AFLP-based genetic relationships in the Mediterranean myrtle (Myrtus communis L.)

Myrtle is an important plant species of the Mediterranean maquis, and is widely exploited for its aromatic properties. It is used in Italy for the production of a typical liqueur, for cut foliage and as an ornamental pot plant. We report the use of amplified fragment length polymorphism (AFLP) profiling to estimate genetic similarities within myrtle germplasm collected from six Italian regions, and from the Botanical Gardens of six other Mediterranean countries (including the outgroup Myrtus communis subsp. tarentina). Five AFLP primer combinations identified 122 polymorphic fragments analysing 92 individual samples, most of them (56%) were informative in discriminating among the populations. The AFLP patterns indicated that the majority of the variation occurs among rather than within populations (G_ST = 0.61). A neighbour-joining (NJ) tree separated the populations into two main branches: the first one grouped some of the Italian populations with those from Spain and Portugal; the second one included a Southern Italian subcluster together with samples from Greece, Israel, France and Croatia. A principal coordinate analysis supported the two major branches identified in the NJ analysis and showed the separation of Western and Eastern Mediterranean populations along the first axis. The Italian populations did not cluster in a single clade, but rather form distinct regional groups. The present analysis suggests that Italy represents a botanical transition zone between the Western and Eastern Mediterranean region in Myrtus communis.     

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.     

Utility of RAPD,ISSR, IRAP and REMAP markers for the genetic analysis of Citrus spp.

The application and informativeness of RAPD, ISSR, IRAP and REMAP markers to study the genetic diversity and relationship among the Citrus and its relative genotypes were investigated. High levels of polymorphism were observed for all four marker systems. The RAPD technique generated the highest number of polymorphic bands and average number of polymorphic band per assay unit. Average limit of the discriminating power was very close to its actual discriminating power of each marker. The highest and the lowest values of effective number of patterns were obtained from the marker REMAP (5.94) and RAPD (4.48), respectively. Correlation between the genetic similarities matrices were estimated from all four markers using the Mantel matrix correspondence test, and results showed significant correlations among the RAPD, ISSR, IRAP and REMAP similarity matrices. The highest correlations were found comparing RAPD and ISSR markers, whereas RAPD and REMAP (r = 0.143) markers were poorly correlated. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and Citrus germplasm classification, cluster analysis was performed. All four techniques, solely or in combination, discriminated the genotypes very efficiently and generated a high similarity in dendrogram topologies, although some differences were observed. The linkage analysis was conducted based on the segregation of 38 RAPD, 13 ISSR, 19 IRAP and 9 REMAP loci in 96 progeny of an intergeneric cross Citrus sinensis and Poncirus trifoliata. Among the 81 studied loci 65 loci distributed on five linkage groups. Comparing the result obtained with RAPD, ISSR, IRAP and REMAP markers in this study, IRAP and REMAP proved to be as a reliable molecular marker for fingerprinting, mapping and diversity study of Citrus and its relatives.     

Comparison of SSR polymorphisms using automated capillary sequencers,and polyacrylamide and agarose gel electrophoresis: Implications for the assessment of genetic diversity and relatedness in almond

In this work we have performed a comparative study of the utilization of three electrophoresis separation methods for the analysis of peach SSR polymorphism in almond and its implications in the assessment of genetic diversity and relatedness. Automated SSR detection and polyacrylamide gel electrophoresis were the most efficient methods and would be able to resolve allelic variation at a finer scale than the electrophoresis in MetaPhor^® agarose. Moreover, automated electrophoresis detection is much more expensive in terms of cost of equipment and cost of the analysis. In addition, SSR detection using polyacrylamide gel electrophoresis showed similar results than automated sequencing, although it is more time-consuming and toxic than electrophoresis in agarose gels. Discrepancies among polyacrylamide and automated capillary, and MetaPhor^® were observed when differences in SSR polymorphisms were between 1 and 5 bp. While the use of MetaPhor^® agarose appears less indicated for genotype characterization, this technique may be the most convenient in other applications, i.e. mapping of population, due to its lower cost and easier routine application. The utilization MetaPhor^® agarose produced a slightly different clustering of genotypes in spite of the big differences observed in the DNA fingerprinting. However, bootstrap values using MetaPhor^® agarose were lower indicating a lower accuracy of this SSR polymorphism analysis method.     

Molecular based assessment of genetic diversity within Barbary fig (Opuntia ficus indica (L.) Mill.) in Tunisia

In this work, we report for the first time on the analysis of genetic diversity within a set of 36 Tunisian Opuntia ficus indica (L.) Mill. ecotypes using RAPD markers.     

基于表型性状和SRAP标记的观赏用辣椒种质资源遗传多样性分析

通过筛选种质资源,为观赏用辣椒的新品种选育提供依据,对20份辣椒材料进行遗传多样性、相关性和SRAP标记分析。结果表明,33个性状的多样性指数范围为0.50~1.97,青熟果色和果形的多样性指数很高。9个数量性状中,单果质量和单株果数的变异系数最大,变异丰富;株幅的变异系数最小,人工选择面窄。果顶形状、商品果纵径、果梗长度与果形显著相关,花冠色、主茎色、叶色与青熟果色4个性状彼此显著正相关。26个SRAP引物组合共扩增出82个多态性条带,每个组合的多态性条带数为1~8条不等,平均3.2个。在相似系数0.64处将20份材料分为5个类群,类群间亲缘关系较远,表现型多样性强。研究结果可为后续筛选和培育果形奇特、色彩丰富的观赏用辣椒品种提供依据。     

Genetic diversity and relationships within Citrus and related genera based on sequence related amplified polymorphism markers (SRAPs)

Sequence related amplified polymorphism (SRAP) markers were used to detect molecular marker polmorphisms among 86 citrus and their relatives in Aurantioidea. Twenty-one SRAP primer combinations produced a total of 376 polymorphic fragments with an average of 17.9 per primer combination and an average polymorphism information content (PIC) of 0.86. The unweighted pair group method arithmetic average (UPGMA) analysis demonstrated that the accessions had a similarity range from 0.28 to 1.00 with a mean of 0.64. The subtribe Clauseninae (tribe Clauseneae) separated from the subtribes of the tribe Citreae. The subtribe Balsamocitrinae (tribe Citreae) was the most distant from the others. In the Citrinae, ‘primitive citrus fruit trees’ and ‘near citrus fruit trees’ groups did not clearly separate from each other but all genera in these groups were distinct. On the other hand, subgenus Papeda and subgenus Citrus were not separated clearly in the dendrogram. C. maxima, C. medica and C. reticulata separated into three distinct clusters in agreement with three ‘true basic species’ thesis. Similarity-based analyses supported the theory of few ancestral species in Aurantioidea.     

RAPD analysis of genetic diversity among and within Portuguese landraces of common white bean (Phaseolus vulgaris L.)

Seventeen landraces of common beans (Phaseolus vulgaris L.) sampled in the North and Centre of Portugal were analyzed at population level for 689 RAPD loci amplified by using forty random primers. The two different parameters used to estimate the genetic variability in and between samples indicate that the inter-population component of the genetic variability is mainly responsible for the diversity found, since only about a 10% would be of an intra-population nature. In addition, the gametic disequilibrium was estimated and reached an average value of 40% for the different combinations of pairs in the 689 loci studied taking the 17 samples as a whole population. Self-pollination, genetic drift and adaptation would thus be favouring the formation of multilocus associations. In addition, the fingerprinting study suggests that each landrace produced unique amplification products allowing it to be distinguished from the other tested genotypes, but, nevertheless, the landraces show a considerable genetic similarity (56% and 70.5% using two different methods). The Neighbour-Joining dendrogram did not show any relation between the geographical distribution of landraces and genetic distance. The results suggest that these Portuguese landraces conserved an important genetic diversity that can be useful to widen the genetic base of currently cultivated beans.     

Comparison of the use of morphological,protein and DNA markers in the genetic characterization of Iranian wild Prunus species

In this study, the genetic diversity of four Iranian wild Prunus species including Prunus eleagnifolia, Prunus hauskenchtii, Prunus scoparia and Prunus lycioides were investigated using morphological, protein and DNA markers. DNA markers included nuclear and chloroplast SSRs and self-incompatibility (S) allele amplification. At the morphological level, leaf width showed significant differences between the four wild Prunus species. Concerning fruit and kernel characters, their values are relatively similar indicating the high degree of homoplasy described in Prunus. Nuclear SSR markers have been the most abundant markers with a higher polymorphism in comparison with morphological, protein and chloroplast SSR markers. Results also indicated the high variability present in the S locus. On the other hand, the correlation between the clustering based on DNA markers and protein were in general low. Dendogram performed using nuclear and chloroplast SSR indicated a more diffuse clustering between the wild almond species probably due to the natural introgression of genes observed in these wild almond species. Data from the analysis of the total protein seems to be more accurate to establish taxonomy relationships in these very close wild species.     

Assessment of genetic diversity among mango (Mangifera indica L.) genotypes using RAPD markers

Knowledge about the extent of genetic diversity/relatedness in mango germplasm is vital for developing coherent strategies for future gains in productivity. The genetic diversity/relatedness among mango cultivars/genotypes developed in Pakistan has not been investigated previously. We have assessed the genetic diversity among 25 mango genotypes/cultivars using randomly amplified polymorphic DNA (RAPD). Sixty random ten-mer primers were surveyed, out of which 45 yielded amplicons in all the genotypes. Genetic similarity between genotypes/cultivars was in the range of 64–89% with an average of 74%. Similarly, the genetic relatedness among all variants derived from a mango cultivar Chaunsa was in the range of 81.18–88.63%. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The genotypes were grouped into three (A, B, C) clusters. Generally, genotypes originating from Pakistan were grouped in cluster ‘A’ while cluster ‘B’ primarily composed of southern India as well as Florida cultivars. Kensington Pride was the most distantly related genotype which grouped with Maya and Yakta, forming a distinct cluster ‘C’.     

Assessment of genetic diversity of Highland bananas from the National Banana Germplasm Collection at Rubona,Rwanda using RAPD markers

The genetic similarities of 49 accessions of bananas from The National Banana Collection at Rubona were investigated using random amplified polymorphic DNA markers. A total of 120 primers were screened for their usefulness in amplifying DNA fragments of four cultivars belonging to the subgroup Mutika–Lujugira. Fifteen random primers were selected for more detailed analysis, on the basis of providing reproducible amplification and revealing a high level of variation between the four cultivars. The genetic similarity was estimated using a simple matching coefficient which showed the lowest value of 0.46 between ‘Ingumba’ and ‘Ishika’ and the highest value of 0.85 between ‘Kirayenda’ and ‘Inyabukuwe’. The data of matrix of coefficient of similarity was subjected to cluster analysis using the unweighted pair group method with arithmetic average (UPGMA). Each accession was clearly separated. The results of this study are important for the curation of the banana germplasm collection in Eastern Central Africa and for future breeding of this crop.     

Using bulked AFLP analysis to assess genetic diversity in Echinacea species

Echinacea is an allogamous genus, thus its cultivars or populations are genetically heterogeneous. Using amplified fragment length polymorphism (AFLP) to estimate the genetic diversity of Echinacea is generally limited by the large number of individual plants and the higher cost that need to be processed. In the present study, effectiveness of several sizes of DNA bulking (10, 15, 20, 25 and 30 individuals) with 20, 36 and 55 primer pairs was compared using AFLP in determining the genetic diversity of Echinacea species. The results indicated that the use of bulked DNA-based AFLP analysis by using the selected eight primer pairs was capable of detecting genetic diversity between the tested Echinacea species, provided that the potential presence of low frequency variants was ignored and a possible bias in the estimates of genetic similarity was accepted. The assessments showed that a bulk of 15 individuals could detect sufficient AFLP variations at most genomic sites. Additionally, 20 primer pairs could generate sufficient polymorphic fragments to achieve high resolving power of AFLP for the tested Echinacea species.     

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.     

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.     

Use of ISSR markers to assess genetic diversity of African edible seeded Citrullus lanatus landraces

We used the twenty primers to evaluate the genetic variability of 80 individuals belonging to four accessions of edible seeded Citrullus lanatus originated from Côte d’Ivoire. Edible seeded C. lanatus, named “egusi” or “pistachio”, had a great importance in nutrition in West Africa. Nevertheless, due to its neglected status no study to our knowledge has been devoted to its genetic variability using DNA markers. The twenty ISSR primers generated 258 bands among which 252 were polymorphic (97.67%). On the whole, the bands generated revealed three types of profile sharply distinct from each other with minor differences within each type. One profile (P1) was most frequent with 65 individuals. Three accessions (NI084, NI127 and NI145) generated the three types of profile and had medium values of genetic diversity (GD = 0.246–0.275, respectively). On the opposite, the accession NI076 only contained individuals of the most represented type of profile (P1) and had the lowest genetic diversity (GD = 0.055 ± 0.017). The pairwise genetic distance between the 80 individuals varied from 0 to 0.61. The Factorial Component Analysis and the dendrogram clearly separated the 80 individuals into three clusters corresponding to the three types of profile. The results showed that clusters were well separated from each other whereas accessions were not. Our results suggest that high number of individuals should be taken into account for sampling missions and conservation strategies because accessions were not well differentiated from each other. Local agricultural practices consisting of frequent seeds exchanges between farmers and the conservation of harvested seeds for next year culture could be one explanation.     

Assessment of genetic diversity of Latvian and Swedish sweet cherry (Prunus avium L.) genetic resources collections by using SSR (microsatellite) markers

Three previously described highly polymorphic SSR (microsatellite) primer pairs were tested on 126 sweet cherry (Prunus avium L.) accessions to adapt a fast, reliable method for preliminary screening of sweet cherry germplasm collections and to compare two sweet cherry germplasm collections: at the Latvia State Institute of Fruit-Growing, Dobele (LIFG-Dobele) and at the Division of Horticultural Genetics and Plant Breeding at Balsgård, Department of Crop Sciences, Swedish University of Agricultural Sciences (SLU-Balsgård). The SSR loci were highly polymorphic with 4–10 different alleles and 5–18 genotypes. Heterozygosity values ranged from 0.431 to 0.809, gene diversity (PIC) values ranged from 0.400 to 0.753, and the discriminating power of each locus varied from 0.631 to 0.894. The combined discriminating power of all loci was highly effective (0.996). Sixteen identical accession groups with the same allele profile were discovered in both collections. This study demonstrated that SSR fingerprinting with the three primer pairs tested, can be used for preliminary characterization of sweet cherry germplasm collections.     

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.     

Phenotypic variability and genetic structure in plum (Prunus domestica L.), cherry plum (P. cerasifera Ehrh.) and sloe (P. spinosa L.)

In the present study, phenotypic variability of 80 plum (Prunus domestica L.) varieties maintained in the French National Plum Collection was evaluated with 19 quantitative traits. In addition, genetic diversity and genetic structure was studied in three plum species (P. domestica L., Prunus cerasifera Ehrh. and Prunus spinosa L.) using chloroplast DNA (cpDNA) markers and five single sequence repeat (SSR) loci. Based on phenotypic traits, some varieties, such as mirabelle plums, grouped together. Bayesian structure analysis was used to identify different genetic groups, whereby damson plums were clearly distinguished from greengage plums. When examining the three species together, a higher level of cpDNA allelic richness was found in P. cerasifera and in P. spinosa than in P. domestica where only five cpDNA haplotypes were detected in the national plum collection, with one main haplotype that accounted for 80% of the varieties studied. P. domestica cpDNA haplotypes tended to group together with P. cerasifera haplotypes whereas most of P. spinosa haplotypes formed a separate cluster. SSR markers were somewhat able to distinguish the three species. These results provide some clues as to the origin of plum and the various plum varieties. Our results also provide useful information for the management of plum genetic resources.     

Genome composition and genetic diversity of Musa germplasm from China revealed by PCR-RFLP and SSR markers

Banana (Musa spp.) is one of the most important crops in the world. In this study, 216 banana accessions, 184 from the National Banana Germplasm Collection of China (NBGCC) and 32 from the International Network for the Improvement of Banana and Plantain (INIBAP), were used to determine the genome composition of banana plants in these collections and to estimate their genetic diversity. The genome composition was examined using PCR-RFLP markers. The molecular data for all but one accession (ITC 1231) from INIBAP were in agreement with the initial records based on phenotypic characteristics. Microsatellite (SSR) markers were used to investigate the genetic variability and relationships among these banana accessions. Ten of the 47 primer pairs tested consistently produced reproducible and discrete fragments. We identified a total of 92 alleles, ranging from 5 to 15 per locus. The genetic similarity between the accessions ranged between 0.1 and 1, when estimated using Jaccard's coefficient. The UPGMA method based on genetic similarities, grouped the NBGCC accessions according to those containing the ‘A’ and ‘B’ genomes. However, this analysis could not separate all the accessions, especially the somatic mutations, using the primers in this study. These data indicated that limited genetic variation exists within these accessions and the collections of NBGCC should include a much wider range of banana plant material.