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.     

Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers

RAPDs and SSRs were used to study the genetic diversity of Iranian almond cultivars and their relationship to important foreign cultivars and three related species. Eight unidentified almond Shahrodi cultivars and three wild almonds (Prunus communis, Prunus orientalis and Prunus scoparia) were also included. Of the primers tested, 42 (out of 80) RAPD and 18 (out of 26) SSR primers were selected for their reproducibility and high polymorphism. A total of 664 polymorphic RAPD bands were detected out of 729 bands. The number of presumed alleles revealed by the SSR analysis ranged from 3 to 10 alleles per locus with a mean value of 6.64 alleles per locus. Both techniques discriminated the genotypes very effectively, but only RAPDs were able to discriminate the cultivars Monagha and Sefied. Results demonstrated an extensive genetic variability within the tested cultivars as well as the value of SSR markers developed in peach for characterization of almond and related species of Prunus. Dice similarity coefficient was calculated for all pair wise comparisons and was used to construct a UPGMA dendrogram. For both markers a high similarity in dendrogram topologies was obtained although some differences were observed. All dendrograms, including that obtained by the combined use of both the marker data, depicted the phenetic relationships among the cultivars and species, depending upon their geographic region and/or pedigree information. Almond cultivars clustered with accession of P. communis showing their close relationship. P. orientalis and P. scoparia were clustered out of the rest of P. dulcis.     

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.     

Genetic characterization of different demes in Prunus persica revealed by RAPD markers

DNAs of 180 accessions in 10 demes in Prunus persica were amplified with twenty-two, 10-base primers selected from 200 arbitrary primers using Randomly Amplified Polymorphic DNA (RAPD) technology. One hundred and eighty loci were observed and recorded. With statistical analyses of the data from the study, genetic diversity of the demes was expressed as follow: yellow peach group > honey peach group > flat peach group > red leaf peach group > crisp peach group > bitao group and juicy peach group > nectarine group > shouxingtao group > weeping peach group. Genetic variations among and within groups by AMOVA analyses were 11.9, 88.1%, respectively. Demes clustered by UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5, the edible peaches of which were combined as a section, while the ornamental species were classified into separate sections. Through analyses of genetic diversity and genetic structure, the results could provide molecular biological evidence for conservation and utilization of P. persica germplasm.     

Re-constructing data of leaf area increment in the greenhouse pot chrysanthemum cultivar ‘Lompoc’

A dynamic model was developed for re-constructing data of shoot leaf area increment in pot chrysanthemum. From specified final areas of individual leaves and time of anthesis of the terminal flower, the model can re-construct dynamic growth of each leaf on side shoots using a modified Richards function. In the model, leaf growth is related to a relative time index ranging from zero to unity, where zero is the start of short day treatment and unity corresponds to anthesis of the terminal flower. Experiments were conducted under greenhouse conditions to collect data for parameter estimation, model verification and model validation. An allometric relationship between leaf length and leaf area was used to estimate leaf area non-destructively from leaf length recordings. When compared with the data used for parameter estimation, the model simulations fitted the data with R² values ranging from 0.979 to 0.998. In the validation trial the model showed a tendency of over estimation early leaf growth but could explain variations in the data with R² values ranging from 0.899 to 0.993.     

The effect of plant growth regulators on somaclonal variation in Cavendish banana (Musa AAA cv. ‘Zelig’)

The effect of the type and concentration of plant growth regulators and sub-culturing on somaclonal variation were studied in Cavendish banana cv. ‘Zelig’ obtained from African Biotechnologies Ltd., South Africa. In vitro grown plants at the fourth multiplication cycle were used for the investigation. Auxins (IAA, IBA and NAA) and cytokinins (BA and TDZ) were used to multiply shoots for 10 generations. Bands generated through RAPD-PCR were scored according to whether they were present (1) or absent (0) to determine the extent of somaclonal variation. Results were then analyzed using cluster analysis. The relationship between multiplication rate and somaclonal variation was assessed using correlation analysis. Results indicated that treatments with higher multiplication rates produced more variants; sometimes as high as 72%. Dwarf off-types accounted for 88% of the variation. A dwarf-specific band, about 1500 kb in size, was amplified by the primer OPC-15. The band appeared consistently in normal plants but was absent in all dwarf plants.     

Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

杨桃遗传多样性的形态特征与RAPD标记的相关性分析

【目的】为指导杨桃种质资源的引进以及良种选育提供科学依据,【方法】采用形态标记数量聚类分析和RAPD分子标记相结合,对广东地区10份杨桃品种资源进行遗传多样性分析,并将形态学聚类和RAPD分子标记聚类结果加以对比。【结果】在所观察的12个形态性状中,变异系数为14.08%~49.71%,平均变异系数为25.38%。从100条RAPD随机引物中筛选出10个引物,共扩增出58条带,其中53条为多态性带,多态率达93.02%。聚类分析结果表明,形态标记和RAPD标记都可依果实风味将供试材料分组,即甜味和酸味。2种标记方法的相关系数为r0.01=0.685 3,达到显著水平。【结论】杨桃具有丰富的遗传多样性,2种方法聚类结果相似,且相关性高,具有较高的可靠性。     

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.     

Genetic diversity of bitter gourd (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits

Bitter gourd or bitter melon (Momordica charantia L.) is considered as minor cucurbitaceous vegetable in spite of having considerable nutritional and medicinal properties. Although some reports on genetic diversity based on morphological characterization are available, no work has been conducted to estimate genetic diversity using molecular markers in this crop. In the present study, 38 genotypes of M. charantia including few commercially cultivars collected from different parts of India based on agro-ecological zones were analysed for diversity study both at morphological and molecular levels. Genomic DNA was extracted from young healthy leaves following the procedure of Doyle and Doyle [Doyle, J.J., Doyle, J.L., 1990. A rapid DNA isolation procedure from small quantity of fresh leaf material. Phytochem. Bull. 119, 11–15]. Pair-wise comparison of genotypes was calculated as per the procedure of Jaccard [Jaccard, P., 1908. Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Sci. Nat. 44, 223–270]. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was done using the computer programme NTSYS-pc Version 2.0 [Rohlf, F.J., 1998. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Version 2.01. Exeter Software, Setauket, NY, USA]. Diversity based on yield related traits and molecular analysis was not in consonance with ecological distribution. Among 116 random decamer primers screened 29 were polymorphic and informative enough to analyse these genotypes. A total of 208 markers generated of which 76 (36.50%) were polymorphic and the number of bands per primer was 7.17 out of them 2.62 were polymorphic. Pair-wise genetic distance (GD) based on molecular analysis ranged from 0.07 to 0.50 suggesting a wide genetic base for the genotypes. The clustering pattern based on yield related traits and molecular variation was different.     

A preliminary genetic linkage map of chrysanthemum (Chrysanthemum morifolium) cultivars using RAPD,ISSR and AFLP markers

A genetic linkage map of chrysanthemum (Chrysanthemum morifolium) was constructed by genotyping 142 F₁ progeny of the bi-parental cross ‘Yuhualuoying’ × ‘Aoyunhanxiao’ with a combination of RAPD, ISSR and AFLP markers in a double pseudo-testcross mapping strategy. A total of 567 polymorphic markers, including 153 RAPDs, 61 ISSRs and 353 AFLPs, were used in linkage mapping. 336 of 567 (60%) markers were grouped on the two parental maps, leaving 231 (40%) markers unlinked. In the ‘Yuhualuoying’ linkage map, 210 markers including 116 testcross and 94 intercross markers were placed in 12 major and 32 minor (8 triplets and 24 doublets) linkage groups, covering 1034 cM with an average map distance of 6.2 cM between adjacent markers. In ‘Aoyunhanxiao’ linkage map, 190 markers consisting of 113 testcross and 77 intercross markers were resolved into 9 major and 24 minor linkage groups, with genome coverage of 1095 cM and a mean inter-marker separation of 6.9 cM between adjacent markers. Six pairs of homologous linkage groups were established on the basis of 64 intercross markers shared by the two parental maps. The maps lay a foundation for further quantitative traits loci (QTL) mapping and marker-assisted breeding of chrysanthemum.     

Development and evaluation of in vitro somaclonal variation in strawberry for improved horticultural traits

Strawberry cultivation is not popular in Bangladesh due to the unpredictable climatic conditions and lack of proper cultivars. Using somaclonal variation, several new promising selections were generated and evaluated for their flowering and fruiting ability, adaptability and sustainability. To induce variation, plants were regenerated using various tissue culture techniques. Our results suggested that a high concentration of BAP in culture medium successfully resulted in the induction of somaclonal variation. Among the tissue culture techniques adopted in this study, meristem culture was most effective for induction of somaclonal variation. Twenty five putative somaclones with better horticultural features were subsequently selected and field evaluated for three clonal generations. Several of the selections reverted back to their original phenotype within 2–3 vegetative propagations. Three of the stable selections were distinct from each other in terms of fruit and other horticultural characters, and have potential for commercial cultivation in Bangladesh.     

Genetics and distribution of fertility restoration associated RAPD markers in inbreds of pepper (Capsicum annuum L.)

Experiments were conducted to study genetics of fertility restoration and to examine distribution of RAPD markers (OPW19₈₀₀ and OPP13₁₄₀₀) linked with fertility restoration gene (Rf) in pepper (Capsicum annuum L.) inbreds. Forty-two hot and five sweet pepper inbreds were crossed on a cytoplasmic male sterile (cms) line CCA-4261 and F₁s were evaluated for fertility restoration under open field conditions. DNA of 5 plants of CCA-4261 and individual plants of 47 inbreds was isolated and PCR reaction was performed using OPW19 and OPP13 primers. The results revealed that most of the hot pepper lines posses Rf gene. The Rf gene associated two markers, viz., OPW19₈₀₀ and OPP13₁₄₀₀ were not frequently distributed in the restorer inbred lines because presence of marker bands often does not coincide with the presence of Rf gene identified in many restorer inbreds. The case specific applications of both the RAPD markers have been described.     

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.     

Genetic diversity of Indonesian cauliflower cultivars and their relationships with hybrid cultivars grown in Australia

The objectives of this study were to investigate genetic variation and relationships between Indonesia-, Australian- and European-based cultivars and to evaluate variation within Indonesia cultivars as all cultivars are open-pollinated. Eight cauliflower cultivars collected from three production regions in Indonesia and four F₁ hybrids cultivars grown in Australia were evaluated using RAPD and ISSR markers. DNA polymorphisms generated from 10 polymorphic RAPD primers were used to construct a dendogram using the unweighted pair-group method with arithmetic averages (UPGMA) and to generate a fingerprinting key. ISSR marker analysis using 14 primers were attempted but DNA polymorphisms could not be clearly identified. The RAPD technique indicated that variation occurred both within and between Indonesian cultivars. Comparison between Indonesian-, Australian- and European-based cultivars showed that Indonesian cultivars have unique genotypes and would be good sources of genes for future crop improvement.     

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.     

中国北方引种的部分越橘品种的RAPD分析

利用RAPD技术对我国北方引种的15份越橘(Vaccinium spp.)栽培品种进行了遗传多样性分析。从70条随机引物中筛选出15条引物,共扩增出94个位点,其中多态性位点83个(88.3%)。材料间遗传相似系数变化为0.5426～0.8936,表明各品种间具有一定的遗传差异。UPGMA聚类分析结果可将供试材料分为2大类、5个亚类。聚类结果与各品种的杂交系谱相对应,从分子水平揭示了品种间的亲缘关系。