Identification of resistant starch related genes in rice by single segment substitution lines

Objective To discover the alleles for high resistance starch in starch-synthesis-related genes SBEIIb, SSIIa and ISA1.

Method The single segment substitution lines (SSSLs) carrying the starch-synthesis-related genes SBEIIb, SSIIa or ISA1 were detected using molecular markers. Then, the resistant starch contents of the SSSLs were measured using an improved AOAC method. Sanger sequencing and sequence alignment were performed to analyze the sequence variations of SBEIIb, SSIIa and ISA1 in different SSSLs. Through linkage analysis of genotypes and phenotypes, the alleles affecting resistance starch content were identified.

Result For SBEIIb gene, a single nucleotide polymorphism (SNP) (Ex4-96G/A) in the coding region results in an amino acid substitution (196-Arg/His), generating two alleles SBEIIb-1 and SBEIIb-2. SBEIIb-1 carrying the Ex4-96G causes Arg at 196th residue, which shows high-resistant starch content of 1.72%. For SSIIa gene, two SNPs (Ex8-334G/A and Ex8-865C/T) in the 8th exon cause two amino acid substitutions (604-Gly/Ser and 781-Leu/Phe), generating three alleles SSIIa-1,SSIIa-2 and SSIIa-3. SSIIa-1 carrying the Ex8-334G and Ex8-865C causes Gly and Leu at 604th and 781th residue respectively, which shows high-resistant starch content of 3.37%. One Indel (AGG/---) and one SNP (C/T) in ISA1 coding region sequence generate three alleles ISA1-1, ISA1-2 and ISA1-3. ISA1-1 carrying AGG-insertion and Ex17-117C causes Glu and Thr at 70th and 717th residue respectively, which shows high-resistant starch content of 2.09%.

Conclusion SBEIIb, SSIIa and ISA1 are key genes regulating resistant starch formation in rice. The SNPs and Indels in coding regions of the three genes lead to amino acid variations, which subsequently affects the resistance starch content. The three alleles SBEIIb-1, SSIIa-1 and ISA1-1 for high-resistant starch content are identified.