Showing papers on "RAPD published in 2012"

DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol

Abstract: Mangroves and salt marsh species are known to synthesize a wide spectrum of polysaccharides and polyphenols including flavonoids and other secondary metabolites which interfere with the extraction of pure genomic DNA. Although a plethora of plant DNA isolation protocols exist, extracting DNA from mangroves and salt marsh species is a challenging task. This study describes a rapid and reliable cetyl trimethylammonium bromide (CTAB) protocol suited specifically for extracting DNA from plants which are rich in polysaccharides and secondary metabolites, and the protocol also excludes the use of expensive liquid nitrogen and toxic phenols. Purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.78 to 1.84 and A260/A230 ratio was >2, which also suggested that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds. DNA concentration ranged from 8.8 to 9.9 μg μL−1. The extracted DNA was amenable to RAPD, restriction digestion, and PCR amplification of plant barcode genes (matK and rbcl). The optimized method is suitable for both dry and fresh leaves. The success of this method in obtaining high-quality genomic DNA demonstrated the broad applicability of this method.

Genetic diversity, population structure and genome-wide marker-trait association analysis emphasizing seed nutrients of the USDA pea (Pisum sativum L.) core collection

Abstract: Genetic diversity, population structure and genome-wide marker-trait association analysis was conducted for the USDA pea (Pisum sativum L.) core collection. The core collection contained 285 accessions with diverse phenotypes and geographic origins. The 137 DNA markers included 102 polymorphic fragments amplified by 15 microsatellite primer pairs, 36 RAPD loci and one SCAR (sequence characterized amplified region) marker. The 49 phenotypic traits fall into the categories of seed macro- and micro-nutrients, disease resistance, agronomic traits and seed characteristics. Genetic diversity, population structure and marker-trait association were analyzed with the software packages PowerMarker, STUCTURE and TASSEL, respectively. A great amount of variation was revealed by the DNA markers at the molecular level. Identified were three sub-populations that constituted 56.1%, 13.0% and 30.9%, respectively, of the USDA Pisum core collection. The first sub-population is comprised of all cultivated pea varieties and landraces; the second of wild P. sativum ssp. elatius and abyssinicum and the accessions from the Asian highland (Afghanistan, India, Pakistan, China and Nepal); while the third is an admixture containing alleles from the first and second sub-populations. This structure was achieved using a stringent cutoff point of 15% admixture (q-value 85%) of the collection. Significant marker-trait associations were identified among certain markers with eight mineral nutrient concentrations in seed and other important phenotypic traits. Fifteen pairs of associations were at the significant levels of P ≤ 0.01 when tested using the three statistical models. These markers will be useful in marker-assisted selection to breed pea cultivars with desirable agronomic traits and end-user qualities.

Construction of intersubspecific molecular genetic map of lentil based on ISSR, RAPD and SSR markers

Abstract: Lentil (Lens culinaris ssp. culinaris), is a self-pollinating diploid (2n = 2x = 14), cool-season legume crop and is consumed worldwide as a rich source of protein (∼24.0%), largely in vegetarian diets. Here we report development of a genetic linkage map of Lens using 114 F2 plants derived from the intersubspecific cross between L 830 and ILWL 77. RAPD (random amplified polymorphic DNA) primers revealed more polymorphism than ISSR (intersimple sequence repeat) and SSR (simple sequence repeat) markers. The highest proportion (30.72%) of segregation distortion was observed in RAPD markers. Of the 235 markers (34 SSR, 9 ISSR and 192 RAPD) used in the mapping study, 199 (28 SSRs, 9 ISSRs and 162 RAPDs) were mapped into 11 linkage groups (LGs), varying between 17.3 and 433.8 cM and covering 3843.4 cM, with an average marker spacing of 19.3 cM. Linkage analysis revealed nine major groups with 15 or more markers each and two small LGs with two markers each, and 36 unlinked markers. The study reported assigning of 11 new SSRs on the linkage map. Of the 66 markers with aberrant segregation, 14 were unlinked and the remaining 52 were mapped. ISSR and RAPD markers were found to be useful in map construction and saturation. The current map represents maximum coverage of lentil genome and could be used for identification of QTL regions linked to agronomic traits, and for marker-assisted selection in lentil.

Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production

Abstract: The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231T, Staphylococcus aurues ATCC 51650T, methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44T and Pseudomonas aeruginosa ATCC 10145T, respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus—polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.

Comparison of three typing methods for Pseudomonas aeruginosa isolates from patients with cystic fibrosis

Abstract: The aim of this study was to compare two traditional pattern matching techniques, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD), with the more reproducible technique of multilocus sequence typing (MLST) to genotype a blinded sample of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. A blinded sample of 48 well-characterized CF P. aeruginosa isolates was genotyped by PFGE, RAPD, and MLST, each performed in a different laboratory. The discriminatory power and congruence between the methods were compared using the Simpson’s index, Rand index, and Wallace coefficient. PFGE and MLST had the greatest congruence with the highest Rand index (0.697). The discriminatory power of PFGE, RAPD, and MLST were comparable, with high Simpson’s indices (range 0.973–0.980). MLST identified the most clonal relationships. When clonality was defined as agreement between two or more methods, MLST had the greatest predictive value (100 %) in labeling strains as unique, while PFGE had the greatest predictive value (96 %) in labeling strains as clonal. This study demonstrated the highest level of agreement between PFGE and MLST in genotyping P. aeruginosa isolates from CF patients. MLST had the greatest predictive value in identifying strains as unique and, thus, has the potential to be a cost-efficient, high-throughput, first-pass typing method.

Analysis of the genetic diversity of physic nut, Jatropha curcas L. accessions using RAPD markers

Abstract: A sum of 48 accessions of physic nut, Jatropha curcas L. were analyzed to determine the genetic diversity and association between geographical origin using RAPD-PCR markers. Eight primers generated a total of 92 fragments with an average of 11.5 amplicons per primer. Polymorphism percentages of J. curcas accessions for Selangor, Kelantan, and Terengganu states were 80.4, 50.0, and 58.7%, respectively, with an average of 63.04%. Jaccard’s genetic similarity co-efficient indicated the high level of genetic variation among the accessions which ranged between 0.06 and 0.81. According to UPGMA dendrogram, 48 J. curcas accessions were grouped into four major clusters at coefficient level 0.3 and accessions from same and near states or regions were found to be grouped together according to their geographical origin. Coefficient of genetic differentiation (Gst) value of J. curcas revealed that it is an outcrossing species.

Molecular Profiling for Genetic Variability in Capsicum Species Based on ISSR and RAPD Markers

Abstract: The taxonomic identity of Capsicum species is found to be difficult as it displays variations at morpho-chemical characters. Twenty-two accessions of six Capsicum species, namely, C. annuum, C. baccatum, C. chinense, C. eximium, C. frutescens, and C. luteum were investigated for phenotypic diversity based on flower color and for genetic differences by molecular makers. The genetic cluster analyses of 27 RAPD and eight ISSR primers, respectively, revealed genetic similarities in the ranges of 23–88% and 11–96%. Principal component analysis of the pooled RAPD and ISSR data further supports the genetic similarity and groupings. Different species showed variations in relation to corolla shade of flower. C. annuum accessions formed a single cluster in the molecular analysis as maintaining their flower characteristic. C. chinense accession shared flower features with the accessions of C. frutescens and were found to be closer at genotypic level. C. luteum was found to be rather closer to C. baccatum complex, both phenotypically and genetically. The only accession of C. eximium presenting purple flowers falls apart from the groupings. The floral characteristics and the molecular markers are found to be useful toward the delineation of the species specificity in Capsicum collection and identification of genetic stock.

Confirmation and characterization of interspecific hybrids of Passiflora L. (Passifloraceae) for ornamental use

Abstract: Three new varieties of Passiflora hybrids were developed from crosses between P. sublanceolata J. M. MacDougal (ex P. palmeri var. sublanceolata Killip) versus P. foetida var. foetida L. Twenty putative hybrids were analyzed. Hybridizations were confirmed by RAPD and SSR markers. The RAPD primer UBC11 (5′-CCGGCCTTAC-3′) generated informative bands. The SSR primer A08FP1 amplified species-specific fragments and heterozygote status was observed with the two parent bands 240 and 280 bp. The molecular markers generated by primers were analyzed in terms of the presence or absence of specific informative bands. The morphological characterization of the hybrids enabled their differentiation into three groups, identified as: (1) Passiflora ‘Alva’, composed of five hybrid plants with white flowers, large corona, light purple filaments at base, white and purple/white banding to apex; (2) P. ‘Aninha’, composed of six hybrid plants with pale pink flowers, corona filaments reddish/purple at base, white, purple/white banding to apex; (3) P. ‘Priscilla’, composed of nine hybrid plants with white flowers, small corona, filaments dark purple at base, white and purple to apex. The genomic homology of parent plants was verified by cytogenetic analysis. Both parents were 2n = 22. Meiosis was regular in genitors and hybrids. Aneuploidy was observed at hybrid groups P. ‘Alva’ and P. ‘Priscilla’ (2n = 20). Other authors had already observed the same number of chromosomes for some P. foetida genotypes. Obtaining valuable interspecific hybrids opens up new perspectives to offer opportunities in agribusiness for producers and to arouse the interest of consumers into using passion flowers in the Brazilian ornamental plant market.

In Vitro Propagation of Ocimum Gratissimum L. (Lamiaceae) and Its Evaluation of Genetic Fidelity Using RAPD Marker

Abstract: An efficient plant propagation system through nodal explants was established in Ocimum gratissimum L, a medicinally important herbaceous perennial herb belonging to the family Lamiaceae. Axillary shoot bud proliferation was initiated from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N6-benzyladenine (BA) (0.5 - 3.0 mg/l), Kinetin (KN) (0.5 - 3.0 mg/l) and 2-isoPentenyladenine (2-iP) (0.5 - 3.0 mg/l). Maximum numbers of shoots (5.17 ± 0.04) with average length (2.50 ± 0.07) were induced on medium containing 1.0 mg/l BA. Shoot multiplication was maintained by repeated subculturing the original nodal explants on shoot multiplication medium after each harvest of newly formed shoots. Histological study shows that the organogenesis occurs directly, without callus formation on epidermal and sub epidermal layer of the explants. Rooting of shoots was achieved on half strength MS medium supplemented with 1.5 mg/1 Indole-3-butyric acid (IBA) and 2% sucrose. Well-developed complete plantlets were transferred to plastic pots containing a mixture of (1:1) soil and vermiculite showed 82.5 % survival rate. Genetic fidelity was assessed by chromosome analysis and DNA fingerprinting using random amplified polymorphic DNA (RAPD) of in vitro and in vivo plants. Nine arbitrary decamers displayed same banding profile showed no genomic alterations, indicating homogeneity among the tissue culture regenerates and genetic uniformity with that of donor plants. The present study provides high fidelity micro-propagated system for efficient and rapid micro-propagation protocol of this important medicinal plant and great use in conserving without risk of genetic instability.

The Use of ISSR and RAPD Markers for Genetic Diversity among South Tunisian Barley

Abstract: Random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) were assayed to determine the genetic diversity of 80 barley specimens from South Tunisia. The ISSR primers showed variation in the percentage of polymorphism, band informativeness (Ib), and resolving power (Rp). The percentage of polymorphism is 66.67%, the average Ib ranged from 0.24 to 0.39, while Rp ranged from 0.74 to 1.16. In RAPD analysis, three primers yielded a total of 17 scorable bands, which are all polymorphic. The three polymorphic primers exhibited variation with regard to average band informativeness (AvIb) and resolving power (Rp). RAPD and ISSR marker systems were found to be useful for the genetic diversity among the barley specimens. The two dendrograms obtained through these markers show different clustering of 80 barely specimens, but we noted that some clusters were similar in some cases. A poor correlation (𝑟=0.12) was found between both sets of genetic similarity data, suggesting that both sets of markers revealed unrelated estimates of genetic relationships. Therefore, the ISSR and RAPD molecular markers show two genetic grouping of studied barely specimens.

Assessment of genetic variations among highly endangered medicinal plant Bacopa monnieri (L.) from Central India using RAPD and ISSR analysis

Abstract: Genetic variations of 15 Brahmi (Bacopa monnieri L.) accessions were evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. During RAPD analysis, amplification of genomic DNA of the 15 accessions by 22 primers generated 197 fragments, of which 187 were polymorphic with an average of 8.95 bands per primer. The amplified products varied in size from 2,200 to 250 bp. Twenty-five selected ISSR primers produced 284 bands across 15 accessions, of which 270 were polymorphic with an average of 10.80 bands per primer. The PIC value ranges from 0.363 to 0.908 for RAPD primers, while 0.419 to 0.836 in case of ISSR. The size of amplified bands ranged from 2,800 to 240 bp. Similarity index values ranged from 0.16 to 0.95 (RAPD), 0.18 to 0.98 (ISSR) and 0.179 to 0.945 for pooled ISSR and RAPD markers data. Mantel test revealed the similar distribution pattern of the polymorphism between RAPD and ISSR markers and the correlation co-efficient (r) was 0.71384. The results indicated that both of the marker systems RAPD and ISSR, individually or combined can be effectively used in determination of genetic relationship among B. Monnieri accessions collected from different parts of Central India. It could be concluded that the information of genetic similarities and diversity among Brahmi accessions is necessary for their conservation and breeding programs.

Mapping of DNA sex-specific markers and genes related to sex differentiation in turbot (Scophthalmus maximus).

Abstract: Production of all-female populations in turbot can increase farmer’s benefits since sexual dimorphism in growth in this species is among the highest within marine fish, turbot females reaching commercial size 3–6 months earlier than males Puberty in males occurs earlier than in females, which additionally slows their growth Thus, elucidating the mechanisms of sex determination and gonad differentiation is a relevant goal for turbot production A ZZ/ZW sex determination mechanism has been suggested for this species, and four sex-related quantitative trait loci (QTL) were detected, the major one located in linkage group (LG) 5 and the three minor ones in LG6, LG8, and LG21 In the present work, we carried out a linkage analysis for several sex-related markers: (1) three anonymous sex-associated RAPD and (2) several candidate genes related to sex determination and gonad differentiation in other species (Sox3, Sox6, Sox8, Sox9, Sox17, Sox19, Amh, Dmrta2, Cyp19a, Cyp19b) We focused our attention on their co-localization with the major and minor sex-related QTL trying to approach to the master sex-determining gene of this species Previously described growth-related QTL were also considered since the association observed between growth and sex determination in fish Amh, Dmrta2, and one RAPD were located in LG5, while Sox9 and Sox17 (LG21), Cyp19b (LG6), and a second RAPD (LG8) co-mapped with suggestive sex-related QTL, thus supporting further analyses on these genes to elucidate the genetic basis of this relevant trait for turbot farming

Genetic diversity and population structure in the Brazilian Cattleya labiata (Orchidaceae) using RAPD and ISSR markers

Abstract: Brazilian orchids are currently threatened with extinction due to habitat loss and, because of their high ornamental value, intense collecting pressure. Genetic diversity can play a key role in the survival of endangered orchid species. Here we provide the first data on genetic diversity and structure of wild populations in the genus Cattleya, in particular C. labiata, using random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. We studied 130 individuals, 117 belonging to Cattleya labiata and 13 from 10 other species in the same genus. Data generated from 12 ISSR and 12 RAPD primers were used to determine genetic variability via a model-based Bayesian procedure (Structure) and molecular variance analysis. In addition, Shannon index, genetic diversity and Jaccard coefficients were also estimated. The marker data indicated that C. labiata has a high level of polymorphism, and five reconstructed populations were identified by Structure. The unweighted pair group method with arithmetic mean dendrogram did not group the samples by origin, which was also confirmed by Bayesian analysis, demonstrating the complex genetic structure of C. labiata. Other Cattleya species showed no relationship with any C. labiata sample. This genetic characterization of Cattleya from northeast Brazil contributes to knowledge of the genetic structure of the species and can be used to define strategies for conservation and breeding programmes.

ISSR and RAPD Based Evaluation of Genetic Stability of Encapsulated Micro Shoots of Glycyrrhiza glabra Following 6 Months of Storage

Abstract: In vitro grown axillary micro shoots of Glycyrrhiza glabra were encapsulated in alginate beads. Following 6 months of normal storage at 25 ± 2°C the re growth of encapsulated G. glabra micro shoots, reached 98% within 30 days of incubation on MS medium supplemented with 0.1 mg/l IAA. Re growth was characterized by the development of both shoot and root from single encapsulated micro shoot. Healthy plants were established to glass house with 95% survival. The genetic fidelity of plants obtained after conversion of alginate beads was ascertained through 10 RAPD and 13 ISSR primers. Of the 10 RAPD primers tested, 6 of them produced 14 clear and reproducible amplicons with an average of 2.3 bands per primer out of which 28.57% were polymorphic generated by only two primers. Eight ISSR primers produced total 37 bands ranging between 300 and 3,500 bp length. Number of scorable bands for each primer varied from 3 to 8 with an average of 4.6 bands per primer. Cluster analysis from ISSR and RAPD showed that all the tested plants including the mother plant distributed in two major groups with similarity coefficient ranging from 0.91 to 0.96 for RAPD and 0.89 to 0.97 for ISSR.

Genetic diversity and relatedness of sweet cherry (prunus avium L.) cultivars based on single nucleotide polymorphic markers.

Abstract: Most previous studies on genetic fingerprinting and cultivar relatedness in sweet cherry were based on isoenzyme, RAPD and SSR markers. This study was carried out to assess the utility of SNP markers generated from 3’UTRs for genetic fingerprinting in sweet cherry. A total of 114 sweet cherry germplasm representing advanced selections, commercial cultivars and old cultivars imported from different parts of the world were screened with 7 SSR markers developed from other Prunus species and with 40 SNPs obtained from 3’UTR sequences of Rainier and Bing sweet cherry cultivars. Both types of marker study had 99 accessions in common. The SSR data was used to validate the SNP results. Results showed that the average number of alleles per locus, mean observed heterozygosity, expected heterozygosity and polymorphic information content (PIC) values were higher in SSRs than in SNPs although both set of markers were similar in their grouping of the sweet cherry accessions as shown in the dendrogram. SNPs were able to distinguish sport mutants from their wild type germplasm. For example, ‘Stella’ was separated from ‘Compact Stella’. This demonstrates the greater power of SNPs for discriminating mutants from their original parents than SSRs. In addition, SNP markers confirmed parentage and also determined relationships of the accessions in a manner consistent with their pedigree relationships. We would recommend the use of 3’ UTR SNPs for genetic fingerprinting, parentage verification, gene mapping and study of genetic diversity in sweet cherry.

Data to the sex determination in Pistacia species using molecular markers

Abstract: Sex identification in Pistacia species dur- ing the long juvenile stage is an economically desirable objective. Due to the lack of morphological methods to identify sex at this stage, the application of molecular markers is expected to facilitate breeding programs. The aim of our study was to identify a marker closely linked to sex loci in Pistacia atlantica Desf subsp. mutica, P. khinjuk, and P. vera subsp. Sarakhs. Samples were collected from both male and female plants of each species, and their band patterns were analyzed according to the presence or absence of specific bands. Thirty random amplified polymorphic DNA (RAPD) primers and a pair of sequence char- acterized amplified region (SCAR) primers were tested as potential markers of sex in wild Pistacia species. Among the RAPD primers, only BC1200 was found to amplify a specific sex band present in female plants. Based on our analysis of all individual samples, a fragment of approximately 300 bp was amplified in female trees but absent in male ones. Although sex determination mechanisms in Pistacia are still unknown, they may be controlled by a single locus that acts as a trigger. The SCAR technique has proved to be a reliable technique in gender determination of pistachio genotypes at the seedling phenophase. This method could reduce both the time and costs associ- ated with breeding programs.

SSR and RAPD analysis of genetic diversity in walnut ( Juglans regia L.) genotypes from Jammu and Kashmir, India

Abstract: In this study, the genetic relatedness of 82 walnut genotypes adapted to the North Western Himalayan region of Jammu and Kashmir, India was analyzed by combination of 13 SSR and 20 RAPD primers A high level of genetic diversity was observed within populations with the number of alleles per locus ranging from one to five in case of SSR primers and two to six in case of RAPD primers, the proportion of polymorphic loci was 100 %, and similarity ranged from 12 % to 79 % with an average of 49 % Dendrogram showed that all the accessions formed four main clusters with various degree of sub-clustering within the clusters These results have implications for walnut breeding and conservation

Early assessment of genetic fidelity in sugarcane (Saccharum officinarum) plantlets regenerated through direct organogenesis with RAPD and SSR markers

Abstract: Early assessment of genetic fidelity of micropropagated plants aids in fine tuning protocol parameters and gauge suitability of regeneration protocol for large scale applications. Induction of direct organogenesis leading to reduced duration in vitro can lead to production of genetically stable plantlets. The present work describes early assessment of clonal fidelity in Sugarcane plants regenerated through direct organogenesis using RAPD and SSR markers. Analysis of RAPD banding patterns generated by PCR amplification using 20 random primers gave no evidences for somaclonal variation and the percent of polymorphic bands in a total of 110 amplicons was 0.02%. RAPD patterns of the plantlets were identical with the original mother plant, indicating that direct adventitious organogenesis did not induce somaclonal variation that can be detected by RAPD. Mean while SSR banding pattern analysis generated with 15 primers (112 amplicons) also gave no evidences for somaclonal variation. The genetic fidelity testing of micro-shoots, based on a RAPD and SSR analysis indicated a strong genetic purity like the parent genotype. Lack of variation confirms the genetic purity of tissue culture plants of sugarcane raised through direct organogenesis in young whorl leaf roll explants and confirms to the suitability of overall regeneration protocol.

Characterization of stress induced by copper and zinc on cucumber (Cucumis sativus L.) seedlings by means of molecular and population parameters.

Abstract: Contamination of plants with heavy metals could result in damage in DNA, such as mutations and cross-links with proteins. These altered DNA profiles may become visible in changes such as the appearance of a new band, or loss of an existing band, in the random amplified polymorphic DNA (RAPD) assay. In this study, various concentrations of copper and zinc salts were applied to cucumber seedlings during germination. Results displayed abnormalities in germination and also changes in root elongation, dry weight and total soluble protein level. All treatment concentrations (40, 80, 160, 240, 320, and 640mg/L) used in the study caused a decrease/delay in germination of the cucumbers to different extents. Inhibition or activation of root elongation was considered to be the first effect of metal toxicity in the tested plants. Application of the metal salts and the combined solutions on cucumber (Cucumis sativus L.) seedlings revealed similar consequences for total soluble protein level, dry weight and ultimately in inhibitory rates as well. The data obtained from RAPD band-profiles and genomic template stability (GTS) showed results that were consistent with the population parameters. In this regard, we conclude that molecular marker assays can be applied in combination with population parameters to measure genotoxic effects of heavy metals on plants.

Characterization of genetic diversity in Jatropha curcas L. germplasm using RAPD and ISSR markers

Abstract: Jatropha curcas L. is a rapidly emerging biofuel crop attracting a lot of interest, triggering large investments and rapid expansion of cultivation areas. In the present investigation, the genetic relationships of 29 J. curcas accessions were assessed based on randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses. A total of 72 polymorphic primers (47 RAPD and 25 ISSR) were used. Amplification of genomic DNA of the 29 genotypes, using RAPD analysis, yielded 552 fragments that could be scored, of which 334 were polymorphic with an average of 7.1 polymorphic fragments per primer. Number of amplified fragments varied from 2 to 23 and ranged in size from 100-3,500 bp. The 25 polymorphic ISSR primers used in the study produced 336 bands across 29 genotypes, of which 201 were polymorphic. The number of amplified bands varied from 7 to 20 with a size range of 100-3,500 bp. Molecular polymorphism was 60.5 and 59.8% with RAPD and ISSR markers, respectively. Mantel test between the two Jaccard’s similarity matrices gave r=0.8623, showing good fit correlation between RAPD and ISSR based similarities. Clustering of genotypes within groups remained more or less similar in ISSR and combined data of RAPD and ISSR.

Development of a coupling-phase SCAR marker linked to the powdery mildew resistance gene ‘er1’ in pea (Pisum sativum L.)

Abstract: Pea powdery mildew is one of the major constraints in pea production worldwide, causing severe seed yield and quality loss. The resistance is governed by a single recessive gene er1 in majority of resistant cultivars, but er2 and Er3 have also been reported. The objective of the study was to find out tightly linked sequence characterized amplified regions (SCAR) markers to er1 gene using NILs. A total of 620 random amplified polymorphic DNA (RAPD) markers were screened for length polymorphism between seven sets of NILs. The 880 bp polymorphic band of the tightly linked RAPD marker OPX 04880 was cloned, sequenced and a SCAR marker ScOPX 04880 was developed. In a population of completely classified 208 F2 plants (supported by phenotypic data from 208 F2:3 and 4,390 F3:4 families) ScOPX 04880 was linked at 0.6 cM in coupling phase with er1 gene in the order ScOPX 04880–er1–ScOPD 10650. ScOPX 04880 will correctly differentiate homozygous resistant plants from the susceptible accessions with more than 99 % accuracy. In combination with repulsion phase marker ScOPD 10650, ScOPX 04880 can help in an error free marker-assisted selection.

Moringa genetic diversity from germplasm bank using rapd markers

Abstract: The Moringa (Moringa oleifera Lam.) is a tree species with the known value of food, medicine and water treatment, and is promising for bioenergy use. Our objective was to evaluate, using RAPD markers, the genetic diversity of sixteen accessions from Germplasm Bank (BAG) of Embrapa Coastal Tablelands, Sergipe, Brazil. We estimate the diversity indices, and genetic similarity between accessions. The Shannon index and the index of genetic diversity (H) were 0.33 and 0.22, respectively. We observed by Jaccard similarity that, the accessions MO1 and MO12 are the most similar (0.27), and MO13 and MO16 are the most divergent (0.69). By UPGMA and PCoA groupings, we identified that MO1, MO2, MO12 and MO13 are genetically isolated. The results are important in designing strategies for conservation, but because of low diversity detected in this investigation, new activities of collection should be realized, with integration and characterization of new accessions, to ensure the ever increasing diversity of the collection.