Prakash G. Patil

A protocol for high-quality genomic DNA extraction from legumes.

Current DNA extraction protocols, which require liquid nitrogen, lyophilization and considerable infrastructure in terms of instrumentation, often impede the application of biotechnological tools in less researched crops in laboratories in developing countries. We modified and optimized the existing CTAB method for plant genomic DNA extraction by avoiding liquid nitrogen usage and lyophilization. DNA was extracted directly from freshly harvested leaves ground in pre-heated CTAB buffer. Chloroform:isoamyl alcohol (24:1) and RNase treatments followed by single-purification step decontaminated the samples thereby paving way for selective extraction of DNA. High molecular weight DNA yield in the range of 328 to 4776 ng/μL with an average of 1459 ng/μL was obtained from 45 samples of cultivated and wild Cajanus species. With an absorbance ratio at 260 to 280 nm, a range of 1.66 to 2.20, and a mean of 1.85, very low levels of protein and polysaccharide contamination were recorded. Forty samples can be extracted daily at a cost between 1.8 and US

New Hypervariable SSR Markers for Diversity Analysis, Hybrid Purity Testing and Trait Mapping in Pigeonpea [Cajanus cajan (L.) Millspaugh]

2.0 per plant sample. This modified method is suitable for most plants especially members of the Leguminosae. Apart from Cajanus, it has been extensively applied in DNA extraction from Cicer and Vigna species.

Novel genic microsatellite markers from Cajanus scarabaeoides and their comparative efficiency in revealing genetic diversity in pigeonpea

Draft genome sequence in pigeonpea offers unprecedented opportunities for genomics assisted crop improvement via enabling access to genome-wide genetic markers. In the present study, 421 hypervariable simple sequence repeat (SSR) markers from the pigeonpea genome were screened on a panel of eight pigeonpea genotypes yielding marker validation and polymorphism percentages of 95.24 and 54.11%, respectively. The SSR marker assay uncovered a total of 570 alleles with three as an average number of alleles per marker. Similarly, the mean values for gene diversity and PIC were 0.44 and 0.37, respectively. The number of polymorphic markers ranged from 39 to 89 for different parental combinations. Further, 60 of these SSRs were assayed on 94 genotypes, and model based clustering using STRUCTURE resulted in the identification of the two subpopulations (K = 2). This remained in close agreement with the clustering patterns inferred from genetic distance (GD)-based approaches i.e., dendrogram, factorial and principal coordinate analysis (PCoA). The AMOVA accounted majority of the genetic variation within groups (89%) in comparison to the variation existing between the groups (11%). A subset of these markers was implicated for hybrid purity testing. We also demonstrated utility of these SSR markers in trait mapping through association and bi-parental linkage analyses. The general linear (GLM) and mixed linear (MLM) models both detected a single SSR marker (CcGM03681) with R2 = 16.4 as associated with the resistance to Fusarium wilt variant 2. Similarly, by using SSR data in a segregating backcross population, the corresponding restorer-of-fertility (Rf) locus was putatively mapped at 39 cM with the marker CcGM08896. However, The marker-trait associations (MTAs) detected here represent a very preliminary type and hence demand deeper investigations for conclusive evidence. Given their ability to reveal polymorphism in simple agarose gels, the hypervariable SSRs are valuable genomic resource for pigeonpea research community, particularly in South Asia and East Africa where pigeonpea is primarily grown.

Characterization of Ty1/copia-like retrotransposon families from pigeonpea genome.

Paucity of molecular markers is hindering molecular breeding programmes for genetic improvement in pigeonpea, which is considered to be among the richest source of dietary protein in Asia and Africa. At the time of the start of this study, only 156 microsatellite markers were available in pigeonpea (Burns et al. 2001; Odeny et al. 2007, 2009). Recently with the publication of draft genome sequence and deep transcriptome studies, the stage has been set to enrich genomic resources to aid molecular breeding in pigeonpea (Dutta et al. 2011; Singh et al. 2012; Varshney et al. 2012). Genic microsatellites or EST-SSRs (simple sequence repeats) derived from expressed sequence tags (ESTs) are useful because these are inexpensive to develop, represent transcribed genes, and often a putative function can be assigned to them. Compared with genomic sequences, genic SSRs have several advantages as genetic markers. First, if an EST marker is found to be genetically associated with a trait of interest, it may represent the gene affecting the trait directly (Chen et al. 2001; Thiel et al. 2003). Therefore, EST-derived markers can provide opportunities for gene discovery and enhance the role of genetic markers by assaying variation in transcribed and known-function genes. Second, EST-derived

Identification, Characterization, and Phylogenetic analysis of Pigeon pea (Cajanus cajan L. Mill sp.) Resistance Gene Analogs using PCR cloning and in silico methods

Retrotransposons contribute significantly to the size, organization, and genetic diversity of their host genomes. To characterize novel retrotransposon families in pigeonpea and develop retrotransposon-based sequence-specific amplification polymorphic markers, in silico homology sequence search was carried out against the whole genome shotgun sequence of pigeonpea variety Asha (ICPL87119). For homology searching, 5 copia-like retro elements belonging to soybean, common bean, mungbean, chickpea, and field pea were used as query sequences. Contigs with at least 80% query coverage and >70% similarity were searched for retroelements using the long terminal repeat finder. A total of 28 copia-like retroelements were identified using this method. Multiple sequence alignment for the reverse transcriptase domain indicated conserved reverse transcriptase domains in all 28 elements compared with other reported elements. Phylogenetic analysis based on reverse transcriptase domains revealed 11 families. The copy number per family ranged from 1 (for B, J, and K family) to 8 (I). The sequence-specific amplification polymorphic marker-based insertion site profiling for one of the retrotransposon families (G) confirmed multiple insertions of this element across the pigeonpea genome. This study showed that our in silico homology search strategy was efficient for identifying and characterizing the Ty1/copia-like retrotransposon. The results of this study are useful for developing retrotransposon-based sequence-specific amplification polymorphic markers for pigeonpea crop improvement.

Comparative efficiency of SRAP, SSR and AFLP-RGA markers in resolving genetic diversity in pigeon pea ( Cajanus sp.)

Pigeon Pea (Cajanus cajan), an important grain legume, is susceptible to Fusarium wilt (FW), sterility mosaic disease (SMD), and Phytophthora blight. Identification of resistance gene analogs (RGAs) is important for development of resistant varieties. In this study, degenerate primers targeting nucleotide binding sites (NBS) of known resistance (R) genes were used to amplify RGAs from two Pigeon Pea genotypes with differing disease resistance profiles. The translated cloned RGAs had high amino acid identity (68–71%) with putative disease resistance proteins in Glycine Max. Five RGA open reading frames were found in the whole Pigeon Pea genome after BLASTN analysis with the cloned sequences. Translated RGA proteins contained several characteristic features such as the NB-ARC domain (characteristic of death-related disease resistance genes) and four NBS motifs. A tryptophan residue at the kinase-2 motif was indicative of the non-TIR-NBS class of proteins. Phylogenetic analysis revealed two major clusters. The seven Pigeon Pea RGAs were in a non-TIR group alongside wilt resistance proteins from tomato. Specific primers were designed against the RGAs identified by BLASTN, and these successfully amplified sequences from all eight Pigeon Pea genotypes. The 40 resultant sequences were combined according to genotype and subjected to phylogenetic analysis. Genotypes clustered according to breeding pedigree. Multiple alignments of the 40 sequences revealed several single nucleotide polymorphisms (SNPs) that are useful in identifying candidate resistance genes associated with FW and SMD.