Identification of RAPD marker linked to fruit skin color in Japanese pear (Pyrus pyrifolia Nakai)

In Japanese pear, fruit skin color is a very important trait for growers because the russet skin protects the fruit against external stress caused by disease, insects, bad weather, and shipping. This report describes the development of a randomly amplified polymorphic DNA (RAPD) marker linked to major genes controlling the fruit skin color in Japanese pear. Two F₁ progenies from the cross of ‘Kousui’ × ‘Kinchaku’ and ‘Niitaka’ × ‘Chikusui’ segregated by fruit skin color were used for bulked segregant analysis. Four kinds of bulked DNA were constructed and used for the polymerase chain reaction in RAPD analysis. After 200 random primers were screened against four bulks, a band named OPH-19₄₂₅ was selected in cooperation with green bulks. The recombination rate between OPH-19₄₂₅ and the green skin phenotype was 7.3%. The RAPD marker (OPH-19₄₂₅) could select green fruit with probability as high as approximately 92%. The marker was apparently useful for the selection of green-skinned individuals in a breeding program. This is the first report on developing a DNA marker closely linked to the fruit phenotype in Japanese pear.     

Identifying the markers and tagging a leucine-rich repeat receptor-like kinase gene for resistance to anthracnose disease in vegetable cowpea [Vigna unguiculata (L.) Walp.]

Anthracnose caused by the fungus Colletotrichum lindemuthianum is the most destructive disease of cowpea. Field-type cowpeas show various levels of resistance, whereas pole-type vegetable cowpeas are highly susceptible. Transfer of resistance available in field types to vegetable types is a major breeding objective in cowpea. This paper details the development of an F₂ mapping population by crossing field-type cowpea variety Kanakamony (Vigna unguiculata ssp. cylindrica) with pole-type vegetable cowpea variety Sharika (Vigna unguiculata ssp. sesquipedalis), screening this population with artificial inoculation and Bulked Segregant Analysis (BSA) with random marker systems Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR); the objective is to identify the markers linked with major resistance-contributing genes. RAPD primer OPA02 has yielded marker at 850 bp in susceptible genome, whereas ISSR primers UBC810 and UBC811 have yielded markers at 1.4 kb and 1.5 kb respectively in resistant genomes. These markers were reproducible and their linkage with resistance and suitability in marker assisted selection (MAS) were confirmed through co-segregation analysis in F₃ population. UBC811 marker was eluted, cloned on pGEM-T, and sequenced. The sequence had shown that this marker is anchored on LRR receptor-like serine/t\hreonine protein kinase gene which could be involved in the resistance mechanism.     

RAPD标记构建芥蓝×甘蓝分子标记连锁图

 以芥蓝C_100-12×甘蓝秋_50-Y7为杂交组合, 构建了F₂ 作图群体, 利用300 个随机引物对两亲本进行了RAPD 检测, 共筛选到150 个RAPD 引物在亲本间表现多态, 多态引物的比率为58. 3 %。共分析了52个引物产生的96 个多态性RAPD 标记在该群体中的遗传。96 个RAPD 标记中有94 个在F2 群体中的分离比符合3∶1 , 说明该群体适合于QTL 分析, 共发现两对共分离的RAPD 标记。利用该作图群体构建了甘蓝的基本遗传图谱。该图谱由9 个主要的连锁图构成, 覆盖基因总长度约为555. 7 cM。     

Assessment of genetic purity of tomato (Lycopersicon esculentum L.) hybrid using molecular markers

Three DNA molecular marker systems, RAPD, ISSR and SSR, were used to test seed genetic purity of two commercial hybrid tomato (Lycopersicon esculentum L.) cultivars ‘Hezuo 903’ and ‘Sufen No. 8’. Genomic DNA from the two F₁ hybrid cultivars and their corresponding parental lines was screened with 218 RAPD decamer primers, 54 ISSR primers and 49 SSR primers. Among the 321 primers, 4 primers for ‘Hezuo 903’ and 3 for ‘Sufen No. 8’, which could produce both female and male parent-specific markers, were selected for testing the genetic purity. A total of 210 hybrid individuals of each cultivar were analyzed using the identified primers. The combined results of the marker analysis showed that eight of the 210 F₁ plants in ‘Hezuo 903’ and 13 of 210 in ‘Sufen No. 8’ were false hybrids, and the overall genetic purity of the two F₁ hybrid seed lots was 96.2 and 93.8%, respectively. This study showed that RAPD and SSR markers could provide a practical and efficient tool in quality control of the tomato commercial hybrid seeds.     

杨桃遗传多样性的形态特征与RAPD标记的相关性分析

【目的】为指导杨桃种质资源的引进以及良种选育提供科学依据,【方法】采用形态标记数量聚类分析和RAPD分子标记相结合,对广东地区10份杨桃品种资源进行遗传多样性分析,并将形态学聚类和RAPD分子标记聚类结果加以对比。【结果】在所观察的12个形态性状中,变异系数为14.08%~49.71%,平均变异系数为25.38%。从100条RAPD随机引物中筛选出10个引物,共扩增出58条带,其中53条为多态性带,多态率达93.02%。聚类分析结果表明,形态标记和RAPD标记都可依果实风味将供试材料分组,即甜味和酸味。2种标记方法的相关系数为r0.01=0.685 3,达到显著水平。【结论】杨桃具有丰富的遗传多样性,2种方法聚类结果相似,且相关性高,具有较高的可靠性。     

Genetic diversity of ash gourd [Benincasa hispida (Thunb.) Cogn.] inbred lines based on RAPD and ISSR markers and their hybrid performance

Ten inbred lines of ash gourd [Benincasa hispida (Thunb.) Cogn.] were crossed to produce 45 F₁ hybrids (without reciprocal) which were evaluated along with the parents for 20 growth- and yield-related traits, in a replicated field trial. High level of heterosis was observed among the hybrids for most of the traits examined, including yield. These inbred lines were analysed by using 42 RAPD primers those produced 282 DNA marker bands. A total of 130 RAPD markers were obtained with a mean of 3.1 per primer, which in combination discriminated all the inbreds from each other. Pair-wise genetic distance measurements ranged from 0.07 to 0.31, suggesting a wide genetic diversity for these inbreds. These inberds were also analysed with five ISSR primers of which four were informative. Twenty-six ISSR marker bands were generated of which 11 were polymorphic with an average of 2.80 per primers. The percentage of polymorphic bands produced were higher in ISSR markers (>80%) than generated through RAPD markers (46%). Although the results indicated significant positive correlations of genetic distance with hybrid performance and heterosis, the RAPD based genetic distance measures and use of limited ISSR markers in this present study could not effectively predict hybrid performance in this crop. The genetic variation among ash gourd inbred lines examined, herein, defined a marker array (combined ISSR and RAPD) for the development of a standard reference for further genetic analyses, and the selection of potential parents for predicting hybrid performance and heterosis.     

雷州半岛菠萝蜜种质遗传多样性的RAPD分析

 用RAPD标记方法对我国雷州半岛的65份菠萝蜜(Artocarpus heterophyllus Lam. ) 实生种质资源DNA的遗传多样性进行了检测, 16个引物共检测到78条带, 其中69条具多态性(占88.4% ) 。聚类分析表明, 65份菠萝蜜材料在遗传距离0.26处可分为3大类。供试种质虽具有丰富的形态性状变异, 但在DNA水平上平均相似性系数为0.7341。干、湿胞类型及引自马来西亚的种质均不能独立聚类。对菠萝蜜品种的干、湿胞分类法及菠萝蜜品种的引种区域进行了讨论。     

Genetic diversity of Indonesian cauliflower cultivars and their relationships with hybrid cultivars grown in Australia

The objectives of this study were to investigate genetic variation and relationships between Indonesia-, Australian- and European-based cultivars and to evaluate variation within Indonesia cultivars as all cultivars are open-pollinated. Eight cauliflower cultivars collected from three production regions in Indonesia and four F₁ hybrids cultivars grown in Australia were evaluated using RAPD and ISSR markers. DNA polymorphisms generated from 10 polymorphic RAPD primers were used to construct a dendogram using the unweighted pair-group method with arithmetic averages (UPGMA) and to generate a fingerprinting key. ISSR marker analysis using 14 primers were attempted but DNA polymorphisms could not be clearly identified. The RAPD technique indicated that variation occurred both within and between Indonesian cultivars. Comparison between Indonesian-, Australian- and European-based cultivars showed that Indonesian cultivars have unique genotypes and would be good sources of genes for future crop improvement.     

核桃早实性状的RAPD分析

 用BSA (Bulked Segregant Analysis) 法获得了与核桃早实性状相关的RAPD 标记OPB208₉₀₀。混合分离群体的混合样来自早实品种辽1 的自然授粉实生后代。用220 个随机引物扩增早实和晚实混合样, 只有引物OPB208 ( 5. GTCCACACGG 3. ) 在早实混合样及其个体上扩增出了约900 bp 的特异带OPB208₉₀₀, 而在晚实混合样及其个体上无此特异带。用OPB208 为引物, 以19 个品种和3 个株系的DNA 为模板进行扩增,特异带OPB208₉₀₀在17 个早实品种中的12 个个体上出现, 而5 个晚实品种( 系) 上均无此带。     

Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

Summary

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.     

豆瓣菜种质资源遗传多样性的RAPD和ISSR分析

以8个豆瓣菜的品种为试材，用筛选出的79个RAPD引物和34个ISSR引物对这8个品种的基因组DNA进行扩增，分别扩增出361条和179条谱带，每个引物扩增出的带在3～10条之间，平均每个引物扩增出约5条带。根据所得的条带进行聚类分析，两种标记产生的聚类图存在一些差异，但它们都可以较好地将8个品种按亲缘关系的远近划分为3个不同的类群。Mantel测试得出相关系数r=0．58155，表明RAPD和ISSR两种分子标记技术的相关度很低。     

30份莲种质资源的RAPD遗传多样性分析

采用RAPD分子标记技术对30份莲(Nelumbo nueifera Gaertn.)种质资源的遗传多样性进行鉴定.筛选出14对有效引物进行PCR扩增,共产生2 146条带,其中有1 516条为多态性条带,各对引物的平均多态性条带为108.29条,扩增条带的多态率达70.64％.对所得到的30份莲种质资源的RAPD图谱...     

Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers

Diversity and genetic relationship in 100 cashew germplasm accessions were analyzed by using RAPD and ISSR markers. Using 10 selected RAPD primers 60 bands were generated, of which 51 bands were polymorphic (85%), and with 10 selected ISSR primers 67 amplified bands were observed with 58 polymorphic bands (86.6%). Though both kinds of markers discriminated the accessions effectively, analysis of combined data of markers (RAPD + ISSR) resulted in better distinction of accessions. By combining markers, a total of 127 bands were detected, of which 109 bands (85.8%) were polymorphic and produced on an average of 5.45 polymorphic bands per primer. Primers with high polymorphic information content and marker index were identified for discriminating accessions. High percentage of polymorphism (>85%) observed with different markers indicated high level of genetic variation existing among the accessions. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pair of accessions varied from 0.43 to 0.94 in RAPD, 0.38 to 0.89 in ISSR and 0.43 to 0.87 with combined markers suggested a diversity (dissimilarity) ranging from 6 to 57%, 11 to 62% and 13 to 57% respectively and the diversity skewed around 50% indicated moderate diversity. The cluster analysis with UPGMA method separated the accessions broadly into 13 clusters and in that three into smaller clusters. Some correspondence between the molecular groupings and the morphological clusters were observed. Among the accessions, NRC-142 and NRC-12 were highly divergent and NRC-231 and NRC-232 were genetically similar.     

Race-specific genetics of resistance to black rot disease [Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson] and the development of three random amplified polymorphic DNA markers in cauliflower

Summary

In order to understand the genetics of resistance to black rot disease caused by Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson in cauliflower (Brassica oleracea var. botrytis L.) and to identify random amplified polymorphic DNA (RAPD) markers that segregate with the resistance genes, susceptible (‘Pusa Himjyoti’, female parent) and resistant (‘BR-161’, pollen parent) plants were crossed. Six generations of plants (30 P₁, 30 P₂,30 F₁, 120 F₂, 90 B₁, and 90 B₂) were evaluated for the presence or absence of black rot disease in a randomised block design with three replications. The pattern of segregation of resistance was tested by the χ² test at the 5% level of significance. All F₁ progeny plants were resistant, and the segregation of resistant and susceptible plants in the F₂ and two backcross generations (B₁ and B₂) showed that a single dominant gene caused resistance to the black rot pathogen in ‘BR-161’. Three polymorphic RAPD markers (OPO-04₈₃₃, OPAW-20₂₅₃₈, and OPG-25₆₂₅) were found by bulk segregant analysis, which produced unique amplicons 833 bp, 2,538 bp, and 625 bp in length, respectively. These markers were associated in coupling phase to the resistance allele. Best fit ratios of 3:1 (resistant:susceptible) in the F₂ plants with the three RAPD markers, suggested that the markers were linked to the single gene controlling black rot resistance. These markers will be useful to identify more closely-linked markers and to develop black rot-resistant hybrid cauliflower varieties.     

Comparative analysis of genetic diversity in Indian bitter gourd (Momordica charantia L.) using RAPD and ISSR markers for developing crop improvement strategies

A genetic analysis of 38 diverse Indian bitter gourd (Momordicacharantia var. charantia, and var. muricata) accessions was performed using 29 RAPD and 15 ISSR markers. RAPD primers yielded 208 amplicons of which 76 (36.5%) were polymorphic providing an average of 2.6 amplicons per primer. RAPD amplicons per primer ranged from 3 (OPE-19, OPW-09) to 15 (OPW-05), and varied in size from 200 bp to 3000 bp. Fifteen ISSR primers provided a total of 125 bands of which 94 (74.7%) were polymorphic. Polymorphic ISSR markers ranged from 0 (UBC-841) to 12 (UBC-890) providing a mean of 6.3 amplicons per primer that ranged in size from 150 bp to 2700 bp. Nevertheless, the concordance among bitter gourd accession groupings after cluster analysis was relatively high (r = 0.77), indicating that RAPD- and ISSR-based diversity assessments in this germplasm array were generally consistent. The M.charantia var. charantia (domesticated) and var. muricata (wild, free-living) accessions examined were genetically distinct, and these differences provided for the development of strategies for genetic analyses and crop improvement in this species.     

番茄抗晚疫病基因Ph-3的RAPD标记

 以番茄抗晚疫病材料CLN2037和感病材料T2-03杂交的F₂分离群体的147个单株为试材, 通过抗病性鉴定, 选择高抗单株和高感单株分别建抗、感病池。采用BSA法筛选了230条RAPD引物, 其中引物CCPB272-03 (序列为GGTCGATCTG) 在抗、感病池扩增出多态性片段CCPB272-03₇₄₀ , 通过F₂单株验证后证明CCPB272-03₇₄₀与抗晚疫病基因Ph-3紧密连锁, 遗传距离为518 cM。     

冬瓜和节瓜种质资源遗传多样性的RAPD分析

采用RAPD分子标记技术对41份冬瓜和节瓜种质资源进行遗传多样性分析。从455条随机引物中筛选出23条能产生稳定多态性扩增谱带的引物用于RAPD分析,共产生143条扩增谱带,其中多态性谱带81条,多态性检测率为56.6%,表明冬瓜和节瓜种质资源在分子水平上具有丰富的遗传多样性。用NTSYSpc软件处理RAPD数据,41份材料间的遗传相似系数在0.60～0.99之间。聚类分析结果可将供试材料分为6大类群9个亚类,主坐标分析可将供试材料分为6大类群10个亚类。两种分类方法的结果基本一致。     

西葫芦种质资源遗传多样性的RAPD分析

利用RAPD技术对国内外47份西葫芦种质材料进行遗传多样性及亲缘关系分析。从520条随机引物中筛选出30条能产生稳定多态性的引物用于RAPD分析,共扩增出367条带,其中302条表现多态性,占总数的82.29%,表明西葫芦种质资源在分子水平上具有丰富的遗传多样性,与形态学研究结果一致。聚类分析将47份西葫芦种质材料分为两大类。在第Ⅰ类群中,可将32份种质分为4个亚类;第Ⅱ类群分成5个亚类。从生长习性来看,矮生与半蔓生的亲缘关系最近;从皮色来看,白色与绿色亲缘关系最近,亲缘关系最远的为白色与花皮。     

Identification of persimmon (Diospyros kaki) cultivars and phenetic relationships between Diospyros species by more effective RAPD analysis

Randomly amplified polymorphic DNA (RAPD) markers were used to identify cultivars of persimmon (Diospyros kaki Thunb.) and to estimate the phenetic relationships between Diospyros species. Long random primers (15 and 20 bases) were used to increase the effectiveness of recognizing polymorphism. Twenty-five of 34 (74%) long random primers amplified polymorphic bands of 25 persimmon cultivars, indicating that the effectiveness improved compared with RAPD analysis using 10-base primers. The 25 primers amplified a total of 444 polymorphic bands, which distinguished all 25 cultivars. RAPD markers (133) clarified the phenetic relationship between the six species and subspecies of Diospyros: D. lotus, D. lotus subsp. glabbra and D. taitoensis formed one group (lotus group), and persimmon was related in order of closeness to the lotus group, D. oleifera, and D. rhombifolia.