mg·L-1GA3培养基播种,待种子萌发后,用流式细胞仪快速检测其倍性,并采用SSR(simple sequence repeat)分子标记鉴定其不同倍性后代植株的来源。【结果】获得了二倍体、三倍体、四倍体、五倍体、六倍体和疑似非整倍体等不同倍性的再生植株各172、52、7、1、1、18株。用13对多态性SSR引物鉴定其来源,结果表明,由二倍体可能三倍体母本所产生的1X配子与周边其他二倍体品种的花粉授粉而来,也可能由三倍体母本产生的重组型2X配子的无融合生殖而来;三倍体大部分由珠心细胞发育而来;四倍体为杂交起源的四倍体;五倍体和六倍体与母本(三倍体葡萄柚)的带型完全相同。【结论】上述活体材料的发掘为柑橘多倍体育种奠定了种质基础。

SSR分子标记在作物遗传育种中的应用

SSR(Simple Sequence Repeat)是建立在PCR技术上的一种广泛应用的分子标记,具有含量丰富、多态性高、共显性等优点。简要介绍了SSR标记技术的发现、原理和特点,并总结了该技术在作物遗传多样性、遗传图谱构建、分子标记辅助选择等方面的应用。

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。

四川主栽小麦品种遗传多样性的SSR标记研究

采用微卫星分子标记 (SSR)对四川省近 5 0年以来年推广面积达 6 6 70 0 hm2 (10 0万亩 )以上的 4 0个主栽小麦品种的遗传多样性进行了研究。结果发现 ,在小麦全基因组 4 2条染色体臂上的 4 6个 SSR位点上 30个 SSR位点 (6 5 .2 2 % )具有多态性。这 4 6个位点共检测到 110个等位变异 ,每个 SSR位点能检测到 1～ 8个 ,平均为 2 .4个。聚类分析表明 ,SSR标记能将 4 0个品种相互区分开。品种间遗传相似系数 (GS)变幅为 0 .4 5 1～ 0 .76 7,平均 GS值为0 .6 0 1。据此认为 ,SSR标记揭示出四川主栽小麦品种具有较高的遗传多样性。各年代间 GS值变化趋势分析表明 ,2 0世纪 70年代后 。

SSR分子标记在作物遗传育种中的应用

SSR(simple sequence repeat)是建立在PCR技术上的一种广泛应用的分子标记,具有含量丰富、多态性高、共显性等优点。本文简要介绍了SSR分子标记技术的原理和特点,重点介绍了SSR分子标记技术在作物遗传育种中的应用,主要在作物遗传多样性、基因定位、分子辅助标记、遗传图谱构建、品种鉴定和纯度鉴定等方面进行阐述。

利用SSR方法鉴定大豆品种纯度

本研究以遗 75 - 14,中黄 14,中品 6 6 2和中品 6 6 1共 4个品种为试验材料 ,利用不同连锁群上的SSR引物对其随机选择个体进行分析 ,以确定分析品种纯度所需的引物数 ,为大豆品种资源的保存及品种指纹图谱的建立提供理论依据。通过 11对SSR引物对遗 75 - 14,中黄 14,中品 6 6 2各 10个单株的检测 ,发现中品 6 6 2纯度最高 ,遗 75 - 14次之 ,而中黄 14的纯度最低。用SSR引物对中品 6 6 1和中品6 6 2每 10个单株混合样品分析并经单株检测验证 ,其纯度分别为 99 0 %和 98 3%,研究结果表明 ,10个植株混合时 ,即使有一个单株为杂合位点也能被检测出来 ,并发现用 3- 5对位于不同连锁群的SSR引物可以对随机或混合样品进行快速、准确的纯度鉴定。

陆地棉品种SSR标记的多态性及用于杂交种纯度检测的研究

:用 48对 SSR引物对 30个棉花栽培品种和4个高优势杂交种的亲本进行了多态性筛选 ,结果只有 2 7对引物检测到了多态性。应用SSR3442、SSR2 4 95、SSR3347和 SSR1 2 31标记 ,区分了湘杂 2号、皖杂 40、中棉所 2 8、南抗 3号的F1杂种和它们的亲本以及其它栽培品种 ;

杨树EST-SSR标记的开发

利用来自美洲黑杨及欧美杨的20023条EST序列,进行EST-SSR标记开发研究。首先对这些EST序列进行序列拼接,然后在非重复序列中发现由二核苷酸至五核苷酸组成的SSR重复序列。共在10816个非重复序列中发现1604个序列含有SSR,占总非重复序列的14.8%。在发现的1918个SSR重复序列中,二核苷酸重复是最丰富的重复类型,占总类型的62.3%。设计合成48对SSR引物,利用6个不同的杨树品种对其进行验证。结果约90%的引物获得扩增产物,58%的引物在6个不同杨树品种中产生多态性分离。另外,还对重复序列的数量及重复类型进行了讨论。

果梅SSR反应体系的优化

以果梅品种小叶猪肝为试材 ,研究了果梅SSR技术中PCR反应体系的主要成分对SSR扩增结果的影响 ,并比较了采用聚丙稀酰胺凝胶及琼脂糖电泳检测扩增产物多态性的差异。结果表明 :在PCR反应体系中 ,Mg2 + 的最适浓度范围为1 89～ 2 83mmol·L-1;dNTP最适浓度为 0 2 4mmol·L-1;引物的最适浓度为 0 38μmol·L-1;Taq 聚合酶在 2 6 5 μL反应体系中宜加入 1U。利用此反应体系 ,对 2 4个果梅代表品种进行了SSR反应 ,用 8%的非变性聚丙稀酰胺凝胶电泳检测 ,扩增产物在 10 0～ 2 0 0bp之间 ,不同品种间DNA谱带多态性丰富。