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.

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.

Genetic analysis of selected Sargassum fusiforme (Harvey) Setchell (Sargassaceae, Phaeophyta) strains with RAPD and ISSR markers

Since the 1980s,Sargassum fusiforme has been cultivated in Zhejiang,South China,and nowadays it becomes one of the important commercial seaweeds in China.With traditions of eating habits in the East Asian countries,this brown alga is used as food,because it contained functional oligo/polysaccharides and chemical components,and was regarded playing roles in antioxidant activities and regulating immunology.Through over 15 years’selection,breeding and cultivation,we obtained three strains with good traits and testified their characters during the production,which included the cultivars with high yield and other two good characters,either all the selected strains were applied in the Sargassum production.To avoid confusion during the selection and nursery,it was preferred to establish one fingerprint for distinguishing the Sargassum cultivars from different strains.Random amplified polymorphic DNA(RAPD)and inter-simple sequence repeat(ISSR)methods were adopted to analyze the genetic diversities of the selected S.fusiforme strains.With that,one fingerprint with RAPD markers was constructed,and one sequence characterized amplifi ed region(SCAR)marker to S.fusiforme was obtained.It is indicated that the applied fingerprint could be valid in S.fusiforme genetic and germplasm justification,and will be positive to molecular marker assistance in its selection and cultivation.

The loss of genetic diversity during captive breeding of the endangered sculpin, Trachidermus fasciatus, based on ISSR markers: implications for its conservation

Abstract Inter-simple sequence repeat （ISSR） markers were used to determine the genetic variation and genetic differentiation of cultured and wild populations of Trachidermus fasciatus, an endangered catadromous fish species in China. Six selected primers were used to amplify DNA samples from 85 individuals, and 353 loci were detected. Relatively low genetic diversity was detected in the cultured population （the percentage of polymorphic loci PPL=73.80%, Nei＇s gene diversity h--0.178 2, Shannon information index I=0.276 9）. However, the genetic diversity at the species level was relatively high （PPL-91.78%; h = 0.258 3, I= 0.398 6）. The UPGMA tree grouped together the genotypes almost according to their cultured and wild origin, showing distinct differences in genetic structure between wild and cultured populations. The pairwise F^t values confirmed significant genetic differentiation between wild and cultured samples. The cultivated population seems to be low in genetic diversity as a result of detrimental genetic effects in the captive population. The results suggest that ISSR markers are effective for rapid assessment of the degree of diversity of a population, thus giving important topical information relevant to preserving endangered species.

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.

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.

Development and Characterization of Microsatellite Markers in Brassica rapa ssp.chinensis and Transferability Among Related Species

Simple sequence repeat （SSR） or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an intersimple sequence repeat （ISSR）-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage （Brassica rapa）. A total of 190 SSRs were obtained. Among these, AG or CT （54.7%） was the most frequent repeat, followed by AC or GT （31.6%） of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content （PIC） value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.

Genome-wide development of interspecific microsatellite markers for Saccharum officinarum and Saccharum spontaneum

Sugarcane has a large,complex,polyploid genome that has hindered the progress of genomic research and molecular marker-assisted selection.The user-friendly SSR markers have attracted considerable attention owing to their ideal genetic attributes.However,these markers were not characterized and developed at the genome-wide scale due to the previously lacking high-quality chromosome-level assembled sugarcane genomes.In this present study,744305and 361638 candidate SSRs were identified from the genomes of S.officinarum and S.spontaneum,respectively.We verified the reliability of the predicted SSRs by using 1200 interspecific SSR primer pairs to detect polymorphisms among 11 representative accessions of Saccharum,including S.spontaneum,S.officinarum,S.robustum,and modern sugarcane hybrid.The results showed that 660 SSR markers displayed interspecific polymorphisms among these accessions.Furthermore,100 SSRs were randomly selected to detect the genetic diversity for 39 representative Saccharum accessions.A total of 320 alleles were generated using 100 polymorphic primers,with each marker ranging from two to seven alleles.The genetic diversity analysis revealed that these accessions were distributed in four main groups,including group I(14 S.spontaneum accessions),group II(two S.officinarum accessions),group III(18 modern sugarcane hybrid accessions),and group IV(five S.robustum accessions).Experimental verification supported the reliability of the SSR markers based on genome-wide predictions.The development of a large number of SSR markers based on wet experiments is valuable for genetic studies,including genetic linkage maps,comparative genome analysis,genome-wide association studies,and marker-assisted selection in Saccharum.

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.

Mapping of a Wheat Resistance Gene to Yellow Mosaic Disease by Amplified Fragment Length Polymorphism and Simple Sequence Repeat Markers

Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.

14份杏种质的ISSR分析

以冀光为试材,研究了PCR反应体系的主要成分及退火温度对杏ISSR扩增结果的影响.同时,从42对ISSR引物中筛选出12对扩增条带清晰、多态性好的引物进行扩增,并运用UPGMA聚类分析法,分析了12份杏种质的亲缘关系及多样性.结果表明:在20 μL的反应体系中,模板DNA含量在10～80 ng均能得到较好的扩增;dNTP用量对扩增无明显影响;而引物、Mg2+的最适浓度分别为0.25 μmol/L、0.25 mmol/L;Taq酶在0.5～4 U均能得到好的扩增条带;退火温度在50～52.1℃范围内均能得到清晰的条带;并在此基础上建立了杏ISSR反应体系.当相似系数在0.444～0.452之间时,将12份杏材料划分为3类:①普通杏(Amentaca vulgaris),西伯利亚杏(A.sibirica),辽杏(A.mandshurica),藏杏(A.holosericea)类型;②仁用杏品种一窝蜂;③紫杏(A. dasycarpa).

ISSR-PCR在石斛种间鉴别中的应用

目的 采用ISSR-PCR方法对石斛属9种植物进行鉴别,探讨不同种石斛在DNA分子水平上的差异。方法 选取10条由SSR组成的引物,对9种石斛进行PCR扩增及琼脂糖凝胶电泳分析。结果 10条引物中有7条扩增出多态性条带。每条引物可检测的多态性位点最少7个,最多14个,扩增片段大小为220-1260 bp。其中,引物UBC-807和UBC-864具有较高的多态性条带比率,均可以独立将所有被测种区分开来。结论ISSR-PCR作为一种简便、可靠的分子标记鉴定技术,可以作为石斛属种间鉴别的方法之一。

云南省苹果棉蚜种群遗传多样性的ISSR分析

苹果棉蚜是世界性检疫害虫,在云南省苹果主要产区造成严重的危害.从100条ISSR(Inter-si mple Sequence Repeat)引物中共筛选出4条多态性和重复性好的引物,研究了云南省昆明市、丽江市、昭通市3个苹果主产区共57个采样点的苹果棉蚜种群间及种群内的遗传多样性.4条引物在苹果棉蚜DNA中共扩增出49个位点,多态位点百分率为75.51%.由Shannon信息指数和Nei s指数估算的种群间遗传分化系数为42.48%和17.53%,种群内遗传分化系数分别为57.52%和83.17%,表明苹果棉蚜大部分遗传变异主要存在于种群内部.各种群的遗传距离聚类分析表明,昆明种群与昭通种群的亲缘关系较丽江种群为近.研究结果为我国苹果棉蚜的遗传防治研究提供了分子依据,为进一步研究苹果棉蚜的分子进化提供了背景资料.

园艺作物的ISSR分子标记研究及应用

ISSR(Inter-simple sequence repeat)是一种基于微卫星序列发展起来的新型分子标记方法,具有无需知道任何靶标序列的微卫星背景信息、遗传多态性高、稳定高效、检测快速等特点。目前,ISSR分子标记技术在园艺作物的遗传多样性研究、品种鉴定、遗传图谱构建、基因定位及分子标记辅助育种等方面得到了广泛应用,文章就ISSR标记的原理、方法、特点及其在园艺作物研究中的应用加以综述。

ISSR分子标记及其在树木遗传育种研究中的应用

在比较ISSR和其它分子标记的原理和特点的基础上,综述了ISSR分子标记在树木遗传多样性研究、亲缘关系及系谱分析、优良种质和品种鉴定及遗传连锁图谱的构建等领域的研究进展,指出了目前ISSR分子标记在树木遗传改良和辅助育种等研究领域存在的问题,并提出今后的研究方向。

ISSR-PCR对我国旋毛虫7个地理株的分子鉴定

目的探讨对不同种和地理株旋毛虫进行分子鉴定。方法利用简单重复序列区间-PCR(inter simple sequencerepeat-PCR,ISSR-PCR)标准引物对5种旋毛虫及我国旋毛虫7个地理株基因组DNA进行扩增,并观察影响扩增效果的因素。结果5种旋毛虫均分别扩增出了各自的特异性条带:旋毛虫(T1)2条(950bp和850bp),乡土旋毛虫(T2)1条(850bp),布氏旋毛虫(T3)3条(1 300bp、950bp和700bp),伪旋毛虫(T4)1条(600bp),纳氏旋毛虫(T7)有4条带(1 700bp、1 450bp、1 050bp、900bp)。我国有6个猪源旋毛虫地理株均扩增出了与T1相同的2个特异性条带(950bp、850bp)。此外,T1、河南株、云南株、哈尔滨株、黑龙江同江株还扩增出了另外2条弱带(500bp和400bp);但湖北株和天津株则无这2条弱带,湖北株扩增出了1 350bp的条带,天津株扩增出了1 600bp的条带。非猪源的广西株与T1相比,缺少950bp的条带。对1条旋毛虫肌幼虫,在80%酒精保存24w的肌幼虫,在10%甲醛、0.2%叠氮钠及0.05%柳硫汞溶液保存9 w的肌幼虫,应用ISSR-PCR均可扩增出其特异性条带。结论ISSR-PCR用于旋毛虫种的分子鉴定具有良好的稳定性和敏感性;我国6个猪源旋毛虫地理株均为T1,其中云南株、河南株、哈尔滨株及黑龙江同江株可归为一类,湖北株和天津株可归为另一类,来自果子狸的广西株分类地位待定。

应用ISSR-PCR对10个菊花品种进行遗传多样性分析

中国的菊花(Chrysanthemum morifolium Ramat.)品种数量众多,形态变异丰富,适应性强,分布广泛,遗传背景非常复杂,这为菊花品种的资源调查、分类鉴定、遗传多样性分析等带来了困难。试验采用分子标记技术,对福白菊(C.morifolium cv.Fubaiju)、杭白菊(C.morifolium cv.Hangbaiju)等10个菊花品种进行了分类鉴定及亲缘关系研究,结果显示,在简单序列重复区间扩增-聚合酶链式反应(Inter-simple sequence repeat-Polymerase chain reaction,ISSR-PCR)分子标记反应体系及引物的筛选方面,选择了条带数量多和清晰度高的菊花反应体系,应用野生福白菊对39条ISSR引物进行了筛选,淘汰了扩增效果差、带型不易辨认的引物,最终确定12条引物用于菊花品种的鉴定。ISSR-PCR聚类分析结果表明,12条ISSR引物扩增出了185条清晰的、重复性好的ISSR条带,其中多态性条带有176条,多态性比率高达95.1%。应用NTSYS 2.10e软件进行UPGMA法聚类分析,在相似系数0.55处可将10个菊花品种大致分为3类,结果福白菊与其他杭菊品种在遗传上有较大的差异。

广西桑树品种遗传多样性ISSR分析

利用ISSR(inter-simple sequence repeat)分析了广西桑树品种的亲缘关系.刘圩7号、灵太3号、恭同5号、大寺12号、池塘1号、刘圩2号、那学8号、那陈2号、那学14号、小董1号、板朝1号、冯屋1号、邕新3号、邕新11号、恭同9号、太平2号、沙油4号、板屯车2号有较近的亲缘关系;大寺特号、冯屋5号、池塘4号、太平新1号、恭城4号有较近的亲缘关系;大寺5号、灵太1号、恭江1号、涠盛4号、灵太2号、板罗1号、那楼14号、恭同4号有较近的亲缘关系;钦州桑、大新白桑、广西鸡桑、全州长穗桑、环江1号、钦州长果桑、桂772、涠州岛白桑、邕新荆桑12号、隆林鬼桑、隆林蒙桑有较近的亲缘关系.白桑种遗传变异大;广东桑种亲缘关系靠近白桑,支持中国植物志、GenBank将广东桑置于白桑的变种.野生桑种与栽培桑种遗传背景明显不同.钦州桑、桂772、邕新荆桑12号遗传背景倾向野生桑,在抗性、速生方面有独特优点,在今后杂交育种中应注意利用.

云南省优良核桃品种三台核桃与普通铁核桃的ISSR分子鉴别

三台核桃是经过长期天然和人工选育的云南省目前主要推广的优良核桃品种之一;铁核桃是云南省天然分布、品质较差的核桃类群,常作砧木。研究采用ISSR-PCR分子标记技术,通过广泛的引物筛选及特征指纹图谱分析,对二者进行分子鉴别,同时对113份三台核桃样本和20份铁核桃样本进行了基于遗传距离的UPMGA聚类。结果表明,引物825对于三台核桃223,329,368和421 bp的4个特异标记,可以将二者区分开,并由此绘制了三台核桃和铁核桃的特征分子指纹图谱。