Detection of somaclonal variation in micro-propagated Echinacea purpurea using AFLP marker

This study used amplified fragment length polymorphism (AFLP) analysis to assess genetic fidelity between primary regenerants of Echinacea purpurea derived from leaf organogenesis and their donor plants. A total of 40 regenerants and 5 donors (T6-28-0, T3-23-0, T5-9-0, T2-15-0 and D7-4-0) were subjected to AFLP analysis using eight primer pairs. The results indicated that a total of 3805 scorable fragments were observed, of which 301 (9.40%) were polymorphic among the tested regenerants and donors probed with eight primer pairs. The percentage of polymorphic fragments within five donor groups averaged from 1.6% to 20.6%. Jaccard's similarity coefficients among regenerants and donors averaged from 0.9508 to 0.9935. However, only two regenerants (T2-15-2 and T2-15-3) had Jaccard's similarity coefficient value of 1 as comparing to their donor, thus they were true-to-type to their donor T2-15-0. It appears that AFLP is a sensitive and reliable molecular marker to detect possible somaclonal variation in micro-propagation system of E. purpurea. In vitro culture-induced somaclonal variation occurs in primary regenerants of E. purpurea derived from leaf organogenesis, though some of regenerants have genetic similarity greater than 0.99 in comparison with their donors.     

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.     

Genetic characterization of asparagus doubled haploids collection and wild relatives

The genus Asparagus is very large consisting of around 150 species found as herbaceous perennials, tender woody shrubs and vines. The cultivated species (Asparagus officinalis L., diploid) is a highly prized vegetable, grown in different environments ranging from cool temperate zones to deserts, Mediterranean climates and tropical areas. As a consequence, Asparagus breeders have developed different cultivars that differ for their morpho-agronomic traits, habit and ploidic status (few triploid and tetraploid cultivars are used). Several breeding methods are used for developing cultivars, among which a well developed in vitro anther culture technique produces homozygous clones useful for F₁ hybrids constitution. A fluorescent based AFLP (amplified fragment length polymorphism) technique were applied with the aim to assess genetic diversity among a collection of 173 doubled haploid (DH) androgenetic clones, five Asparagus wild species and interspecific hybrids obtained among the cultivated species and two wild relatives. The average number of AFLP fragments generated per primer set was 105, varying in size from 50 to 550 bp. A total of 1054 AFLP fragments were detected, 20% of which were polymorphic. Genetic similarity based on DNA polymorphisms, showed that a few number of AFLP primer combinations are able to distinguish the cultivated DH clones from the wild species. Indeed, from one DH clone for each anther donors and the wild species were used to construct a dendrogram using Dice's coefficient and the unweighted pair group method with the arithmetic mean (UPGMA). Genetic distances among all DH clones were calculated using the C.S. Chord distance; and a neighbour-joining (NJ) consensus tree was constructed in order to support the breeder for parental genotype choice for asparagus hybrid constitution.     

部分板栗品种遗传多样性的AFLP分析

利用荧光标记AFLP技术,采用7对M+3和E+3引物组合对30份板栗和日本栗栽培品种进行了总基因组DNA水平上的多态性检测,共获得962条可统计的条带,其中852条呈多态性,多态性带百分率达89％。揭示了板栗丰富的遗传多样性。7组引物在30个品种中检测到数目不等的品种特异带型,对供试板栗品种具有一定的鉴别价值。7对引物能将30个板栗和日本栗品种完全区分开。聚类分析结果表明,多数来源地相同的板栗品种资源表现出较为密切的亲缘关系。     

Development of novel chloroplast microsatellite markers for Dendrobium officinale, and cross-amplification in other Dendrobium species (Orchidaceae)

Nine pairs of polymorphic chloroplast microsatellite primers were developed for Dendrobium officinale Kimura et Migo, an endangered herb. Levels of polymorphism were tested across a total of 55 individuals from four natural populations (12–15 individuals per population). Allele numbers varied from two to four per locus, while the number of haplotypes ranged from four to six per population. Transferability of the nine polymorphic chloroplast microsatellite primers was checked on an additional set of 51 Dendrobium individuals (belonging to 17 different species). Three markers could be transferable to all the species tested, while the remaining six markers successfully cross-amplified in most species tested. Moreover, polymorphism of the nine chloroplast microsatellite primers was tested across Dendrobium moniliforme (L.) Sw. and Dendrobium loddigesii Rolfe. All of them were polymorphic in D. moniliforme, while seven of which were polymorphic in D. loddigesii. These polymorphic chloroplast microsatellite primers developed for D. officinale will be a useful tool for the study of genetic diversity, population genetic structure, evolution of D. officinale and establishment of effective conservation strategies.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.     

Analysis of the taxonomic subdivision within the genus Helleborus by nuclear DNA content and genome-wide DNA markers

Helleborus is a genus of herbaceous perennials belonging to the family Ranunculaceae. Within this genus six sections with a total of 22 species are found. The largest section Helleborastrum contains 16 species for which genetic relationships are still unclear. This study represents the first genetic analysis in the genus Helleborus, including the two newly described species H. liguricus and H. abruzzicus based on multilocus amplified fragment length polymorphism (AFLP) markers with a genome-wide distribution in combination with nuclear DNA content data. Chromosome analyses of roots tips revealed a number of 2n = 32 for the selected species, which was congruent with previous observations. The nuclear DNA content of Helleborus was estimated by flow cytometry applying propidium iodide staining and varied between 18 and 33 pg/2C, depending on the species. For AFLP analyses, 19 out of the 22 Helleborus species were studied using 10 AFLP primer combinations, resulting in a total of 1109 polymorphic bands among all species including the outgroup. The genetic distances between species varied between 0.034 and 0.330. Based on genetic distances a phenogram using the Neighbor-joining cluster method with bootstrap analysis was calculated. The results support the previously suggested division of the genus into six sections and thereby approve AFLP data to be applicable for phenetic analyses. Moreover, this genetic information is significant for the development of future Helleborus breeding strategies.     

广东果梅种质资源遗传多样性的AFLP分析

为了从DNA分子水平上探讨广东境内果梅种质资源遗传多样性状况,利用荧光标记AFLP技术,对采自广东境内的57份果梅种质进行研究,筛选出适于果梅AFLP分析的8对Mse Ⅰ和EcoR Ⅰ引物组合,这些引物可以将供试材料完全分开.利用8对引物组合对57份果梅种质进行总基因组DNA水平上的多态性检测,得到了清晰的多态性指纹图...     

Genetic diversity in ecotypes of Tunisian date-palm (Phoenix dactylifera L.) assessed by AFLP markers

Summary

Amplified fragment length polymorphisms (AFLP) were used successfully to survey genetic diversity in 40 ecotypes of date-palm (Phoenix dactylifera L.) collected from oases in Tunisia. Six primer pairs were screened to assess their ability to detect polymorphism in this tree crop. As a result, a total of 428 AFLPs have been generated and used to estimate genetic distances which ranged from 0.07 – 0.63. A large, and typically continuous, range of genetic diversity characterises Tunisian date-palm germplasm. In addition, the UPGMA dendrogram derived from these data exhibited two clusterings of ecotypes independent of their geographic origin or the sex of the trees. These data corroborate the hypothesis of the origin of date-palm domestication being in Mesopotamia. Moreover, taking into account the high percentage of polymorphic bands, together with their resolving power (Rp), all the primer pairs tested contributed to the discrimination of date-palm genotypes, suggesting that the AFLP method is efficient in assessing genetic diversity in this crop. The data are discussed in relation to the use of AFLP molecular markers in the management and improvement of date-palm.     

Amplified fragment length polymorphism (AFLP) markers for fingerprinting of Argyranthemum frutescens cultivars

Argyranthemum frutescens is a commercially important ornamental species with extensive breeding programmes in several countries. Since it is vegetatively propagated there is a great need for tools for identification of cultivars. Vegetatively propagated species require clean-up from diseases, often performed through meristem-tip cultures. Forty-three cultivars of A. frutescens propagated by meristem-tip culture and traditional cuttings were analyzed for genetic relatedness and possible somaclonal variation using AFLP. Five primer combinations resulted in a relatively high degree of polymorphism. Ten molecular markers generated by one primer combination could distinguish between all 43 cultivars. Differences in fingerprints between meristem-tip culture and cuttings from the same cultivars were found, but the proportion of fragments being specific for either tissue culture or cuttings was relatively low. Some cultivars that did not display somaclonal variation as judged by the AFLP-fingerprints may still be genetically unstable since some morphological variation was observed in the true to type test.     

Comparative analysis of genetic diversity in Prunus L. as revealed by RAPD and SSR markers

RAPD and SSR markers were used for genetic diversity evaluations among 15 genotypes selected from the genus Prunus L. Altogether 40 RAPD primers and 21 primer pairs designated for microsatellite loci were applied on the whole group of genotypes.     

Functional characterization of watermelon (Citrullus lanatus L.) EST–SSR by gel electrophoresis and high resolution melting analysis

The present work was conducted to characterize the functionality of 257 watermelon EST–SSR primer pairs for their PCR amplification and polymorphisms. EST–SSR markers were tested on DNA sample panels of six watermelon cultigens and two related species of Citrullus lanatus var. citroides and Citrullus colocynthis based on agarose gel electrophoresis and high resolution melting (HRM) analysis. Successful PCRs were shown for 240 primer pairs (79%), and 173 primer pairs (67%) were polymorphic in a watermelon DNA sample panel on agarose gel electrophoresis. In addition, HRM analysis of 24 EST–SSR markers that were monomorphic on agarose gel separation identified an additional 19 polymorphic markers, indicating that HRM is an efficient tool for the rapid screening of sequence variations and allele discrimination. In the assessment of genetic relationships, six watermelon cultivars were closely related together (0.91–0.97) and demonstrated a narrow genetic base in the watermelon genetic pool. A high level of genetic dissimilarity (0.36–0.97) was shown between watermelon species and other related species. Marker transferability to melon species (Cucumis melo L.) was examined by cross-species PCR amplification and genetic diversity assessment in eight melon cultigens. Of the 257 EST–SSR primer pairs, 79 (32.9%) showed successful PCR amplification from melon DNA samples. A dendrogram of the genetic relationship based on 22 EST–SSR markers showed a clear classification of melon genotypes in accordance to fruit characteristics. The EST–SSR markers characterized in this study will contribute to diverse genomic investigations and breeding efforts, including comparative genome mapping, marker-assisted selection, and DNA fingerprinting for genetic diversity and cultivar identification in many cucurbit crops.     

Development of specific primers for cpSSR analysis in caper,olive and grapevine using consensus chloroplast primer pairs

Chloroplast SSR (cpSSR) markers have demonstrated utility in studying genetic relationships. DNA sequence information of the chloroplast genome is necessary for the development of cpSSR primer pairs. To overcome this limitation, “consensus” primers have been developed to amplify the homologous regions in plants where chloroplast sequences are not available. However, 80% Pinus thunbergi and Nicotiana tabacum developed “consensus” primers tested with grapevine, olive and caper showed multi-locus patterns. The presence of multi-locus patterns requires the use of agarose gel electrophoresis followed from isolation and sequencing of the bands. Herein, a PCR-strategy is proposed to construct specific cpSSR primer pairs without genomic sequence information, giving single-band amplifications that can be directly sequenced. Twelve new specific cpSSR primer pairs were developed for Capparis spinosa L., Olea europea L. and Vitis vinifera L. PCR products were sequenced to confirm the presence of microsatellite sequences, and their transportability was tested on six V. vinifera cultivars. Both single-nucleotide polymorphisms and polymorphic cpSSR were observed in the six grapevine cultivars using the specific cpSSR primers.     

Identification of parental species of the Alstroemeria cv. ‘Jubilee’ using AFLP marker technique

Twelve Alstroemeria species, two hybrids, one cv. ‘Jubilee’, an anther-cultured plant from cultivar ‘Jubilee,’ and Bomarea salsilla and Leontochir ovallei (the latter two were chosen as outgroup) were evaluated using the AFLP marker technique in order to identify putative parental genotypes of the Alstroemeria cv. ‘Jubilee’ and of known interspecific hybrids, and to estimate their genetic relationships within the genus Alstroemeria. A total of 297 AFLP markers were scored by using the primer combination (E + ACCA/M + CTAG). In order to discriminate all Alstroemeria genotypes, cluster analysis (UPGMA) and principal coordinates analysis were performed. The Alstroemeria cv. ‘Jubilee’, of which the parents are unknown, had genetic distance (GD) 0.54 from Alstroemeria exserens, GD 0.57 from Alstroemeria garaventae, GD 0.62 from Alstroemeria gayana, and GD 0.66 from Alstroemeria hookeri cumminghiana. Thus, these four species are considered as putative parental genotypes. An interspecific hybrid (Alstroemeria aurea × Alstroemeria inodora), showed the smallest genetic distance from A. aurea (GD 0.56) and A. inodora (GD 0.45). The Alstroemeria ligtu group was distantly allocated from other Chilean species. We conclude that the AFLP marker technique appears to be a satisfactory tool for identifying the parental genotypes of interspecific hybrids in Alstroemeria.     

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.     

萱草部分野生种和栽培品种亲缘关系的AFLP分析

 借助AFLP标记对35份萱草野生种和栽培品种进行亲缘关系研究, 结果表明, 7对引物组合对萱草共扩增出条带380条, 其中多态性条带357条, 平均多态性达到93.39% , 单对引物扩增条带19～85条, 平均每对引物组合扩增多态性条带51条。7对引物扩增出的多态性条带均超过90.0%。种质资源相似系数为0.3822～0.9656, 平均相似系数为0.7039。UPGMA聚类结果将供试材料分为3类, 即早花、中花和晚花类, 同一产地的品种基本能聚在一起。AFLP标记技术能较好地从分子水平揭示萱草种质资源的亲缘关系。     

Genetic diversity of Dimocarpus longan in China revealed by AFLP markers and partial rbcL gene sequences

Amplified fragment length polymorphism (AFLP) and partial rbcL gene sequencing were used to investigate genetic diversity among various longan (Dimocarpus longan Lour) accessions as well as a presumed closely related species Dimocarpus confinis How et Ho and litchi (Litchi chinensis Sonn). No significantly shared AFLP fragment was found between the three species, indicating that D. confinis and litchi are very far in genetic distance from any longan accession studied. Partial rbcL sequences of 501 bp from the first coding site in these species were obtained, which revealed several substitutes. One such DNA base pair substitute resulted in an amino acid difference between longan and litchi. Furthermore, another 4 bp resulted in a two amino acid difference between longan and D. confinis, which was consistent with AFLP results and indicated that D. confinis should be excluded from the longan genus, Dimocarpus. Within the longan species, no DNA substitute was found. Using nine primer combinations, a total of 66 AFLP markers were obtained from 41 longan accessions. One non-Chinese longan accession ‘Miaoqiao’ was distinctly different from all other longan cultivars collected in China, indicating that more genetic resources of longan might be collected also from longan production regions outside of China. AFLP markers might be developed to identify longan cultivars as well as expedite progeny screening in breeding programs of this perennial fruit tree.     

EST-PCR markers developed for highbush blueberry are also useful for genetic fingerprinting and relationship studies in rabbiteye blueberry

The pedigrees of most rabbiteye blueberry (Vaccinium virgatum) cultivars can be traced back to four wild selections, ‘Ethel’, ‘Clara’, ‘Myers’, and ‘Black Giant’; thus, they result from a very narrow germplasm base and are highly related. Until now randomly amplified polymorphic DNA (RAPD) has been the only type of molecular marker used in rabbiteye blueberry. Here we have tested whether a type of sequence-tagged site (STS) marker which utilizes specific ∼20-mer primers from expressed sequence tags (ESTs) of highbush blueberry (V. corymbosum), called EST-PCR markers, are useful for genetic fingerprinting and relationship studies in rabbiteye blueberry. Of 44 EST-PCR primer pairs, from an assortment of genes expressed in flower buds of cold acclimated and non-acclimated plants, and shown to amplify polymorphic fragments among a collection of highbush genotypes, 40 (91%) resulted in successful amplification, and 33 of those (83%) amplified polymorphic fragments among the rabbiteye genotypes. The average number of scorable bands per primer pair was two. A dendrogram constructed from genetic similarity values, based on the EST-PCR marker data, tended to group siblings and parent/progeny together, generally agreeing with pedigree information. A group of 20 markers from five EST-PCR primer pairs distinguished all the genotypes in this study. These markers are as easy to generate and as affordable as RAPDs, but are based on actual gene sequences, and should have general utility for DNA fingerprinting, genetic diversity, and mapping studies.     

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

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

大花蕙兰遗传多样性及亲缘关系的AFLP分析

对来源于日本、韩国和美国的42个大花蕙兰品种和两个国产兰属原生种进行了遗传多样性和亲缘关系的AFLP分析, 9对多态性引物在50～500 bp内共扩增出1 597条带, 其中多态性带1 565条, 多态性比率9810%。单引物对扩增的带数156～193条, 平均每对引物扩增带数177条。42个品种具特征带或缺失带。大花蕙兰品种间的遗传多样性丰富, 品种间的相似系数0.3399 ～0.8223, 平均相似系数0.5783。UPGMA聚类结果将供试品种分为4大类, 与根据花枝类型或花径大小、花色等形态指标分类的结果相吻合, 同一产地来源甚至同一育种公司选育出的品种能基本上聚类在一起, 反映出了品种间的亲缘关系。