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.

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。

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.

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.

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.

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。

Cluster Analysis on Japonica Rice(Oryza sativa L.) with Good Eating Quality Based on SSR Markers and Phenotypic Traits

Diversity of 60 conventional japonica rice accessions with good eating quality at home and abroad was analyzed using SSR molecular markers, agronomic traits and taste characteristics. A total of 290 alleles were detected in the 60 accessions at 72 SSR loci with the high similarity coefficients varying between 0.600 and 0.924. The loci on chromosome 5 showed the greatest value in average allele number. Additionally, most of the SSR loci could detect 3 to 4 alleles. An UPGMA dendrogram based on the cluster analysis of the genetic similarity coefficients showed that the grouping trend of part of the rice accessions was geographic-related and most of the rice accessions in Jiangsu Province, China were clustered together. Furthermore, many domestic accessions from south and north origins in China were close to the foreign japonica rice varieties, as proved by their pedigree origin from the foreign high-quality sources. For taste characteristics, part of the accessions with excellent taste were clearly clustered into one category though they came from different geographical regions, which indicates that taste characteristics of some varieties were mainly genetically determined. In addition, the agronomic traits of japonica rice with good taste might be closely related with their geographical origins, but the relationship between superior taste characteristics and agronomic traits should be further clarified.

Analysis of SSR in Citrus Sequences from EMBL Database

Abundance of simple sequence repeat (SSR) in Citrus sequences from EMBL database was investigated by usingcomputer program MISA (MIcroSAtellite), which aimed to provide useful information for the development of SSR markers.Among 32 896 sequences of Citrus, 4 987 SSRs were found in 4 167 sequences and the average distance between SSRs wasapproximately 3.5 kb. Mononucleotide repeats (50.6%) were the most abundant repeats. And di-, tri-, tetra-, penta- andhexa-nucleotide repeats were 22.8, 25.2, 1, 0.08, and 0.36%, respectively. The most abundant motif was A/T followed indescending order by AG/CT, AC/GT, AT/TA. AAT/ATT, AAG/CTT, AGC/CGT, ACG/CTG and C/G. They comprised about90% of all microsatellites. Ten primer pairs were designed, and three of them produced clear visible bands among Citrusand its related genera.

De novo assembly of the seed transcriptome and search for potential EST-SSR markers for an endangered, economically important tree species: Elaeagnus mollis Diels

Seeds of Elaeagnus mollis(Elaeagnaceae) produce an edible oil and contain more vitamin E(Ve) than major oil-seed crops. Despite its economic value, there is no information on its genome sequence. Here, we used the Illumina platform to determine the seed transcriptome of E.mollis to identify the genes related to Ve biosynthesis and potential simple sequence repeat(SSR) markers. In total, 100,999 unigenes were obtained with an average length of 605 bp and N50 of 985 bp. Of these unigenes,52,256(51.7%) were annotated in at least one public database(NT, NR, PFAM, Swiss Prot, KOG, KO, and GO)in searches using blastn/x. The unigene annotation identified 15 unigenes encoding six enzymes(GGR, HPPD,HPT/VTE2, MPBQ-MT/VTE3, TC/VTE1, and c-TMT/VTE4) putatively involved in Ve biosynthesis. In addition,16,810 SSRs distributed in 14,057 unigenes were mined.Of these, 2820, 583, and 3423 SSRs were located in the 50–UTR, coding sequence(CDS), and 30–UTR regions,respectively, while the remaining 9984 SSRs had undetermined physical locations. The largest group of repeat motifs comprised mononucleotide repeats(70.76%), followed by dinucleotide(15.59%) and trinucleotide(12.10%)repeats. AG/CT(8.69%) and AAG/CTT(4.15%) were the main dinucleotide and trinucleotide repeats, respectively.Furthermore, 9597 SSR-specific primer pairs were designed. Among 100 primer pairs selected randomly to determine their usefulness, 53 proved to be efficient. To the our best of knowledge, this work is the first study of the E. mollis transcriptome and constitutes valuable genomics data for future genetic engineering studies to alter the amount of Ve. The identified potential EST-SSR markers can be used for population genetics studies and assistedbreeding of E. mollis.

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.

Novel in silico EST-SSR markers and bioinformatic approaches to detect genetic variation among peach(Prunus persica L.)germplasm

Because there are thousands of peach cultivars,cultivar classification is a critical step before starting a breeding project.Various molecular markers such as simple sequence repeats(SSRs)can be used.In this study,67 polymorphic primers produced 302 bands.Higher values for SI index(1.903)suggested higher genetic variability in the genotype under investigation.Mean values for observed alleles(Na),expected heterozygosity(He),effective alleles(Ne),Nei’s information index(h),and polymorphic information content(PIC)were 4.5,0.83,5.45,0.83,and 0.81,respectively.The dendrogram constructed based on Jaccard’s similarity coefficients outlined four distinct clusters in the entire germplasm.In addition,an analysis of molecular variance(AMOVA)showed that70.68%of the total variation was due to within-population variation,while 29.32%was due to variation among populations.According to this research,all primers were successfully used for the peach accessions.The EST-SSR markers should be useful in peach breeding programs and other research.

Identification of necrophagous fly species from 12 different cities in China using ISSR and SCAR markers

Objective:To identify necrophagous fly speies from different regions in China using inter simple sequenc repeat(ISSR) and sequence-characterized amplified region(SCAR) melocular markers and to analyze their gene difference and genetic relationship.Methods:Five carrion fly species were collected from 12 cities and regions in China,including Musca domestica(M.domestica), Lucilia sericata(L.sericata),Chrysomya megacephala(C.megacephala),Helicophagella melanura(H.melanura),Boethcherisca peregrina,and they were studied using ISSR and SCAR markers.Results:Eight ISSR primers were used for amplification of 121 samples.679 clear and stable bands were identified,of which 516 bands were polymorphic.Several species-specific ISSR fragment were cloned and sequenced as an initial effort to derive the SCAR markers.Using M.domestica SCAR specific primers,SCAR-PCR amplification was performed for 8 M.domestca population sample DNA from different regions in China as well as L sericata,C.megacephala, H.melanura and Lucillia cupirina.The result showed only M.domestica produced specificalty 600 bp fragment,but L sericata,C.megacephala,H.melanura and Lucillia cupirina did not produce the same specific fragment.Clustering analysis showed clustering of most flies of M. domestica,C.megacephala and L sericata.M.domestica samples from different regions in China yielded different banding patterns.Conclusions:Application of ISSR-PCR and SCAR markers to identify necrophagous fly species from 12 cities and regions in China is first reported.ISSR-PCR and SCAR markers provide a quick reliable molecular marker technique for the identification of different species of necrophagous fly.

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。

Genetic diversity of Oryza rufipogon Griff. in Hainan Province analyzed by ISSR and SSR markers

Assessment of genetic diversity is an essential component in germplasm characterization and conservation.There are three wild rice species in Hainan Province,including Oryza rufipogon Griff.In order to detect the genetic diversity of different populations of Oryza rufipogon in Hainan,ISSR(inter-simple sequence repeat)and SSR(simple sequence repeat)markers were used to investigate 180 accessions from six localities in Hainan.Fourteen ISSR primers amplified 185 alleles with 171(92.43%)polymorphic,the number of alleles ranged from 8 to 17,with an average of 13.14 alleles per locus.Thirty-eight pairs of SSR primers used in this study amplified 213 alleles with 190(89.20%)polymorphic,the number of alleles ranged from 2 to 14,with an average of 5.66 alleles per locus.Both ISSR and SSR analyses revealed a high level of genetic diversity in the wild populations.The population with the highest genetic diversity is Wanning(WN),and the population with lowest genetic diversity is Wenchang(WC).The results of a UPGMA cluster using the NTSYS program showed that each population has a low degree of genetic differentiation.Furthermore,the Mantel test revealed that the genetic similarities detected by ISSR and SSR were significantly correlated(r=0.8634,t=93.67)when detecting genetic diversity at the species level.The two molecular marker systems were able to determine the genetic diversity among Oryza rufipogon,and the two groups of indexes obtained by using the two markers have a high level of consistency.

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。

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。

Development and mapping of SSR markers linked to resistance-gene homologue clusters in common bean

Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.