"Development and characterization of genome wide SSR markers in bottle gourdAnd their synteny study with other related cucurbits.Abstract Next generation sequencing has revolutionized the approaches for discovery, assessment andvalidation of molecular markers. In this study, we have used bottle gourd, a medicinally andculinary important crop, for microsatellites (SSR) marker survey and characterization. Despitebottle gourd worldwide importance still accessibility of enormous informative co-dominantmicrosatellite markers are very restricted, impeding genetic improvement, phylogenetic study inbottle gourd. As SSR markers are ideal genetic markers due to their high-level of polymorphismand co-dominant character and ease of use. In view of this, we conducted a genome wideanalysis, for developing SSR markers by utilizing recently available restriction site-associatedDNA sequencing (RAD-Seq) data. By performing in silico mining of microsatellite repeat- motifs, we developed 45,066 perfect SSR markers. Of which 105 (~0.2%) markers weresuccessfully validated among them 21.3% primers showing polymorphism in 20 accessions of bottle gourd. The mined SSRs were widely distributed across the bottle gourd genome, aligningto 365,668 different contigs and 48,262 different scaffolds. Tetranucleotide repeats ( ~34.3%)were the most prevalent followed by trinucleotide repeats ( ~30.73%), further 21.03%, 9.6% and4.3% of di-, penta- and hexa- nucleotide repeats in the bottle gourd genome. The generated SSRmarkers provides a valuable tool for germplasm characterization, genetic linkage mapconstruction, studying synteny, gene discovery and for breeding in bottle gourd and othercucurbits species.Keywords: Bottle gourd, Microsatellites, Genome-wide, SSR marker development, RAD-Seq,Synteny.Key Message: Development of 45,066 perfect microsatellite markers as a valuable tool formarker assisted selection (MAS) in plant breeding. IntroductionMicrosatellites or simple sequence repeats (SSRs) are small (1-6 bp) tandem repeats,which are ubiquitously found in the genome of both prokaryotic and eukaryotic organisms (Tothet al. 2000). High mutation rate, are the consequences of the change in array length ofallele, these repeats signify a rich cause of hyper-variable co-dominant markers (Morgante et al.2002; Powell et al. 1996). Consequently, microsatellite markers are consider as important markerin various research such as population genetics (Innan et al. 1997), linkage mapping (McCouchet al. 2002; Somers et al. 2004), phylogenetic, structural, functional or comparative genomicsresearch (Garza et al. 1995; MacHugh et al. 1997). Due to their high reproducibility, multi- allelic variation, co-dominant inheritance and abundance in the genome (Tautz et al. 1984), theyare also widely used for marker-assisted breeding and parentage analysis (Bowers et al. 1997).Microsatellites are believed to play significant roles in genome evolution, by producing geneticvariability (Kashi et al. 1997), in study regulation of gene expression (Santi et al. 2003; Savelievet al. 2003), acclimatizing the development of the cell cycle (Li et al. 2002) and manymore. Microsatellite are distributed across the genome in unequal and non-random fashion, assubmitted in various SSR data obtained from plants (Morgante et al. 2002), humans(Subramanian et al. 2003) and other eukaryotic organisms (Toth et al. 2000), with bothsimilarities and differences detected across texas. The distribution of microsatellites in thegenome has useful inferences with respect to their usage as molecular markers, genomic SSRmarkers have higher transferability amongst related species, which eases their use as anchormarkers for comparative physical mapping (Varshney et al. 2005).Development in technology like, genotyping-by-sequencing (GBS) or reduced- representation libraries sequencing (RRLS) are reflected to be the most auspicious technical substitutes (Altshuler et al. 2000; Elshire et al. 2011; Helyar et al. 2011). Most importantlyrestriction site-associated DNA sequencing (RAD-Seq), one of the approaches known as agenome ‘complexity reduction’ protocol, has been demonstrated to be predominantly valuable innon-model species as it pools the benefits of low cost and high output (Rowe et al. 2011). RAD- Seq known to be used in about 20 species without using any reference genome in many phases ofgenomic/genetic research, comprising effective marker development approach (Pujolar et al.2013), comparative mapping studies (Yang et al. 2013), QTL mapping (Hegarty et al. 2013),phylogenetic analyses (Nadeau et al. 2013) and genome-wide studies (Hecht et al. 2013). Inspecies with existing reference genomes RAD-Seq has been a worthwhile substitute to WGRS(Whole Genome Re-Sequencing) for population genomic studies (Bruneaux et al. 2013;Varshney et al. 2013). In this view, recent available RAD sequencing data provide newopportunities to develop SSR marker in bottle gourd. As in spite of the significanceof microsatellite markers in many research practices, genome-wide characterizationof microsatellite sequences in the available bottle gourd genome have not yet be accompanied. Bottle gourd [Lagenaria siceraria (Mol.) Standl.] (2n = 2x = 22), is an edible, medicinal,container and a grafting stock plant cultivated all over the tropics (Heise et al. 1979). The bottlegourd is considered to be one of the first crops to be domesticated (>10000 years ago) (Whitakeret al. 1971; Erickson et al. 2005). Amazingly, high genetic variability exists in bottle gourd,especially in fruit size and shape, with various medical properties were also reported includingits anti-cancerous, cardio-protective (Fard et al. 2008), diuretic, purgative, cooling effects(Badmanaban et al. 2010). It can also cure ulcers, pectoral cough, asthma and other bronchialdisorders (Upaganlawar et al. 2010). Till now few molecular markers like, 20 ISSR (Bhawna etal. 2014), 3226 SNP (Pei et al. 2013), SSRs (Xu et al 2011; Bhawna et al. 2015a, b), 54 RAPD (Decker-Walters et al. 2001), two chloroplast and five nuclear markers (Andrew et al. 2006)were available in bottle gourd to assess its genetic diversity. But still available resources are stillinsufficient for such beneficial crop. Considering the efficacy of whole genome sequencebased microsatellite marker data, we made an effort to develop microsatellite markersfrom RAD sequence data, along with distribution and density of perfect microsatellites in bottlegourd genome sequences. The work will prove to be useful for not only genetic level research,but also to study phylogenetic, DNA fingerprinting, breeding for other cucurbits crop species,which have very less or no genomic markers are available. "