Bioinformatics a Tool for Biosystematics Studies of Lagenaria siceraria (Mol.) Standl. Complex

Bioinformatics has been a major tool in the revolution of plant systematics in recent times. The diversity of fruit shapes in Lagenaria siceraria (Mol.) standl. species has been of great concern because of its fruit complexity. This study is based on the application of rubisco enzyme using rbcL marker because of its conservativeness and its ability to discriminate below the specific level hence its usage to sequence the chloroplast genome with ABI, PRISM 377 DNA sequencer. The sequences obtained were viewed using MEGA X software and subsequently subjected to validation through National Center for Biotechnology Information (NCBI) using Nucleotide Basic Local Alignment Search Tool (BLAST N). The result obtained showed that all the sequences belong to Lagenaria siceraria (Mol.) standl. with percentages ranging from 95% to 100% for query cover sequences and 98% to 100% for identity sequences. From the taxonomic report obtained sequence A, C, G, H, J, Q has the highest hits of 44 on Lagenaria siceraria out of 109 total value, sequence O and R has the highest hits of 44 on Lagenaria siceraria out of the total value of 111, sequence V has the highest hits of 44 on Lagenaria siceraria out of 119 total value and sequence X finally has the highest value hits of 44 on Lagenaria sceriania out of 105 total value, based on this report, phylogenetic tree was constructed to show the level of relatedness of the different fruit diversity of L. siceraria complex. This work therefore has aided in the molecular characterization of Lagenaria siceraria (Mol.) standl. landraces found in Nigeria.


Introduction
The species Lagenaria siceraria (Molina) Standl is a member of the Cucurbitaceae family, Cucurbitaceae subfamily and Benincaseae tribe [1]. The genus Lagenaria consists of five other wild species, namely Lagenaria breviflora (Benth) Roberty, Lagenaria rufa (Gilg) C. Jeffrey, Lagenaria sphaerica E. May, Lagenaria abyssinia (Hook F) C. Jeffrey, Lagenaria guinensis (G. Den) C. Jeffrey and Lagenaria siceraria (Molina) Standley is the most cultivated within the species of siceraria; two morphologically distinct sub-species of bottle gourd have been recognized thus Lagenaria siceraria subspecies asiatica and Lagenaria siceraria subspecies siceraria [2] [3] [4]. It is universally agreed fact, that different species are systems of population which exhibit variation and not fixed entities, therefore no individual is identical as developed by [5]. Systematics as defined by [6], states that systematics is a natural science that deals with the study of individual, population and taxon relationship for the purposes of classification. Plant systematics is therefore based on the premise that in the enormous variation in the plant kingdom there exist discrete units called species that can be identified classified and named with further logical relationships developed among these units. Over the years, plant groupings have been redefined as more information is accumulated from newer approaches and different recent sources such as molecular systematics, and taximetrics. Variety of Lagenaria is known throughout West Africa where they serve different economic purposes but these cultivars are largely unrecorded in terms of micromorphological characteristics [7] and diversity of fruit shape and sizes continue to create serious taxonomic difficulties in the delimitation of the taxon Lagenaria siceraria (Mol) Standley below the specific level.
The emerging field of molecular systematics deals with the exclusive utilization of molecular data [6]. A molecular marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify species, which detect variation at the nuclear, Mitochondrial, and chloroplast DNA. Molecular markers have been developed and utilized to provide the requisite landmarks for clarification of genetic variation [8]. DNA is more stable macromolecule than RNA and is found in all plant tissues hence, DNA based markers are preferred for precise identification of plant species [9]. An understanding of the extent of genetic diversity within and amongst the landraces of the gourd is important for strategic breeding and conservation [10].
Plants are generally more tolerant of variation, hence genetic variation may result from mutation or recombination [6] [11]. According to [12], effective utilization of plant genetic resources depends on a detailed understanding of their genetic variability. Interestingly, substantial variation exists in the ability of plants to tolerate gene imbalance both between different plant species and between varieties of the same species [13]. However, despite the widespread interest in molecular systematics, there is limited understanding of the molecular mechanisms that lead to phenotypic alterations in plant species as well as gene interactions involved in the global genomic level [14], went further to explain that the molecular systematic studies give deeper insight into genetic structures. It has performed an important role in molecular biologies, such as analyzing genetic diversity by classification of cultivars and germplasm collections. It has also aided in clearing phylogenetic relationships among groups or closely related species. It is further used to determine the sequence of nucleotides in the DNA of plants, conclusively, has increased significantly, understanding of plant evolution.

Study Map
The species were planted in the faculty of Agricultural science research farm, of the University of Port Harcourt (Figure 1). The amplification products from land races of Lagenaria siceraria (Mol.) Standl. were separated using 1.5% agarose electrophoresis and the expected size of bands were excised using scapel and purified by Spin Column Purification method. Purified amplicons were sequenced using BigDye ® Terminator v3.1 Cycle Sequencing Kit protocol in ABI Prism 377 DNA Sequencer (Applied Biosciences, NY, USA).

Sequence Alignment and Phylogenetic Analysis
Lagenaria siceraria sequences in FASTA format were aligned and used in the analysis of protein content of the twenty three species, the process of sequence analysis was advanced through pair wise alignment, construction of a distance matrix using Mega X. The sequences were compared to the NCBI database using Nucleotide Basic Local Alignment Search Tool (BLASTN). The results of distance matrix were sent to Excel 2007, which was finally analyzed using PAST 3.14 software for the construction of phylogenetic tree showing similarity and distance relationship between the twenty-three species. The chloroplast sequences of L. siceraria from NCBI data base were analysed using CHLOROBOX.   The quantity of DNA extracted is read using spectrophotometer as shown in Table 1. The value ranges from 1.13 to 1.82
The phylogenetic tree ( Figure 4) shows great diversity and similarity in Long handle dipper -U and Microphone gourd-X are out group from the root, and meets at similarity scale of 0.7, while Kettle gourd-B and Caveman club gourd-C are out group from the second root of the tree meeting at a similarity scale of 1.0. The second root has two main clusters that meets at different similarity scale. The first cluster of the second root (R2a), African bottle gourd-A is seen as out group at 0.65 while Snake gourd-W and Powder horn gourd-V clustered together at 1.0 similarity scale. Swan gourd-S and palmwine gourd-T meets at 0.89 while Mini dipper gourd-P and Goose neck gourd-Q clustered at 1.0, Nigerian gourd-R is observed as an out group at 0.85 similarity scale. In the second cluster of the second root (R2b), Cup gourd-I was an out group at 0.85 while Baseball gourd-D, Bird house gourd-G, Water jug gourd-H, Penis shield gourd-J, Warted bushel gourd-K, Extra-large pawpaw gourd-L, Long siphon gourd-M, Indian gourd-N and Chinese gourd-O met at 1.0 similarity scale.

Discussion
According to [6], the concentration of macromolecular studies was geared towards DNA and RNA hence, molecular systematics deals with the utilization of nucleic acid data. Genetic materials have been used in the understanding of the     (Table 3) had an absorbance ratio of 1.2 to 1.8 showing the purity of the DNA, which is in line with the work of [16] [17]. The conserved rbcL gene has been utilized in PCR amplification of chloroplast gene sequences for determining and ratifying phylogenies [18] [19]. The sequence alignment of the twenty-three landraces from Nigeria shows great variation in the arrangement of the nucleotide bases (Figure 4), which is due to gene recombination and mutation.
The sequences were subjected to validation through National Center for Bio-

Conclusion
The use of bioinformatics tools in biosystematics studies of Lagenaria siceraria landraces (biotypes) found in Nigeria has served as a tool in resolving the quagmire in the diversity of L. siceraria complex and the classification of the Landraces into proper taxa. Hence molecular systematics of the species demonstrates differences in sequence arrangement that is due to gene recombination and the functional effect of heterologous genes expressed phenotypically on the fruit shape thereby resulting in the diversity of in fruit shapes of Lagenaria siceraria (Mol.) Standl. found in Nigeria.