Molecular marker-based characterization in candidate plus trees of Pongamia pinnata, a potential biodiesel legume

Vigya Kesari 1 Vinod Madurai Sathyanarayana 0 Ajay Parida 0 Latha Rangan 1 Background aims 0 M S Swaminathan Research Foundation , 3rd Cross Street, Institutional Area , Taramani, Chennai 600113, India 1 Department of Biotechnology, Indian Institute of Technology , Guwahati, Assam 781039, India Pongamia pinnata, a legume tree, has many traditional uses and is a potential biodiesel plant. Despite its importance and the availability of appropriate molecular genetic tools, the full potential of Pongamia is yet to be realized. The objective of this study was to assess genetic diversity among 10 systematically characterized candidate plus trees (CPTs) of P. pinnata from North Guwahati. The application and informativeness of polymerase chain reaction-based molecular markers [random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP)] to assess the genetic variability and relatedness among 10 CPTs of P. pinnata were investigated. Polymorphism rates of 10.48, 10.08 and 100 % were achieved using 18 RAPD, 12 ISSR and 4 AFLP primer combinations, respectively. Polymorphic information content (PIC) varied in the range 0.33 - 0.49, 0.18 - 0.49 and 0.26 - 0.34 for RAPD, ISSR and AFLP markers, respectively, whereas the corresponding average marker index (MI) values for the above markers were 7.48, 6.69 and 30.75. Based on Nei's gene diversity and Shannon's information index, interpopulation diversity (hsp) was highest when compared with intra-population diversity (hpop) and the gene flow (Nm) ranged from a moderate value of 0.607 to a high value of 6.287 for the three DNA markers. Clustering of individuals was not similar when RAPD- and ISSRderived dendrogram analyses were compared with that of AFLP. The Mantel test cophenetic correlation coefficient was higher for AFLP (r 0.98) than for ISSR (r 0.73) and RAPD (r 0.84). Molecular markers discriminated the individuals efficiently and generated a high similarity in dendrogram topologies derived using unweighted pair-group arithmetic average, although some differences were observed. The three-dimensional scaling by principal coordinate analysis supported the result of clustering. Comparing the results obtained with the three DNA markers, AFLP indicated higher efficiency for estimating the levels of genetic diversity and proved to be reliable for fingerprinting, mapping and diversity studies in Pongamia in view of their suitability for energy production purposes. Methodology Principal results Conclusions Introduction Pongamia pinnata also referred to as Pongam, is a biodiesel tree legume well adapted to arid zones and has many traditional uses (Meera et al. 2003; Brijesh et al. 2006; Scott et al. 2008; Mukta et al. 2009). The pongam tree has the rare property of producing seeds with high oil content (Sarma et al. 2005; Kesari et al. 2010). Despite the importance of this versatile plant and the availability of appropriate molecular genetic tools, the full potential of Pongamia is far from being realized. The natural constraints that limit its large-scale production and availability, to meet the demand for biodiesel production, are its long gestation period (4 7 years), plant height, seed storage behaviour, insect pests, and the seed oil yield and quality (NOVOD 2010). Until recently, the identification of elite individuals of P. pinnata was mainly described in terms of morphological and agronomic traits which are known to be deeply influenced by environmental factors (Kaushik et al. 2007; Kesari et al. 2008). The cross-pollinating nature of P. pinnata contributes to its wide germplasm biodiversity. Thus, it becomes an important step to examine the genetic variations among naturally growing elite individuals of P. pinnata at inter- and intra-population levels, and to prepare strategies for its specific exploitation by plant breeders in promoting it as a versatile biodiesel plant. With the large array of molecular analytical techniques available, it has become possible to provide an accurate and unambiguous tool for the evaluation of genetic diversity and identification of germplasm (Meudt and Clarke 2006; Simmons et al. 2007; Li et al. 2008). In the last decade, several polymerase chain reaction (PCR)-based DNA marker classes, viz. random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP), contributed to this goal, and may extend and complement the assessment of genetic variation within the species based on morphological and polypeptide descriptions, providing more speed, accurate and detailed information (Williams et al. 1990; Zabeau and Vos 1993; Zietkiewicz et al. 1994; Surekha and Larson 2005). In addition, it is independent of the growth, stage, season, location and agronomic practices, which gives them a high value (Lombard et al. 2001). RAPD markers have been widely used in plant research for phylogenetic studies, genome mapping and genetic variation analysis both at intra- and inter-population levels (Li et al. 2008). The technique has several advantages including simplicity, low cost, speed and lack of requirement for DNA sequence information (Williams et al. 1990; Lopes et al. 2007). However, RAPD technology has several limitations including dominance, uncertain locus homology, sensitivity and reliability. Inter-simple sequence repeat represents the marker of choice for varietal identification studies as they are transferable, hypervariable, highly polymorphic, multiallelic dominant markers, relatively simple to interpret and show high information content (Souframanien and Gopalkrishna 2004). The AFLP technique has been used for DNA fingerprinting in a number of tree species such as almond (Sorkheh et al. 2007), cashew (Archak et al. 2003) and Jatropha (Tatikonda et al. 2009). The choice of primer pairs used in AFLP amplification is critical for obtaining high levels of polymorphism. Therefore, it would be useful if few informative primers are identified such that they amplify markers with high levels of polymorphism, which are representative of the whole genome. There is a strong need to assess and document the extent of genetic diversity in naturally growing, systematically characterized P. pinnata populations to expedite its use in different germplasm-related studies and breeding programmes. In continuation of our studies on candidate plus trees (CPTs), the present study was undertaken with the objective of screening the untapped genetic variability or relatedness among the 10 CPTs of P. pinnata tagged from North Guwahati, Assam, at the inter- and intra-population levels using PCR-based DNA markers (RAPD, ISSR and AFLP). Candidate plus trees are individual trees of P. pinnata possessing superior morphological and reproductive characters compared with other individuals of the same species identified on the basis of morphometric markers (Kesari et al. 2008). The study further involve (...truncated)