Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Identification of Cotton Hybrid through the Combination of PCR Based RAPD, ISSR and Microsatellite Markers

Hybrid cotton H ‘6’ and its parents G.Cot.10 (male) and G.Cot.100 (female) were studied for identification with three PCR based molecular markers, RAPD, ISSR and microsatellite. Twenty RAPD primers, nineteen ISSR primers and twenty-five JESPR cotton microsatellite loci were used. RAPD primer OPA 11 was found to be useful in differentiating parents and hybrid. Two ISSR primers, IS4 and IS7 showed polymorphism in the parents. IS4 identified a female-specific amplicon of about 500bp and IS7 identified two female-specific amplicons of about 500 and 1200bp in the hybrid H ‘6’. Microsatellite loci JESPR-2 and JESPR-17 were found to be heteroallelic for parents. JESPR-2 identified one male-specific repeat of about 850bp, while JESPR-17 detected two male-specific repeats of about 800bp and 700bp in the hybrid H ‘6’. Results indicated that using all three markers - RAPD, ISSR and SSR - in combination is faster and more reliable than using the three in isolation, for the identification of cotton hybrid.     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.     

Development of DNA markers for identifying chrysanthemum cultivars generated by ion-beam irradiation

Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ (Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.     

扬子鳄鞣制皮革和鳞片的DNA提取方法

运用一种改进的提取方法 ,作者从鞣制皮革中成功地提取了总DNA ,同时还对尾尖皮、鳞片、盐腌生皮等皮质进行了DNA提取 ;用 12SrRNA基因扩增的通用引物、扬子鳄鉴别引物、微卫星引物及RAPD引物进行PCR扩增 ,并对部分扩增结果进行测序 ,以检验提取效果。结果证明 ,几种皮质标本都可提取出DNA ,其中尾尖皮和鳞片的提取效果较好 ,用四种引物都可扩增出明显亮带 ;盐腌生皮和鞣制皮提取结果也很好 ,并且用12SrRNA通用引物、扬子鳄鉴别引物扩增的亮带较明显 ,可进行扬子鳄皮质用品等的分子鉴定及部分序列的扩增和测序研究     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

一种新的分子标记方法－随机微卫星扩增多态DNA (RMAPD)

随机微卫星扩增多态DNA（RMAPD）是利用随机引物和微卫星的上游或下游引物一起作为该扩增的引物,在Taq DNA聚合酶、MgCl2、dNTPs和模板DNA等共同作用下进行PCR扩增的一种新型分子标记方法。其核心是RMAPD引物的有效性问题。通过对西农萨能奶山羊群体RMAPD电泳检测、数据统计分析及验证实验等证明RMAPD的引物是有效的。通过与微卫星和RAPD标记比较,发现RMAPD标记在扩增引物、扩增程序和重复性等方面区别于微卫星和RAPD标记;它是RAPD标记的一种广义的延伸,但又不完全等同于RAPD标记。因此,确定RMAPD是一种新的分子标记方法。该方法也具有DNA标记的特点,在群体遗传结构和亲缘关系分析以及标记辅助选择等遗传育种领域具有广阔的应用前景。     

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

 A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8₄₀₀, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8₄₀₀ marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8₄₀₀ were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp. Received: 16 January 1998 / Accepted: 30 April 1998     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

香蕉33个品种的RAPD研究

利用RAPD技术对香蕉（Musa nana Lour.)33个品种的遗传变异进行了研究,从249个随机引物中筛选出18个有效引物,用它们共扩增出192条DNA带,其中183条为多态性带,占95．31％,平均每个引物扩增的DNA带数为10．67条,利用18个有效引物扩增的192条DNA带对香蕉33个品种间的亲缘关系进行UPGMA聚类分析,计算出33个品种间的平均遗传距离为0．3412。在此基础上建立了香蕉33个品种的DNA分子系统树状图。该系统将香蕉33个品种划归A,B,C和D4个群,其中A群20个品种,B群5个品种,C群2个品种,D群6个品种;A群又可以分为3个亚群。对香蕉遗传多样性分子基础进行了探讨。     

RAPD Analysis of Lathyrus sativus Somaclones

RAPD patterns were studied in seven somaclones of Lathyrus sativus having contrasting characteristics alongwith the parent cultivar P-24. Out of 81 decamer random primers used, 5 did not amplify and 24 revealed DNA polymorphism while the rest generated monomorphic banding patterns. Eight unique bands were amplified with different primers in four different somaclones. With most of the informative primers differences were observed between somaclones and also between some of the somaclones and parent cultivar P-24. More than 90% similarity in the RAPD patterns was evident among the somaclones and the parent cultivar P-24. Though it was not possible to identify a particular somaclone with a single primer, a combination of two or more primers could be employed to identify a somaclone.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

Distinction between cold-sensitive and -tolerant jute by DNA polymorphisms

Jute is the principal coarse fiber for commercial production and use in Bangladesh. Therefore, the development of a high-yielding and environmental-stress tolerant jute variety would be beneficial for the agro economy of Bangladesh. Two molecular fingerprinting techniques, random-amplified polymorphic DNA (RAPD) and amplified-fragment length polymorphism (AFLP) were applied on six jute samples. Two of them were cold-sensitive varieties and the remaining four were cold-tolerant accessions. RAPD and AFLP fingerprints were employed to generate polymorphism between the cold-sensitive varieties and cold-tolerant accessions because of their simplicity, and also because there is no available sequence information on jute. RAPD data were obtained by using 30 arbitrary oligonucleotide primers. Five primers were found to give polymorphism between the varieties that were tested. AFLP fingerprints were generated using 25 combinations of selective-amplification primers. Eight primer combinations gave the best results with 93 polymorphic fragments, and they were able to discriminate the two cold-sensitive and four cold-tolerant jute populations. A cluster analysis, based on the RAPD and AFLP fingerprint data, showed the population-specific grouping of individuals. This information could be useful later in marker-aided selection between the cold-sensitive varieties and cold-tolerant jute accessions.     

Genetic variation of petaloid male-sterile cytoplasm of carrots revealed by sequence-tagged sites (STSs)

The mitochondrial DNA of various carrot lines was characterized by random amplified polymorphic DNA (RAPD) analysis, and six sequence-tagged sites (STSs) led to identification of the petaloid type of cytoplasmic male sterility (CMS). Using six STS primer combinations, we were able to classify five CMS lines into two groups and eight fertile carrots into six groups. Both the STS1 and the STS4 primer combinations differentiated CMS cytoplasms from the fertile cytoplasms, and the STS2 primer combination revealed two different types of CMS cytoplasms – of Wisconsin Wild and Cornell origins. Cybrid carrot lines with petaloid flowers which had been obtained by asymmetric cell fusion could also be separated from fertile cybrids by the STS1 primer combination. The STS1 fragment contained a homologous sequence with the orfB gene. DNA gel blot analysis indicated that homologous regions to the STS1 fragment existed in fertile types as well as the CMS types, although the restriction fragment size patterns differed. These observations demonstrate that rearrangements involving this region occurred in the mitochondrial genome. The STS4 fragment had a more complicated gene structure, including retrotransposon-like sequences and small segments of chloroplast genome. Received: 10 September 1998 / Accepted: 24 February 1999     

Primer R108 performs best in the RAPD strain typing of threeAspergillus species frequently isolated from patients

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.     

Genetic variation among European strains of Saccharomyces paradoxus: results from DNA fingerprinting

We used microsatellite fingerprinting and RAPD analysis to characterize 28 wild European strains of Saccharomyces paradoxus. In contrast to our results from a previous allozyme survey [Naumov et al. Int. J. Syst. Bacteriol. 47: 341-344 (1997a)], these methods revealed extensive genetic variation. The RAPD primers 5'AATCGGGCTG and 5'GGGTAACGCC and the microsatellite primer (GTG)5 yielded reproducible amplification patterns of sufficient clarity and variability to distinguish individual strains from the wild. UPGMA analysis tended to group the strains according to climatic and geographic origin. A comparative study of Ty1 sequence having multiple chromosomal location was also done. Each wild S. paradoxus isolate shows a unique hybridization pattern allowing discrimination to the strain level.     

Comparative Evaluation of RAPD, ISSR and Anchored-SSR Markers in the Assessment of Genetic Diversity and Fingerprinting of Oilseed Brassica Genotypes

Forty-two genotypes representing oilseed Brassica species were analyzed for the level of genetic diversity and molecular identity using Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat (ISSR) and 5'-Anchored Simple Sequence Repeat (ASSR) markers. DNA profiles revealed high degree of interspecific polymorphism, while the level was considerably low within a species, particularly in B. juncea. The UPGMA clusters clearly delineated genotypes of the respective Brassica species. Comparison of cophenetic matrices indicated a high degree of correspondence between dendrograms generated by different marker systems. A minimum of 10 random primers (approximately 105 bands) were required for the RAPD profiles to generate the expected cluster. Comparatively less number of primers was required to do the same in case of ISSR (4 primers) and ASSR (3 primers). The principal component analysis revealed similar genetic relationship among the genotypes as in cluster analysis. Although none of the DNA profiles could individually identify all the B. juncea genotypes, a combined DNA profile consisting 125 markers from the informative primers of all the three DNA marker systems could do the same. A positive correlation was found among the marker utility parameters (calculated for individual primers of different marker systems) such as marker index (MI), resolving power (Rp) and discrimination coefficient (D) with the number of genotypes identified by each primer with a few exceptions. Single plant analysis for a set of five B. juncea varieties revealed absence of intra-varietal heterogeneity in case of ASSR profiles, thereby suggesting its utility in varietal identification and differentiation.     

Microsatellite DNA and RAPD fingerprinting,identification and genetic relationships of hybrid poplar (<Emphasis Type="Italic">Populus x canadensis</Emphasis>) cultivars

Microsatellite DNA markers of ten SSR loci and 248 RAPD loci (resolved by 26 RAPD primers) were used for DNA fingerprinting and differentiation of 17 widely grown Populus x canadensis syn. Populus x euramericana (interspecific Populus deltoides x Populus nigra hybrids) cultivars ("Baden 431", "Blanc du Poitou", "Canada Blanc", "Dorskamp 925", "Eugenei", "Gelrica", "Grandis", "Heidemij", "I-55/56", "I-132/56", "I-214", "Jacometti", "Ostia", "Regenerata", "Robusta", "Steckby" and "Zurich 03/3"), and determination of their genetic interrelationships. Informativeness of microsatellite and RAPD markers was also evaluated in comparison with allozyme markers for clone/cultivar identification in P. x canadensis. High microsatellite DNA and RAPD genetic diversity was observed in the sampled cultivars. All of the 17 P. x canadensis cultivars could be differentiated by their multilocus genotypes at four SSR loci, and were heterozygous for their parental species-specific alleles at the PTR6 SSR locus. Except for "Canada Blanc" and "Ostia", which had identical RAPD patterns, all cultivars could also be differentiated by RAPD fingerprints produced by each of the two RAPD primers, OPA07 and OPB15. For microsatellites, the mean number of alleles, polymorphic information content, observed heterozygosity, observed number of genotypes and the number of cultivars with unique genotypes per locus was 5.2, 0.64, 0.67, 5.7 and 2.2, respectively. For RAPD markers, the number of haplotypes per locus, and the number of cultivars with unique RAPD profiles per locus were 1.06 and 0.72, respectively. Overall, microsatellite DNA markers were the most informative for DNA fingerprinting of P. x canadensis cultivars. On the per locus basis, microsatellites were about six-times more informative than RAPD markers and about nine-times more informative than allozyme markers. However, on the per primer basis, RAPD markers were more informative. The UPGMA cluster plots separated the 17 cultivars into two major groups based on their microsatellite genotypic similarities, and into three major groups based on their RAPD fragment similarities. Both the microsatellite and RAPD data suggest that the cultivars "Baden 431", "Heidemij", "Robusta" and "Steckby" are genetically closely related. The inter-cultivar genetic relationships from microsatellite DNA and RAPD markers were consistent with those observed from allozyme markers, and were in general agreement with their speculated origin. Microsatellite DNA and RAPD markers could be used for clone and cultivar identification, varietal control and registration, and stock handling in P. x canadensis.