RAPD and ISSR molecular markers in <Emphasis Type="Italic">Olea europaea</Emphasis> L.: Genetic variability and molecular cultivar identification

Thirty Portuguese and eight foreign olive (Olea europaea L.) cultivars were screened using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers. Twenty RAPD primers amplified 301 reproducible bands of which 262 were polymorphic; and 17 ISSR primers amplified 204 bands of which 180 were polymorphic. The percentage of polymorphic bands detected by ISSR and RAPD was similar (88 and 87%, respectively). The genetic variability observed was similar in the Portuguese and foreign olive cultivars. Seven ISSR and 12 RAPD primers were able to distinguish individually all 38 olive cultivars. Twenty specific molecular markers are now available to be converted into Sequence Characterised Amplified Region (SCAR) markers. Relationships among Portuguese and foreign cultivars is discussed.     

AFLP Fingerprinting in Pigeonpea (Cajanus cajan (L.) Millsp.) and its Wild Relatives

Detection of DNA polymorphism in cultivated pigeonpea (Cajanus cajan) and two of its wild relatives Cajanus volubilis and Rhynchosia bracteata is reported here for the first time using amplified fragment length polymorphism (AFLP) fingerprinting. For this purpose, two EcoRI (three selective nucleotides) and 14 MseI (three selective nucleotides) primers were used. The two wild species shared only 7.15% bands with the pigeonpea cultivars, whereas 86.71% common bands were seen among cultivars. Similarly, 62.08% bands were polymorphic between C. volubilis and pigeonpea cultivars in comparison to 63.33% polymorphic bands between R. bracteata and pigeonpea cultivars, and 13.28% polymorphic bands among pigeonpea cultivars. The cluster analysis revealed low polymorphism among pigeonpea cultivars and very high polymorphism between cultivated pigeonpea and its wild relatives. The AFLP analysis also indicated that only one primer combination (EcoRI + ACT and MseI + CTG), at the most any four primer pair combinations, are sufficient for obtaining reliable estimation of genetic diversity in closely related cultivars like pigeonpea material analyzed herein. AFLP analysis may prove to be a useful tool for molecular characterization of pigeonpea cultivars and its wild relatives and for possible use in genome mapping.     

Studying the extent of genetic diversity among Gossypium arboreum L. genotypes/cultivars using DNA fingerprinting

Genetic diversity is an area of concern for sustaining crop yield. Information on genetic relatedness/diversity among Gossypium arboreum L. cultivars/genotypes is scanty. We have used random amplified polymorphic DNA (RAPD) analysis to assess the genetic divergence/relationship among 30 genotypes/cultivars of G. arboreum. Of 45 primers surveyed, 63% were polymorphic. Out of the total number of loci amplified, 36% were polymorphic. The calculated genetic similarity between the cultivars/genotypes was in the range of 47.05–98.73%. Two genotypes, HK-244 and Entry-17, were the most distantly related. The average genetic relatedness among all the genotypes was 80.46%. However, most of the cultivated varieties showed a close genetic relationship, indicating a narrow genetic base in comparison to the non-cultivated germplasm. The calculated coefficients were used to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA) algorithm, which grouped the genotypes/cultivars into two major and three smaller clusters. The study is the first comprehensive analysis of the genetic diversity of G. arboreum germplasm and identifies cultivars that will be useful in extending the genetic diversity of cultivated varieties and future genome mapping projects.     

Exploration of intra- and inter-population genetic diversity in <Emphasis Type="Italic">Hedysarum coronarium</Emphasis> L. by AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to characterize the genetic diversity within and among natural populations and cultivars of Hedysarum coronarium. Twelve populations within Tunisia were evaluated with three AFLP primer combinations. A total of 207 reproducible bands was detected of which 178 (86%) were polymorphic. The great discriminative power of AFLP markers and their ability to represent genetic relationships among Hedysarum plants was demonstrated. Genetic diversity within and among populations was assessed through Principal Component Analysis (PCA) and cluster analysis by using the Neighbor-joining clustering algorithm. AFLP technology has provided evidence of a high degree of intra- and inter-population genetic diversity in H. coronarium. AFLP banding patterns provided molecular markers correlated with the plants’ geotropism. In addition, AFLP markers can differentiate wild accessions from cultivars. Moreover, geographical origins did not correspond to population clustering.     

Genetic diversity of European pear cultivars (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) and wild pear (<Emphasis Type="Italic">Pyrus pyraster</Emphasis> (L.) Burgsd.) inferred from microsatellite markers analysis

The aim of this study was to identify the group of highly polymorphic microsatellite markers for the identification of six pear cultivars (P. communis) and two individuals of wild pear (P. pyraster). From among 40 tested SSR markers, 19 were selected to profile genetic diversity in pear genotypes due to high polymorphisms. These markers showed high heterozygosity levels (0.5–1) and, on average, 6.4 alleles per marker were found. The set of microsatellite markers employed in this study demonstrated usefulness of microsatellite markers for the identification of pear genotypes. The examined wild forms were represented in this study by only two individuals of P. pyraster. It can be assumed that these forms were distinctly different from the cultivated pear cultivars.     

Application of random amplified polymorphic DNA to study genetic diversity in Paspalum scrobiculatum L. (Kodo millet, Poaceae)

Summary Genetic diversity and patterns of geographic variation among collections of Paspalum scrobiculatum (kodo millet) and P. polystachyum were studied using molecular markers generated through the random amplified polymorphic DNA (RAPD) method. A high level of polymorphism in RAPD markers was observed among the individual accessions, demonstrating the high genetic diversity of the crop. The markers obtained from the RAPD method were analyzed with the cluster analysis, principal coordinates and minimum spanning tree methods. Three major groups were resolved, one representing the African accessions, and two for the Indian accessions. The accessions of the north African kodo millet and P. polystachyum (considered conspecific with P. scrobiculatum) were quite distinct. The Australian kodo millet showed higher affinity to the African types. The study demonstrated that the RAPD technique can be applied to resolving degrees and patterns of genetic variation at the population and species levels, identifying cultivars, and defining gene pools of this crop.     

Assessment of genetic diversity in cultivars and wild accessions of Luohanguo (<Emphasis Type="Italic">Siraitia grosvenorii</Emphasis> [Swingle] A. M. Lu et Z. Y. Zhang), a species with edible and medicinal sweet fruits endemic to southern China,using RAPD and AFLP markers

Genetic variation of wild populations and cultivars of Luohanguo (Siraitia grosvenorii), a plant species endemic to southern China, was assessed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. Based on the results for 130 individuals from seven populations, a high level of genetic diversity of Luohanguo was observed at the species level. The percentage of polymorphic loci (P) was 89.4%, Nei’s gene diversity (H _e) was 0.239, and Shannon’s information index (H _o) was 0.373 based on the combined AFLP and RAPD data. There was a high degree of genetic differentiation, with 45.1% of the genetic variation attributed to differences between the populations. The genetic diversity of the Luohanguo cultivars is much lower than that of wild populations (P = 41.8%, H _e = 0.141, H _o = 0.211), and a distinct genetic differentiation is observed between the cultivars and wild accessions. The pool of genetic variation in the wild populations provides an excellent gene resource for Luohanguo breeding.     

Genetic diversity and similarity of pear (<Emphasis Type="Italic">Pyrus</Emphasis> L.) cultivars native to East Asia revealed by SSR (simple sequence repeat) markers

Simple sequence repeat (SSR) markers were used to assess genetic diversity and relationship of Pyrus L. cultivars native mainly to East Asia. A total of 168 putative alleles were generated from six primer-pairs (BGA35, KU10, BGT23b, NH004a, NH011b and NH015a). All the SSR markers showed a high level of genetic polymorphism with a mean of 28 putative alleles per locus and the heterozygosity of 0.63. The Dice’s similarity coefficient between cultivars ranged from 0.02 to 0.98 and Occidental pears generally had low affinities to Asian pears. Ten major groups were generated from all the accessions by UPGMA clusters analysis. Chinese sand pears consisted of four groups with Chinese white pears and Japanese pears, of which Chinese sand pears occurred in all four groups, presenting a large genetic diversity, Chinese white pears were included in three groups, and Japanese pears only fell into one group. In the dendrogram, Chinese sand pears and Chinese white pears did not form discrete group, even subgroups. Some Japanese pear cultivars had high affinities to Chinese sand pear cultivars. These findings supports the authors’ previous viewpoints of Chinese white pears as a variety or an ecotype of Chinese sand pears (P. pyrifolia var. sinensis (Lindley) Y. Teng et K. Tanabe) and the progenitor of Japanese pears coming from China. Cultivars of P. ussuriensis Maxim. were clustered together with one clone of P. hondoensis Nakai et Kikuchi, a relative species of P. ussuriensis. Cultivars of P. communis L. and other Occidental species formed three independent groups and were distant from most Asian pears, except for P. betulaefolia Bge.     

Identification of Chinese white pear cultivars using SSR markers

109 Pyrus accessions including 92 local Chinese accessions of P. bretschneideri were identified genetically using nine microsatellite loci developed from apple and pear. The nine SSR loci revealed 129 alleles in 109 pear accessions and 114 alleles in 92 Chinese white pears. Among the 92 local Chinese accessions of P. bretschneideri, 70 could be differentiated successfully except for 10 sets of synonymous or mutants. For the 92 accessions, the number of putative alleles per locus ranged from seven to 18, with an average of 12.67; the average values of observed heterozygosity and Shannon’s Information index were 0.60 and 1.85, respectively. A phenogram based on the SSR (simple sequence repeat) genotypes was obtained. The 109 accessions clustered into 11 groups based on geographical origin. The European pears and the Asian pears did not form independent two groups, but three P. communis cultivars grouped together independently. The Japanese P. pyrifolia cultivars mingled together with the Chinese P. bretschneideri cultivars, but four P. ussuriensis cultivars except for one (Jianbali) grouped together independently. The results indicated that the relationship of P. bretschneideri cultivars and P. pyrifolia cultivars was much closer than others.     

Genetic Diversity among Syrian Cultivated and Landraces Wheat Revealed by AFLP Markers

Genetic diversity among some important Syrian wheat cultivars was estimated using Amplified Fragment Length Polymorphism (AFLP) markers. Five Triticum aestivum L. and 10 Triticum turgidum ssp. durum were analyzed with 11 EcoRI–MseI primer pair combinations. Of the approximately 525 detected AFLP markers, only 46.67% were polymorphic. Cluster analysis with the entire AFLP data divided all cultivars into two major groups reflecting their origins. The first one contained T. aestivum L. cultivars, and the T. turgidum ssp. durum cultivars and landraces were grouped in the second. Narrow genetic diversity among all cultivars was detected with an average genetic similarity of 0.884. The lowest similarity index (0.9) was found between Cham5 and Hamary (durum wheat), whereas this value was 0.93 between Salamony and Bouhouth 4 (T. aestivum L.). The narrow genetic diversity level indicates that these genotypes could be originated from the same source. AFLP analysis provides crucial information for studying genetic variation among wheat cultivars and provides important information for plant improvement.     

Pear (Pyrus Species) Genetic Resources in Iwate, Japan

Iwateyamanashi (Pyrus ussuriensis Maxim. var. aromatica (Nakai et Kikuchi) Rehd.) is one of the Pyrus species grown wild in Japan. The origin of the Japanese pear (P. pyrifolia) is uncertain but it has been suggested that Iwateyamanashi is the possible progenitor of the modern Japanese cultivar. During the last few decades, the number of Iwateyamanashi trees has been decreasing and therefore, conservation is urgently needed. After 13 explorations in the northern Tohoku region of Japan, 615 pear trees and 30 local names were recognized mainly in Iwate Prefecture. The center of the distribution seemed to be somewhere around Mt. Hayachine to the northern area of Kitakami highlands (from lat. 39 °20′ to 40 °10′N, and from long. 141 °20′ to 141 °50′E). Four morphological characters concerning fruit shape, measured for 85 trees, showed a wide range of continuous variation. For the skin colour of fruit, 51% of trees bore russet type fruits, 22% smooth and 27% intermediate ones. Most of the fruits had five loculi but Sanenashi fruits (seedless pear), one of the old cultivars, had three, and fruits of another two trees had four. More than 80% of trees tended to produce fruits with a calyx but some trees bore fruits without a calyx. These observations indicate a wide range of genetic diversity in Pyrus species which is caused, not only by high heterogeneity in Iwateyamanashi itself, but also by the coexistence of Iwateyamanashi, P. pyrifolia (Burm.) Nakai and hybrid progeny in this area. Already 250 trees have been conserved as genetic resources by grafting at Kobe University.     

基于SSR标记的梨资源遗传多样性分析

本研究利用SSR标记技术对56份梨资源进行了遗传多样性分析。利用筛选出的6对SSR引物共扩增40条谱带,其中多态性位点38个,多态性位点比例为95%,每对引物产生有效等位基因6.3个。各位点期望杂合度H值在0.0354～0.491,平均为0.1964;有效等位基因Ne值在1.0367～1.9648,平均值为1.2958;香农指数I值平均值为0.3256,说明了供试梨材料的遗传多样性较低。利用SSR标记可将44个栽培品种区分开,但无法区分芽变和原种。根据SSR标记揭示的多态性,采用NTSYS-pc软件,以UPGMA法进行聚类分析,结果显示所检测的56份梨材料在相似系数0.71处可分为4组,其中中国的白梨、秋子梨和砂梨相互交错在一起,没有独自各自成组。     

Identification and evaluation of <Emphasis Type="Italic">Forsythia</Emphasis> germplasm using molecular markers

This study identifies Forsythia germplasm and evaluated the genetic relationships of F. ×intermedia hybrids, cultivars and their putative parental species. Leaf samples of F. ×intermedia cultivars and species, such as F. koreana and F. suspensa, were collected in the Netherlands, Korea, and USA. Total genomic DNA was extracted and evaluated by randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Dendrograms were constructed by using the neighbor-joining (NJ) clustering algorithm applying the interior branch (IB) test or analyzed by STRUCTURE. In the dendrogram generated by RAPD markers, two major clusters were observed. One cluster (CL-I) contained most of the F. ×intermedia cultivars, F. suspensa, and F. koreana. The other cluster (CL-II) included F. europaea, F. ovata, F. densiflora, F. mandshurica, F. japonica, F. viridissima, and cultivars derived from F. ovata. In the AFLP dendrogram, the placement of F. ×intermedia cultivars with F. suspensa was similar, forming cluster CL-A I. The RAPD and AFLP results clearly separated most F. ×intermedia cultivars from F. ovata derived cultivars. The full range of genetic diversity of F. suspensa and F. viridissima should be investigated to verify whether these two species are truly parental taxa for F. ×intermedia. Placement of F. viridissima, F. ovata, and F. japonica in different sub-clusters requires further investigation regarding genetic diversity in the species, and their close relationship with F. koreana, F. mandshurica, and F. saxatalis.     

Relationships between an Italian Strawberry Ecotype and its Ancestor using RAPD Markers

Random-amplified polymorphic DNA (RAPD) markers were used to evaluate genetic variability among populations of an Italian strawberry ecotype, and to determinate genetic relationships between genotypes and their putative ancestor. A total of 65 selections and one cultivar ‘Madame Moutot’ (MM), were analysed to evaluate genetic variability present in Etna mountain area and to confirm as MM was one of the cultivars that originated the ecotype. A total of 222 RAPD markers was obtained using 16 decamer primers and 6 longer primers, 90.8% of the markers obtained by selected primers resulted polymorphic at least within analysed genotypes. RAPDs were used to calculate genetic similarity coefficients and to generate dendrograms representing genetic relationships among genotypes analysed. Cluster analysis displays as RAPD polymorphisms were able to characterize the genotype variability among closely related groups. The data show as MM could be considered the ancestral genotypes introduced in that area. The results obtained confirm that RAPD markers could be used as reliable markers to perform phylogenetic studies in Fragaria×ananassa Duch. ex Rozier. Giuseppe Bertino and Piero Spada - Coauthor involved in genotype selection and field management     

Species delimitation,genetic diversity and structure of the European indigenous wild pear (<Emphasis Type="Italic">Pyrus pyraster</Emphasis>) in Saxony,Germany

The present study investigated genetic diversity, structure and hybridization in a collection of the endangered wild pear species Pyrus pyraster (L.) Burgd. A total of 278 putative ‘true type’ P. pyraster trees originating from seven populations in the federal state of Saxony in Germany were analyzed along with 35 pear cultivars commonly cultivated in Saxony. The genetic analysis was performed using nine nuclear microsatellite markers (ncSSR) and two paternally inherited chloroplast marker (cpDNA) amplifying in the intergenic spacer region trnQ–rps16 and the intron region rps16. On basis of the ncSSR dataset after STRUCTURE analysis 80 % of the wild pear individuals were assigned as ‘true type’ P. pyraster genotypes. The cpDNA analysis showed shared haplotypes in P. pyraster and P. communis but with an unequal frequency in both species. The analysis of molecular variance resulted in a moderate (ncSSR) and great (cpDNA) variation among ‘true type’ P. pyraster and the pear cultivars. The genetic diversity in the ‘true type’ P. pyraster populations was still high and the genetic structure between the populations low (ncSSR and cpDNA) indicating a genetic exchange between the populations by pollen and seeds. The clear discrimination between the P. pyraster and P. communis confirms our expectation of the existence of ‘true type’ P. pyraster individuals in the study area. The existing genetic integrity and the high genetic diversity argue for the implementation of preservation measures in P. pyraster.     

Genetic diversity of Pakistan wheat germplasm as revealed by RAPD markers

Wheat breeding in Pakistan started in 1930s before partition in the United India and so far has released more than 68 cultivars, but no systematic analyses of the genetic diversity of Pakistan wheat have been made. Twenty Pakistan wheat cultivars released from 1933 to 2002 were examined for genetic diversity and relationships using random amplified polymorphic DNA (RAPD) markers. Forty-two RAPD primers were applied and 184 polymorphic bands were generated for each cultivar. Most of the cultivars were genetically interrelated, although six of them displayed some genetic distinctness. The RAPD variation observed among these cultivars was low. Only 40.7% of the total scorable bands were polymorphic, and 26.1% of the polymorphic bands were observed most frequently (f = 0.95) among the 20 cultivars. The proportions of polymorphic bands for each cultivar ranged from 0.67 in ‘Yecora’ to 0.84 in ‘C-250’ with an average of 0.76. About 1.4% of the RAPD variation might have been fixed over the 69 years of wheat breeding, but such fixation was not statistically significant. These results are significant for future improvement and conservation of Pakistan wheat.     

Assessing genetic diversity of <Emphasis Type="Italic">Pyrus</Emphasis> spp. in the central Zagros mountains based on morphological characters

The morphological diversity and relationship among 138 pear genotypes, including 11 commercial cultivars and 127 wild pear accessions from central Zagros mountains located in the west of Iran, based on 64 morphological characters were studied. The higher coefficient of variation indicated that high level of variation was belonged to the petiole stipule, fruit the relative area of the skin color, fruit relative area periphery rust dents end, fruit relative blight on an area of skin, fruit curved tail, fruit dents end, fruit depth dents end, fruit relative area periphery patina tail connection characters. Pyrus communis L. and Pyrus glabra Boiss. species were the highest and the shortest tree, respectively. According to the cluster analysis, 138 genotypes were clustered into nine main groups. Principal components analysis revealed that the first three components accounted for 68.77 % of the total variation in data. Moreover, 19 factors justified 72.57 % of the total variation based on factor analysis. The research results showed that the Zagros mountains are the centers of diversity of pear. Therefore, it is necessary to use the Iranian genotypes as the genetic resources for improving the morphological characters of pear.     

Molecular phylogeny of banana cultivars from Thailand based on HAT-RAPD markers

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the wild progenitors of the cultivated banana, they are highly variable in Thailand. The genetic system is relatively unknown and complicated due to interspecific hybridization, heterozygosity and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, especially when sterile. The high annealing temperature-random amplified polymorphic DNA (RAPD) technique was used to estimate the genetic relationship between 22 selected banana cultivars, utilizing 14 random primers. Phylogenetic relationship was determined by unweighted pair group method with arithmetical averages cluster analysis. The dendrogram constructed from the similarity data showed that all the 22 cultivars analysed were closely related with a narrow genetic base. There were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram grouped all the AA, BB, AAA, AAB and ABB genomes into a major cluster. Several subgroups are recognized within the major clade. As expected, Ensete glauca Roxb. (Musaceae) and Strelitzia reginae Banks (Strelitziaceae) were clearly differentiated from the analysed edible bananas. Our study showed that RAPD markers are sufficiently abundant to classify and readily dissect genetic differences between the closely related Musa germplasm and provide a basis for the selection of parents for improvement of this germplasm.     

Study of clonally propagated cassumunar ginger (<Emphasis Type="Italic">Zingiber montanum</Emphasis> (Koenig) Link ex Dietr.) and its relation of wild <Emphasis Type="Italic">Zingiber</Emphasis> species from Thailand revealed by RAPD markers

The genetic relatedness among 51 accessions, 14 species of the genus Zingiber and genetic variability of a clonally propagated species, Zingiber montanum (Koenig) Link ex Dietr., from Thailand were studied using random amplified polymorphic DNA (RAPD) profiles. Twenty-nine random primers gave reproducible amplification banding patterns of 607 polymorphic bands out of 611 scored bands accounting for 99.40% polymorphism across the genotypes. Jaccard’s coefficient of similarity varied from 0.119 to 0.970, indicative of distant genetic relatedness among the genotype studied. UPGMA clustering indicated eight distinct clusters of Zingiber, with a high cophenetic correlation (r = 1.00) value. Genetic variability in Z. montanum was exhibited by the collections from six regions of Thailand. High molecular variance (87%) within collection regions of Z. montanum accessions was displayed by AMOVA and also explained the significant divergence among the sample from six collection regions. Our results indicate that RAPD technique is useful for detecting the genetic relatedness within and among species of Zingiber and that high diversity exists in the clonally propagated species, Z. montanum.     

Comparative analysis of genetic diversity using molecular and morphometric markers in Andrographis paniculata (Burm. f.) Nees

Andrographis paniculata is a medicinal plant of immense therapeutic value. The present study was aimed to elucidate its genetic diversity based on morphochemical and RAPD markers from 53 accessions belonging to 5 eco-geographic regions. Analysis of variance and D ² statistics revealed significant differences in all the metric traits and sufficient inter-cluster distances indicating considerable diversity among the accessions. The complementary approach of RAPD was used to evaluate the genetic dissimilarities among all the accessions using 6 highly polymorphic primers. The average proportion of polymorphic loci across primers was 96.28%. The molecular genetic diversity based on Shannon index per primer averaged 5.585 with values ranging from 3.08 to 8.70 indicating towards wide genetic base. RAPD based UPGMA and D ² cluster analysis also revealed that various accessions available in different eco-geographic regions might have originated from native places of wild abundance. Similarity matrices were generated for molecular markers and morphometric data to determine the degree of congruence between the two. A highly significant but low correlation (r = 0.547, P < 0.001) was obtained thus implying the correspondence between the two. The species is hermaphroditic and a habitual inbreeder. The present study yielded a typical triangular congruence between its breeding system, morphometric traits and RAPD markers thus elucidating the usefulness of complementary approaches to make diversity analysis more explanatory and purposeful for optimum genetic amelioration and effective conservation of its genotypic variability.