Showing papers on "RAPD published in 2017"

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques.

Abstract: In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random aplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific targets is also mentioned. The fast progresses achieved during the last years in the DNA-based classification of this pathogen are described and discussed and specific primers designed for the different methods are listed, in order to provide a concise and useful tool to all the researchers working in the field.

Molecular Identification and Genetic Characterization of Macrophomina phaseolina Strains Causing Pathogenicity on Sunflower and Chickpea

Abstract: Macrophomina phaseolina is the most devastating pathogen which causes charcoal rot and root rot diseases in various economically important crops. Three strains M. phaseolina 1156, M. phaseolina 1160, and M. phaseolina PCMC/F1 were tested for their virulence on sunflower (Helianthus annuus L.) and chickpea (Cicer arietinum L.). The strains showed high virulence on both hosts with a disease score of 2 on chickpea and sunflower. The strains also increased the hydrogen per oxide (H2O2) content by 1.4- to 1.6-fold in root as well as shoot of chickpea and sunflower. A significant increase in antioxidant enzymes was observed in fungal infected plants which indicated prevalence of oxidative stress during pathogen propagation. The M. phaseolina strains also produced hydrolytic enzymes such as lipase, amylase, and protease with solubilization zone of 5-43 mm, 5-45 mm, and 12-35 mm, respectively. The M. phaseolina strains were identified by 18S rRNA and analyzed for genetic diversity by using random amplified polymorphic DNA (RAPD) markers. The findings based on RAPD markers and 18S rRNA sequence analysis clearly indicate genetic variation among the strains collected from different hosts. The genetically diverse strains were found to be pathogenic to sunflower and chickpea.

RAPD and ISSR marker assessment of genetic diversity in Citrullus colocynthis (L.) Schrad: a unique source of germplasm highly adapted to drought and high-temperature stress

Abstract: Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) shows high levels of variation in fruit color, fruit stripe pattern, seed coat color, and size. Thirty-eight accessions of C. colocynthis plants from different parts of semi-arid Rajasthan were collected and genetic diversity was assessed using random-amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 65 RAPD decamer primers, 50 primers produced 549 scorable bands of which 318 were polymorphic. Polymorphic banding patterns with the number of amplified fragments varied from 5 (OPA-08 and OPF-9) to 19 (OPT-20) in the molecular size range of 150–6000 bp. Percent polymorphism ranged from 22.2% (OPA-09) to 83.3% (OPE-12) with 55.14% polymorphism. Out of the 20 ISSR primers screened, 13 primers produced 166 amplification products, of which 99 were polymorphic. The number of bands amplified per primer varied between 9 (UBC-807, 802) and 16 (UBC-803, 812) with average band size between 250 and 4000 bp. Percent polymorphism ranged from 45.4% (UBC-815) to 73.3% (UBC-814) with 65.05% polymorphism. Dendrogram constructed on the basis of RAPD + ISSR polymorphism separated the accessions into four distinct clusters at 72% variation with Jaccard’s similarity coefficient ranging from minimum 0.64 to 0.95. The matrices for RAPD and ISSR were also compared using Mantel’s test and obtained correlation value (r = 0.7947). Discriminating power of RAPD and ISSR markers was assessed by calculating polymorphic information content, multiplex ratio, marker index, and resolving power. Approx. 50% RAPD and ISSR markers showed PIC value and heterozygosity (H) ≥ 0.50, indicating marker as informative. The primers that showed higher polymorphism had higher RP, MR, and MI values.

Plant regeneration from cell suspension culture in Saccharum officinarum L. and ascertaining of genetic fidelity through RAPD and ISSR markers

Abstract: The aim of this study was to produce sugarcane plantlets from cell suspension culture and study its genetic fidelity using molecular markers. The study was carried out using sugarcane varieties Co 86032 and Q117. Callus cultures of both the varieties were optimized using six different callus induction media. After screening the growth response of callus on six different callus induction media, it was observed that medium no. VI supplemented with 500 mg l−1 of each PVP, Casein hydrolysate and MES buffer showed high amounts of callus in Co 86032 (79.66 ± 0.44%) and Q117 (82.83 ± 1.69%). Addition of PEG 8000 at 2.5% to this medium had a profound impact on inducing somatic embryogenesis in Co 86032 (54.66 ± 1.76%) and Q117 (66.66 ± 2.60%) as compare to control (24.33 ± 1.76%) and (27.33 ± 2.73%), respectively. Cell suspension cultures were established by culturing embryogenic calli in liquid medium showed well established suspension cultures with fever cell aggregates. There was negligible cell division during initial 2 days of incubation and cell count increased rapidly between 2 and 8 days. Further incubation beyond 8 days resulted in a decrease in cell viability. Enhanced callus proliferation in Q117 while enhanced shoot regeneration in Co 86032 was observed from cell suspension culture. The clonal fidelity of in vitro regenerated plants was assessed by using RAPD and ISSR markers. Analysis of the ten RAPD markers indicated that 90.48 and 86.95% true-to-type regenerated plantlets in Co 86032 and Q117, respectively. However, in the ISSR markers, Co 86032 did not show any polymorphism and in the Q117, 92.18% true-to-type plantlets were found. These results confirmed that somaclonal variation occurs during the process of indirect organogenesis and RAPD and ISSR marker based molecular analysis is a suitable method for an early detection of variation in sugarcane.

In vitro clonal propagation and evaluation of genetic fidelity using RAPD and ISSR marker in micropropagated plants of Cassia alata L.: a potential medicinal plant

Abstract: Present study reports successful in vitro clonal propagation of a potential medicinal plant, Cassia alata using mature nodal explants. Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) of 6-benzyladenine (BA), kinetin and 2-isopentenyl adenine (2-iP) singly as well as in combination with different auxins, α-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) or Indole-3-acetic acid (IAA) (0.1, 0.5 and 1.0 μM) were used. MS medium enriched with 7.5 μM BA and 0.5 μM NAA yielded the highest regeneration frequency (92 %) with maximum multiple shoots (12.3 ± 0.6) and shoot length (4.7 ± 0.1 cm) after 12 weeks of culture. Shoots were rooted best on full MS containing 0.5 μM IBA. Ex vitro rooting of in vitro derived microshoots was also achieved in 150 μM IBA treatment for 20 min followed by transfer to thermocol cups containing sterile Soilrite™. About 85 % plantlets survived acclimatization procedure to the field. The genetic fidelity of in vitro regenerated plants was analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Of the 20 RAPD primers, 18 primers produced clear, reproducible and scorable bands while out of 13 ISSR primers screened, only ten generated well-defined and scorable bands in all the tested plants. A total of 69 and 71 bands were scored with an average of 3.8 and 7.1 bands per primer for RAPD and ISSR primers respectively. All banding profiles from micropropagated plants were monomorphic and similar to of the mother plant, thus confirming the true-to-type nature of the in vitro-raised clones.

Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments

Abstract: // Shangyi Fu 2, * , Jingliang Cheng 1, * , Chunli Wei 1, * , Luquan Yang 1 , Xiuli Xiao 3 , Dianzheng Zhang 4 , M. David Stewart 2, 5, 6 and Junjiang Fu 1, 7 1 Key Laboratory of Epigenetics and Oncology, the Research Center for Preclinical Medicine, Southwest Medical University, Luzhou, Sichuan 646000, China 2 Honors College, University of Houston, Houston, TX 77204, USA 3 Department of Pathology, Affiliated Hospital of Southwest Medical University, Southwest Medical University, Luzhou, Sichuan 646000, China 4 Department of Bio-Medical Sciences, Philadelphia College of Osteopathic Medicine, Philadelphia, PA 19131, USA 5 Department of Biology & Biochemistry, University of Houston, Houston, TX 77204, USA 6 Texas Heart Institute at St. Luke's Episcopal Hospital, Houston, TX 77030, USA 7 Judicial Authentication Center, Southwest Medical University, Luzhou, Sichuan 646000, China * These authors have contributed equally to this work Correspondence to: Junjiang Fu, email: fujunjiang@swmu.edu.cn , fujunjiang@hotmail.com Dianzheng Zhang, email: dianzhengzh@pcom.edu Keywords: high-GC primer, RAMP, random amplified polymorphic DNA (RAPD), sequence-characterized amplified region (SCAR), genomic instability Received: February 24, 2017 Accepted: March 19, 2017 Published: March 30, 2017 ABSTRACT Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas.

Genetic diversity of Lactobacillus plantarum strains from some indigenous fermented foods in Nigeria

Abstract: Lactobacillus plantarum has been found to be commonly associated with Nigerian indigenous fermented foods. The intraspecies differentiation of L. plantarum using different molecular techniques is essential for the selection of functional strains. In the present study, 48 L. plantarum isolates from some Nigerian indigenous fermented foods; (gari, fufu and ogi) were phenotypically characterized. The intraspecies diversity of 17 selected L. plantarum strains with good acidification rates, hydrogen peroxide production and variation in carbohydrate fermentation patterns were carried out using molecular techniques, 16S-23 rDNA intergenic transcribed spacer and restriction fragment length polymorphism (ITS-PCR and ITS-RFLP), randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). The ITS-RFLP-HaeIII, RAPD-OPA5, OPA20 and PFGE-Sfi1 analysis showed genetic diversity among the strains of L. plantarum isolated from the different fermented foods, and it can be established that these molecular tools are useful for differentiation of L. plantarum strains. The molecular techniques used in this study may be considered useful tools for characterization of isolates and for in-depth examination of the strain diversity as the various strains isolated in this study can be used as adjunct and/or starter cultures in food fermentation processes.

Phylogenetic analysis among some pome fruit trees of Rosaceae family using RAPD markers.

Abstract: To evaluate the phylogenetic relationships among subtribe Pyrinae of Rosaceae, 50 different genotypes and cultivars of pome fruit trees were collected from various locations in Iran, and analysed using random amplified polymorphic DNA (RAPD) markers. Altogether, 85 polymorphic fragments were produced by 11 random 10-mer primers. The number of produced bands varied from 4 to 12 for each primer, 7.72 on average. The lowest Jaccard's genetic similarity coefficient was scored between apple cv. ‘Akan’ and a wild Pyrus syriaca pear (0.04), and the highest similarity was observed between two genotypes of P. syriaca (0.97). Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) properly separated the accessions and divided them into three major groups, including Malus, Pyrus, Mespilus and Crataegus. Members of Mespilus and Crataegus were further separated and formed two subgroups. Analysis of the genetic structure, using STRUCTURE and phylogenetic relationship using TreeView ...

Comparative assessment of genetic diversity in Sesamum indicum L. using RAPD and SSR markers

Abstract: Sesame (Sesamum indicum L.) is an ancient oilseed crop known for its nutty seeds and high-quality edible oil. It is an unexplored crop with a great economic potential. The present study deals with assessment of genetic diversity in the crop. Twenty two RAPD and 18 SSR primers were used for analysis of the 47 different sesame accessions grown in different agroclimatic zones of India. A total of 256 bands were obtained with RAPD primers, of which 191 were polymorphic. SSR primers gave 64 DNA bands, of which all of were polymorphic. The Jaccard's similarity coefficient of RAPD, SSR, and pooled RAPD and SSR data ranged from 0.510 to 0.885, 0.167 to 0.867, and 0.505 to 0.853, respectively. Maximum polymorphic information content was reported with SSRs (0.194) compared to RAPDs (0.186). Higher marker index was observed with RAPDs (1.426) than with SSRs (0.621). Similarly, maximum resolving power was found with RAPD (4.012) primers than with SSRs (0.884). The RAPD primer RPI-B11 and SSR primer S16 were the most informative in terms of describing genetic variability among the varieties under study. At a molecular level, the seed coat colour was distinguishable by the presence and absence of a group of marker amplicon/s. White and brown seeded varieties clustered close to each other, while black seeded varieties remained distanced from the cluster. In the present study, we found higher variability in Sesamum indicum L. using RAPD and SSR markers and these could assist in DNA finger printing, conservation of germplasm, and crop improvement.

Reconstruction of molecular phylogeny of closely related Amorphophallus species of India using plastid DNA marker and fingerprinting approaches.

Abstract: Plastid DNA markers sequencing and DNA fingerprinting approaches were used and compared for resolving molecular phylogeny of closely related, previously unexplored Amorphophallus species of India. The utility of individual plastid markers namely rbcL, matK, trnH–psbA, trnLC–trnLD, their combined dataset and two fingerprinting techniques viz. RAPD and ISSR were tested for their efficacy to resolves Amorphophallus species into three sections specific clades namely Rhaphiophallus, Conophallus and Amorphophallus. In the present study, sequences of these four plastid DNA regions as well as RAPD and ISSR profiles of 16 Amorphophallus species together with six varieties of two species were generated and analyzed. Maximum likelihood and Bayesian Inference based construction of phylogenetic trees indicated that among the four plastid DNA regions tested individually and their combined dataset, rbcL was found best suited for resolving closely related Amorphophallus species into section specific clades. When analyzed individually, rbcL exhibited better discrimination ability than matK, trnH–psbA, trnLC–trnLD and combination of all four tested plastid markers. Among two fingerprinting techniques used, the resolution of Amorphophallus species using RAPD was better than ISSR and combination of RAPD +ISSR and in congruence with resolution based on rbcL.

RAPD analysis of Leuconostoc mesenteroides strains associated with vegetables and food products from Korea

Abstract: Lactic acid bacteria play a very important role in food fermentation. For strain identification, characterization and protection, it is important to use highly discriminatory identification methods. In the present study, 37 strains isolated from vegetables and food products of Korea were identified using 16S rDNA gene sequencing and were found to be Leuconostoc mesenteroides. Further, molecular characterization of all the strains was performed using two RAPD primers, i.e. 239 and KAY3. All strains have shown RAPD profiles and the amplified products of the profiles ranged from 300 to 4000 bp with both the primers and only small differences in banding pattern were observed. Out of 37, maximum bands were observed in strain 11436 with 239 and strain 11260 with KAY3. With both the primers, the phylogenetic analysis revealed seven clades which could be further subdivided into groups. The dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA). The results showed that 16S rDNA sequencing and RAPD-PCR are suitable preliminary molecular tools for identification and characterization of bacteria.

Assessment of genetic diversity in Costus pictus accessions based on RAPD and ISSR markers.

Abstract: Costus pictus, belonging to the family Costaceae, is one of the valuable medicinal plants with its anti-diabetic property. Despite ever-increasing demand from the pharmaceutical industry, this species is being less exploited at molecular level. Hence, an effort has been made in the present study to characterize the 15 accessions of C. pictus collected from different geographical regions of India through random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. A total of 25 RAPD and 20 ISSR primers were used in the present study. The RAPD analysis generated 343 loci, of which 124 were polymorphic with an average of 4.96 loci per primer. While, ISSR primers produced 177 loci, of which 77 were polymorphic with an average of 3.85 loci per primer. The similarity coefficients ranged from 0.86–0.99, 0.84–0.95 and 0.86–0.96 for RAPD, ISSR and combined RAPD-ISSR, respectively. The UPGMA dendrogram generated using these data showed low level of divergence among the accessions from South and West regions. Further, accession-specific bands were also revealed by RAPD and ISSR markers which might be contributed to specific trait. This investigation was an understanding of genetic variation within the C. pictus accessions. The present finding indicates that both the marker tools RAPD and ISSR combined or individually can be used in determining the genetic relationship between the accessions. It may be concluded that data of hereditary differences appeared among the C. pictus accessions could be utilized for their conservation and reproducing programs.

Cytogenetic and molecular evidences revealing genomic changes after autopolyploidization: a case study of synthetic autotetraploid Phlox drummondii Hook

Abstract: Polyploidy is known to be common in plants; indeed most of the world’s economically important crop plants are polyploids. Recent studies revealed extensive genomic changes in synthetic polyploids after genome doubling, although most of the information available is with regards to allopolyploids and little information have been generated in autopolyploids. In the present study, we used Phlox drummondii Hooker (2n = 2x = 14) as a model plant to observe genomic changes, if any, in synthetic autopolyploids. Colchitetraploids were produced and followed through different generations (C0, C1, C2 and C3). Male meiosis analysis showed differences between the frequency of both quadrivalents and bivalents from C0 to C2 generations. RAPD analysis revealed 2.8, 1.6, 2.1 and 3.2% polymorphism in C0, C1, C2 and C3 colchitetraploids respectively. The polymorphic fragments were further characterized after cloning. Dot blot assay was performed to confirm high copy/low copy nature of fragments showing variation. The analysis revealed changes in both repetitive and non-repetitive regions. Out of the six fragments only one fragment T01 was found to be of high copy, while four fragments were of the moderate copy and one fragment of the low copy nature.

Molecular identification of bamboo genera and species based on RAPD-RFLP markers

Abstract: Bamboo species have a very significant ecological and economic impact. Determining morphological and genetic differences among bamboo genera and species are crucial to explore desirable traits for breeding purposes. Several advances have been made in the taxonomy of bamboos by using molecular fingerprinting tools and next generation sequencing technologies. Nevertheless, classical molecular markers such as RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism) and ISSR (Inter Simple Sequence Repeats) also provide an accurate discrimination among genera and species. Moreover, the RAPD-RFLP (Random Amplified Polymorphic DNA, Restriction Fragment Length Polymorphism) method, in which amplification products from RAPD are digested with restriction enzymes, is a reliable, fast and cost-effective method for fingerprinting. RAPD-RFLP has been scarcely used in the literature and no report regarding bamboo taxonomy is available with this method. Here we explored the molecular (RAPD, RAPD-RFLP) variation among genera (Bambusa, Dendrocalamus, Guadua and Phyllostachys) and species of bamboo cultivated in Brazil. Both molecular markers allowed clear distinction among the genera studied. Moreover, high cophenetic correlation values in UPGMA clusters indicated their potential for discriminating bamboo species. The digestion of RAPD products (RFLP) resulted in high number of polymorphic bands and produced very characteristic profiles for each genus with three enzyme combinations (HindIII/HaeIII, HinfI/RsaI, and single digestion with MspI). We recommend RAPD-RFLP as a reproducible and informative method for screening differences among genera, species and varieties of bamboos. Providing a cost-effective and accurate method for species identification and characterization is straightforward for bamboo conservation, management and breeding.

Assessment of genetic variation among nineteen turmeric cultivars of Northeast India: nuclear DNA content and molecular marker approach

Abstract: Turmeric (Curcuma longa L.) is a domesticated cultivar belonging to the family Zingiberaceae and is widely used as spice, medicine and cosmetics. In the current paper, the genetic structure of nineteen commercial cultivars of turmeric of northeast (NE) India was assessed by molecular markers and their genome size. The average polymorphism, polymorphic information content, marker index were found to be 98.14%, 0.34, 33.66, and 86.48%, 0.22, 19.57, for RAPD- and ISSR-based markers, respectively. Based on RAPD and ISSR markers, at the inter-population level, effective number of alleles, Nei’s gene diversity and Shannon’s information index values were 1.46, 0.28, 0.44 and 1.50, 0.28, 0.41, respectively. As inferred using flow cytometry, the genome size varied between 2.59 ± 0.03 pg (cultivar of Assam) to 2.95 ± 0.04 pg (cultivar of Meghalaya) with a 1.14-fold variation. Terminal positioning of higher genome size containing turmeric cultivars on the dendrogram represents geographical isolation leading to evolutionary younger origin.

Assessing Date Palm Genetic Diversity Using Different Molecular Markers.

Abstract: Molecular marker technologies which rely on DNA analysis provide powerful tools to assess biodiversity at different levels, i.e., among and within species. A range of different molecular marker techniques have been developed and extensively applied for detecting variability in date palm at the DNA level. Recently, the employment of gene-targeting molecular marker approaches to study biodiversity and genetic variations in many plant species has increased the attention of researchers interested in date palm to carry out phylogenetic studies using these novel marker systems. Molecular markers are good indicators of genetic distances among accessions, because DNA-based markers are neutral in the face of selection. Here we describe the employment of multidisciplinary molecular marker approaches: amplified fragment length polymorphism (AFLP), start codon targeted (SCoT) polymorphism, conserved DNA-derived polymorphism (CDDP), intron-targeted amplified polymorphism (ITAP), simple sequence repeats (SSR), and random amplified polymorphic DNA (RAPD) to assess genetic diversity in date palm.

Construction of a genetic linkage map based on RAPD, AFLP, and SSR markers for tea plant (Camellia sinensis)

Abstract: Based on double pseudo-testcross theory, a population of 79 F1 clones, which was derived from a crossing of two tea cultivars using a Japanese tea cultivar “Fushun” (Camellia sinensis) as female parent and a Korean tea cultivar “Kemsull” (C. sinensis) as male parent, was applied to construct a genetic linkage map with different molecular markers. Previously, 143 random amplified polymorphism DNA (RAPD) markers and 11 simple sequence repeat (SSR) loci developed from 41 decamer random primers and 60 published SSR primer pairs, respectively, were used into our mapping analysis. In the study of amplified fragment length polymorphism (AFLP), 2439 bands were generated from 27 primer combinations with an average number of 90.3 bands for each, of which 495 (20.3%) were polymorphic. The majority of those polymorphic markers segregated in accordance with Mendelian segregation ratios at p < 0.01 for 400 (80.8%) or at p < 0.05 for 382 (77.2%), of which 136 (34.0%) or 131 (34.3%) were at 3:1 segregation ratios and 264 (66.0%) or 251 (65.7%) were at 1:1 segregation ratios at p < 0.01 and p < 0.05, respectively. For developing more SSR markers, the transcriptome sequences of flowers and leaves of two parents were obtained using high throughput RNA sequencing and compared. Afterwards, 1800 potential polymorphic SSR markers were successfully developed and 296 of them were selected and experimentally validated with a subset of tea plants (including two parents and six F1 offspring), from which 75 (25.3%) were repeatably amplified and polymorphic between two parents. From that, 29 (38.7%) newly mined SSR markers were heterozygous in “Fushun” and/or “Kemsull” and showed segregant genotypes in F1 seedlings and were adoptable by JoinMap 4.0. Totally, 678 markers including 143 RAPDs, 11 public SSRs, 495 AFLPs, and 29 newly mined SSRs were conjointly used to construct a combined linkage map for tea plant. The new genetic map located 79 RAPDs, 5 public SSRs, 214 AFLPs, and 11 new SSRs developed from RNA seq technique and covered 1441.6 cM with an average distance of 4.7 cM between two adjacent markers. This map will lay a foundation for qualitative or quantitative trait loci (QTLs) analysis of important agronomic traits for tea plant in the future study.

Genetic diversity of sugarcane hybrid cultivars by RAPD markers

Abstract: Genetic diversity among sugarcane hybrids (Saccharum spp) is pre-requisite for sugarcane improvement through breeding. Twelve decamer oligonucleotide random-amplified polymorphic DNA (RAPD) markers were utilized to investigate the genetic potential among 24 sugarcane cultivars. A total of 120 fragments were originated by 12 RAPD primers. An average number of fragments were obtained as 11.42 fragments per cultivar, which ranged from 4 to 21 fragments. The genetic similarity among 24 sugarcane cultivars ranged from 0.236 to 0.944 with the mean similarity value of 0.508. On the basis of phylogenetic analysis based on dendrogram, the cultivars were clustered into five groups. Two varieties Co 0118 and CoS 07250 were found as highly diverse sugarcane cultivars. Three most popular cultivars viz, Co 0238, Co 1158, and CoS 08272 were clustered a diverse among particular group. These clusters with their diverse genealogy indicated the influence of parental genome contribution to clustering. Diverse varieties developed for east region were grouped in the separate clusters which indicated the influence of adaptation of varieties to particular agro-climatic condition. Hence, these five diverse hybrid cultivars would be used in further breeding program to get the prominent sugarcane clones which may produced higher cane yield and sugar content.