A Novel Regulatory Region for Amylose Synthesis in Rice Grains Identified by Systems Genetics Approach.

A deeper understanding of the genetics of rice grain starch structure is crucial in tailoring grain digestibility and ensuring cooking quality to meet consumer preferences. Significant association peaks on chromosomes 6 and 7 were identified through genome-wide association study (GWAS) of debranched starch structure from grains of a 320 indica rice diversity panel using genotyping data from the high-density rice array. A systems genetics approach that interrelates starch structure data from GWAS to functional pathways from a gene regulatory network identified known and novel genes with high correlation to the proportion of amylose and amylopectin. A novel SNP in the promoter region of Granule Bound Starch Synthase I (GBSS I) was identified along with seven other SNPs to form haplotypes that discriminate samples into different phenotypic ranges of amylose. A novel GWAS peak on chromosome 7 between LOC_Os07g11020 and LOC_Os07g11520 indexed by a non-synonymous SNP mutation on exon 5 of a bHLH transcription factor was found to elevate the proportion of amylose at the expense of reduced short-chain amylopectin. Linking starch structure with starch digestibility by determining the kinetics of cooked grain amylolysis of selected haplotypes revealed strong association of starch structure with estimated digestibility kinetics. Combining all results from grain quality genomics, systems genetics, and digestibility phenotyping, we propose novel target haplotypes for fine-tuning starch structure in rice through marker-assisted breeding that can be used to alter the digestibility of rice grain, thus offering rice consumers a new diet-based intervention to mitigate the impact of nutrition-related non-communicable diseases. A microarray experiment was conducted strictly following the One-Color Microarray-Based Gene Expression Analysis protocol using a custom 8 x 60K microarray slide for rice (Agilent,Germany). The cDNA synthesis and cRNA labelling were done using a single-color Low Input Quick Amp Labeling Kit. Microarray hybridization was conducted in a SureHyb chamber assembly using the large-volume Hi-RPM Gene Expression Hybridization Kit.Raw data were generated from the TIFF file using Agilent Feature Extraction software. The quality of the data was judged per batch using the PDF QC report and as a group using QC Validation software. Differentially expressed genes (DEGs) between haplotype 8 (highamylose) and haplotype 4 (waxy) were analyzed.The microarray data sample for haplotype8 highamylose are GQ00086,GQ00125,GQ00170 and haplotype4 waxy are GQ00205,GQ00212.