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.     

Molecular characterisation of Afghan pistachio accessions by amplified fragment length polymorphisms (AFLPs)

Summary

In Afghanistan, pistachio (Pistacia vera L.) is found mainly in the wild in natural forests. In this study, 17 wild Afghan pistachio accessions and three cultivated genotypes were characterised using amplified fragment length polymorphisms (AFLPs). Material was sampled mainly from the Kunduz and Takhar regions. A total of 288 AFLP fragments were generated using eight AFLP primer combinations. The number of amplified fragments varied from 18 – 48 per primer combination, and 136 bands were polymorphic, with an average of 17 polymorphic bands per primer combination. The percentages of polymorphic bands ranged from 26.2 – 79.2 per primer pair. According to UPGMA clustering of the Nei and Li distance matrix, the 17 wild accessions were grouped according to their region of origin and were distinct from the three cultivars. The AFLP technique was found to be effective for characterising wild P. vera material, which was genetically the closest to cultivated pistachio.     

Evaluation of genetic relationships among Iranian pistachios using microsatellite markers developed from Pistacia khinjuk Stocks

To evaluate the genetic relationships among wild and cultivated Pistacia species grown in Iran and the analysis of genetic variation among Iranian pistachio genotypes, two DNA libraries enriched for dinucleotide (AG)_n and trinucleotide (ATG)_n microsatellite motifs were developed from Pistacia khinjuk genome. Following screening of clones by colony PCR technique, 44 clones were sequenced and 27 pairs of primers designed from flanking regions of the repeats. The examination of primer pairs, designed from P. khinjuk sequences, showed successful cross-species amplification within the genus Pistacia. A dendrogram constructed on the basis of the Minimum Evolution clustering algorithm revealed that Pistacia vera has closer relationships with P. khinjuk, than with Pistacia integerrima, Pistacia palaestina, Pistacia atlantica and Pistacia mutica. The dendrogram further distinguished the wild Sarakhs pistachio from the rest of P. vera genotypes suggesting that the domesticated genotypes of P. vera are evolved from P. vera var. Sarakhs and then this wild genotype likely develops to other local pistachios. Hence, it seems that the wild Sarakhs pistachio plays an important role in evolutionary trend of the edible pistachios in Iran. The results indicated that microsatellites developed in P. khinjuk are distributed in the genome of indigenous pistachio species including P. vera genotypes and therefore they will be useful in characterization of Iranian pistachio genotypes.     

Morphological and molecular analyses of Rosa damascena × R. bourboniana interspecific hybrids

Rosa damascena Mill is the most important scented rose species cultivated for rose oil production. Rosa bourboniana L. (Edward rose), a related species, is popular on account of its longer blooming period and ease of propagation. With an aim to combine the oil quality of R. damascena and recurrent flowering habit of R. bourboniana, two cultivars (Jwala and Himroz) of R. damascena were crossed with R. bourboniana. The F1 hybrids obtained were evaluated using morphological, random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers. Twenty-two selected RAPD and three SSR primer pairs were utilized for hybrid identification. According to presence or absence of bands RAPD and SSR markers were classified into seven types of markers. The bands specific for the pollen parent and occurring in the hybrids were good markers to confirm the hybridity. The non-parental bands expressing uniquely in hybrids were effective in distinguishing the hybrids from each other. Cluster analysis, based on Jaccard's similarity coefficient using unweighted pair group method based on arithmetic mean (UPGMA), reliably discriminated the hybrids into two main clusters. These results indicate the practical usefulness of RAPD and SSR markers in hybrid identification in scented roses. The approach is advantageous for its rapidity and simplicity, for identification of hybrids at the juvenile stage. One of the studied morphological traits – prickle density, can also complement in the identification of interspecific hybrids between R. damscena (♀) and R. bourboniana (♂).     

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.     

黄瓜栽培种、近缘野生种、种间杂种及其回交后代的RAPD 分析

 黄瓜栽培种、黄瓜野生变种、近缘野生种、种间杂交种及其与黄瓜回交自交的后代以及甜瓜为试材, 采用RAPD 方法探讨其亲缘关系。对23 份试材进行扩增, 筛选出31 条随机引物的扩增产物进行结果分析, 共产生375 个位点, 具多态性位点占90. 0 % , 平均每条扩增出6. 3 条。通过UPGMA 聚类分析,以遗传距离0. 37 为阀值, 可将23 份试材归为黄瓜、近缘野生种、种间杂交种及甜瓜亚属种4 类。     

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.     

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.     

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.     

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

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

适合我国南方地区栽培的枣优良品种亲缘关系研究

利用荧光AFLP分子标记技术,对适合我国南方地区栽培的26个枣优良品种亲缘关系进行研究。结果表明,8对引物共扩增得到886条带,其中多态性条带为817条,多态性比例达到了92.2%,体现了非常丰富的遗传多样性。系统聚类结果表明大部分来源于北方地区的鲜食品种聚为一类,彼此间亲缘关系较近;制干、兼用品种和南方本地品种表现出较近的亲缘关系。酥脆枣与孔府酥脆枣亲缘关系很近,而河南鸡蛋枣与湖南鸡蛋枣之间存在着较远的遗传距离。     

New microsatellite markers developed from reported Ipomoea trifida sequences and their application to sweetpotato and its related wild species

Ipomoea trifida belongs to the family Convolvulaceae and is closely related to sweetpotato. In this study, we reported the microsatellites in I. trifida sequence database, and tested their transferability and polymorphisms for both sweetpotato and its related wild species.In the DNA database, 1425 sequences were registered for I. trifida. Sixty-one independent sequences were found to have microsatellite motifs and PCR primers were designed to amplify 15 microsatellites loci identified. Twelve primer pairs could amplify the expected product size and nine primer pairs showed polymorphisms among the three genotypes of I. trifida. These 12 functional primer pairs were used to assess the transferability and the level of polymorphism between sweetpotato cultivars and its related wild species. The transferability showed, 100% for I. batatas, 83.3% for I. tiliacea, 75% for I. triloba and 66.7% for I. Lacunosa, respectively. These markers also revealed high level of polymorphism between wild species and sweetpotato cultivars.     

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.     

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.     

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。     

Syrian pear (Pyrus syriaca) as a pollinator for European pear (Pyrus communis) cultivars

In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work.     

Genetic variation and differentiation in tea (Camellia sinensis) germplasm revealed by RAPD and AFLP variation

Summary

Genetic variation and resulting variable groupings in tea (Camellia sinensis) and its wild Camelia relatives were assessed using Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphic (AFLP) markers. Numerous polymorphic bands were generated, of which 266 unambiguous ones were scored. The highest level of polymorphism as determined by the expected heterozygosity (H_av) was detected with AFLPs. Three major groups were recognized in the germplasm based on the parsimony method of cluster analysis. Two of the groups corresponded to varieties assamica and sinensis while the third group consisted of a heterogeneous mix of tea cultivars and related wild species. The Taiwan yamacha, unique tea cultivars grown predominantly in the highlands of Taiwan for the production of pan fired semi-fermented (Oolong and Pouchong) tea clustered in this group. Analysis of phenotypic diversity based on the generated RAPD and AFLP profiles revealed that population diversity (H), decreased in the order; Wild Camellia >India > China > Kenya > Sri-lanka > Vietnam > Japan > Taiwan. Analysis of molecular variance (AMOVA) revealed that most variation resided among individuals within populations (72%). Calculation of genetic distances and nested AMOVA further revealed a significant degree of structuring among the populations based on the country of origin.     

Effect of increasing climatic water deficit on some leaf and stomatal parameters of wild and cultivated almonds under Mediterranean conditions

The prevailing environmental conditions, temperature in particular, drive seasonal changes both in leaf development and stomatal characteristics. In order to ascertain the effect of increases in climatic water deficit on some leaf and stomatal parameters under field conditions, a study was carried out on two sets of leaves (spring and summer) on a large sample of Amygdalus communis L. cultivars in comparison with several Amygdalus webbii Spach seedlings, a species more adapted to arid environments and probable ancestor of cultivated almonds. Observations were performed between spring and summer of a particularly hot season. The results showed a significant and general reduction of both leaf area and stomatal frequency and an increase in stomatal size. Nevertheless, there were evident differences between cultivated and wild almonds. A stronger reduction of leaf area was observed in A. webbii (−31%) with respect to A. communis (−14%); on the contrary, the latter reduced stomatal frequency more than the former (−25% and −19%, respectively). The examined cultivated almonds, in response to the increase in climatic water deficit, tended to arrange their stomatal structures like those of wild almonds. Finally, increasing the climatic water deficit, the slope of the linear regressions between stomatal frequency and size did not change in either species, leading to a better understanding of the mechanisms of almond acclimation to environmental stresses.     

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.     

Suitability of isozyme,RAPD and AFLP markers to assess genetic differences and relatedness among fig (Ficus carica L.) clones

Sarilop is the main and standard cultivar for commercial dried fig (Ficus carica L.) production in Turkey. Eleven of the most promising Sarilop clones and one clone of Sarizeybek, all selected from a former agronomic evaluation, were analysed by three molecular marker techniques, isozymes, RAPDs and AFLPs. The resolution power and the accuracy of these three analytical techniques, in distinguishing among fig clones, were determined. The analysis of five isozyme systems permitted the discrimination between the two cultivars, Sarilop and Sarizeybek. Besides the discrimination between the two fig cultivars, the use of 31 10-mer primers in RAPD analysis allowed splitting the 11 Sarilop clones into two groups of genetic similarity, but not to distinguish between all the clones. The AFLP™ technology showed a much higher multiplex ratio than the RAPD technique (42.4 vs. 6.1) and eight combinations of EcoRI/MseI primers were enough to clearly distinguish between all the Sarilop clones.