Development and validation of novel EST-SSR markers in black pepper
| dc.contributor.advisor | Sujatha, R | |
| dc.contributor.author | Sherin Jose | |
| dc.contributor.author | KAU | |
| dc.date.accessioned | 2020-02-07T09:00:38Z | |
| dc.date.available | 2020-02-07T09:00:38Z | |
| dc.date.issued | 2017 | |
| dc.description | PG | en_US |
| dc.description.abstract | The study entitled ―Development and validation of novel EST-SSR markers in black pepper (Piper nigrum L.)‖ was carried out at College of Agriculture, Padannakkad, Kasaragod, Kerala during 2015-2017. The main objectives of the study were to develop EST-SSR markers in black pepper, validation of the developed EST-SSR markers in different genotypes of black pepper and to study the genetic relationship among different species of Piper. Different genotypes (53 numbers) including nineteen varieties and sixteen cultivars of black pepper and eighteen different species of Piper were included in the study. The plant materials were collected from Hi-Tech black pepper nursery in College of Agriculture, Padannakad; Indian Institute of Spices Research (ICAR- IISR), Kozhikode; the Experimental farm of IISR, Peruvannamuzhi and Pepper Research Station, Panniyur. Simple sequence repeats (SSR) were mined from the expressed sequence tags (EST) obtained in a previous study about the genes expressed during berry development stage of black pepper. The microsatellite identification tool, MISA was used for detecting SSRs from 1048 unigenes having a total size of 518179 bp and the results were confirmed using another microsatellite identification tool, GMATo. Seventy SSRs were detected from 68 unigene sequences which accounts for 6.49 per cent of total sequences. There were 62 mononucleotides (88.6 per cent), two dinucleotides (2.8 per cent) and six trinucleotides (8.6 per cent) identified from the sequences. Ten microsatellites including six trinucleotides, two dinucleotides and two compound SSRs were selected and primers were designed based on the corresponding unigene sequences to amplify these regions in the genomic DNA. Nine primers (which consist of forward and reverse primer) were designed, viz., PNS1, PNS2, PNS3, PNS4a, PNS4b, PNS5, PNS6, PNS7 and PNS8. Primers were designed for eight repeat motifs including six trinucleotide repeats, one dinucleotide repeat and one compound repeat. The developed primers were screened using genomic DNA isolated from black pepper variety Panniyur-1. Annealing temperature of the primers was standardized through gradient PCR. Based on the specificity of the amplification, five primers (PNS1, PNS3, PNS4a, PNS6 and PNS8) were selected for further validation. Genomic DNA was isolated from the 53 genotypes of Piper spp. and amplified with the selected primers for validation. In total, 34 alleles were obtained for the five loci amplified by the five primer sets. The average number of alleles per locus was 6.8. Polymorphism Information Content (PIC) value of different primers was calculated based on the number of polymorphic bands obtained using each primer set. All the five primers were successful in amplifying the corresponding locus in P. nigrum genotypes as well as in other Piper spp. Among the 34 alleles, seven were found only in P. nigrum, 16 were specific to other Piper spp. and 11 were shared by both groups. PIC value was in the range of 0.16 to 0.83. The maximum PIC value was given by PNS6 with 10 alleles bringing out the difference between all the species and minimum was shown by PNS8. For cluster analysis using the software DARwin version 6, scoring was carried out based on the presence (1) or absence (0) of the alleles in each genotype using each primer. Dissimilarity between the genotypes was found out using dice matrix. Dissimilarity values were in the range of 0.08 to 0.87. The genotypes were grouped into different clusters based on the similarity between genotypes. Different Piper species were present in two clusters, Cluster II with seven species and Cluster III with eleven species. In Cluster III-B, one species, Piper sp. (North East Fragrance) was present along with six black pepper cultivars indicating that this may be a closely related species of P. nigrum. Some of the parental type and corresponding varieties derived from them (Aimpiriyan and Panchami, Balankotta and Panniyur-2) were present in single cluster which indicates the genetic similarity between these genotypes. Allelic size of 400 bp obtained using EST-SSR marker, PNS 1 was unique to Piper sp. (North East). The marker PNS 3 also showed a unique allele in Piper sp. (North East) with two alleles having a size of 230 bp and 210 bp. Allele with a size of 180 bp was given by Piper sp. (Anand) using PNS 3 EST-SSR marker. Allelic size of 400 bp, 320 bp and 300 bp was unique to Piper chaba, Piper ornatum and Piper betle using PNS 6 marker. The markers developed will be linked with a gene, as they are derived from the expressed region of the genome. In the present study also the limited number markers could bring out preliminary information about the existing genetic diversity between certain groups which was reported earlier using other molecular markers such as ISSR and RAPD. However this cannot be generalized, as further information is needed based on more number of SSR markers. | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810142693 | |
| dc.keywords | Black pepper, DNA marker, SSR, Species, Protein markers, Morphological markers | en_US |
| dc.language.iso | en | en_US |
| dc.pages | 80 | en_US |
| dc.publisher | Department of Plant Breeding and Genetics, College of Agriculture, Padannakkad | en_US |
| dc.sub | Plant Breeding | en_US |
| dc.subject | null | en_US |
| dc.theme | novel EST-SSR markers in black pepper | en_US |
| dc.these.type | M.Sc | en_US |
| dc.title | Development and validation of novel EST-SSR markers in black pepper | en_US |
| dc.type | Thesis | en_US |