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Computational Molecular Biology 2014, Vol. 4, No. 12, 1-8
http://cmb.biopublisher.ca
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adequate due to its complex property of related
components like grain number per panicle, grain
weight, tillers per plant, etc. Of these factors, grain
number was shown to be highly correlated with yield
(Hua et al., 2002). Three hundred sixty nine QTLs
distributed across all over the 12 chromosomes of rice
controlling grain number per-panicle have been
identified (http://www.gramene.org) using various
mapping populations derived from inter-specific,
indica-japonica, indica-indica
and
japonica-japonica
crosses
.
Some of them were fine mapped to less than 1
cM intervals and few have been cloned using
QTL-based near isogenic lines (NIL). Genes
controlling grain number per-panicle directly or
indirectly, i.e.
Gn1a
,
Ghd7, Dep1, fz, Sp1, rcn2, lax1
and
Apo1
also have been isolated from rice (Li et al.,
2003; Jin et al.,2008; Tan et al., 2008; Li et al.,2009;
Piao et al., 2009; Terao et al.,2010; Zha et al.,
2009).The recent development in molecular biology,
genomics and bioinformatics has helped in the fine
mapping and cloning of major genes/QTLs associated
with rice yield traits at a rapid pace for the past 25
years. With the information on the cloned genes and
closely linked markers associated with agronomic and
yield traits in rice, accumulating beneficial
alleles/genes using MAS has become a straight
forward approach for improving target traits in rice
(Wang et al., 2012). Pyramiding of genes in the elite
genetic backgrounds has been reported to result in
higher yield potential, longer grains, and a more
suitable heading date. The novel genes are preserved
in many related wild species, land races and cultivated
varieties of rice. Therefore, pyramiding genes for
grain number is highly indispensable in rice
improvement programs. Here we have considered four
different high grain number genes
APO1, DEP1,
GHD7, GN1A
. The main objective of the work is to
find out the exons and promoters of genes, to predict
their restriction sites, comparative homology
modeling of corresponding proteins, designing and
validation of primers for high grain number for use in
molecular breeding in rice.
1 Material and Methods
1.1 Retrieval of nucleotide sequence
The nucleotide sequences of the high grain number
genes i.e
Apo1, Dep1, Ghd7,
were retrieved from
oryzabase, RAP-DB and gramene databases and from
NCBI (http://www.ncbi.nlm.nih.gov/) by searching
against nucleotide.
1.2 Similarity search for high grain number genes
Then the sequences were subjected for similarity
search by using BlastP (http://blast.ncbi.nlm.nih.gov/
Blast.cgiPROGRAM=blastp&PAGE_TYPE=BlastSea
rch&LINK_LOC=blasthome).
1.3 Gene prediction
The coding and non-coding regions were predicted
using Genscan tool (http://genes.mit.edu/GENSCAN.
html). From this we could find out the exon
(functional region) and intron (non-functional region).
1.4 Protein structure prediction
The secondary structures were predicted by using
SOPMA server (http://www.rcsb.org/pdb/home/home.do).
From this prediction it was easy to find out the helices,
beta and turns. Then the comparative homology
modeling was done by using modeller9.12 tool. The
templates were identified from BlastP after similarity
search and collected from PDBin fasta format. Then
the align2d.py, model-single.py and evaluate model.py
files were run on modeller9.12. The best model was
selected on the basis of lowest DOPE score.
1.5 Model optimization and evaluation
Then the final models were visualized by using
visualization tools i.epymol, rasmol and discovery
studio visualiser. Then the backbone confirmation of
the proteins were predicted by using PROCHECK
Saves server (http://services.mbi.ucla.edu/SAVES/
Ramachandran/), which showed the allowed and
disallowed regions of proteins from where it was easy
to find out the suitable protein for further study.
1.6 Primer designing
Primers were designed to amplify the particular
regions of the genes which can be further used for
cloning purpose leading to high grain production in
rice. Primer3 tool (http://primer3.ut.ee/) was used to
find out the full length primers, and genomic primers,
exon primer tool (ttp://ihg.gsf.de/ihg/ExonPrimer.html)
was used to find the exon specific primers and
multiple primer design with primer3tool