Introduction
Luffa cylindrica is an annual vine commonly called sponge gourd, loofah, vegetable sponge, bath sponge or dish cloth gourd (2n= 2x= 26) (Chandra, Reference Chandra1995). It is the most cultivated species in India (Prakash et al., Reference Prakash, Pandey, Radhamani and Bisht2013a, Reference Prakash, Radhamani, Pandey and Yadavb). It has wide industrial applications (Oboh and Aluyor, Reference Oboh and Aluyor2009; Partap et al., Reference Partap, Kumar, Sharma and Jha2012) but considered an underutilized crop as primarily grown for domestic consumption and commercial cultivation is secondary (Malik et al., Reference Malik, Ellington, Wehner and Sanders2001; Silva et al., Reference Silva, Ranil and Fonseka2012; Jaenicke and Höschie-Zeiedon, Reference Jaenicke and Höschie-Zeiedon2006). Prakash et al. (Reference Prakash, Pati, Arya, Pandey and Verma2014) conducted genetic diversity in Luffa species; however, knowledge about the genetic diversity and population structure of L. cylindrica is scarce. Therefore, there is an urgency of characterization of genetic structure for effective conservation, management and utilization of L. cylindrica. Sequence-related amplified polymorphism (SRAP) marker (Li and Quiros, Reference Li and Quiros2001) that amplifies the open reading frames is becoming the marker of choice for genetic diversity, gene mapping and genotypes fingerprinting (Li et al., Reference Li, Gao, Yang and Quiros2003) due to their simplicity, reliability, reasonable throughput rate and easy isolation of bands for sequencing. The objectives of this study were: (1) to evaluate SRAP marker effectiveness in cultivar identification; (2) to study genetic diversity in L. cylindrica for use in breeding programmes; (3) to determine the genetic structure of L. cylindrica population.
Experimental
The sampling strategy consisted of assemblage of 45 accessions from different geographical regions of India. (Table S1, available online). These accessions were selfed and maintained as pure lines in the fields of IARI, New Delhi during March to May 2013.
For morphological characterization, three seed characters, viz. seed coat colour, seed surface and seed wing's presence or absence based on Bal et al. (Reference Bal, Hari, Radha, Madhusudan, Bhuwon and Madhusudan2004) descriptor with some modifications (Table S1, available online) were documented.
For SRAP marker analysis, total genomic DNA was extracted from young leaves using the Cetyl Trimethyl Ammonium Bromide (CTAB) method (Saghai-Maroof et al., Reference Saghai-Maroof, Soliman, Jorgesm and Allard1984). A total of 60 different SRAP primer combinations (ten forward and six reverse primers) were used (Table 1). 12.5 μl reaction contained 50 ng DNA, 1X Taq buffer, 0.2 mM deoxyribo Nucleoside Tri Phosphates (dNTPs), 1.5 mM MgCl2, 8 μM each of forward and reverse primer and 0.5 U Taq DNA polymerase (Thermo-Scientific, Waltham, Massachusetts, USA). The PCR amplification was carried out in Bioer-Gene Procycler using the following thermal profile: 5 min at 94°C followed by five cycles of 1 min at 94°C, 1 min at 35°C, and 2 min at 72°C; 35 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C and a final extension of 5 min at 72°C. The amplification products were run in 2% (w/v) agarose gel in 1X Tris Borate EDTA (TBE) buffer with 50 bp and 1 Kb ladder (Thermo-Scientific).
Table 1 The SRAP forward and reverse primers used for Luffa cylindrica accessions
The marker performance was investigated by calculating polymorphism information content (PIC), marker index (MI), resolving power (Rp) and percent polymorphism (P%) (Powell et al., Reference Powell, Morgante, Andre, Hanafey, Vogel, Tingey and Rafalski1996; Prevost and Wilkinson, Reference Prevost and Wilkinson1999; De Riek et al., Reference De Riek, Calsyn, Everaert, Bockstaele and Loose2001). Population genetic diversity parameters were determined by the POPGENE 1.31 (Yeh and Boyle, University of Alberta, Canada; Yeh and Boyle, Reference Yeh and Boyle1997) (Table 2). Dendrogram and principal coordinate analysis (PCoA) was carried out through the NTSYS-pc 2.20 (Rohlf, Setauket, New York; Rohlf, Reference Rohlf2000) and population structure by using the STRUCTURE 2.3.4 (Dr. Sung-Chur Sim, Ohio State University; Pritchard et al., Reference Pritchard, Stephens and Donnelly2000).
Table 2 Genetic diversity within populations of Luffa cylindrica
N a= observed number of alleles, N e= effective number of alleles, H e= Nei's (Reference Nei1973) gene diversity, G st= coefficient of differentiation, N m= estimate of gene flow, N m= 0.5 (1 − G st)/G st (McDermott and McDonald, Reference McDermott and McDonald1993), I= Shannon's information index.
Discussion
SRAP along with morphological markers provide valuable information about the existing diversity of L. cylindrica. Variation in seed traits was reported earlier by Prakash et al. (Reference Prakash, Pandey, Radhamani and Bisht2013a, Reference Prakash, Radhamani, Pandey and Yadavb). Hence, we studied within species variation in seed traits; however, insignificant diversity was observed which might be due to few traits and more traits should be incorporated in future studies. The primers Me3-Em5, Me5-Em6, Me9-Em5, Me3-Em6 and Me4-Em1 had higher Rp, PIC, MI and P% values, and were more informative for genetic diversity and cultivar identification (Table S2, available online). Nei's gene diversity (H e= 0.120) was comparable to dicotyledons (H e= 0.0960) (Hamrick et al., Reference Hamrick, Godt and Sherman-Broyles1992) and similarity coefficient value of 0.77 suggests moderate genetic diversity. Our results were in conformity with previous studies on L. cylindrica (Cruz et al., Reference Cruz, Tolentino, Altoveros, Villavicencio, Siopongco, dela Vina and Laude1997; Marr et al., Reference Marr, Bhattarai and Xia2005). L. cylindrica is an annual, wild, monoceious and self-compatible crop (Sujatha et al., Reference Sujatha, Chithakari, Raghuvardhan, Prasad, Khan, Sadanandam and Christopher2013; Silva et al., Reference Silva, Ranil and Fonseka2012; Sinnot and Bloch, Reference Sinnot and Bloch1943). As long-lived perennials have higher genetic diversity as compared with annual or short-lived perennials (Hamrick and Godt, Reference Hamrick and Godt1996; Nybom and Bartish, Reference Nybom and Bartish2000; Nybom, Reference Nybom2004) and self-incompatibility maintains high genetic diversity (Borba et al., Reference Borba, Semir and Shepherd2001), thus supports our results. West Bengal and Uttar Pradesh population exhibit highest genetic diversity (~0.190), which might be due to large number of accessions in both populations (Table 2) as compared with other six populations ( < 0.154). Heterozygosity measured by molecular markers is significantly correlated with population size (Frankham, Reference Frankham1996). Small groupings were observed in sub-clusters in both Unweighted pair group method with arithmetic average (UPGMA) and PCoA; however, no association was found between genetic distance and geographic region (Figs S1 and S2, available online). Regional isolation may not reproduce the genetic make-up of diverse accessions due to dispersal of seeds from their native cultivation places (Kumar and Shekhawat, Reference Kumar and Shekhawat2009) and thus no association.
Self-pollinated crops have low diversity within populations than cross-pollinated (Huang et al., Reference Huang, Zhang and Li2009) and small animals promote gene flow between populations (Levin and Miller, Reference Levin and Miller2005). L. cylindrica is a cross-pollinated crop (Jayaramappa et al., Reference Jayaramappa, Pattabhiramaiah and Bhargava2011; Bhardwaj and Srivastava, Reference Bhardwaj and Srivastava2012) and N m value was comparable to animal-pollinated species (Huang et al., Reference Huang, Zhang and Li2009) indicating high gene flow among populations, thus high genetic diversity within populations and low among populations (Table 2). The genetic differentiation (G st) among populations was estimated to be 0.4073, which indicate that 40.73% of the genetic variability was distributed among populations, and 59.27% of the variation existed within populations. Maximum genetic distance was observed between Maharashtra and Madhya Pradesh populations (Table S3, available online) and accessions of these populations could be utilized for Luffa improvement programmes. Population structure revealed three clusters contrary to two (UPGMA and PCoA) and not geographically distributed (Fig. S3, available online). The genetic data are multivariate and population structure can have complex pattern without correspondence in the different methods utilized (Kalinowski, Reference Kalinowski2009). Although this study provides a foundation for gauging the inherent genetic diversity and structure in L. cylindrica, there is an urgent need to evaluate population structure in more accessions in Asia as well as Africa using varied molecular markers to signify the diversity at the species level.
Supplementary material
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S1479262115000258
Acknowledgements
The authors are grateful to the Head, DGR and the Director, NBPGR for permitting access to the facilities. They thank D. K. Pokhriyal (Technical Assistant) for his assistance in conducting the experiments.
