Functional Characterization And Over-expression Of Gh?A07G1537 Gene To Improve Fiber Quality In Cotton

The precise information about candidate genes involved in complex quantitative trait is a major obstacle in the improvement of fiber quality of cotton.As a result,the overall genetic gain in conventional phenotypic selection is low.In recent years,advancements in large-scale genome sequencing have led to the development of high-throughput molecular markers,quantitative trait loci(QTL)fine mapping and single nucleotide polymorphisms(SNPs).The genetic identification has resolved the existing bottlenecks in trait-specific breeding,and molecular interventions have helped to explore the genes involved in cotton fiber length,fiber strength,and other quality parameters with improved characteristics.The chromosome segment substitution lines(CSSLs)are considered an ideal and efficient material for QTL analysis,fine mapping,and map-based cloning.Just similar to other existing studies,the significant chromosome 7 carrying SNPs for effect-stable QTLs related with fiber length was identified in this study,which provides important reference information for the further targeted research on synchronously improving cotton yield and fiber quality.Based on the previous results,the function of Gh?A07G1537,a key candidate gene for regulating cotton fiber length and identified from RNA-seq data of a superior CSSL MBI7747,was further verified in this study.The recombinant vector of Gh?A07G1537 derived from the MBI7747 line was over-expressed in a genotype CCRI24 with a low profile of fiber quality parameters through Agrobacterium-mediated transformation.After shedding,cotton seeds were sterilized,of which germination index was recorded as 63.33%.The isolated embryos were incised and co-cultivated with Agrobacterium culture harboring gene of interest in MS medium.Putative transformants were selected on MS medium containing hygromycin(25 mg/m L),acclimatized,and shifted to a greenhouse for further growth and proliferation.Transgene integration was validated through Polymerase Chain Reaction(PCR)and Southern blot analysis.Stable integration of the transgene(?Gh?A07G1537)was validated by tracking its expression in different generations(T₀,T₁,and T₂)of transformed cotton plants.The presence of significant In Del of 55 bp and an SNP was detected in the sequencing of the genotypes of three materials,and these findings strengthen the hypothesis of the study and supported the previous findings.PCR and Southern blot analysis proved the 1314 bp of full CDS length of this candidate gene.The preparation of gene construct and restriction digestion analysis confirmed the integration of the gene into plant expression vector p CAMBIA2300.The detection of the gene using full length and short length primers confirmed the presence of the gene with the size of 1314 bp and 128 bp respectively.It was found to be 2.97-,2.86-and 2.92-folds higher expression in T₀,T₁,and T₂plants respectively of transgenic compared with non-transgenic cotton plants by real time PCR.Fiber quality parameters such as fiber length were observed to be improved by 4.4%(30.1mm to 31.5 mm),fiber strength by 3.0%(32.2 c N/tex to 33.2 c N/tex),and uniformity index by 4.2%(82.6%to 86.1%),compared to those in the non-transgenic plants,whereas micronaire value was found to be improved by7.7%(4.3 to 4.0),and boll weight by 12%(5.12g to 4.6g)in the engineered cotton line than the non-engineered cotton.In addition,the transformation efficiency of Agrobacterium-mediated method was counted to be 1.48%.Genetic modifications of Gh?A07G1537 support the improvement in fiber quality parameters and should be appreciated for the textile industry.The current findings would be a great prospect for the researchers to exploit the gene Gh?A07G1537 under various environments incorporated with the advanced gene editing technology to improve the fiber quality parameters in cotton.