International Journal of Horticulture & Agriculture

With recent advances in the field of genome sequencing, analysis and availability of large genomic data in the public domain, we made an attempt to survey the presence of the conserved domains, super families and multi domains having putative functions identified from green gram [Vigna radiata (L.) R. Wilczek] Genomic Survey Sequences (GSS) using computational tools. In this study we have been identified the various conserved domains, super families having putative functions for fundamental, metabolic, developmental, evolutionary processes and physiogenic nature from the query sequences. This study was beneficial in the area of comparativ e genomics for the identification of important genes and also development of functional molecular markers in identified genes for green gram and its related crops improvement.

Keywords: Conserved domains; Genomic Survey Sequences (GSS); Green gram; Multi domains; Putative functions; Super families

Sequenced crop plants are the good resources in identification of important genes for quality, insect pest and disease resistance, resistance to abiotic stresses like temperature, drought, salinity etc. which are possible to transfer to cultivable background by combining traditional and molecular breeding methods [28]. Genome Survey Sequences (GSS) are nucleotide sequences similar to EST’s that the only difference is that most of them are genomic in origin, rather than mRNA, While Expressed Sequence Tags sequences represent the expressed region of the genome [4]. These GSS and EST sequences used for the identification of “Functional Molecular Markers” (FMM) which are associated with trait of interest and may be transferable in closely related genera [3]. Genomic Survey Sequences of Green gram [Vigna radiata (L.) R. Wilczek] available online in public domain from NCBI (http://www.ncbi.nlm.nih.gov) for public use. Due to generation and availability of huge genomic information online of the crop plants, the computational studies i.e. performed on computer or via computer simulation are important areas of interest for genomics researchers for comparative genomics study [31, 21, 15].

Domains can be thought of as distinct functional and/or structural units of protein. The identification of a conserved domain footprint may be the only clue towards cellular or molecular function of a protein, as it indicates local or partial similarity to other proteins, some of which may have been characterized experimentally. Conserved Domains (CD) contain conserved sequence patterns or motifs, which allow for their detection in polypeptide sequences (www.ncbi.nlm.nih.gov/ Structure/cdd). It has been suggested that domain combinations are evolutionarily conserved and evolution creates novel functions predominantly by combining existing domains [13]. The Conserved Domain Database (CDD) is a compilation of multiple sequence alignments representing protein domain conserved in molecular evolution [24]. Keeping all above points in view the computational identification study was carried out to identify the conserved domains having putative function like insect pest resistance, improved quality parameters or for resistance to abiotic stresses like temperature, drought, salinity or any other evolutionary functions from Green gram GSS and their possible use in Green gram and also its related crops improvement.

The Genome Survey Sequences (GSS) of Green gram [Vigna radiata (L.) R. Wilczek] available online in public domain from NCBI (http://www.ncbi.nlm.nih.gov). These were downloaded in FASTA format to be used for further analysis.

Search for conserved domains within query GS sequence (nucleotide sequence) was analyzed using conserved domain search service (CDD search) available online (http://www.ncbi. nlm.nih.gov). The GS query sequences of more than 170 bp in length were used for the analysis. If a specific hit is not found on a query sequence, but the nucleotide sequence has an otherwise statistically significant hit (E-value cutoff of 0.01) to any domain model in CDD, the domain model is regarded as a non-specific hit. The E-value threshold used for filtering results was kept at 0.001,maximum number of hits for CD search kept at 500 and the result mode kept as standard default settings.

A total of eighty two GS sequences which were chosen deliberately from the Green gram [Vigna radiata (L.) R. Wilczek] were analyzed for the identification of conserved domains, super families and for multi domains. The distribution of 82 hits in CDD search is represented in fig 1. As the online CDD search is inbuilt for identification of low complexity region search as determined by the SEG program of [38] or for BLASTN, by the DUST program of Tatusov and Lipman (ftp://ncbi.nlm.nih.gov/pub/tatusov/ dust/versionl/), we used GS query sequences as such with more than 170 bp in length for analysis. After analysis of 82 GSS, we observed 30 non-specific hits, which contribute about 20 % sequence analyzed. The various conserved domains, super families having putative functions for fundamental, metabolic, developmental, evolutionary processes and physiogenic nature have been identified from the query sequences. From the specific hits a total of 16 conserved domains (11%) and 34 super families (23%) were observed, respectively and 29 multi domains (20%) were identified. It is interesting to note that out of 82 query sequence hits, 39 hits (26%) did not identify any specific CD or super family or multi domains indicating all these sequences are unique (unique GSS) to the Green gram species. A specific hit is a high confidence association between a nucleotide query sequence and a conserved domain, resulting in a high confidence level for the inferred function of the query sequence. In the GS query sequence AZ254242.1 (i.e sequence number 2) (Table l) the specific hit identified CD LRR_4 and have LRR_4 super family (Fig 2) with E-value of 2.62e-04. This leucine rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. These repeats are usually involved in protein-protein interactions. Each Leucine Rich Repeat is composed of a beta-alpha unit. These units form elongated non-globular structures. Leucine Rich Repeats are often flanked by cysteine rich domains. LRR-containing proteins from plants have diverse overall structures and functions. Several classes contain LRR-containing receptor-like kinases (LRR-RLKs) [1, 16], LRR-containing receptor-like proteins (LRR-RLPs) [10], nucleotide binding site LRR (NBS-LRR) proteins [7, 26] and Poly- Galacturonase Inhibiting Proteins (PGIPs) [8, 9, 30]. They provide an early warning system for the presence of potential pathogens and activate protective immune signaling in plants [20, 18, 35]. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. Evolution of plant, disease resistance (R) genes that encode an LRR region has been studied by many researchers [1, 26, 27, 6, 23, 14, 25, 39, 17, 12, 32, 37, 22, 2, 40, 30, 11, 19]. The generations of R genes are proposed to be mainly due to gene duplication, genetic recombination, diversifying selection, and sequence divergence in the intergeneric region, composition of the transposable elements, gene conversion, and unequal crossover [40, 30, 11]. Another conserved sequence in the GS query sequence AZ254272.1 (i.e. sequence number 5) (Table l) the specific hit identified AspRS_cyto_N: N-terminal, anticodon recognition domain of the type found in Saccharomyces cerevisiae and human cytoplasmic aspartyl-tRNA synthetase (AspRS). This domain is a beta-barrel domain (OB fold) involved in binding the tRNA anticodon stem-loop. The enzymes in this group are homodimeric class2b aminoacyl-tRNA synthetases (aaRSs). aaRSs catalyze the specific attachment of Amino Acids (AAs) to their cognate tRNAs during protein biosynthesis. This 2-step reaction involves i) the activation of the AA by ATP in the presence of magnesium ions, followed by ii) the transfer of the activated AA to the terminal ribose of tRNA. In the case of the class2b aaRSs, the activated AA is attached to the 3’OH of the terminal ribose. Eukaryotes contain 2 sets of aaRSs, both of which are encoded by the nuclear genome. One set concerns with cytoplasmic protein synthesis, whereas the other exclusively with mitochondrial protein synthesis. This gene malfunctioning might be results in the hinderance in the protein synthesis of cytoplasm and mitochondria [34]. One more conserved domain in the GS query sequence AZ254277.1 (i.e sequence number 6) (Table l) the specific hit identified TVP38. The protein Tvp38 is conserved in yeasts and higher eukaryotes and potentially involved in

vesicle transfer processes at the Golgi membrane. Members of the so-called “SNARE-associated proteins of the Tvp38-family” have also been identified in prokaryotes and those belong to the DedA protein family. Tvp38/DedA proteins are also conserved in cyanobacteria and chloroplasts. While only a single member of this family appears to be present in chloroplasts, cyanobacterial genomes typically encode multiple homologous proteins. Mainly based on our understanding of the DedA-homologous proteins of Escherichia coli, it appears likely that the function of these proteins in chloroplast and cyanobacteria involves stabilizing and organizing the structure of internal membrane systems. Another domain name SANT (AZ254291.1 table 1) found in regulatory transcriptional repressor complexes where it also binds DNA. Based on the putative function of the conserved domains for the gene or gene families identified from the nucleotide query sequence, they could be used for the identification of genetargeted markers (GTMs) (Varshney and Tuberosa, 2007) and for the development of Functional Markers (FM) (Andersen and Lu” bberstedt, 2003). After validation, these markers can be used in the Marker Assisted Breeding (MAB) while transferring the gene of interest from wild species under the cultivated background of Green gram. In the present analysis, it has been observed that most of the identified CD regions are common in large groups of living beings viz., rice, maize, human, arabidopsis, tortula, fungus, bacteria, protozoan, drosophila etc. Thus during the evolution process, these sequences have been highly conserved in nature among different living organisms and additional information can be generated for the identified CDs or gene families or for the gene of interest by means of comparative genomics tools. The generated information for the genes of interest can be utilized for the improvement of Green gram and this new approach may help the plant breeders for identification of functional markers in crop plants.

from this study we observed the various conserved domains, super families having putative functions for fundamental,metabolic, developmental, evolutionary processes and physiogenic nature have been identified from the query sequences which can be further utilized for the development of functional markers in green gram and its related crop plants.

We express s Agricultural University, Hyderabad for providing the opportunity to work on the above topic.

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