"3.1 SSR-enriched library SSR enriched libraries were constructed from the genotype ‘Pusa Santushti’ following a slightlymodified protocol of Bloor et al. (2001). Enrichment of the libraries was done by using SSRrepeat probes (CA, CT, TG, AG and GA) and from the constructed libraries, 220 clones wereselected from the 96-well plates. Sequence analysis of the clones provided 60 sequences thatcontained inserts in the size range of 200 bp to 1400 bp with an average size of 350 bp. Out ofthe 60 sequences, 65.31% contained insert of moderate size ranges from 120 bp-400 bp while22.45% clones had an insert size in between 400 bp-600 bp and 12.24% clones contained insertsof >600 bp. Sequence data of the above mentioned 60 clones were analyzed to reveal 48.33% clones withone or more SSRs regions. Similar results were reported by He et al. (2003), with 61% of clonescontaining SSR regions, Gimenes et al. (2007) reported 56% SSRs clones andlow enrichmentefficiency was shown by Wang et al. (2007) i.e., 43.7% clones with SSRs. 10% to 31%, cloneswere found in some otherSSRenrichedlibraries(Fergusonetal.,2004;Moretzsohnetal., 2005). Enrichment effectiveness depends on various factors, comprising the selection of therestriction enzymes used for library construction, the SSR probes used for enrichment, etc., themethodology used in the present study proved to be effective for SSR isolation in bottle gourd.Sixteen per cent of the sequences were redundant. Clones having redundant sequences have beenreported in other plant species,e.g.,onion(AlliumcepaL.,24.3%),groundnut(Arachis hypogaea,upto67%) (Ferguson et al., 2004; Gimenes et al., 2007; Moretzsohn et al., 2005)and the olive tree (Olea europaea L., 16.6%)(Rallo et al., 2000). The strategy used in the currentstudy seems to be successful to isolate a higher percentage of unique and novel SSRs. The biasdetected for certain SSRs may be due to repeated multiple clones which can also be due to the enrichment process, single-strand enriched DNA amplification or growth of bacteria beforeplating. During the enrichment process the addition of excess adapter, during the initial ligation stepproduced 7.2% clones that were concatenates, due to the presence of internal RsaI, Sau3A andHaeIII restriction sites in them. Another type of concatenation may be produced during the PCRstep of the procedure (Koblizkova et al., 1998). Such chimeras usually remain hidden and mayaffect the failure of amplification of genomic DNA by using such primer pairs. 3.2 Occurrence and features of SSRsSequenceanalysisof60clonesshowedthepresenceofoneormoreSSRswithdifferentproportion of SSRs repeats in 37 clones having total 52 SSR repeats with different type, repeatand size (Table 3, Fig 2). Among these 52 SSR repeats, 86.53% of the SSRs identified wereperfect and 13.46% were compound repeats. In terms of the repeat motifs, the CT accounted for23.07%, after that TGG with 9.61%, followed by GA, GTC, CTT with 7.69% and AG, TC, TG,GC, GTTTT, GAA repeats with 3.85%. The use of CT, GA, GTC, CTT, AG, TC, TG, GC,GTTTT, GAA filters might increase the efficiency of retrieving perfect repeat motifs. There are various repeats like AC, CCA, TTG etc. having only 1.92% occurrence in the libraries.However the present study reported around 13.46% of compound SSRs which were retrieved byusing a mixture of different SSR oligos. The maximum repeatnumberofdinucleotidemotifs, TCandGAwere28and26units, respectively; overall 3 to 28 repeat motif numbers arefound in the sequences. Some sequences show variation in the number of SSR. In addition to TCand GA repeats, several other SSRs containing the repeat motifs (AAG) n, (TGG) n, (CCA) n, (TTG) n,(GAA) n, (CTT) n, (TCT) n, (GTC) n, (GTG) n, (TCGG) n, (ACAT) nand (AATT) n and (GTGTTT) nwith 3–11 repeat numbers werealsofound. Maximum numbers of the clonescomprising repeats were not completely complementary sequences to the oligo nucleotide probesused in the study. In the reported study, only 100 clones sequenced were selected randomly fromthe set of 220 clones, possibly sequencing of more number of clones can produce more numberof SSR repeat motifs. 3.3 Marker developmentFrom a total of 100 sequenced clones 40 sequences were usedtodesignprimers,outof which30primersproducedclearamplicons.SSRrepeats (16.67%)producedwerehigher thanseveralreportedstudies;Arachishypogaea(10.5%;Moretzsohn et al.,2005), whilelesser than some other reports 21.3%, Ferguson et al. (2004). Only seven(23.33%)primers producedpolymorphicbands,thatis relatively lower than the Moretzsohn et al. (2005) andFerguson et al. (2004) reporting 81.6%and 84.9%,respectively.63.3% markers were genebased;amplifying regions — NOD26-like membrane integral protein, large subunit GTPasehomolog, C-repeat/DRE binding factor 1 (cbf1) mRNA, 17S rRNA, 5.8S rRNA, and 25S rRNAgene region, resistance gene analog (RGA) partial sequences,26S ribosomal RNA gene, Satgene for serine acetyltransferase, tRNA-Arg (trainer) gene, ycf15-like gene, Prv splice variant B(Prv) gene and whereas 13.34% amplified genomic regions from chloroplast and mitochondria(Table 2). Among the 30 SSR markers, 20 SSRs markers fell within genes, whereas 3 primerswere of non-genic regions and 7 showed no hits either with genic or non-genic region of thegenome (Table 2). For many of the sequences, primer designing was not possible as in manycases SSRs repeats were too near the start or end of the insert.This may be due to differencesinthemethodology,sizerangeofinserts,therestrictionenzymeusedinthe construction ofgenomic DNA libraries (Gupta and Varshney, 2000)."