Lentil (Lens culinaris Medik. subsp. culinaris) is a self-pollinated crop (2n = 2x = 14) belonging to the Vicieae tribe in the Fabaceae family. Lentil is a rich source of protein and micronutrients and is grown mainly in the Indian subcontinent, Middle East, North Africa, southern Europe, North and South America, Australia, and West Asia. Lentil has varied uses for consumption as a main dish, salads, or infant foods, and mixed with cereals to make bread and cakes. Although lentil is a highly nutritious food legume, its conservation and breeding potential is largely limited by a lack of molecular markers available for the crop.
Microsatellites are well-known genetic markers because of their codominant inheritance, polymorphism, and abundant coverage. They have become the markers of choice for many crops for studying genetic relatedness, diversity analysis, and constructing framework genetic maps. A limited number of microsatellite markers (approx. 100) have been published in L. culinaris to date. Hamwieh et al. (2005) developed 35 simple sequence repeat (SSR) markers, but these have reported no amplification or limited polymorphism, creating a major bottleneck to gene tagging and mapping studies in this crop. Kaur et al. (2011) developed 51 SSRs in L. culinaris, but these are less polymorphic and not sufficient to be used for genetic studies or for marker-assisted selection. An effective strategy for enrichment of microsatellite markers is the screening of expressed sequence tags, thereby reducing the time and cost for microsatellite development. The objective of this study was to develop new microsatellite markers using this strategy and characterize them in 18 L. culinaris accessions. The amplification success of these markers was also investigated in five wild Lens Mill. species (L. nigricans (M. Bieb.) Godron, L. odemensis Ladiz., L. tomentosus Ladiz., L. ervoides (Brign.) Grande, L. orientalis Popow) for potential genetic application or improvement of cultivated lentil (L. culinaris).
METHODS AND RESULTS
A total of 9513 L. culinaris expressed sequence tags (ESTs) were downloaded from the dbEST/GenBank database as of 15 January 2012 ( http://www.ncbi.nlm.nih.gov). The ESTs were trimmed for poly(A) tails, and vector sequence contamination were removed using the SeqClean program (Masoudi-Nejad et al., 2006). The trimmed EST sequences were assembled into unigenes with the Cap3 program (Huang and Madan, 1999) to reduce redundancy. The unigenes containing 951 contigs and 3092 singletons produced a total of 251 putative SSRs using the software Troll (Martins et al., 2009). The fragments with inappropriate flanking sequences or with less than 500 bp were excluded and 82 SSRs were designed using Primer3 software (Rozen and Skaletsky, 2000).
The newly identified microsatellite markers were screened on 32 individuals representing six Lens species including 18 accessions from cultivated species and 14 wild accessions comprising five species. Voucher specimens were sourced from the International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria, and the lentil breeding program at the Indian Agricultural Research Institute (IARI), New Delhi, India (Appendix 1). The genomic DNA from each individual was isolated using the cetyltrimethylammonium bromide (CTAB) method as described by Murray and Thompson (1980). PCR mixtures of 20 µL consisted of 2.0 µL, 10 × buffer (100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin), 200 µM each dNTP, 0.5 µM each of forward and reverse primers, 1 U Taq DNA polymerase (PCR reagents and primers procured from Sigma-Aldrich, St. Louis, Missouri, USA), and ∼40 ng DNA and were performed in a Veriti Thermal Cycler (Applied Biosystems, Life Technologies, Singapore). The PCR protocol consisted of one denaturation cycle at 94°C for 4 min followed by 30 cycles of 94°C for 1 min, annealing at 59–62°C (depending upon the primer) for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 8 min. The amplification fragments were separated on 3% MetaPhor Agarose gels (Lonza, Rockland, Maine, USA) and visualized by ethidium bromide staining. The band size was obtained in comparison to a 100-bp DNA ladder (MBI, Fermentas, Vilnius, Lithuania). Among these 82 SSRs, 62 amplified successfully in most of the species, providing an amplification success rate of 75.6%, and 19 of them showed more than two clear scorable bands in the Lens species (Table 1). Primer sequences and related information for 43 monomorphic EST-SSR primers are available as Appendix 2. The conservation of SSRs across species has been validated by sequencing one amplicon from each species. The EST-SSR markers amplified one to four alleles among the six species. The expected heterozygosity was determined on the basis of the number of genotypes amplified per species and ranged from 0 to 0.875 (Table 2). The putative functions of SSR-associated unigenes were determined by using BLASTX (Altschul et al., 1997) against the nonredundant GenBank database.
The EST-SSR markers identified and characterized in this study have enriched the limited microsatellite marker resources in Lens species. The markers developed will be helpful in saturating Lens genetic maps and for tagging and mapping of genes and quantitative trait loci associated with important traits to be further used in marker-assisted breeding for enhancing productivity and quality. These markers would also be helpful in studying genetic diversity and detecting interspecies polymorphisms for marker-based introgression of genes from related species.
Characteristics of the 19 polymorphic EST-SSRs identified in Lens culinaris.
Total number of alleles (A) and expected heterozygosity (He ) of EST-SSRs in six Lens species.