Highly variable ‘Arhar’ simple sequence repeat markers for molecular diversity and phylogenetic studies in pigeonpea [Cajanus cajan (L.) Millisp.]

Abstract

Pigeonpea is an important tropical grain legume widely cultivated in South and South‐East Asia for versatile end usage as food, feed, fodder and fuel. Recent publication of draft genome sequence of pigeonpea (‘Arhar’) has allowed mining of large numbers of genomic simple sequence repeat (SSR) markers most of which are either not validated or show very little polymorphism in simple agarose gel–based assays. Here, we describe a special category of 370 validated highly variable ‘Arhar’ genomic SSR (HASSR) markers, which provide much higher level of polymorphism than a random set of SSR markers. These markers were validated for consistent amplification in a set of eight pigeonpea varieties and 152 of these (41%) showed allelic polymorphism on agarose gel electrophoresis. Twenty‐four highest polymorphic HASSR markers were used on a larger set of forty pigeonpea varieties and eight wild Cajanus species for the analysis of genetic diversity and phylogenetic relationship. Genomic HASSR markers described here are highly suitable for genetic diversity and phylogenic studies in pigeonpea.

Leaf Transcriptome Sequencing for Identifying Genic-SSR Markers and SNP Heterozygosity in Crossbred Mango Variety ‘Amrapali’ (Mangifera indica L.)

“…A total of 5,465 SSR loci of different categories (mono, di, tri, tetra, penta, hexa and complex) were identified in 4,912 contigs, representing 7.01% of the total 70,057 unigenes. We excluded mononucleotide repeats, complex SSR and those having total lengths of <10 bp because SSR marker based mono-nucleotide repeats are not reproducible due to recombination slippage and PCR amplification problems, whereas complex SSRs show the least polymorphism [ 39 ]. This exclusion left only 1,481 Type I SSRs for primer design.…”

“…The most abundant SSRs were with five reiterations, the frequency of a given SSR structure and the number of repeat units in it showed an inverse relationship ( S2 Fig ). Hence, SSR loci with less than five repeats are expected to be even more abundant but were not included in the present investigation because they would be less useful in the study of detectable polymorphism [ 39 ]. SSR motifs showing more than ten reiterations were rare with a frequency range of 1.28%–0.06%.…”

“…The SSR loci containing repeat units of 2–6 nucleotides only were considered. The criteria for minimum SSR length were defined as 6 reiterations for di-nucleotide SSR and 5 reiterations for tri-, tetra-, penta- and hexa-nucleotide SSRs, mono-nucleotide repeats and complex SSR were excluded [ 39 ]. The parameters for designing of primers from SSR flanking sequence were: primer lengths = 20–25 bp; PCR product size = 100–250 bp; annealing temperature = 65°C; GC content = 40–60% with an optimum of 50%; only single consecutive bases of Gs and Cs at the 3’ end of both primers were specified.…”

Novel chloroplast microsatellite markers in pigeonpea (Cajanus cajan L. Millsp.) and their transferability to wild Cajanus species

“…The three major pigeonpea producing regions are the Indian sub-continent, central-America and eastern Africa. It is well known as a protein-rich crop with 21% protein content in the seeds along with the presence of various vital amino acids (Dutta et al, 2013). Pigeonpea is globally cultivated on 5.32 Mha land area with an overall annual production of 4.32 Mt (FAO 2013).…”

PineElm_SSRdb: a microsatellite marker database identified from genomic, chloroplast, mitochondrial and EST sequences of pineapple (Ananas comosus (L.) Merrill)

“…[6]. Among all the markers till date, Simple Sequence Repeats (SSRs) are the most ideal, powerful and reliable markers for molecular plant breeding applications because of their high abundance, co-dominant inheritance and multiple alleles [7]. In addition, BES-SSR markers serve a useful resource for integrating genetic and physical maps [8,9].…”