Cultivar identification and genetic diversity analysis of broccoli and its related species with RAPD and ISSR markers

A total of 18 genotypes of broccoli were subjected to random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) marker analysis. Seventy-four RAPD and eight ISSR primers generated 344 and 67 polymorphic bands, respectively. All broccoli genotypes could be distinguished with two-primer combinations, indicating that RAPD and ISSR markers can be used to efficiently identify broccoli cultivars. These 18 broccoli genotypes could be separated into two major sub-groups. The first major sub-group (A) included 13 genotypes and the second major sub-group (B) was comprised of five genotypes belonging to early-maturating cultivars. Genetic diversity analysis was performed on the 18 broccoli genotypes, one radish genotype, and six related Brassica accessions. All accessions could be clustered into two groups (radish and Brassica) based on the unweighted pair group of arithmetic average (UPGMA) cluster analysis. The UPGMA analysis indicated that broccoli is most closely related to cauliflower, than to cabbage and Chinese cabbage.     

Genetic diversity analysis in date palm (Phoenix dactylifera L.): a comparative assessment using ISSR and RAPD marker assays

SUMMARY

A comparative study was conducted to evaluate genetic diversity in 45 genotypes of date palm (Phoenix dactylifera L.), including both male and female plants, employing RAPD and ISSR marker systems. The data were analysed to calculate the total number of bands, the number of polymorphic bands, the percentage polymorphism, the average number of bands per primer, the effective multiplex ratio (EMR), the polymorphic information content (PIC), the marker index (MI), and genetic similarity coefficients. The 37 RAPD and 53 ISSR primers used generated 363 and 608 scorable amplified products, respectively, of which 95.0% and 90.9% were polymorphic. The ISSR markers produced more information than the RAPD markers due to their higher EMR and MI values. Jaccard similarity values among male plants, female plants, and between all male and all female plants varied between 0.72 – 0.80. The results indicate the effectiveness of these two marker systems for demonstrating genetic relationships among date palm genotypes.     

SRAP、ISSR和RAPD分子标记技术在银耳菌株鉴别上的应用

Three types of molecular markers (SRAP, ISSR and RAPD) were used to identify four Tremella fuciformis strains, T6 (white), T7 (white), T8 (yellow) and T9 (light yellow). Twelve SRAP primer pairs, ten ISSR primers and eight RAPD primers were screened, and identification data obtained using the three molecular markers were consistent in that the four T. fuciformis strains were divided into three groups with T7 and T9 clustered together in a single group. Each RAPD primer generated a higher average number of polymorphic bands than either the SRAP or ISSR primers, and the average similarity between the four strains was 81.34%. SRAP markers reflected more genetic information compared with the two other markers, and the average similarity was 68.98%. Genetic information reflected by ISSR markers was intermediate between SRAP and RAPD, and the average similarity was 77.48%.     

Genetic diversity of Rehmannia glutinosa cultivars based on sequence-related amplified polymorphism markers

SRAP analytic system was used to assess genetic diversity of Rehmnnia glutinosa. Twenty-three Rehmnnia glutinosa cultivars were screened with 288 primer combinations, of which 13 produced stable and reproducible amplification patterns in three repetitive experiments. Among a total of 338 amplified fragments, 306 (90.5%) were polymorphic, with an average of 23.5 fragments for each primer combination. The percentage of polymorphic bands for each primer combination varied from 58.3 to 100%. The cultivars had a similarity ranging from 0.335 to 0.713 with a mean of 0.518. Shannon's diversity index and expected heterozygosity were 0.3217 and 0.2008, respectively. Based on the cluster, which were conducted on the similarity matrix of SRAP marker data, the cultivars were divided into four groups at the 20 rescaled distance cluster combine. The results demonstrated that SRAP is a stable marker technique for the assessment of genetic diversity of Rehmannia glutinosa cultivars, and that the level of genetic diversity among them from different production areas was relatively high.     

山楂（Crataegus pinnatifida Bge.)遗传多样性的RAPD和ISSR标记分析

 利用RAPD和ISSR标记对35份山楂（Crataegus pinnatifida Bge.）资源进行了DNA多态性分析。12个RAPD引物共扩增出110条清晰的谱带,其中89条显示多态性,平均每个引物扩增出7.4条多态性谱带。13个ISSR引物共扩增出110条清晰的谱带,其中94条显示多态性,平均每个引物扩增出7.2条多态性谱带。基于RAPD和ISSR标记,利用UPGMA分别构建了35份山楂资源的聚类树状图。距离系数分别为0～0.62（RAPD）和0～0.64（ISSR）,表明山楂具有较高的遗传多样性。     

Molecular identification and genetic analysis for 24 turf-type Cynodon cultivars by Sequence-Related Amplified Polymorphism markers

Bermudagrass (Cynodon ssp.) germplasm is genetically diverse and widely distributed in the world. The study was conducted to identify and assess the molecular variation and relationship among 24 cultivars developed in China, Australia and the USA. Sequence-Related Amplified Polymorphism (SRAP) was applied to cultivars identification in this study for the first time. Thirty of the 90 SRAP primer combinations generated a total of 274 clearly bands encompassing 249 (91%) polymorphic. Each bermudagrass cultivar has its unique binary code and can be distinguished from the others. Three distinct clusters were obtained by unweighted pair-group method with arithmetic averages based on the polymorphic markers. Coefficients of genetic distance among the genotypes ranged from 0.57 to 0.97. The results demonstrated that SRAP marker is a stable molecular marker technique for the identification of bermudagrass cultivars and their genetic relationships.     

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.     

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.     

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。     

SRAP在葱栽培品种遗传多样性研究中的适用性分析

为评价SRAP技术对葱品种进行鉴定和遗传关系分析的适用性, 对20个葱栽培品种的表型特征进行了观察记载, 利用256个SRAP引物组合对其进行了遗传多样性研究。结果表明: (1) 256个SRAP引物组合中有161个引物组合产生多态性条带, 占所用引物组合数的62.9%。161个引物组合共产生336条多态性条带, 不同引物组合产生的多态性条带数为1～6个, 平均2.1个。20个葱栽培品种遗传相似系数变幅为0.464～0.938, 平均0.703。(2) 依据SRAP进行聚类分析的分类结果与依据表型特征分类的结果一致。上述结果说明SRAP标记可以在葱栽培品种的鉴定和遗传多样性研究中应用。     

基于SRAP的三倍体无子西瓜品种指纹分析和杂种纯度鉴定

为了解三倍体无子西瓜品种SRAP(Sequence-Related Amplified Polymorphism)扩增特点及其在三倍体西瓜杂种纯度鉴定中的适应性,利用SRAP引物组合对当前生产上推广的9个三倍体无子西瓜品种进行了指纹分析和杂种纯度鉴定研究。结果表明,1)30对引物组合中筛选出多态性引物组合24对,共产生109条多态性带,平均每对引物组合产生多态性带4.5条,单个组合的多态性比率在12.5%～80%。2)结合多对SRAP引物组合,可以鉴别不同品种。3)在黑蜜5号及其父母本的指纹分析中,发现有1对引物组合(ME4/EM3)在黑蜜5号杂交种上扩增出了特异的条带,可以区分母本和杂交种,试验证明该引物组合能够用于黑蜜5号的杂种纯度鉴定。     

Use of ISSR,SRAP, and RAPD markers to assess genetic diversity in Turkish melons

The genetic relationships among 63 melon (Cucumis melo L.) genotypes collected from various regions of Turkey were determined by comparing their molecular ISSR, SRAP, and RAPD markers with those of 19 foreign melon genotypes to investigate the taxonomic relationships and genetic variation of Turkish melon germplasm. Total 162 polymorphic markers (69, 18, and 75 obtained from ISSR, SRAP, and RAPD primers, respectively) were used to define the genetic similarity among the melon genotypes by dendrogram or two and three dimensional scalings. The average similarity (SM coefficient) between any two pairs of accessions examined as estimated by molecular variation was 0.73 ± 0.48. Within-group genetic similarities ranged between 0.46 and 0.96. Related genotypes or genotypes collected from similar regions were partitioned to similar clusters. Southeastern Anatolian genotypes were distinctly apart from group inodorus and group cantalupensis (sweet) genotypes. This reinforced the position of Turkey in the secondary genetic diversity center of melon. The genetic diversity among Turkish genotypes (H = 0.28 and I = 0.42) was only a little less than that of the world accessions (H = 0.30 and I = 0.45). On the other hand, the percentage of polymorphic loci among Turkish melon genotypes (90.7%) was even higher than that of the world accessions (87.6%).     

贵州野生枇杷资源遗传多样性的ISSR分析

采用ISSR标记,构建了贵州39份野生枇杷种质的DNA指纹图谱,并对其遗传多样性进行评价。结果表明,以筛选出的15条引物进行PCR扩增,获得清晰、重复性好的谱带125条,其中多态性谱带108条,多态性比例为86.4%;应用引物815、825和841的组合,构建了39份枇杷种质的ISSR指纹图谱,可以有效区分所有供试材料;15条引物的谱带数据聚类分析表明,供试种质的遗传相似性系数为0.40～0.98,在相似性系数为0.70时,可将供试种质分成五大类;聚类结果与种质表型相关,与地理分布的相关性不明显。     

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.     

ISSR．PCR技术在桂花品种分类研究中的应用

 利用ISSR．PCR方法对桂花的19个品种进行了基因组多态性分析，从74个ISSR引物中筛选出13个多态性引物用于正式扩增，共扩增出9o条DNA片段，其中多态性DNA条带79条，占总扩增片段的87．8％ ，平均每个引物扩增的DNA带的数目为6．92条。根据ISSR扩增结果，应用RAPDistance软件进行Nei相似性系数和遗传距离计算，利用UPGMA法构建聚类树状图。聚类分析的结果把供试桂花的l9个品种分为8个大类，并对4个品种群的遗传关系和种下分类系统进行了探讨。     

桃早熟芽变品种‘大观一号’的RAPD分析及其特异片段的克隆

利用140个单个和混合随机引物对桃布日早生及其早熟芽变品种'大观一号'基因组DNA的多态性进行RAPD分析。其中有一个引物能检测出两者之间DNA的多态性，在1．1kb处'大观一号'多了一条特异性条带，将该片段克隆到pUC19载体，并作了酶谱分析。     

Molecular assessment of polymorphism among local Jordanian genotypes of the common fig (Ficus carica L.)

This research was conducted to assess polymorphism among local genotypes of common fig available in Jordan (one of countries of origin). Leaf samples were collected in spring for DNA isolation from 20 different local genotypes (5 cultivars and 15 landraces). Two more wild types and one foreign cultivar were included. The genotypes were assessed using the randomly amplified polymorphic DNA (RAPD) technique. Six of the 19 screened primers showed reproducible polymeric profiles. Out of 62 amplified bands, 48 were polymorphic (77%). They generated 1104 data entries (591 for present and 513 for absent bands). After determining Jaccard similarity index, some genotypes showed high genetic similarity (90% between F20 and F22), while other were less similar (3–18% between F11 and all other genotypes). Moreover, the primers were evaluated for their discriminating power, where primer RAPD06 showed the weakest power (0.431), while highest values of 0.989 and 0.996 were achieved for primers RAPD02 and RAPD13, respectively.     

ISSR和RAPD分子标记技术在不同君子兰品种遗传多样性上的应用

采用ISSR标记和RAPD标记技术研究了15个君子兰品种的遗传多样性。结果表明:从55条寡聚核苷酸引物中筛选出10条简单重复序列引物,共扩增出65条带,平均每条引物扩增出6.5条带,其中5.4条多态性带,多态性比率为81.95%;从55条寡聚核苷酸引物中筛选出11条多态性引物,共扩增出47条带,平均每条引物扩增出4.27条带,其中32条多态性带,多态性比率为67.88%。POPGEN软件计算出君子兰品种间遗传距离为0.1123~0.6330,平均值为0.3263。基于遗传相似系数的UPGMA聚类分析将15个品种明显聚为二组,国兰系列品种为一组,日本兰系列品种组成另一组。     

Genetic diversity and phylogenic relationships in date-palms (Phoenix dactylifera L.) as assessed by random amplified microsatellite polymorphism markers (RAMPOs)

Random amplified microsatellite polymorphisms (RAMPOs) were used to assess genetic diversity among 30 date-palm cultivars and 10 male trees. Using 18 primers combinations, 197 bands were scored and 186 were polymorphic suggesting the high level of polymorphism among studied cultivars. Moreover, taking into account the high percentage of polymorphic bands (ppb), the resolving power (R_p) together with the polymorphism information content (PIC) scored values, all the tested primer sets contribute strongly in the discrimination of date-palm genotypes. In addition, the topology of the derived UPGMA dendrogram exhibited cultivars’ clustering made independently both from the geographical origin and/or from the sex of trees. The present data support the Mesopotamian origin of the date-palm domestication. Thus we assume that the used method is efficient to assess genetic diversity within date-palm cultivars. Data are discussed in relation with the opportunity of the RAMPO method to provide additional molecular markers suitable in the improvement of the date-palms germplasm characterisation.     

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle.