Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic (Allium sativum L.)

Here we characterized eight novel polymorphic SSR markers, developed from an enriched genomic library of garlic (Allium sativum L.). These SSRs produced a total of 64 alleles across 90 garlic accessions, with an average of 8 alleles per locus. Values for observed (H_O) and expected (H_E) heterozygosity ranged from 0.16 to 0.77 (mean = 0.44) and from 0.22 to 0.86 (mean = 0.65), respectively. Six loci deviated significantly (P < 0.05) from Hardy–Weinberg equilibrium (HWE). The averages of gene diversity and PIC values were 0.65 and 0.62, respectively. The mean genetic similarity coefficient was 0.4380, indicating that among garlic accessions existed wide genetic variation. Based on 64 alleles obtained by 8 SSRs, a phenogram was constructed to understand the relationships among the 90 accessions. These newly developed SSRs should prove very useful tools for genotypes identification, assessment of genetic diversity and population structure in garlic.     

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.     

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.     

Inter- and intra-specific genetic diversity among cyclamen accessions investigated by RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity of 26 Cyclamen persicum and Cyclamen com accessions. Eighty-four arbitrary primers tested, among which nine primers showed reliable polymorphic banding patterns and yielded 104 polymorphic markers. Jaccard's similarity coefficient among accessions ranged from 0.99 to 0.08. At a similarity of 68%, accessions were divided into three clusters. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. The RAPD analysis offered a rapid and reliable tool for the estimation of inter- and intra-specific variability in cyclamens. The wide genetic variation observed for cyclamens within Iran guarantees a promising future of breeding.     

Genetic Relationship of Iranian Pear Genotypes with European and Asian Pears as Revealed by Random Amplified Polymorphic DNA Markers

Genetic variation within specific fruit tree germplasms is an important tool in fruit tree breeding programs. In the present work, the genetic relationship of 31 European and Iranian (Pyrus communis L.) and Asian (Pyrus serotina Rehd) genotypes of pear were studied using 19 randomly amplified polymorphic DNA (RAPD) primers. Fifteen out of the 19 primers used in this study amplified 3373 clear and reproducible bands associated with 150 loci and many of them were polymorphic. The dendrogram resulting from the unweighted pair-group method of arithmetic cluster analysis separated the cultivars into eight groups. The correlation coefficient between the cophenetic matrix and the similarity matrix was 0.82 (r = 0.82). There was a significant difference between populations and most studied genotypes clustered closely together based on their geographic origin and Iranian pears placed between two groups of pears. Results showed the suitability of RAPD analysis in genetic diversity study of pear.     

Genetic diversity of Iranian and some European grapes as revealed by nuclear and chloroplast microsatellite and SNP molecular markers

Genetic and chloroplast haplotype variations of 35 Iranian genotypes and 10 European grape cultivars were investigated using 9 nuclear simple sequence repeats (nSSRs), 4 chloroplast simple sequence repeats (cpSSRs) and 46 single nucleotide polymorphism (SNP) markers. In total, 83 alleles were detected at nine nSSRs, giving a mean of 15.66 alleles per locus and polymorphism information content (PIC) values ≥0.75 ranged from 0.75 to 0.90. For SNP markers, PIC values varied from 0.30 to 0.39 with an average of 0.34. Analysis of molecular variance revealed 97 and 93% of partitioned genetic diversity within populations using nSSRs and SNPs markers, respectively. Un-weighted neighbour-joining (NJ) cluster analysis grouped grapes into 10 and 9 major clusters using SSR and SNP markers, respectively. Synonyms and homonyms were identified among the Iranian genotypes. Close genetic relationship among Farkhi and Bidane-Sefid genotypes may probably propose a common ancestor and mutational evolution. Most European cultivars were differentiated from Iranian genotypes, however, clustering of some Iranian genotypes with European cultivars in the same clusters suggests that clonally propagated materials have probably been exchanged from the Middle East to West or vice versa. C and D chloroplast haplotypes were the most frequent within the Iranian genotypes, while A chloroplast haplotype was exclusively observed among European cultivars.     

Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It is native to Iran and spread from Iran to other areas. In this study amplified fragment length polymorphism (AFLP) was used to detect intra- and inter-population genetic diversity of pomegranate. A group of 67 accessions belonged to 4 populations from Iran was studied using eight primer combinations. A total of 221 scorable bands were amplified, of which, 118 (54.13%) were polymorphic. Resolving power (Rp) ranged from 5.70 to 9.21, and the average of polymorphism information content (PIC) per primer pair was 0.40. According to Nei's gene diversity and allelic statistics, Isfahan population had a highest genetic diversity (H = 0.3646, I = 0.5327, N_e = 1.6467). Coefficient of gene differentiation between populations (GST) was 0.124, indicated that mainly proportion of genetic variation (87.6%), was within populations and the remaining (12.4%) of the variation was among populations that, also supported by analysis of molecular variance (AMOVA). The gene flow (Nm) varied from 0.969 to 10.404 between pair-wise populations and was 3.504 among all of the populations. The Jaccard similarity coefficient between individuals ranged from 0.26 to 0.88. The UPGMA dendrogram clustered all 67 accessions into 6 groups. In some cases accessions from same region were grouped together but in most cases, there was gene exchange. To study the genetic relationships among populations, a principal coordinate analysis (PCoA) based on Nei's genetic distances was performed. Results of this study showed that AFLP marker can be a useful tool for investigating the genetic diversity of pomegranate genotypes.     

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.     

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%).     

Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. sesquipedalis) using morphological characters,SSR and ISSR analysis

Genetic diversity and relatedness of 23 yardlong bean (Vigna unguiculata spp. sesquipedalis) accessions and 7 accessions of a hybrid between cowpea (V. unguiculata spp. unguiculata) and yardlong bean (dwarf yardlong bean) in Thailand were estimated using morphological characters, simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers. In addition, two mungbean (Vignaradiata (L.) Wilczek) and two blackgram (Vigna mungo (L.) Hepper) accessions were also used as outgroup species for molecular analysis. Five morphological characters were diverse among most accessions. However, five groups of 2–3 accessions could not be distinguished from one another based on these morphological characters alone. Unweighted pair-group arithmetic average (UPGMA) analysis of these characters separated these 30 accessions into 2 major groups; the yardlong bean group and the dwarf yardlong bean group. Eleven of the sixteen SSR primers yielded clear SSRs, ten of which were polymorphic (90.91% polymorphism), detecting a total of 54 alleles with an average of 4.91 alleles per locus. These 10 polymorphic SSR markers successfully distinguished 28 yardlong bean and dwarf yardlong bean accessions. The polymorphic information content (PIC) among genotypes varied from 0.251 to 0.752 with an average of 0.597. Among the 16 ISSR primers used, a total of 312 ISSR fragments were amplified for these three Vigna species, revealing the polymorphism percentage of 91.03%. The average ISSR PIC value (0.197) with the range of 0.137–0.276 was lower than that of SSR. Nevertheless, the average marker index of this multilocus marker was 3.495, which was higher than that of SSR (0.669), owing to the differences in the effective multiplex ratio. In addition, Mantel test cophenetic correlation coefficient was higher for ISSR (0.566) than that of SSR (0.198). These results indicated higher efficiency of ISSR for estimating the levels of genetic diversity and relationships among yardlong beans and dwarf yardlong beans in this study. Pair-wise coefficients of SSR- and ISSR-based genetic similarity among all yardlong bean and dwarf yardlong bean accessions averaged 0.87 and 0.91, respectively, suggesting a narrow genetic base that emphasizes the need to broaden genetic diversity to ensure continued breeding success. Clustering of genotypes within groups was not similar when SSR and ISSR derived dendrograms from UPGMA analysis were compared. It appeared that ISSR was the most effective marker system in determining the genetic variability and relationships among yardlong bean and dwarf yardlong bean accessions and differentiating three Vigna species. In addition, ISSR was also most useful for variety identification since all 30 yardlong beans and dwarf yardlong bean accessions can be effectively distinguished by only four ISSR primers with the highest PIC values.     

Genetic diversity in fruiting-mei,apricot, plum and peach revealed by AFLP analysis

Summary

High-throughput amplified fragment length polymorphism (AFLP) analysis, employing a two-dye automated DNA sequencer, was used to genotype accessions of four important Prunus spp. fruit crops in China, fruiting-mei (P. mume), apricot (P. armeniaca), plum (P. salicina, and P. ussuriensis) and peach (P. persica). Nine primer pairs generated a total of 2,089 AFLP bands, of which 94.4% were polymorphic. Levels of polymorphism and the genetic relationships among the crops were studied.The fixation index (F_st) of the four different fruit trees ranged from 0.53 – 0.82, and the average genetic diversity (AGD) in these species ranged from 3.2% to 12.8%. These four stone fruit species could be clustered into four clear groups on a phylogenetic tree based on bootstrap analysis. Phylogenetically, fruiting-mei was shown to be closest to apricot and most distant from peach.     

RAPD markers reveal polymorphism among some Iranian pomegranate (Punica granatum L.) genotypes

In this study RAPD markers were used to determine the diversity level among 24 Iranian pomegranate genotypes. One hundred decamer random primers were used for PCR reactions, among which 16 showed reliable polymorphic patterns. These primers produced 178 bands, of which 102 were polymorphic. Cluster analysis of the genotypes was performed based on data from polymorphic RAPD bands, using Jaccard's similarity coefficient and UPGMA clustering method. The highest and lowest similarities detected between genotypes were 0.89 and 0.29, respectively. At a similarity of 60%, the genotypes were divided into four sub-clusters. Cophenetic correlation coefficient between similarity matrix and cophenetic matrix of dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. RAPD markers showed to be a useful tool for studying the genetic diversity of pomegranate.     

Detection of genetic diversity among populations of sweet cherry (Prunus avium L.) by AFLPs

Summary

The high degree of polymorphism of AFLPs provides an efficient system for identification and genome analysis of sweet cherry (Prunus avium) cultivars and selections. The cultivars of sweet cherry have usually been characterized by assessment of phenotypic and pomological traits. AFLP markers were employed to identify 38 sweet cherry accessions and estimate the genetic diversity among this material. Ten of 18 tested primer combinations were informative with up to 80 bands per primer combination. Seven to 33% of the amplfied bands were polymorphic depending upon primer combination. Allcultivars and selections tested could be clearly identified. The objective of this work was to demonstrate the usefulness of molecular markers in revealing the genetic diversity among different sweet cherry genotypes.     

Assessment of genetic diversity in common bean (Phaseolus vulgaris L.) germplasm using amplified fragment length polymorphism (AFLP)

AFLP technique was applied to assess genetic diversity among 44 common bean accessions that included 6 exotic accessions, 15 Indian land races and 23 released varieties. Eight AFLP primer pairs were used that produced 820 products of which 698 were polymorphic (85.12%). Wide variations were observed among all the accessions for the number of amplification products, percent polymorphism and average polymorphism information content (PIC). The Jaccard's similarity indices (J) based on the AFLP profiles were subjected to UPGMA cluster analysis. The dendrogram generated revealed seven major groups. Seventeen out of 23 released varieties were restricted to clusters VI and VII. The value of r = 0.934 in Mantel's test for cophenetic corrlelation applied to the cluster analysis indicated the high fitness of the accessions to a group. The germplasm used in the present study had narrow genetic base, although moderate to high genetic diversity was observed. The details of diversity analysis and the potential use of Indian common bean accessions in common bean breeding programme are provided in the present study.     

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.     

Commercial Iranian olive cultivars: morphological traits,molecular diversity,and genetic structure

Iran is considered to have a unique gene pool of different fruit and nut species including olive (Olea europaea L.). In this study, we used 22 previously developed microsatellite (simple sequence repeat; SSR) markers for olive to evaluate the level of genetic variation and to produce identification keys for 63 Iranian accessions of olive belonging to 17 groups of cultivars. Based on morphological features, the number of flowers per inflorescence, fruit weights, endocarp weights, oil percentages, and flesh weights per endocarp had the highest coefficient of variation values, indicating the large extent of morphological variability among the 63 Iranian olive cultivars studied. All 22 microsatellite (SSR) markers revealed a high level of polymorphism, with a mean polymorphic information content (PIC) value of 0.511. Analyses of genetic structure among the 63 olive accessions were carried out using model-based methods, which showed a tendency for geographical clustering. Ten SSRs out of the 22 were successful for the identification of unique ID keys for 52 of the 63 accessions. In most cases, there was disagreement between the molecular data and the morphological data. These results could be used to reconstruct and maintain a collection of olive for future breeding programmes.     

Genetic divergence among gerbera accessions evaluated by RAPD

Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a total of 42 accessions of Barberton daisy (Gerbera jamesonii) consisting of 29 commercial and 13 wild accessions. A total of 74 polymorphic bands were obtained employing a set of 12 primer pairs. The average genetic similarity coefficient for the 42 accessions, evaluated by Jaccard index was 0.55 ranging from 0.28 to 1.00. The genetic structure found among Barberton daisy accessions was evaluated by hierarchic classification analyses and UPGMA modeling, revealing six clusters of genotypes where two of them include the wild accessions and the remaining four including commercial material, except for wild genotype number 9. Shannon (H′) index was calculated using the molecular markers to investigate the genetic variation among the Gerbera accessions and showed higher values for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster, namely 0.34 versus 0.27, respectively. Therefore, both calculated indices (Jaccard and Shannon) indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics.     

Assessment of genetic variability in Italian heritage peach resources from Emilia-Romagna using microsatellite markers

Summary

Heritage peach (Prunus persica L. Batsch) varieties in Emilia-Romagna, Italy’s leading peach-producing region, are largely marked by melting, juicy, aromatic white-fleshed fruit with a short shelf-life and susceptibility to bruising.While these varieties have rapidly been replaced since the 1950’s by yellow-fleshed peaches from US breeding programmes with improved handling resistance, the Fruit Research Unit of the Agricultural Research Council, at Forlì, has begun an effort to safeguard and characterise the heritage varieties of Emilia-Romagna. The programme has screened 26 local heritage accessions using a set of 16 highly polymorphic microsatellite (simple sequence repeat; SSR) markers to assess genetic diversity, to elucidate inter-relationships, and to resolve any cases of homonymy. Seven international peach cultivars have been included in order to standardise allele scoring. This study resulted in 19 unique molecular profiles among the heritage accessions, and a relatively high mean observed heterozygosity value of 0.49 for a germplasm pool from a restricted region, an indicator of potential genetic diversity. The main morphological features of the heritage peaches are reported, and potential benefits for future breeding programmes discussed.     

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.