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.

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.

Development of Starfruit Simple Sequence Repeat (SSR) Using Next Generation Sequencing

Starfruit (Averrhoa carambola L.) is an important fruit for Malaysian export and great attention has been made to improve starfruit fruit quality at Malaysian Agricultural Research and Development Institute (MARDI). The current study used next generation sequencing (NGS) technologies to develop starfruit simple sequence repeat (SSR) from 2 varieties namely B11 and B17 using Illumina HiSeq. The pre-processed reads were de novo assembled to generate approximately 75,000 and 74,000 scaffolds respectively. Total genome size for B11 and B17 were around 345 Mbp and 342 Mbp based on K-mer distribution analysis. In-silico microsatellite mining of each variety has identified more than 17,000 SSR in B11 and B17 respectively. Dinucleotides were the most abundant, accounting for more than 70% of all SSR and repeat motif GA (49%) was most common. A total of 239 SSR primer pairs were designed from contigs larger than 350 nucleotides and tested for amplification. The 30 polymorphic SSRs were used to DNA fingerprint of 12 starfruit hybrids. Polymorphism information content (PIC) ranged from 0.1411 to 0.6838, with an average of 0.3919. The Unweighted Pair-Group Method for Arithmetic Averages (UPGMA) dendrogram clustered 12 starfruit accessions into 2 groups.

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。

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。

辐射诱变小兰屿蝴蝶兰叶片生长与表型差异品种间ISSR分析

使用60Co-γ射线对小兰屿蝴蝶兰进行辐射处理,分别对单唇瓣、三唇瓣品种采用15 Gy和20 Gy剂量的处理,采用简单重复序列区间扩增多态(ISSR)分子标记技术对经辐射处理的小兰屿蝴蝶兰材料进行遗传多样性和亲缘关系分析.筛选出8条引物,共扩增出89条清晰的谱带,其中72条表现出多态性,多态比例为80.9%.单唇瓣品种之间遗传相似范围在0.54~0.97,三唇瓣品种之间遗传相似范围在0.54~0.91,说明辐射突变品种之间有丰富的遗传多态性.单唇瓣小兰屿蝴蝶兰经过辐射后,在遗传距离L=0.65处可分为4组,三唇瓣小兰屿蝴蝶兰经过辐射后,在遗传距离L=0.72处可分为7组.观察生长表型发现:经辐射处理的小兰屿蝴蝶兰出现生长快速表型,部分经辐射处理的单唇瓣株系生长率比对照组更高,而经辐射处理的三唇瓣株系生长率普遍比对照组高.非加权组平均法(UPGMA)聚类分析表明:小兰屿蝴蝶兰经过辐射诱变后有不同程度的突变,突变程度最大的品种在遗传距离图谱上自成一支.以上结果为培育优质蝴蝶兰品种奠定了材料基础.

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.

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.

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.

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。

基于滇黄精转录组序列的SSR标记开发及其在黄精属资源分析中的应用

本研究基于滇黄精转录组序列开发简单重复序列(SSR)标记并将其应用于黄精属资源分析。设计合成了45对SSR引物,经PCR扩增验证,选择其中20对SSR引物对75份黄精属资源进行分析。结果表明,共在46416个Unigene中检出含有二核苷酸~六核苷酸重复类型的SSR位点60238个,序列SSR发生频率为22.78%,平均分布距离约7.07 kb;SSR位点中的主导类型是二核苷酸和三核苷酸重复,分别占50.06%和34.89%。测试的45对SSR引物中有34对(75.56%)可扩增SSR条带。筛选的20对引物共扩增出153个条带,多态率为98.69%,每对引物扩增条带4.00~14.00个,平均7.65个,不同SSR标记的多态性信息含量为0.626~0.973,平均为0.870。75份材料的等位基因数和遗传相似系数分别为7.00~52.00个和0.531~0.941,平均值分别为24.65个和0.689,显示出丰富的遗传多样性。基于SSR标记分析的聚类图显示,在遗传相似系数0.666处可将供试材料分为4类,较好地反映了供试材料的分类归属。此外,还发现5份多花黄精材料具有特异性的SSR条带扩增或缺失,可作为不同多花黄精材料鉴定的重要分子依据。本研究开发的SSR标记多态性较高,能够有效揭示黄精属种质资源的遗传多样性,对于丰富黄精分子标记种类、构建遗传图谱、促进种质资源的评价与育种应用、开展特定性状的辅助选择等研究都具有重要的意义。

抗根肿病大白菜材料SSR分子标记的初步筛选

为了明确抗根肿病大白菜材料CCR12049中的抗病基因,进一步开发分子标记。以高抗根肿病的高代自交系大白菜CCR12049、高感根肿病的高代自交系大白菜CM12081、CCR12049和CM12081杂交得到的F_(1)及F_(1)自交构建的F_(2)分离群体为试材,通过人工接种鉴定、聚丙烯酰胺凝胶电泳和序列比对。结果显示,该抗病材料中的根肿病抗性由显性单基因控制;36对引物在2个亲本和F_(1)中初步筛选出4对有多态性的引物,在F_(2)中进一步验证,发现只有1对(cr-26)在F_(2)中的扩增结果与表型鉴定一致;2个亲本及F_(1)的PCR产物测序比对发现,在95~111 bp这个位置,抗病亲本和F_(1)的序列完全相同,但是感病材料在这个位置出现了17个碱基(TCTCTATCTCTTACGCA)的缺失。可以推断抗病材料可能是由于这17个碱基的插入从而表现出抗病性,该标记可以将抗感材料区分开,该标记可以作为一个初步筛选白菜抗根肿病的SSR分子标记来利用。

白三叶转录组SSR位点特征分析及引物开发

本研究旨在了解白三叶(Trifoliumrepens)SSR位点信息和序列特征,开发多态性SSR引物,区分20份白三叶材料。对白三叶不同颜色的花瓣进行转录组(RNA-seq)测序,并根据转录组测序结果中的SSR位点批量设计引物,随机选择60对引物进行多态性验证,选择多态性高的引物指纹图谱构建。结果表明,白三叶转录组中含有SSR位点的序列有24960个,出现频率为13.25%,平均约5.29 kb出现1个SSR位点;白三叶转录组SSR重复碱基类型有6种,其中三碱基重复占比最高(33.70%),其次为双碱基(28.26%)和单碱基重复(25.02%),其他碱基重复类型较低,仅占总SSR的9.01%。A/T(24.85%)、AG/CT(16.48%)和AAG/CTT(8.89%)分别为单碱基到三碱基的优势重复单元;单碱基至六碱基各基元重复次数集中在4~21次,占总数量的98.11%;序列长度变化在12~35 bp,占总数的96.43%;根据SSR位点序列设计出18594对引物,占总SSR的74.50%;60对随机引物中能扩增出条带的有52对,多态性引物17对,分别占86.7%和32.7%;以多态性较高的5对引物构建的白三叶指纹图谱,能够有效地将20个不同白三叶品种区分开。本研究中白三叶转录组SSR位点分布频率高,类型丰富,具有较高的多态性,在白三叶遗传多样性分析、分子标记辅助育种和指纹图谱构建中具有较大的应用潜力。

基于SSR标记的江西省枫香古树遗传多样性评价

采用14对SSR引物对江西省9个枫香古树群体的222个单株进行毛细管电泳测序,利用GenAIEx、CERVUS和Structure软件,进行遗传多样性与聚类分析。结果表明14个SSR位点平均观测等位基因数(Na)为8.143个,平均有效等位基因数(Ne)为2.819个,Shannon信息指数(I)平均值为1.009,平均期望杂合度(He)和观测杂合度(Ho)分别为0.504和0.470,多态信息含量(PIC)平均值为0.513。宁都(ND)群体具有最高的遗传多样性(He=0.551),湾里(WL)群体的遗传多样性最低(He=0.394)。在地区水平,赣南(He=0.534,n=8)和赣北(He=0.505,n=14)地区具有较高的遗传多样性和特有等位基因;赣中地区具有最低的遗传多样性(He=0.473)和最少的特有等位基因(n=2)。分子方差分析(AMOVA)结果表明枫香群体内变异为92%,显著高于群体间变异8%,与遗传分化系数(Fst=0.133)一致,有可能是较高的基因流造成的(Nm=2.995)。主成分分析和聚类分析表明9个群体可分成3大类群,不同枫香古树群体间存在较为强烈的基因渐渗。研究结果为枫香古树的利用及保护提供科学依据,在今后的枫香育种工作中,要加强对枫香古树个体的保护,可以加强在赣北、赣南地区群体内开展优树选择,有可能含有特定的基因类型资源,可以获得更大的遗传增益。

葡萄品种钟山红玉与钟山红杂交后代的SSR鉴定及葡萄果实性状遗传倾向分析

以母本钟山红葡萄、父本钟山红玉葡萄及其50株杂交F_(1)代植株为试验材料,通过简单重复序列(SSR)分子标记技术分析亲本与子代间的亲缘关系并绘制遗传关系聚类图,通过测定杂交F_(1)代与父母本的果实基本品质及酚类物质含量等指标,以进一步了解钟山红玉、钟山红及其杂交F 1代的遗传规律。结果表明,通过SSR分子标记技术检测,发现含有父母本特征性条带的杂交F_(1)代占比不低于86%,杂交F_(1)代与父母本的遗传相似系数为0.857~1.000,果实基本品质性状呈广泛分离的现象,符合数量性状的遗传特点。F_(1)代果皮中的总花色苷含量呈低于双亲遗传的特点,果肉中总类黄酮含量、原花色素含量呈超高亲遗传的特点,但是果实颜色的遗传受到多基因的调控,还需要进一步研究。

疣吻沙蚕转录组SSR位点鉴定及特征分析

【目的】鉴定疣吻沙蚕转录组中简单重复序列(SSR)位点,并分析其分布规律,为该物种多态性SSR分子标记的高效开发提供理论依据。【方法】利用高通量测序技术获得疣吻沙蚕的转录组数据,经过滤、组装后使用MISA搜索鉴定SSR位点,分析其序列特征及分布规律,初步验证SSR引物的有效性,并对有效扩增引物进行多态性分析。【结果】从转录组数据组装获得79420条Unigenes,总长度为104411064 bp,N50值和N90值分别为1902和331 bp。从79420条Unigenes中鉴定到9932个SSR位点,分布在8707条Unigenes上,其中1029条至少包含1个SSR位点,SSR出现频率为8.49%,发生频率为7.43%,丰度为58.85个/Mb(未计入单核苷酸重复)。SSR重复类型共133种,重复次数为4~26次,主要集中在4~15次,长度≥20 bp的SSR约占SSR总数的20%。SSR优势重复基序为二核苷酸、单核苷酸和三核苷酸,发生频率分别为40.29%、32.12%和21.76%,其中二核苷酸重复基序以AT/TA为主,占比79.88%,单核苷酸和三核苷酸分别以A/T和AGC/GCT为主,占比分别为65.92%和29.06%。SSR位点以中等多态性的Ⅱ型为主,共有7296条,占SSR总数的80%,而高度多态性的Ⅰ型有1813条,占SSR总数的20%。筛选到11对多态性引物,每对引物平均产生3.73个多态性片段。【结论】疣吻沙蚕转录组SSR类型丰富,且具有较高的多态性,可用于SSR分子标记开发,对其种质资源评价与保护利用、种群遗传学及分子育种研究等具有实用价值。

Development and utility of EST-SSR markers in Ulva prolifera of the South Yellow Sea

Ulva species can grow rapidly in nutrient-rich habitats causing green tides and marine fouling. A more complete understanding of the reasons behind these outbreaks is urgently required. Accordingly, this study attempts to use microsatellite markers based expressed sequence tag（EST） to analyze the genetic variation of several Ulva prolifera populations in the South Yellow Sea of China. Two hundred and thirty-eight SSRs were identified from 8 179 unique ESTs（6 203 newly sequenced and 1 976 downloaded from NCBI database） and 37 primer pairs were successfully designed according to the ESTs; 11 pairs were selected to detect the genetic diversity and relationship of 69 attached U. prolifera samples and 13 free-floating samples collected from coastal and off-coast areas of the South Yellow Sea. The results of cross-species transferability showed that six of the 11 EST-SSR primers could give good amplification in other five Ulva species and the average allele number was 4.67. Genetic variation analysis indicated that all 82 U. prolifera samples were clearly divided and most samples collected from the same site clustered together as a group in the dendrogram tree produced by unweighted pair-group mean analysis（UPGMA） method and the cluster results showed some consistency with the geographical origins. In addition, 13 free-floating samples（except HT-001-2） were grouped as a single clade separated from the attached samples.