Assessment of genetic fidelity of micropropagated plants of <Emphasis Type="Italic">Simmondsia chinensis</Emphasis> (Link) Schneider using RAPD and ISSR markers

RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) markers were screened to test the genetic integrity of jojoba (Simmondsia chinensis) plants multiplied through axillary bud multiplication from nodal segments. The in vitro raised plantlets were maintained for up to 12 in vitro subcultures. During the study a total of 48 (32 RAPD and 16 ISSR) primers were screened, out of which 24 RAPD and 13 ISSR primers produced a total of 191 (126 RAPD and 65 ISSR) clear, distinct and reproducible amplicons. The amplified products were monomorphic across all the selected micropropagated plants and were similar to the mother plant. The micropropagation protocol developed by our group for rapid in vitro multiplication is appropriate for clonal propagation of jojoba. The outcome supports the fact that axillary bud multiplication can also be used as one of the safest modes for the production of true-to-type plants.     

Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.     

Genetic homogeneity of in vitro raised plants of grapevine cv. Crimson Seedless revealed by ISSR and microsatellite markers

The present study was conducted to test the clonal homogeneity of six month old tissue culture raised plants of grapevine cv. Crimson Seedless using Inter Simple Sequence Repeat (ISSR) and Simple Sequence Repeat (SSR) markers. Visible assessment of these in vitro raised plants maintained in polyhouse did not show any morphological differences among themselves. However, to test the genetic homogeneity of these plants, we screened 50 ISSR primers out of which, 22 primers produced scorable and repeatable bands. These 22 primers were used further for assessing genetic homogeneity of in vitro raised plants of Crimson Seedless. These 22 ISSR primers generated 134 distinct band classes with a total of 3216 scorable bands. All the primers showed uniform banding pattern for all the in vitro raised plants and the mother plant. In case of 5 SSR primers (VS1, VVMD5, VVS2, VMCNG4c8 and VVMD31) used, a total of 288 scorable bands were obtained. The allele sizes ranged from 98 to 254 bp. Allelic composition of 23 in vitro raised plants and the mother plant at 5 SSR loci did not show any polymorphism. The results of the two marker systems in the present study revealed the genetic uniformity among the in vitro raised plants demonstrating the reliability of in vitro propagation system used for the cultivar.     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

Efficiency of different PCR-based marker systems for assessment of Iris pumila genetic diversity

We investigated informativeness and effectiveness of different marker types (ISSR, IRAP, REMAP, RGAP and LP-PCR that employ primers based on the conservative sequences of abiotic stress response genes) to study genetic diversity of Iris pumila L. By the number of amplicons per primer, number of polymorphic amplicons per primer and resolving power index (Rp), ISSR-markers were the most efficient followed by LP-PCR-markers. In order of decreasing value of indicators of genetic diversity “the percentage of polymorphic bands”, and “the average Jaccard? genetic distance between plants”, marker systems may be arranged as follows: ISSR > RAPD > LP-PC > RGAP ≈ IRAP. For ISSR-markers, the percentage of polymorphic bands was 1.3–1.7 times higher than for the others, and the average genetic distance was 1.2–1.3 times higher. Different marker systems were ranked by the value of Nei? gene diversity and the Shannon? index as follows: ISSR > RAPD ≈ LP-PCR > RGAP ≈ IRAP, with the highest and the lowest values differing 1.4 times. Genetic population structure was investigated with program Structure 2.3. The data of all marker systems suggest that all genomes under study belonged to one population. The PCoA and cluster analyses based on genetic distances showed distinctions in clustering generated from different markers data and summarized data, as well as the lack of strong clusters. Mantel test revealed significant positive correlation between the matrices of genetic distances generated by the data of almost all marker systems. The strongest correlation was found between RGAP- and IRAP-markers (r = 0.452, p = 0.01) and between RGAP and ISSR (r = 0.430, p = 0.01). ISSR, RAPD and LP-PCR proved to be more effective for the study of I. pumila genetic diversity, nevertheless, joint use of different marker systems will provide a more comprehensive assessment of variation in different genomic regions.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.     

The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers

Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identification of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson's diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets.     

Molecular Characterization of Brinjal (Solanum melongena L) Cultivars using RAPD and ISSR Markers

Molecular characterization of 19 advanced cultivars and landraces of brinjal was carried out using RAPD and ISSR markers. Twenty-nine RAPD primers generated a total of 240 amplified fragments, while 23 anchored and non-anchored ISSR primers produced 299 fragments. Of these, 66 (27.5%) RAPD and 56 (18.73%) ISSR fragments were polymorphic. All the cultivars could be distinguished based on RAPD and/or ISSR profiles. A set of two RAPD primers, OPW 11 and OPX 07, was adequate to distinguish all the 19 cultivars. On the other hand, a minimum of ten ISSR primers were required to achieve the same result. Eleven cultivars could be identified by the unique presence or absence of one to four markers. The correlation between primer Rp and the number of cultivars distinguished by RAPD was r = 0.873, while that for ISSR it was r = 0.327. The correlation between PIC of primer and the number of cultivars distinguished was r = 0.324 for RAPD, while for ISSR primers it was r = ? 0.066. The probability of chance identity between two cultivars for RAPD and ISSR markers was calculated as 8.94×10^?4 and 2.25×10^?2, respectively. The average Jaccard’s similarity coefficient between cultivars based on combined RAPD and ISSR data was estimated to be 0.919. The UPGMA analysis grouped the cultivars into three main clusters with significant bootstrap support. While the cultivars bred at Indian Agricultural Research Institute, New Delhi formed one sub-cluster; others did not show a prominent region-based clustering.     

Molecular diversity analysis of Rhizoctonia solani isolates infecting various pulse crops in different agro-ecological regions of India

Genetic diversity of 89 isolates of Rhizoctonia solani isolated from different pulse crops representing 21 states from 16 agro-ecological regions of India, 49 morphological, and 7 anastomosis groups (AGs) was analyzed using 12 universal rice primers (URPs), 22 random amplified polymorphic DNA (RAPD), and 23 inter-simple sequence repeats (ISSR) markers. Both URPs and RAPD markers provided 100?% polymorphism with the bands ranging from 0.1 to 5?kb in size, whereas ISSR markers gave 99.7?% polymorphism with the bands sizes ranging from 0.1 to 3?kb. The marker URP 38F followed by URP13R, URP25F, and URP30F, RAPD marker R1 followed by OPM6, A3 and OPA12 and ISSR3 followed by ISSR1, ISSR4, and ISSR20 produced the highest number of amplicons. R. solani isolates showed a high level of genetic diversity. Unweighted pair group method with an arithmetic average (UPGMA) analysis grouped the isolates into 7 major clusters at 35?% genetic similarity using the three sets of markers evaluated. In spite of using three different types of markers, about 95?% isolates shared common grouping patterns. The majority of the isolates representing various AGs were grouped together into different sub-clusters using all three types of markers. Molecular groups of the isolates did not correspond to agro-ecological regions or states and crops of the origin. An attempt was made for the first time in the present study to determine the genetic diversity of R. solani populations isolated from different pulse crops representing various AGs and agro-ecological regions.     

Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Schult. & Schult. F.) Backer ex K. Heyne using DNA-based markers

Dendrocalamus asper, an edible bamboo is valued for its tender edible shoots in the food industry. However, overexploitation of natural stands of D. asper coupled with minimal conservation and reforestation efforts has led to its rapid depletion in nature. Therefore protocol for rapid multiplication of D. asper via direct regeneration using nodal segments from mature clumps was standardized and more than 25,000 plants were transferred to the field (Singh et al. 2012a). However, genetic fidelity of these in vitro raised plants needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plants. This study report the genetic fidelity analysis of in vitro raised plants of D. asper for the first time using arbitrary (Random Amplified Polymorphic DNA, RAPD), semi-arbitrary (Inter-Simple Sequence Repeat, ISSR; Amplified Fragment Length Polymorphism, AFLP), and sequence-based (Simple Sequence Repeat, SSR) markers. Bulked DNA samples of 20 in vitro raised shoots (collected after every three subculture cycles starting from 3rd to 30th passage) and field transferred plantlets were compared with the mother plant DNA using 90 primer combinations (25 each of RAPD, ISSR, SSR, and 15 AFLP) and scorable bands were produced by 78 (22 RAPD, 24 ISSR, 21 SSR, and 11 AFLP) primers. A total of 146 distinct and scorable bands were produced by 22 RAPD primers with an average of 6.6 bands per primer while the number of bands for ISSR primers varied from 3 (ISSR-4 and 9) to 13 (ISSR-17), with an average of 7.1 bands per primer. Similarly, SSR markers also showed wide variation in number of bands, ranging from 2 (RM 261) to 12 (RM 44, 140, and 224) with an average of 7.8 bands. AFLP primer combinations could generate 35–72 bands with an average of 48.7 bands per primer pair. Amplification of monomorphic bands with all primer combinations authenticated the true to type nature of the in vitro raised plants of D. asper which underwent up to 30 subculture passages over a period of approximately 2 years thereby supporting the commercial utilization of the developed micropropagation protocol.     

Assessment of genetic fidelity of micropropagated <Emphasis Type="Italic">Swertia chirayita</Emphasis> plantlets by ISSR marker assay

Inter simple sequence repeat (ISSR) marker assay was employed to validate the genetic fidelity of Swertia chirayita plantlets multiplied in vitro by axillary multiplication upto forty-two passages. Sixteen ISSR primers generated a total of 102 amplicons among the tissue-cultured plants. Forty-eight amplicons were amplified in the outlier (a Swertia species). The outlier (negative control) was employed to rule out the possibility that the invariant fingerprint was due to chance alone and that the ISSR technique employed was not discriminatory enough to detect the off-types. A homogenous amplification profile was observed for all the micropropagated plants. The results confirmed the clonal fidelity of the tissue culture-raised S. chirayita plantlets and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers

To evaluate genetic homogeneity of 1-year-old guava (Psidium guajava L.) plants developed from in vitro somatic embryogenesis, DNA from leaf tissues of seven randomly selected plants along with the mother plant, was isolated and subjected to molecular analysis. A total of six Simple Sequence Repeat (SSR) primer pairs, producing reproducible and clear bands ranging from 100 to 300?bp in size, resulted in amplification of single band (allele), corresponding homozygous individuals. Moreover, of 10 different inter-simple sequence repeat (ISSR) primers screened, six produced resolvable, reproducible and scorable bands. All these ISSRs produced a total of 25 bands, ranging between 300 and 1,200?bp length, and the number of scorable bands, for each primer varied from three to six with an average of 4.1 bands per primer. The amplification products were monomorphic across all the micropropagated plants produced by all SSR and ISSR primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for guava.     

Genetic divergence and allelic-specificity in relation to expression of voltinism in silkworm using ISSR and RAPD fingerprinting

The silkworm B. mori is a multicellular organism revealing genetic resources which makes an ideal model for lepidoptera for the present investigation. With the objective of targeting distinctive markers for utilization in future breeding programmes, Bivoltine and Polyvoltine silkworm strains were used by inter-simple sequence repeats (ISSR) and random amplified polymorphic-DNA (RAPD) fingerprinting to detect their genetic versatility and volatility. Six ISSR primers generated 99 markers, of which 76.76% were found to be polymorphic with an average number of observed alleles (N _a) (1.86 ± 0.40), an effective number of alleles (N _e) (1.43 ± 0.30) as well as six RAPD primers that produced a total of 95 bands, developing 61.05% polymorphism with N _a (1.93 ± 0.51) and N _e (1.18 ± 0.30). The dendrogram produced by UPGMA analysis, based on Dice’s coefficient, clustered four races into two major groups which accurately segregated them according to their inheritance of voltinism. In this research, the ISSR markers were more accurate than the RAPD markers and ISSR also displayed better polymorphism. The outcome showed that the bivoltine strains exhibited higher allelic expressions with ISSR primers when compared to the polyvoltine strains. Despite exhibiting their unique race by certain DNA markers, most of the primers represented voltinism-specificity. Hence molecular marker amplification is a beneficial approach to reveal genetic divergence among closely related strains, and molecular characterization of phylogenetic relationships in addressing evolutionary evidences of individuals.     

Assessment of Genetic Diversity and Identification of Informative Molecular Markers for Germplasm Characterization in Caribbean Stylo (Stylosanthes hamata)

Caribbean stylo (Stylosanthes hamata) is a tropical fodder and cover crop. Along with four other Stylosanthes species (S. scabra, S. humilis, S. viscosa, S. guianensis), it was introduced in India. It became well adapted in certain parts of the country and has been recommended for the improvement of range and degraded lands. A collection of 63 S. hamata accessions was fingerprinted with RAPID, ISSR and STS markers. Though the mean discriminating power of these marker systems ranges from 0.65 to 0.71, high values of marker index (2.91), resolving power (14.92) and effective number of patterns per assay unit (50.65) makes ISSR as a better marker system in comparison to other two markers used in this study. Thirteen RAPD and eleven STS primers could differentiate a maximum of 42 and 17 accessions, respectively, whereas two ISSR primers produced distinct fingerprints of all the S. hamata accessions. Mean genetic similarities of accession ranged from 0.83 (ISSR) to 0.91 (RAPD). Two RAPD, two STS and four ISSR primers generated a set of 12 diagnostic markers which could be useful for germplasm characterization and management.     

Study of genetic variation among parsley (<Emphasis Type="Italic">Petroselinum Crispum</Emphasis> (Mill.) Nym.) samples using RAPD and ISSR markers

RAPD and ISSR markers have been used to classify 32 samples of parsley (Petroselinum Crispum (Mill.) Nym. ex A. W. Hill) including the root and leaf plants. In total, 51 RAPD and 66 ISSR markers were amplified by five RAPD and six ISSR primers. On average, the ISSR and RAPD spectra had 11 and 10.2 bands, respectively. The matrix of genetic distances between the samples was constructed using Jaccard coefficients. The dendrogram constructed by the UPGMA method graphically shows the values of genetic distances. The distance between root and leaf forms of parsley was 0.292.     

Clonal diversity in the rare Calamagrostis porteri ssp. insperata (Poaceae): comparative results for allozymes and random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers

Four populations of the rare, highly clonal grass Calamagrostis porteri ssp. insperata were examined using allozymes and the two polymerase chain reaction (PCR)-based markers, random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) bands. Only one of the 15 allozyme loci was variable and two alleles were detected, both of which were found in two populations, while only one genotype was detected in the other two populations. ISSR and RAPD markers detected more genotypes within populations than did allozymes. ISSR markers detected more diversity than RAPD markers in three of the four populations examined. In one population, no RAPD diversity was found whereas eight different genotypes were found among the 10 plants with ISSR markers. This diversity is present despite rare flowering, no documented occurrence of seed set in natural populations and very low seed set with experimental pollinations, all of which suggest that sexual reproduction rarely occurs. The subspecies is self-compatible, but seed initiation is lower in selfed ovules; also, there is high embryo abortion regardless of pollen source. Variation detected by RAPD and ISSR primers may reflect higher levels of sexual reproduction in the past, very rare sexual reproduction in extant populations, somatic mutations, or a combination of the three. Although the PCR-based markers identify several multilocus genotypes within populations, it is not known whether these all represent distinct genets generated by sexual reproduction or result from somatic mutations in the old, perennial and highly clonal plants.     

Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD,ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5 %) RAPD, 211 (74.6 %) ISSR, and 98 (74.8 %) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.     

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.     

利用RAPD和ISSR分子标记分析怀地黄种质遗传多样性

用RAPD与ISSR技术对怀地黄的8个品种和2个脱毒品系进行了种质遗传多样性分析。分别从80条RAPD引物和44条ISSR引物中筛选出适合怀地黄种质分析的17条RAPD引物和10条ISSR引物,用于RAPD和ISSR分析。17条RAPD引物共扩增出177条带, 多态性位点数为109; 多态性位点比率为61.58%;平均多样性指数(I)为0.3135;每个位点的有效等位基因数（Ne）是1.3641; 10条ISSR引物共扩增出110条带. 多态性位点数为79; 多态性位点比率为71.58%;平均多样性指数(I)为0.3577;每个位点的有效等位基因数（Ne）是1.4037。 基于扩增条带数据库建立了各自的Jaccard遗传相关系数矩阵,构建了相似的分子树状图,将10个供试材料分为2类:一类群含组培85.5、大田85.5、组培9302、大田9302、金状元和金白6个材料;另一类群含北京1号、大红袍、地黄9104和野生地黄4个材料。两种分子标记的分析结果呈极显著正相关(r＝0.649)。结果表明,RAPD与ISSR标记适合于怀地黄种质遗传多样性分析,ISSR标记技术是一种多态性和重复性优于RAPD技术的实用技术。