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.

油菜简单重复序列SSR(simple sequence repeat)研究进展

简单重复序列(simple sequence repeat, SSR)是重复单元少于6个核苷酸重复序列,广泛分布于动植物基因组中,呈孟德尔遗传,已被作为一种理想的共显性标记应用于动植物遗传研究中。本文重点介绍了SSR分类、特点,及近几年来油菜SSR标记的开发和SSR技术在油菜基因定位、品种鉴定中的应用,并对SSR标记在油菜中的应用进行了探讨。

Genetic Diversity of Chinese and Swedish Rapeseed (Brassica napus L.) Analyzed by Inter-Simple Sequence Repeats (ISSRs)

We have compared genetic diversity of 24 Chinese weak-winter, Swedish winter and spring B. napus accessions by inter-simple sequence repeats (ISSRs). By cluster analysis (UPGMA) based on 125 polymorphism bands amplified with 20 primers, the 24 accessions were divided into three groups. Six Swedish winter lines and eight Chinese weak-winter lines were in the group I and the groupⅡwere two Chinese weak-winter lines XiangyoulS and Bao81. The third group contained eight Swedish spring lines. Principal co-ordinates analysis (PCO) showed similar groupings to cluster analysis. Results from cluster analysis and PCO analysis showed very clearly that Chinese weak-winter, Swedish spring and winter accessions were distinguished from each other and Chinese weak-winter accessions in this study were genetically closer to Swedish winter accessions than to Swedish spring accessions. The Chinese weak-winter accessions had larger diversity than Swedish spring or winter accessions did. This study indicated that ISSR is a suitable and effective tool to evaluate genetic diversity among rapeseed germplasm.

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.

Identification and Characterization of Simple Sequence Repeats (SSR) Markers Associated with Downy Mildew Resistance Locus “<i>RPV1</i>” in Grapes

The cultivation of grapes is severely impacted by the emergence of downy mildew (DM) disease which negatively affects quality and yield possibly resulting in heavy losses. Due to certain shortcomings in the usage of fungicides and the development of new cultivars by plant breeding, marker assisted selection (MAS) will be an efficient alternative method to introduce desired genes into the cultivated varieties in a short time period. The Simple sequence repeats (SSR) markers seem to be the most popular genetic marker of choice for MAS. In the present study, we identified 14 new SSR markers in RPV1 locus that are associated with downy mildew resistance in grapes. The characterization of the identified markers was carried out on the basis of various parameters such as types of repeat motifs, number of repeats, different classes and structure of microsatellites. Additionally, SSR genotyping in 56 different grape accessions was done to determine the susceptibility or resistance of these accessions to DM.

基于ISSR-PCR体系鉴别樟芝单核体交配型

通过原生质体单核化技术获得樟芝单核体,基于内转录间隔区(internal transcribed spacer,ITS)序列进行鉴定,采用14个引物对简单重复序列区间(inter-simple sequence repeats,ISSR)进行多态性扩增,筛选条带清晰、重复性好的引物用于樟芝单核体的交配型鉴定。结果表明,通过原生质体单核化技术获得31个樟芝单核体,经ITS序列分析确定获得的单核体为樟芝。以单核体S2、S10、S14、S27的DNA为模板,对14条ISSR引物进行初步筛选,得到4个条带清晰、重复性好的引物P7、P9、P21、P25用于交配型鉴定。单核体S9和S25为同一交配型,两者与S2为不同交配型,通过镜检进一步验证S9、S25可以与S2形成具有锁状联合的双核菌株。该方法可明显缩短樟芝单核体交配型的鉴定时间。

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。

Mapping a Rice Glabrous Gene Using Simple Sequence Repeat Markers

Inheritance analysis and gene mapping of the glabrous trait were conducted using the crosses between a pubescent rice material Chuanxiang 29B （an aromatic elite maintainer line of hybrid rice）, and two glabrous rice materials, Lemont and R2 （R2 is a new restorer line of hybrid rice）. All F1 plants from the two crosses had pubescent leaves, confirming that the pubescence trait in Chuanxiang 29B is dominant over the glabrous trait. In F2 individuals, the segregation ratio of three pubescent to one glabrous plant suggests that a single glabrous gene was present in Lemont and R2. Using the bulked segregant analysis, the linkage analysis of the simple sequence repeat markers showed that the glabrous gene, gl-1, was flanked by GL311 and GL8 with the genetic distances of 0.19 and 0.92 cM on chromosome 5, respectively.

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 Diversity among Parents of Hybrid Rice Based on Cluster Analysis of Morphological Traits and Simple Sequence Repeat Markers

The genetic diversity of 41 parental lines popularized in commercial hybrid rice production in China was studied by using cluster analysis of morphological traits and simple sequence repeat （SSR） markers. Forty-one entries were assigned into two clusters （i.e. early or medium-maturing cluster; medium or late-maturing cluster） and further assigned into six sub-clusters based on morphological trait cluster analysis, The early or medium-maturing cluster was composed of 15 maintainer lines, four early-maturing restorer lines and two thermo-sensitive genic male sterile lines, and the medium or late-maturing cluster included 16 restorer lines and 4 medium or late-maturing maintainer lines. Moreover, the SSR cluster analysis classified 41 entries into two groups （i.e, maintainer line group and restorer line group） and seven sub-groups. The maintainer line group consisted of all 19 maintainer lines, two thermo-sensitive genic male sterile lines, while the restorer line group was composed of all 20 restorer lines. The SSR analysis fitted better with the pedigree information. From the views on hybrid rice breeding, the results suggested that SSR analysis might be a better method to study the diversity of parental lines in indica hybrid rice.

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。

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.

Evaluation of Population Structure, Genetic Diversity and Origin of Northeast Asia Weedy Rice Based on Simple Sequence Repeat Markers

Weedy rice exerts a severe impact on rice production by competing for sunlight, water and nutrients. This study assayed the population structure, genetic diversity and origin of Northeast Asia weedy rice by using 48 simple sequence repeat markers. The results showed that weedy rice in Northeast Asia had a high genetic diversity, with Shannon＇s diversity index （I） of 0.748 and the heterozygosity （He） of 0.434. In each regional population, I value varied widely. The widest range of I （0.228-0.489） was observed in the weedy rice of Eastern China, which was larger than that of Northeast China and Korea （0.168-0.270）. The F-statistics of regional populations （Fis, Fit and Fst） also showed higher values in the weedy rice of Eastern China than those of Northeast China and Korea All weedy rice accessions were grouped into two clusters in the unweighted pair group method with arithmetic mean cluster analysis dendrogram, namely Eastern China branch and Northeastern China plus Korea branch. There was significant differentiation in genetic characteristics in weedy rice of northeastern and eastern Asia, especially in Eastern China.

基于SSR构建中国主栽草莓品种指纹图谱

【目的】利用SSR分子标记构建中国主栽草莓品种的DNA指纹图谱,为保护育种者权益和实现草莓资源的高效鉴定提供基础数据。【方法】以中国目前主栽的草莓品种(70个)为材料,采用荧光毛细管电泳技术对28个SSR分子标记进行评估,利用筛选后的高效SSR标记构建草莓品种的DNA指纹图谱。【结果】从28对引物中选出了5对多态性较高、重复性较好的核心引物。核心引物共检测出40个等位位点,平均每对引物检测出8个等位位点;共检测到142个带型,平均每对引物检测出28.4个带型;各标记的多态信息含量(PIC)为0.79~0.87,平均PIC值为0.83;聚类分析结果表明,草莓品种之间的相似系数范围为0.65~0.97。【结论】成功构建了具有唯一对应关系的70个草莓品种的DNA指纹图谱,为未来草莓品种的资源保护和利用推广提供数据支撑。

蓝莓自花和异花授粉花柱转录组SSR位点分析

为鉴定蓝莓自交不亲和基因,本研究对“杰兔”蓝莓品种自花和异花授粉花柱进行了转录组测序,对简单重复序列(SSR)位点进行分析,对含SSR位点且差异表达的非冗余序列(Unigene)进行基因本体论(GO)功能富集和京都基因与基因组数据库(KEGG)注释分析,并开展SSR标见引物设计。结果表明,蓝莓花柱转录组共获得85890条Unigene,其中有21962个Unigene含有26557个SSR位点,发生频率为25.57%。在所有SSR位点中包含有297种重复基元,以单核苷酸和二核苷酸重复为主要重复类型,占总SSR的89.52%;其中A/T类型和AG/CT类型SSR位点数占SSR总数的83.44%。针对所有SSR位点,共设计出17212对引物,成功率78.37%。差异转录组筛选到58、27、16、3、2和1条含有SSR位点的差异表达Unigene,分别与植物激素信号转导、细胞骨架、蛋白质泛素化、花粉管生长、核糖核酸酶T2家族和细胞程序化死亡等代谢途径相关。本研究丰富了蓝莓的EST-SSR分子标记,为蓝莓自交不亲和的分子机制研究提供了参考。

基于ISSR标记分析连云港地区野生灵芝种质资源遗传多样性及亲缘关系

为分析连云港地区野生灵芝种质资源之间的亲缘关系和多样性水平,以连云港地区15株野生灵芝为研究对象,采用ISSR分子标记进行遗传多样性及亲缘关系分析。15株野生灵芝中扩增出多态性条带为40条,片段大小在250~2000 bp之间,平均等位基因位点1.8个,平均有效等位基因数1.2454,平均Nei's遗传多样性0.1742,平均Shannon信息指数0.2925,有效等位基因数(Ne)的变化范围为1.0000~1.6423,Nei's遗传多样性(He)的变化范围为0.1031~0.3911,Shannon信息指数(I)的变化范围为0.2235~0.5799,根据ISSR分子标记的遗传聚类图,15株灵芝相似系数范围在0.415~0.866之间,相似系数为0.63时,可以将15株野生灵芝分为四组。研究结果为连云港地区灵芝种质资源分类、保护和品种创制提供了参考依据。

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.