Bottle gourd [Lagenaria siceraria (Mol.) Standl.] (2n = 2x = 22), also known as calabash or opo squash, is a diploid belonging to the genus Lagenaria of the Cucurbitaceae family . Phylogenetically, bottle gourd is close to many economically important cucurbit species including cucumber and melon that belong to the genus of Cucumis, as well as watermelon that belong to the genus Citrullus. Worldwide, bottle gourd is grown for its fruit either being harvested young and used as a vegetable or harvested mature and used as a bottle, utensil, or pipe. The fresh fruit, which usually has a light green smooth skin and a white flesh, is frequently used in many regions of Asia and Africa as either a stir-fry or soup vegetable ingredient . Another recent utilization of bottle gourd is as rootstocks for watermelon against soil-borne diseases and low soil temperature [3, 4].
Bottle gourd was one of the first crops to be domesticated. Based on archaeological evidence, bottle gourd is presumed to have been domesticated in Africa [5, 6], and might have dispersed to the New World by ocean currents or by human migration in pre-historic times [7, 8]. Africa is believed to be the centre of genetic diversity for bottle gourd, although wild progenitors of bottle gourd have not been identified there . Substantial morphological variation for fruit and seed size, shape, color and rind hardness exists in the bottle gourd gene pool [8–10]. Yetisir et al. observed a wide range of morphological variation among Turkish bottle gourd accessions despite the fact that this region is not a center of origin of the crop .
At present, very few molecular genetic/genomic resources are publically available for bottle gourd. Achigan-Dako et al. measured the genome size of bottle gourd and showed that the nuclear 2C-value of DNA was around 0.734 pg, which is estimated to be equal to ~ 334 Mb . In spite of the relatively small genome size of bottle gourd, there are only dozens of bottle gourd DNA sequences available in the public DNA database, making it unfeasible to identify bottle gourd genes or to analyze their functions. A limited number of anonymous random amplified polymorphic DNA (RAPD) markers have been described [10, 13], but there has been no locus specific DNA markers such as microsatellite (SSR), sequence tagged site (STS) or single nucleotide polymorphism (SNP) markers available for bottle gourd so far. Also unclear is the extent of genome conservation/diversification between bottle gourd and other important cucurbit species such as the model cucumber (Cucumis sativus L.), which serves as the basis for comparative genomic analysis across cucurbit species.
Microsatellites, or simple sequence repeats (SSRs), are short repeat motifs usually associated with a high level of frequency of length polymorphism. With the advantages of being stable, PCR-based and relatively low-cost, SSR markers are one of the best choices for genetic research and molecular breeding. SSR markers can be developed, in case of the availability of large number of DNA sequences, in silico , or experimentally . Traditionally, the experimental approach requires the construction of a genomic library enriched for repeated motifs, hybridization and isolation of microsatellite containing clones, sequencing of positive clones and primer design . Most of these steps, especially the hybridization/isolation step, are expensive and time-consuming. Recent emerging 'next generation' sequencing technique, for instance, the 454 Genome Sequencer FLX (GS-FLX Titanium) shotgun System (Roche, Penzberg, Germany), provides a powerful alternative for generating a tremendous number of DNA sequences for genomics study and marker development. Instead of creating a conventional genomic library enriched for microsatellites, GS-FLX Titanium system sequences a shotgun library in a high-throughput manner, producing tens of thousands of reads around 300-400 bp. By mining the sequence reads, SSR-containing sequences can be identified. Using this technology, we partially sequenced the bottle gourd genome. Through assembling and annotating the sequence reads, tens of thousands of genes with broad range of functional types were recognized. Moreover, hundreds of microsatellite markers were developed using the sequencing data, which are invaluable in future marker assisted breeding and phylogeny analysis. The markers were then applied to a range of bottle gourd accessions to assess genetic diversity to enable more efficient parental line selection for breeding purposes and to dissect the genetic factors underlying morphological variations.