Development and validation of novel EST-SSR markers in black pepper
Loading...
Date
2017
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Department of Plant Breeding and Genetics, College of Agriculture, Padannakkad
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.
Description
PG
Keywords
null