Madan Singh Negi

Assessment of Genetic Diversity of Biodiesel Species Pongamia pinnata Accessions using AFLP and Three Endonuclease-AFLP

Efficacy of two dominant molecular markers, namely, amplified fragment length polymorphism (AFLP) and three endonuclease (TE)-AFLP, were assessed in 20 individuals of the biodiesel species Pongamia pinnata. Four primer combinations generated a total of 254 and 194 bands in AFLP and TE-AFLP, respectively. Both techniques could unequivocally identify each accession used in this study. The Jaccard’s similarity coefficient ranged from 0.30 to 0.90 for AFLP and from 0.25 to 0.85 for TE-AFLP. The correlation coefficient between AFLP and TE-AFLP dendrogram was 0.56 which was low but significant (P < 0.001). Values of effective multiplex ratio, marker index, and resolving power were markedly higher in AFLP than in TE-AFLP. However, the band intensities across different lanes were uniform in TE-AFLP leading to easy and accurate scoring of gels which resulted in slightly higher bootstrap values with TE-AFLP data as compared to AFLP data. Inferences based on TE-AFLP data had similar level of biological relevance as compared to AFLP data when location and diameter of trees were taken in to consideration. However, the easy scorability of TE-AFLP profiles is extremely important and especially desirable in studies requiring genotyping of large number of individuals distributed across many gels.

Isolation and characterization of a first set of nine polymorphic microsatellite loci in Pongamia pinnata (Fabaceae)

Pongamia pinnata (L.) Pierre, is nonedible oil producing tree legume, which has been recognized as a major biodiesel species in India (Tewari 2003). P. pinnata (synonym Millettia pinnata) is indigenous to India and Southeast Asia from where it has spread to other parts of the world. Its seed oil content ranges from 24–40% which is at par with other biodiesel species such as Jatropha. However, availability of any improved and characterized planting stock has been the major bottleneck in harnessing the biofuel potential of this plant. A large proportion of existing trees do not flower at all and commercially attractive levels of fruiting are observed in only a small fraction of trees. However, there is a large phenotypic diversity in this species, thus providing an opportunity for genetic improvement (Kaushik et al. 2007). More recently, initiatives have been taken towards identification of superior genotypes and their characterization. Characterization of genetic diversity is a prerequisite for efficient conservation and utilization of genetic resources. A number of studies have been conducted on Pongamia using dominant markers such as RAPD, ISSR, AFLP and TEAFLP (Kesari et al. 2010; Sahoo et al. 2010; Sharma et al. 2011). However, the information content of these dominant markers is less due to presence of only two allelic states. Codominant markers such as microsatellites can display high number of allelic states thus providing higher information per data point. However, these markers need to be developed in species of interest through generating sequence information and primer development. To best of our knowledge, no microsatellite markers have been reported in this species till date. The aim of the present study was to isolate and characterize polymorphic microsatellite markers as to facilitate genetic diversity, clonal identification and conservation in P. pinnata germplasm.

First identification of core accessions of Jatropha curcas from India based on molecular genetic diversity

We report on identification of core collection from 192 accessions collected from 12 Indian states and five other countries based on 109 polymorphic amplified fragment length polymorphism (AFLP) markers. Pairwise Jaccards similarity coefficient for accessions varied from 0.25 to 1 with a maximum genetic distance of 0.75 obtained between accessions Jc428 (from Mexico) and J204 (from Madurai, Tamil Nadu). Both UPGMA (Unweighted Pair Group Method of Arithmetic Averages) clustering and principal coordinate analyses showed similar grouping of accessions in three major clusters in which Mexican accessions clustered separately from Indian, Chinese and African accessions. Results obtained from analysis of molecular variance indicated that 59% of the genetic variation was distributed among the populations, while 41% of variation was within the populations. A total of 16 (8.3% of the entire collection) core accessions were identified, which contained the entire allelic diversity of 192 accessions with respect to the sampled AFLP loci. The core accessions would be highly useful for future genetic improvement of Jatropha . To the best of our knowledge, this is the first report on identification of core accessions in Jatropha .

Development of Novel Microsatellite Markers in Jatropha curcas and Evaluation of Their Cross-Species Transferability

Jatropha curcas has been a species of interest all over the world due to its potential as a renewable biodiesel crop. In the present paper, a set of 40 novel genomic microsatellites from Jatropha developed through cloning and sequencing are reported. The microsatellite markers were evaluated at intra-specific, inter-specific and inter-generic levels. All forty markers successfully amplified fragments from J. curcas and J. integerrima showing their potential in linkage mapping using inter-specific breeding populations involving these species. The average number of alleles within 12 core accessions of J. curcas was 3.083. Polymorphic information content ranged from 0.15 to 0.80 within J. curcas accessions and from 0 to 0.37 among different genera. There was no significant deviation from Hardy–Weinberg equilibrium or linkage disequilibrium. Ten of these microsatellites showed polymorphism within Emblica officinalis while thirteen were polymorphic within Phyllanthus niruri and Ricinus communis accessions. These new set of microsatellite markers are expected to be especially useful in future for linkage mapping in J. curcas and for varietal identification and genetic diversity studies in other genera mentioned above.

Rapid development and characterization of fifteen novel microsatellite loci in vulnerable tree aquilaria malaccensis using next generation ion torrent PGM™ sequencing

Aquilaria malaccensis is one of the 15 tree species of the genus Aquilaria widely distributed in the Indo-Malaysian region. Its resin (Agar or Gaharu) is widely known for its use in the production of highly valued incense and traditional medicine throughout Asia. The species has been listed as ‘Vulnerable’ in the IUCN red list of threatened species due to its over exploitation. In the present study, for the first time, next generation sequencing platform (Ion Torrent PGM™) was used to obtain a large number of sequences containing microsatellites for A. malaccensis. A total of 527, 425 high quality reads were identified, which contained 4138 SSRs. In these SSRs, the number of dinucleotide repeats were the most frequent one (74.77%). Primers were developed for 93 loci; of these 82 loci were successfully amplified and 47 were polymorphic across a panel of 42 genotypes. Fifteen loci with at least three or more alleles were chosen for detailed characterization. The observed heterozygosity ranged from 0.25 to 0.90, whereas the expected heterozygosity ranged from 0.24 to 0.89, with 5.9 alleles per locus on average. Simple matching dissimilarity coefficients and cluster analysis revealed significant diversity among the genotypes. The study suggested that PGM™ sequencing is a rapid technique for developing microsatellite markers. The microsatellite markers developed in the present study would be useful in comprehensive genetic diversity studies aimed towards the conservation and germplasm management of Aquilaria.

Changes in DNA methylation levels during seed development in Jatropha curcas

Jatropha curcas has attained significant economic importance during the last decade due to its potential as nonedible oil that can be used for production of biodiesel (Openshaw 2000). The species was presumably taken from Central America and Mexico to tropical parts of Africa and Asia by Portuguese seafarers (Heller 1996). The endosperm of the seed contains 24–40% of oil (Kaushik et al. 2007). Methylation of cytosine (5-methylcytosine), as part of epigenetic modification, regulates gene expression during developmental and stress stimuli (Reyna-Lopez et al. 1997). These changes are often heritable but do not alter the primary sequence of DNA. As a result, they are not detected with genotyping methods such as simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP) and single-nucleotide polymorphisms (SNPs). The methylation sensitive amplification polymorphism (MSAP) is a reliable, inexpensive and relatively simple method for the identification of genomewide putative changes in DNA cytosine methylation (Fulnecek and Kovarik 2014). MSAP has been used in Jatropha to investigate methylation levels in different accessions (Kanchanaketu et al. 2012; Mastan et al. 2014) and during salinity stress (Mastan et al. 2012). Most of the MSAP markers are inherited as epialleles following Mendelian segregation (Yi et al. 2010). However, so far, no study on methylation levels during seed development has been reported in J. curcas. In the present study, MSAP was used to investigate the changes in methylation levels during seed development and their possible involvement with processes related to oil biosynthesis.