Identification of Genetic Purity of Bitter Gourd Hybrid by ISSR Markers

Objective] This study was conducted to verify the feasibiIity of ISSR marker for identifying genetic purity of bitter gourd hybrid, and thus to provide an effective method for seed purity test in production practices. [Method] The DNA fin-gerprints of a bitter gourd cuItivar Xiuyu 1 and its parents were analyzed using IS-SR marker with 91 primers. [Result] Two primers ISSR-845 and ISSR-891 which ampIified two DNA bands of 510 and 300 bp respectiveIy from F1 generation and its parents were screened out from 91 primers. ISSR-845 couId distinguish the male parent from F1 hybrid and the female parent, whiIe ISSR-891 couId distinguish the female parent inbred Iine from Xiuyu 1. Seed purity test with the specific markers gave the same resuIt with fiIed trials based on morphoIogical identification. [Conclu-sion] ISSR marker is an accurate, simpIe and effective method for seed purity test bitter gourd hybrid, and thus can be used in production practices.

ISSR Marker and ITS Sequence Study of Melampsora Larici-populina

To compare the differences in intertranslation space of ribosomal DNA （ITS） of Melampsora larici-populina, between the isolates from China and isolates from other countries, this study investigated ITS sequences and ITS polygenetic tree based on 11 isolates that were collected from 5 races in different parts of China. The results indicated that there was no difference among the ITS sequences of 11 isolates from China. The ITS sequence of isolates from China was more homogeneous with that of isolates from Britain compared with France, Germany, and Canada. Intersimple sequence repeat （ISSR） markers were also used to study the genetic division of Melampsora larici-populina, and the results showed that the 11 tested isolates could be divided into Western population and Northern population. Genetic diversity index of race C2 was significantly different from that of races C4, C3, and C1, and no significant differences were observed among the other races. Pathogenicity division of races must not harmonize with their genetic division, except race C2. The ITS region is conservative, and ITS sequence is not fit for studying the differences that existed among the races. ISSR marker can be used for intraspecies population study, and Melampsora larici-populina in China can be divided into two populations.

The Application of ISSR Markers to Identify the Fertility Restorer Gene Rf6 in T. timopheevii Cytoplasmic MaleSterile Wheat(Triticum aestivum)

Inter-simple sequence repeat (ISSR) analysis was carried out on a F2 population of 147 plants derived from a cross between a wheat male fertility restorer line 2114 and a male sterile line ND44A. Out of 43 primers examined, 18 primers produced distinguishable, polymorphic bands between the two parents. Linkage analysis in the mapping population showed that two markers UBC-808 and UBC-848 were closely linked with the restorer gene Rf6 of the Triticum timopheevii CMS system. The distance between the two markers and the restorer gene was 7.9 cM and 4.9 cM, respectively. Also two parents were screened with 181 pairs of SSR primers, of which, 34.3% showed polymorphisms. But no locus was found linked with the restorer gene. Compared with the SSR technique, the ISSR approach used in the experiment provided more information and proved to be a valuable method to identify alien fragments.

Study on Genetic Diversity of Turf Bamboo Based on ISSR Marker

[ Objective ] This study aimed to analyze the genetic diversity of turf bamboo species by using ISSR molecular marker technology. [ Method] Excellent turf bamboo species imported from France and domestic ornamental turf bamboo species were used as experimental materials for ISSR analysis, cluster analysis was conducted on 10 species of turf bamboo materials based on the obtained ISSR molectfiar ma^kers. [ Result] A total of 201 clear bands with good repeatability and high polymorphism were amplified with 21 ISSR primers, with a polymorphism rate of 93.1% ; similarity coefficients between different turf bamboo species ranged from 0.275 to 0.571, with an average similarity coefficient of 0. 357 ; according to the results of ISSR markers, 10 different ornamental turf bamboo species were di- vided into three categories by using UPGMA cluster analysis method. [Conclusion] Turf bamboo with different sources had relatively high genetic diversity, this study had provided theoretical and technical basis for the breeding, cultivation and vromotion of ornamental turf bamboo.

Population genetic structure of Culex quinquefasciatus in India by ISSR marker

Objective:To characterize the genetic structure of various populations of Culex quinquefasciatus (Cx.quinquefasciatus) from India representing different geoclimatic locations.Methods:Inter simple sequence repeat(ISSR) markers were used.A set of 20 primers were screened with the laboratory populations of mosquito species.Finally the IS 40 primer was chosen based on the scorable banding pattern showing 100 percent polymorphism among the various populations.The statistical analysis was done using POPGENE 1.31 software.The consensus tree was generated based on UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5.Results:The cluster analysis shows the main cluster which is divided into two sub cluster representing all the populations separated as per their phylogeographic and geoclimatic condition.Conclusions: The findings will be helpful in understanding the population variation under different ecological conditions and development of effective vector management strategies.

Identification of ISSR markers linked to flowering traits in a representative sample of Eucalyptus cladocalyx

Early flowering and flower abundance have long been considered desirable traits in eucalypt breeding programs. In particular, flowers of Eucalyptus cladocalyx provide a nectar source for the production of honey in arid ecosystems. To identify inter-simple sequence repeat （ISSR） markers that are associated with early flowering and flower abundance in the southern Atacama Desert, we used a sample of 47 trees, representing five Australian prove- nances of E. cladocalyx. A unified mixed linear model （which considered the effect of genetic structure and the kinship relationship among trees） revealed that three loci were significantly associated with early flowering, which accounted for 10-16 % of the phenotypic variation, while two loci accounted for 11-13 % in flowering intensity. Locus ISO1-500 bp was associated with both flowering traits. This result is consistent with our previous findings indicating that marker-assisted selection on early flowering should have significant and positive impact on flowering intensity. The application of marker-assisted selection to identify trees that flower early and intensively may increase honey production, a resource that generates additional income for the local farmers of the southern Atacama Desert.

基于表型性状和ISSR分析的5个核桃品种亲缘关系鉴定

[目的]选择四川省栽培面积最大、产量最多的核桃品种“盐源早”(Juglansregia“Yanyuanzao”),探索“盐源早”的亲缘关系。[方法]选择“盐源早”、“三台泡核桃”(Juglanssigillata‘Santai’)、“漾濞泡核桃”(Juglanssigillata“Yangbi”)、“温185”(Juglansregia“Wen185”)、“新新2”(Juglansregia“Xinxin2”)5个核桃品种为试验材料,测定单果重、果仁重、出仁率、坚果三径、果形指数、种壳厚以及果仁成分(粗蛋白、粗脂肪)等指标,通过方差、相关性、聚类与主成分分析等探索5个核桃品种的表型性状和果仁成分多样性;利用ISSR分子标记技术探究“盐源早”等5个核桃品种的遗传多样性和遗传关系。[结果]果实的表型性状与果仁成分多样性分析结果表明,5个核桃品种的单果重、果仁重、出仁率、侧径、横径、纵径、果形指数、种壳厚及果仁成分(粗脂肪、粗蛋白含量)之间差异极显著或显著(P<0.01或P<0.05)。聚类分析和主成分分析结果表明,可将5个核桃品种聚为三大类群,第Ⅰ类为“温185”,第Ⅱ类为“新新2”“盐源早”“漾濞泡核桃”,第Ⅲ类为“三台泡核桃”。利用ISSR分子标记筛选出10条图谱条带清晰,背景明亮,重复性强,多态性较好,稳定性强的引物。通过对扩增图谱进行多态性分析结果表明,5个核桃品种的有效等位基因数均值为1.3370,Nei’s基因多样性指数均值为0.1935,Shannon信息指数均值为0.2858。聚类分析和主成分分析表明,“盐源早”和“漾濞泡核桃”聚为一类,表明亲缘关系表现较近,且相同地区的“温185”和“新新2”聚为一类,“三台泡核桃”聚为一类。[结论]通过对5个核桃品种的果实表型性状、果仁成分及ISSR分子标记的聚类分析得出,“盐源早”与“漾濞泡核桃”亲缘关系较近。

Primer Screening on Germplasm Resources of Mallotus oblongiolus by ISSR Molecular Marker

[ Objective] To provide effective primers for the rapid and accurate ISSR analysis of the germplasm materials of Mallotus oblongiolus （Miq.） Muello-Arg.. [Method] The modified CTAB method was used in the extraction of the genomic DNA. 99 ISSR primers were used in the ISSR-PCR amplification for 20 germplasm materials from 10 populations in Hainan Island, so that some primers, which were suitable to all gerplasm materials of M. oblongiolu, could be selected. [ Result] 15 effective primers with characteristics of rich polymorphism, clear bands, and good repeatability were selected from 99 test primers. The 15 primers selected were used in the ISSR-PCR amplification for 66 germplasm materials of M. oblongiolus. From all of which the abundant and distinct DNA fingerprintings could be obtained. 286 DNA bands were obtained, and of which 231 bands were polymorphic, which amounted to 80.77% of the total bands amplified. And 19.1 bands could be obtained with each primer, averagely. [ Conclusion] The 15 primers selected could be effectively applied to ISSR analysis of the germplasm resources of M. oblongiolus.

Morphological and ISSR molecular markers reveal genetic diversity of wild hawthorns (Crataegus songorica K. Koch.) in Xinjiang, China

The wild hawthorn species, Crataegus songorica K. Koch., is an important wild germplasm resource in Xinjiang, China that has been endangered in recent years. The genetic diversity of C. songorica K. Koch. germplasm in five populations from Daxigou, Xinjiang, China were evaluated based on phenotypic traits and ISSR molecular markers to provide basic infor- mation on resource protection, rational utilization and genetic improvement. The F-value for the phenotypic differentiation coefficient of the 33 traits measured ranged from 0.266 to 15.128, and mean value was 13.85%. The variation among populations was found to be lower than that within population. A total of 303 loci were detected within the five populations by 12 primers. Within 298,polymorphic loci, the polymorphism was 98.35%, showing a high genetic diversity in C. songorica K. Koch. The gene diversity within population, total population genetic diversity, genetic differentiation coefficient and gene flow were 0.2779, 0.3235, 0.1408, and 3.0511, respectively. Our results showed that C. songorica K. Koch. from Xinjiang has a high level of genetic diversity at both the phenotypic and molecular levels. Significant genetic differentiation existed within population and the differentiation trend showed a regional association. And in this study, in situ and ex situ conser- vation approaches were raised for wild hawthorn protection utilization.

Genetic variation of the genus Kengyilia by ISSR markers

We investigated the genetic variation within 32 accessions distributed to 14 species and one variety by using ISSR(inter-simple sequence repeat)markers.The results showed that genetic variation was relatively higher among the accessions.A total of 593 bands were amplified by 12 ISSR primers,of which 535 bands(90.2%)were polymorphic.Eleven to 80 polymorphic bands were amplified from each prime,with an average of 44.6 bands.The interspecies GS(genetic similarity)value ranged from 0.430 to 0.866,and the average was 0.620.Cluster analysis showed that all accessions could be classified into 4 groups by ISSR markers.The different accessions in a species were clustered together,but they had genetic variation in molecular levels.There was obvious interspecies genetic variation.Species with similar morphological characteristics and from the same areas or neighboring geographical regions were clustered together and had close relationships.ISSR markers are useful in analyzing interspecies variation in Kengyilia.

基于ISSR标记的96份兰属种质资源遗传多样性分析及指纹图谱构建

为加强我国兰属种质资源的保护利用,本研究通过ISSR分子标记对96份兰属种质进行多样性分析和指纹图谱构建。结果显示,共筛选出11条可扩增清晰条带的多样性引物,在96份材料共检测67条多态性条带,平均多态性条带比例为73.63%;等位基因数(Na)为1.925,有效等位基因数(Ne)为1.450,Nei′s遗传多样性指数(H)为0.277,Shannon多样性指数(I)为0.427,多态性位点百分比(PPL,percentage of polymorphic loci)为92.54%;种群内基因多样性(Hs)为0.1934,基因分化度(Gst)为0.3009,总遗传多样性指数(Ht)为0.2767,种群间的平均基因流(Nm)为1.1619,种群间的两两遗传分化固定指数值范围为0.002~0.527,平均值为0.325。系统聚类结果表明,兰属种群间遗传分化程度高,8个种群可分为3类,春兰和墨兰为一大类,寒兰、春剑、蕙兰、莲瓣兰、建兰为第二类,杂交种独为一类,与其他两类种群之间的遗传距离较大。主坐标分析表明,莲瓣兰和春兰表现出较远的亲缘关系。本研究筛选出6对引物构建了96个品种的指纹图谱二维码。本研究结果可为今后兰属新品种选育及品种鉴定提供重要依据。

基于形态标记和ISSR分子标记的长瓣铁线莲遗传多样性分析

为快速区分鉴定市场上繁多的长瓣铁线莲(Clematis macropetala)品种,了解长瓣铁线莲间的遗传多样性水平及亲缘关系,本试验利用形态标记结合ISSR分子标记对25个长瓣铁线莲品种和1个野生种进行遗传多样性研究。结果表明:14个数量形状的变异系数在0.24%~2.32%之间;15个假质量性状的信息多样性指数H在0~2.29%之间,遗传多样性指数D在0~0.94%之间;在欧式距离为15时,可将29个表型性状划分为七类,在欧式距离为15时,可将26个长瓣铁线莲划分为五类。12条引物共扩增出144个条带,多态性条带占比为100%。利用引物UBC815、UBC824和UBC836构建26个长瓣铁线莲的指纹图谱,可快速区分鉴定26个长瓣铁线莲。UPGMA聚类结果显示,在遗传相似系数为0.68时,可将26个长瓣铁线莲划分为八大类。形态标记结合ISSR分子标记可有效鉴别长瓣铁线莲种质资源,以期为长瓣铁线莲种质资源收集保存、创制新品种等提供理论基础。

Assessment of Genetic Diversity in Contrasting Sugarcane Varieties Using Inter-Simple Sequence Repeat (ISSR) Markers

Sugarcane is an important tropical crop, responsible for two thirds of the world sugar production, gaining actually importance as a source of biofuel. Drought tolerance is a very important feature considering the actual climate change scenario throughout the world. This study aimed to determine the genetic diversity between sugarcane varieties with contrasting features under drought. For this purpose, twelve ISSR primers were used to characterize nine sugarcane varieties under cultivation in different countries including selected drought resistant material from Northeast Brazil and two varieties from India as contrasting genotypes. 317 scorable bands were generated, among which 301 comprised polymorphic markers, with an average of 25 polymorphic bands per primer. In the generated dendrogram the accessions were placed in clusters, where cluster A included two varieties from India (Co331 and Co419), and B comprised plants eight Brazilian accessions and a ‘Barbado’ variety. Within this clade, drought tolerant and susceptible varieties were clearly separated. The present evaluation revealed important contrasting parental candidates regarding their drought response, very promising for future mapping approaches aiming the identification of quantitative trait loci (QTLs) associated to drought in sugarcane. The selected primers were used for the first time in sugarcane, representing valuable tools for future evaluations, with emphasis to diversity characterization and genetic mapping.

31份藜麦种质农艺性状及ISSR遗传多样性分析

【目的】筛选西北干旱及半干旱地区不同利用价值藜麦种质,为藜麦种质资源的保护与利用奠定基础。【方法】以种植于甘肃临夏东乡县的31份藜麦种质资源为材料,分析其农艺性状,利用ISSR引物进行分子标记PCR扩增,阐明其遗传多样性及特点。【结果】株高、茎粗、鲜干比、茎叶比、单株产量5个农艺性状的变异系数在14.41%~63.30%,其中株高与茎粗、鲜干比呈极显著正相关,与单株产量呈显著正相关。茎粗与单株产量呈极显著正相关,与鲜干比呈显著正相关。鲜干比与单株产量呈极显著正相关。筛选出8条多态性高且清晰的ISSR引物,扩增出63条条带,其中多态性条带55条,多态性位点比率为83.70%,有效等位基因数(Ne)为1.4515,基因多样性(H)为0.2731,香农信息指数(I)为0.4174。根据UPGMA聚类分析结果,在遗传相似系数为0.72时,31份藜麦种质可分为5个类群。第1类群为已选育的陇藜1号、陇藜7号,抗倒伏性、抗病虫害好;第2类群株高较低,可作为抗倒伏材料进一步选育;第3类群株高高、单株产量大、鲜干比适中、茎叶比小,可作为鲜喂材料进一步选育;第4类群鲜干比最大,且与台湾红藜LQ-223的亲缘关系相近;第5类群具有茎叶比最大、中熟品种的特性。【结论】31份藜麦种质资源具有较高的遗传多样性,可为藜麦种质资源创新、遗传育种提供优质材料和科学依据。

基于ISSR标记的西藏22份葱属材料的遗传多样性分析

采用ISSR标记对收集自西藏多地的22份野生和地方葱属(Allium Linn.)材料进行遗传多样性分析,并探讨了材料间的遗传关系。结果显示:基于筛选出的20个引物,22份葱属材料共扩增出211个条带(位点),其中多态性位点208个,多态性比率达到98.6%,平均每个引物扩增出10.6个位点;每个位点的观测等位基因数均值为2.0,其中有效等位基因数均值为1.6,Nei's基因多样性指数均值为0.37,Shannon's信息指数均值为0.54,表明供试22份葱属材料具有较高的遗传多样性水平。供试22份葱属材料的遗传相似系数在0.43~0.87之间,遗传距离在0.14~0.84之间,其中,15号香葱(A.schoenoprasum Linn.)与18号香葱、12号青甘韭(A.przewalskianum Regel)与13号青甘韭以及13号青甘韭与14号青甘韭间的亲缘关系较近。聚类分析结果显示:在遗传相似系数0.35处,供试22份葱属材料可聚为6组,青甘韭和韭葱(A.porrum Linn.)与韭菜(A.tuberosum Rottler ex Spreng.)和香葱间的亲缘关系较近,遗传相似系数在0.83~0.86之间,遗传距离在0.15~0.19之间;粗根韭(A.fasciculatum Rendle)与韭菜、10号香葱和大花韭(A.macranthum Baker)间的亲缘关系较远,遗传相似系数在0.64~0.68之间,遗传距离在0.39~0.44之间。综合研究结果表明:供试22份葱属材料间的变异程度较大,具有较丰富的遗传多样性,部分材料体现出一定的来源地域一致性特征,其遗传关系基本符合传统分类结果。

基于ISSR和TRAP分子标记的东北地区栽培黑木耳遗传多样性研究

为探究东北地区黑木耳栽培菌株的遗传多样性及亲缘关系,采用ISSR和TRAP分子标记对50株栽培黑木耳菌株进行遗传多样性分析。利用22条高多态性、高分辨率的ISSR和TRAP分子标记引物,对栽培黑木耳进行了DNA扩增。结果表明,黑木耳ISSR和TRAP标记谱带多态性较高,分别占89.58%和84.71%,遗传相似系数为0.53~1.00;在ISSR标记的聚类图中相似系数大于0.96的菌株占44.0%,TRAP标记的聚类图中相似系数大于0.96的菌株占54.0%;STRUCTURE聚类结果显示,50株菌株可分为四大类;遗传多样性分析结果表明,东北地区黑木耳栽培菌株的遗传多样性主要来源于群体间,黑木耳栽培菌株的遗传多样性欠丰富,生产菌种资源的同质化较为突出。

23份香蕉特色种质资源的ISSR分类

对中国热带农业科学院南亚热带作物研究所香蕉种质资源圃所保存的23份特色香蕉种质(野生蕉和地方香蕉种质为主),进行ISSR分子标记及遗传关系分析,以期明晰这些香蕉种质的亲缘关系,为后续香蕉高产、抗病杂交育种研究奠定基础。提取23份香蕉种质嫩叶DNA,利用筛选得到的ISSR引物对其进行分子标记筛选和遗传多样性聚类分析。结果表明,从40个ISSR引物中筛选出12个ISSR引物,对供试23份种质扩增清晰、可重复的条带共126条,多态性条带占比均达到100%。其中,UBC807、UBC812和UBC820扩增的条带数最多,均为13条;UBC821扩增的条带数最少,为6条。聚类分析结果显示,23份香蕉材料可分为8组,孟加拉大蕉、正果野生蕉、南亚指天蕉;高脚遁地雷、怒江野蕉5、志满粉蕉、怒江野蕉6、假金手指;天宝988、金手指、红河矮蕉、海南红蕉;宝山粉蕉、台湾8号、增城野生蕉、兴隆红蕉、黎母野生蕉;紫茎蕉、宝山野蕉;紫花野生蕉、宝山野蕉1;山芭蕉1号;中山龙芽蕉分别聚为一组。其中,中山龙芽蕉与其他种质的相似系数低,亲缘关系远,遗传多样性较丰富;而其他组间的相似系数高,亲缘关系近,遗传背景相似。说明筛选的12个ISSR引物可对23份香蕉种质进行有效分类。

冰薰2号薰衣草ISSR-PCR反应体系优化及遗传特征分析

为探究冰薰2号薰衣草的遗传特征,采用ISSR分子标记技术在优化反应体系的基础上对冰薰2号薰衣草亲代及F1代个体进行遗传多样性分析。结果显示,优化的最佳ISSR-PCR扩增体系为:在20μL体系中,dNTP(每种2.5 mmol/L)用量为1.4μL,引物(10μmol/L)用量为0.7μL,DNA模板量为20 ng,Taq DNA聚合酶用量0.75μmol/min。选取8个ISSR引物对冰薰2号薰衣草亲代及F1代个体进行试验,根据扩增结果进行计算,并分析亲代及F1代个体间遗传多样性指数。结果表明,冰薰2号薰衣草遗传多样性水平低,遗传变异度低。

Assessment of Genetic Variation in Soybean (<i>Glycine max</i>) Accessions from International Gene Pools Using RAPD Markers: Comparison with the ISSR System

Soybean (Glycine max) is one of the most important crops in the world in terms of total production and usage. It is also among the least diverse species. The main objectives of the present study were to 1) assess the level of genetic variation among soybean (G. max) accessions from different countries using Random Amplified Polymorphic DNA (RAPD) markers and 2) compare Inter Simple Sequence Repeats (ISSR) and RAPD marker systems in detecting polymorphic loci in soybeans (G. max). Genomic DNAs from 108 soybeans (G. max) accessions from 11 different gene pools were analyzed using several ISSR and RAPD primers. The average level of polymorphic loci detected with the RAPD primers was 35%. The soybean accessions from the China, Netherlands, and Canada gene pools were the least genetically variable with 25%, 26%, and 30% of polymorphic loci, respectively. Accessions from Hungary (43%) and France (48%) showed the highest level of polymorphism based on the RAPD analysis. Overall, RAPD data revealed that the accessions from different countries are closely related with 64% genetic distance values below 0.40. The levels of polymorphic loci detected with the RAPD and ISSR marker systems were in general moderate and similar even if they target different regions of the genome. A combination of different marker systems that include RAPD/ISSR, microsatellites (SSR), and SNPs should provide the most accurate information on genetic variation of soybean (G. max) accessions.

基于ISSR分子标记的苗药朱砂根遗传多样性研究

为了探究不同居群朱砂根遗传多样性水平及亲缘关系,以24份不同居群朱砂根为材料,采用ISSR分子标记技术对其进行筛选分析,结果从100条引物中筛选到14条扩增效果好、多态性高的ISSR引物,对24个朱砂根居群进行遗传多样性分析,共检测到75条扩增位点,平均每条引物扩增5.4个位点,74个为多态性位点,多态性比率(PPB)为98.67%;其中,UBC841扩增位点最多(8个),UBC820、UBC821、UBC844、UBC887扩增位点最少(4个)。观测等位基因数为2,有效等位基因数为1.0868~2.0000,Nei's基因多样度为0.0799~0.5000,Shannon's指数最大,为0.6931;遗传距离与地理距离无显著相关性。研究表明,朱砂根种质资源遗传多样性高,ISSR分子标记可用于朱砂根亲缘鉴定。