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%).     

Microsatellite markers based assessment of genetic diversity in Iranian olive (Olea europaea L.) collections

To study the genetic variation in Iranian olive collections and some foreign olive cultivars, 47 accessions of 18 local cultivars from 6 olive collections of Iran (Roudbar, Zanjan, Ahvaz, Dezful, Kazeroon and Shiraz), were analyzed along with 30 imported cultivars using 16 microsatellite primer pairs. All the used microsatellite loci revealed polymorphism in the studied genotypes, except GAPU14 and GAPU113 markers. Fourteen microsatellite primers amplified 126 polymorphic alleles in the 87 selected olive accessions. The average number of alleles per locus was 9, ranging from 3 to 14. Polymorphic information content (PIC) was 0.85. The genetic similarity based on Jaccard coefficient ranged from 0.15 to 1. The genetic relationships among accessions were investigated using cluster analysis and principal component analysis (PCA). Most of the accessions with the same name were grouped together; some exceptions were also observed. As expected, close relationship was observed among accessions within same cultivar. Most of the Iranian olive accessions were clustered to a main distinct group. Two-dimensional scatter plot of principal component analysis revealed a clear separation of most of the Iranian olives from Syrian and other introduced cultivars. These suggest that Iranian cultivars have different origin related to West Mediterranean basin cultivars and have evolved independently from the others. Between and within Iranian and foreign cultivars (cultivars including three or more accessions) genetic diversity was analyzed using analysis of molecular variance (AMOVA). AMOVA revealed higher within cultivar genetic variation (62.76%) as compare to that between cultivar variations (37.24%). The intra- and inter-cultivar variance tested by permutation test showed significant genetic variation at both levels. The high level within cultivar genetic variance could be due to mislabeling and presence of homonyms in cultivars produced by vegetative propagation from original plants.     

Genetic diversity of Chinese natural bermudagrass (Cynodon dactylon) germplasm using ISSR markers

Bermudagrass (Cynodon dactylon L.) is an important warm-season turfgrass. Although it is widely distributed in China, studies on the genetic variation and relationship among the large-scale indigenous bermudagrass were relatively insufficient, especially at molecular level. The purpose of this study was to assess the molecular variation and relationship among one commercial cultivar ‘Tift3’ and 95 wild bermudagrass accessions collected from 11 provinces in China by ISSR marker technique. The results indicated that 29 ISSR primers generated a total of 248 bands among which 242 (97.6%) were polymorphic bands. The genetic similarity coefficients among accessions ranged from 0.51 to 0.97 with an average of 0.74. All accessions could be clustered into 11 groups with UPGMA method. Accessions from the same or adjacent regions generally were clustered into the same group or subgroups. A few accessions, however, were greatly different from the majority of all accessions. The results suggested that ISSR marker is an effective tool for the study of genetic variation in Chinese natural bermudagrass.     

Analysis of genetic diversity among wild pomegranates in Western Himalayas,using PCR methods

The genus Punica (Punicaceae) is distributed in Iran, Afghanistan, India and Mediterranean countries. Iran is considered to be its primary center of origin. In India Punica granatum is found in wild only in Western Himalayan regions comprising, Jammu and Kashmir, Himachal Pradesh and Uttarakhand states. However, there is little information available about the genetic variation present in pomegranates in the regions. In this paper we describe the use of DAMD and RAPD methods that generate the profiles, to study genetic diversity in wild genotypes of the P. granatum in India. Forty-nine accessions representing two regions of Western Himalaya were analyzed. Similarity coefficient value varied from 0.08 to 0.79 across different accessions. The results indicate that DAMD (97.08%) revealed more polymorphism in comparison to RAPD (93.72%). The results show that these methods are sufficiently informative to unravel the genetic variations in wild pomegranates in Western Himalayas.     

Genetic relatedness (diversity) and cultivar identification by randomly amplified polymorphic DNA (RAPD) markers in teasle gourd (Momordica dioica Roxb.)

Genetic relationship and variation of 29 accessions of teasle gourd (Momordica dioica Roxb.) and 1 accession of Momordica cochinchinensis Spreng. (wild relatives of teasle gourd) were examined by RAPD analysis using 44 dodecamer oligonucleotide primers. A total of 496 fragments were produced by 44 primers of which 95% bands were polymorphic. Using presence or absence of specific RAPD markers or combination of primers, 23 out of 30 accessions were identified. The genetic relatedness or genetic distance based on Nei and Li's genetic similarity varied from 0.86 to 0.65 with an average of 0.74 among 29 M. dioica accessions (when M. cochinchinensis excluded). In the phenetic dendrogram developed from cluster analysis using UPGMA method, M. cochinchinensis was out grouped as single accession, while others showing relatively weak grouping formed four groups. Clustering pattern did not demonstrate any relationship between geographical origin and genetic diversity. A DNA extraction method has been standardized. This is the first report of using RAPD techniques in teasle gourd. It was concluded that RAPD analysis is a useful tool for genotypic identification and estimation of genetic similarity in teasle gourd.     

Assessment of diversity in Podophyllum hexandrum by genetic and phytochemical markers

For successful conservation and domestication of a species, evaluation of its genetic diversity by different markers is important. Morphological characteristics, phytochemical variation and random amplified polymorphic DNA (RAPD) profiles were generated in different accessions of Podophyllum hexandrum in order to determine the genetic diversity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity among the accessions used in the study. There was also high diversity in the concentration of marker compounds in the collected samples as revealed by HPLC analysis. It is shown that the approaches used in the work successfully discriminate between the accessions of this species and thus they constitute interesting tools to analyze molecular, biochemical and phenotypic diversity within this species. Similarity measurement using UPGMA followed by cluster analysis resulted in formation of many groups based on geographical distribution that generally reflected expected trends between the genotypes. There were also some important exceptions like PW-S, an accession from Wastoorwan, Khrew showing close resemblance to PG-S and PG-B collected from Gulmarg but grown at two different gene banks at Srinagar and Bonera. Further an accession PSH-B from Keller was significantly diverse from the rest of the native genotypes phytochemically, morphologically and at molecular level. RAPD data analysis was found to be significant predictor of phytochemical markers in cultivated P. hexandrum germplasm. Twelve accessions grown in gene bank repository were subjected to RAPD analysis and were assessed for content of podophyllotoxin and podophyllotoxin β-d-glycoside by HPLC. Individual regressions of podophyllotoxin and podophyllotoxin β-d-glycoside by RAPD analysis against HPLC has been found to determine linear values. Strong correlation and a strong association of values of the phytochemical variables and the DNA polymorphism data has been recorded.     

Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA

The taxonomy and phylogeny of Indian Citrus is revisited using PCR-RFLP of the trnD-trnT and rbcL-ORF 106 regions as well as sequence data analysis of the trnL-trnF intergenic spacer region of cpDNA. The study was based on 50 accessions of Citrus genotypes, collected from wild, semi-wild and domesticated stocks. Of the 13 restriction enzymes (RE) used for restriction digestion of the polymerase chain reaction (PCR) amplicons, four (Hinf I, Msp I, Alu I, Hae III) generated 47 restriction fragments, of which 24 (51%) were polymorphic. PCR-RFLP data showed a genetic distance ranging from 0 to 0.79 among 50 accessions of Citrus, and a cluster analysis, based on Neighbor-Joining (NJ) method, placed all the accessions in eight major clusters. Analysis of trnL-trnF sequences from 23 representative accessions of Citrus showed a pair-wise sequence divergence rate in the range of 0–0.064. NJ, minimum evolution (ME) and maximum parsimony (MP) analyses of trnL-trnF sequences produced phylogenetic trees, which placed all the 23 accessions in five clusters. PCR-RFLP analysis resulted in a well resolved phylogenetic tree with branches supported by moderate to high bootstrap values, while the trnL-trnF sequence-based trees showed only moderate to low bootstrap support for the internal tree branches, indicating uncertain origin of some Citrus genotypes. This study shows that the trnL-trnF spacer sequence data can detect genetic variation in Indian Citrus genotypes, but the utility of the data in inferring phylogeny at intra and inter-specific levels is limited probably by factors such as hybridization, bud mutations, apomixis and polyploidy. However, PCR-RFLP and trnL-trnF data supported the recognition of C. maxima, C. medica, and C. reticulata as the basal species of edible Citrus.     

Genetic variability in wild vs. cultivated Eruca vesicaria populations as assessed by morphological,agronomical and molecular analyses

In this study, the genetic diversity of 50 individuals of rocket, Eruca vesicaria, from five accessions, four of them wild type collected from different parts of Spain and one commercial, were evaluated using morphological, agronomical and inter simple sequence repeat DNA (ISSR) data. Molecular analysis was carried out using the ISSR technique with 20 primers. Out of these 20 primers, nine were polymorphic, producing a total of 395 DNA bands, 247 of which were polymorphic among the accessions. A dendrogram drawn on the basis of a similarity matrix using the UPGMA algorithm revealed that the 50 samples of rocket plants could be classified into three major clusters at a Nei's genetic distance of 0.36. The experiment shows that molecular markers such as ISSR are a good instrument for distinguishing and selecting rocket accessions to group different wild populations. In general, a high variation was observed for most of the 16 morphological and 6 agronomical traits showing significant differences. Some morphological traits such as leaf length, petiole length and lamina width explained 69.1% of the whole variation observed in the populations, and some agronomical traits such as leaf area, nitrate and chlorophyll contents accounted for 65.7%, but the clusters generated by means of agronomical and morphological variables were less evident than when ISSR markers used. Some accessions showed good qualities, such as small leaves, high chlorophyll content, late-flowering or low nitrate content. All these parameters, together with the high degree of genetic homogeneity found, could make the local accessions good candidates for a future breeding programme.     

Wide genetic diversity of Rosa damascena Mill. germplasm in Iran as revealed by RAPD analysis

The genetic relationships among 41 Rosa damascena accessions from various cultivation areas of Iran and one accession from Bulgaria were analyzed using 31 RAPD (Randomly Amplified Polymorphic DNA) primers. Each primer exhibited 3–12 banding patterns for a total of 343 scorable and 184 polymorphic bands. The combination of 11 primers was found optimal for discrimination of 42 accessions with very low values of cumulative confusion probability (9.7 × 10⁻⁵); indicating that only one pair from over 10,000 distinct pairs of accessions would be indistinguishable. Unweighted pair group method cluster analysis based on similarity values revealed 10 groups at the distance of 0.85. The Bulgarian genotype grouped with the majority of the Iranian genotypes in a main cluster. Results of molecular variance analysis (AMOVA) indicated that the major proportion (65.7%) of the total genetic variation was within collecting provinces rather than between them. The wide genetic variation seen for R. damascena in Iran indicates that Iran is a center of genetic diversity for this species and that there is a promising future for the breeding.     

丝瓜种质资源遗传多样性的形态和RAPD标记分析

应用形态标记和RAPD标记对26份丝瓜种质材料进行遗传多样性和亲缘关系分析。所观察的44个形态性状变异系数为5.27 %～107.00 %,平均变异系数为32.75 %。从200个随机引物中筛选出16个引物,共扩增出145条带,多态性带有125条,多态性比率为86.21 %,平均Shannon多样性信息指数为 0.325。基于形态标记的聚类分析将26份丝瓜种质分为普通丝瓜和有棱丝瓜两大类。基于RAPD标记的聚类分析将26份丝瓜种质也分为两大类,但有2份有棱丝瓜种质和普通丝瓜聚为了一类,与形态标记聚类结果不一致。     

龙眼24个品种的SCoT遗传多样性分析

利用SCoT标记对24份龙眼品种资源进行了检测。从80条引物中筛选出24条重复性好,条带清晰的引物进行PCR扩增,共扩增出211条带,其中181条具有多态性,多态性带比率为85.8%。24个龙眼品种间遗传相似系数在0.65 ~ 0.86之间,说明SCoT标记能够揭示材料间较高的遗传多样性。UPGMA聚类分析显示,SCoT标记能将24份龙眼品种完全区分开,并分成两类。     

Genetic diversity of bitter gourd (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits

Bitter gourd or bitter melon (Momordica charantia L.) is considered as minor cucurbitaceous vegetable in spite of having considerable nutritional and medicinal properties. Although some reports on genetic diversity based on morphological characterization are available, no work has been conducted to estimate genetic diversity using molecular markers in this crop. In the present study, 38 genotypes of M. charantia including few commercially cultivars collected from different parts of India based on agro-ecological zones were analysed for diversity study both at morphological and molecular levels. Genomic DNA was extracted from young healthy leaves following the procedure of Doyle and Doyle [Doyle, J.J., Doyle, J.L., 1990. A rapid DNA isolation procedure from small quantity of fresh leaf material. Phytochem. Bull. 119, 11–15]. Pair-wise comparison of genotypes was calculated as per the procedure of Jaccard [Jaccard, P., 1908. Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Sci. Nat. 44, 223–270]. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was done using the computer programme NTSYS-pc Version 2.0 [Rohlf, F.J., 1998. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Version 2.01. Exeter Software, Setauket, NY, USA]. Diversity based on yield related traits and molecular analysis was not in consonance with ecological distribution. Among 116 random decamer primers screened 29 were polymorphic and informative enough to analyse these genotypes. A total of 208 markers generated of which 76 (36.50%) were polymorphic and the number of bands per primer was 7.17 out of them 2.62 were polymorphic. Pair-wise genetic distance (GD) based on molecular analysis ranged from 0.07 to 0.50 suggesting a wide genetic base for the genotypes. The clustering pattern based on yield related traits and molecular variation was different.     

DNA content,morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus.     

Use of chloroplast microsatellite markers as a tool to elucidate polymorphism,classification and origin of Tunisian grapevines

Chloroplast microsatellite markers were used in this study to genotype 43 grapevines accessions grown in Tunisia. Size variation was observed for the three cpSSR loci, both in the sample of cultivars and in wild accessions. The seven alleles observed in the sample of cultivars for the three loci are present in wild accessions except that their distribution is different. Levels of genetic diversity obtained for the Tunisian grapevines either in wild or cultivated gene pools are high and comparable with values obtained with other studied samples of Vitis vinifera. The distribution of haplotypes within the two samples is differential. Indeed, the chlorotype A is most abundant in the wild sample, whereas the chlorotype C is majority in the sample of cultivars. Haplotypes frequencies for cultivated grapevine distinguish haplotypes B and C as the most frequent (28% and 44% respectively) and haplotypes A and D as the least frequent (16% and 12% respectively). For wild grapevines, the seven alleles combined in three haplotypes, A, C and D. The haplotype A is the most frequent (44%) in the analyzed sample of wild accessions while haplotypes C and D show a frequency of 28%. Chlorotype distribution in Tunisian cultivars is comparable with that of cultivars in the Eastern Region representing the primary centre of domestication of the species. These results agree with the higher relevance of table grape cultivars in Tunisian viticulture and support an oriental origin of a large part of autochthons cultivars. Our results agree with other studies based in nuclear and chloroplast microsatellite markers and suggest independent domestication events for V. vinifera L. species.     

Assessment of genetic relationships among 29 introduced and 49 local sweet cherry accessions in Turkey using AFLP and SSR markers

Summary

The characterisation of sweet cherry (Prunus avium L.) genetic resources in Turkey may help to increase their use in breeding programmes worldwide, as Turkey is the centre of origin of sweet cherry. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were therefore used to analyse genetic diversity among a total of 78 local and introduced sweet cherry cultivars. Four AFLP primer combinations, and six SSR primer pairs for sweet cherry were used for genetic diversity analysis. A genetic similarity matrix was calculated using the combined data from AFLP and SSR analyses with simple matching coefficient. Genetic similarities among the sweet cherry genotypes studied were higher than 42%. No two accessions had an identical AFLP and SSR marker profile, indicating that all 78 genotypes were unique. An UPGMA dendrogram, based on the similarity matrix, revealed 18 separate Groups at or above the 70% similarity level. While some Groups consisted of both introduced and local genotypes, other Groups had only local genotypes. This result suggests that there was broad genetic diversity among the local Turkish sweet cherry genotypes, which was not present in the introduced sweet cherry accessions. The genetic variation present in local Turkish sweet cherry genotypes may be useful for future breeding programmes. We found that the use of both SSR and AFLP marker systems was effective for distinguishing between genetically-close sweet cherry genotypes. These marker systems can be used to complement pomological and morphological markers during the characterisation and identification of sweet cherry genotypes.     

云南蔷薇属部分种质资源的SSR遗传多样性研究

利用简单重复序列SSR（Simple Sequence Repeat）标记技术对42份蔷薇属（Rosa L.）种质资源（包括13份野生种、变种、变型及29份栽培品种）的遗传多样性进行了研究。用筛选出的18对SSR引物对42份材料DNA进行PCR扩增,在18个位点共检测到148个等位基因,每一位点的等位基因变幅为6~14个,平均8.2个。材料间遗传相似系数变化范围为0.282~0.892,表明在分子水平上云南省蔷薇属植物具有丰富的遗传多样性。本研究发现,在相似系数为0.456时,基于SSR标记的聚类分析可以将 13个蔷薇野生种明显分为5个组,这与植物形态学分类结果大体一致。在遗传相似系数为0.43水平上,聚类分析将42份供试材料分为5大组群;同时初步探讨了野生种之间以及野生种与栽培品种之间的遗传亲缘关系。     

DNA profiling of Capsicum landraces of Manipur

Seven chilli landraces of Manipur belonging to three cultivated species of Capsicum (Capsicum annuum, Capsicum frutescens, and Capsicum chinense) form economically important food crops of the region. The genotypes were characterized using ten random amplified polymorphic DNA (RAPD) markers. The cluster analysis based on Jaccard's similarity coefficient calculated by UPGMA method differentiated the genotypes into two main cluster groups. One cluster represented the C. annuum genotypes while the other cluster represented the C. frutescens and the C. chinense genotypes. C. chinense genotypes were more close to C. frutescens genotypes. Genetic variation between the C. frutescens genotypes was more than among the C. annuum genotypes and the C. chinense genotypes were the least similar ones.