In silico and molecular characterization of GA2ox genes involved in rice tillering.
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Date
2022-11
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College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam
Abstract
The introduction of semi dwarf varieties (Oryza sativa L.) led to a massive increase in food production. This was possible due to identification of mutant gene called sd-1 which encodes for GA20ox enzyme, involved in biosynthesis of endogenous gibberellin (GA). Loss of function of this gene resulted in reduced GA level with visible phenotypic changes like reduced plant height, increased tiller number, increased harvest index and lodging resistance. Another strategy to achieve semi-dwarf varieties is to target GA2ox genes which are involved in degradation of bioactive GAs. Previous studies suggest that over expression of GA2ox genes results in reduced GA with phenotypic changes like increased tiller number, increased adventitious root growth and reduced plant height. Additionally, GA2ox gene family was one of the candidate gene identified for tiller number (TN) in a Meta-QTL analysis conducted using 1052 QTLs reported for yield related traits. Hence, for in silico and molecular characterization of GA2ox gene family in rice, three genes i.e., OsGA2ox5, OsGA2ox6, OsGA2ox7 were selected. In silico analyses using databases like Gramene, Oryzabase, RGAP revealed information regarding gene annotation and expression. Using tiller number (TN) data on a set of 199 genotypes from Indica panel of 3K rice germplasm grown across two seasons in lowland acidic soils and available sequence data from Rice SNP Seek database for the three genes marker-trait association was carried out. Statistical analyses (chi square and t-test) revealed that eleven and ten SNPs in OsGA2ox5 and OsGA2ox7, respectively significantly associated with TN at 0.05 level of significance, whereas no significant SNPs were identified for OsGA2ox6. A total of eight InDels (seven in intron of 5′ UTR and one in the gene) were identified in OsGA2ox5 and InDel 219243- 219255 lying at the 3′ end was significantly associated with TN at 0.1 level of significance (P=0.0856). Previously, role of intron lying in promoter in regulating gene expression has been reported. Therefore, InDels present in the 5′ UTR of OsGA2ox5 were evaluated further. The effect of these InDels in acting as binding site for cis acting regulatory elements (CRE), was analysed using NewPLACE and different CRE elements like TATABOX, BIHDIOS, WRKY71, HEXMOTIFTAH3H4, ASF1MOTIFCAMV, TGACGTVMAMY, ACGTATERD1, POLLEN1LELAT52 and INRNTPSADB were identified. Among these ASF1MOTIFCAMV and WRKY71 are involved in auxin/salicylic acid and GA hormonal pathways, respectively and can be studied further. Three haplotypes (based on combination of Indels present in 5′ UTR) Hap1, Hap2 and Hap7 showed significant association with TN. The SNP 219257 (P=0.0996) and the SNP 5970563 (P=0.0139) of OsGA2ox5 and OsGA2ox7, respectively, were targeted for designing ARMS-PCR primers (amplification refractory mutation system-polymerase chain reaction) to select individuals with high TN using gel-based assay. Molecular evaluation for candidate-gene based markers designed in the present study (targeting GA2ox5 and GA2ox7) and other SSR markers reported in previous studies was done by testing for parental polymorphism in parents i.e., Kasalath and UR29 contrasting for TN. Among the markers evaluated seven markers were polymorphic and these were further tested in 30 extreme progenies derived from above mentioned biparental population (Kasalath X
UR29). Among the 30 progenies, only one line carried the UR29 allele for markers GA2ox5FGR, GA2ox5R-AF and OsAAP. This could be due to segregation distortion and needs further confirmation by testing these markers on a larger population. The markers GA2ox7F-R, HvSSR02-14 and RM 12557 showed significant association with TN at 0.1 level of significance. Therefore, the newly designed marker GA2ox7F-R can be used for marker assisted selection of high tillering genotypes. Expression analysis for genes OsGA2ox5 and OsGA2ox7 in leaves of four contrasting rice genotypes at 20 days after germination was carried out by semiquantitative PCR. OsGA2ox5 was expressed in high tillering genotypes Kasalath and 2803 and also in low tillering genotype ARR but not in UR29. OsGA2ox7 was expressed only in high tillering genotype Kasalath. Further expression analysis can be done at different stages of tillering to study expression level of these selected genes. Due to occurrence of nucleotide variation, the 3′ end of OsGA2ox5 was cloned using TA cloning technique from two rice genotypes contrasting for TN. However, in these two genotypes no amplicon size difference was found. Role of intron present in 5′ UTR of OsGA2ox5 in gene expression, if any can be a part of future studies.
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Keywords
Rice, Genetic techniques