A Putative Gene <em>sbe3-rs</em> for Resistant Starch Mutated from <em>SBE3</em> for Starch Branching Enzyme in Rice (<em>Oryza sativa</em> L.)

<div><p>Foods high in resistant starch (RS) are beneficial to prevent various diseases including diabetes, colon cancers, diarrhea and chronic renal or hepatic diseases. Elevated RS in rice is important for public health since rice is a staple food for half of the world population. A <em>japonica</em> mutant ‘Jiangtangdao 1’ (RS = 11.67%) was crossed with an <em>indica</em> cultivar ‘Miyang 23’ (RS = 0.41%). The mutant <em>sbe3-rs</em> that explained 60.4% of RS variation was mapped between RM6611 and RM13366 on chromosome 2 (LOD = 36) using 178 F₂ plants genotyped with 106 genome-wide polymorphic SSR markers. Using 656 plants from four F_3∶4 families, <em>sbe3-rs</em> was fine mapped to a 573.3 Kb region between InDel 2 and InDel 6 using one STS, five SSRs and seven InDel markers. <em>SBE3</em> which codes for starch branching enzyme was identified as a candidate gene within the putative region. Nine pairs of primers covering 22 exons were designed to sequence genomic DNA of the wild type for <em>SBE3</em> and the mutant for <em>sbe3-rs</em> comparatively. Sequence analysis identified a missense mutation site where Leu-599 of the wild was changed to Pro-599 of the mutant in the <em>SBE3</em> coding region. Because the point mutation resulted in the loss of a restriction enzyme site, <em>sbe3-rs</em> was not digested by a CAPS marker for <em>Spe</em>I site while SBE3 was. Co-segregation of the digestion pattern with RS content among 178 F₂ plants further supported <em>sbe3-rs</em> responsible for RS in rice. As a result, the CAPS marker could be used in marker-assisted breeding to develop rice cultivars with elevated RS which is otherwise difficult to accurately assess in crops. Transgenic technology should be employed for a definitive conclusion of the <em>sbe3-rs</em>.</p> </div

Sequence confirmation of the PCR products.

<p>A: Nucleotide ‘T’ of the wild type was substituted by ‘C’ of the mutant Jiangtangdao 1; B: The substitution resulted in a missense from Leu-599 of the wild in <i>SBE3</i> coding region for 599 amino acid to Pro-599 of Jiangtangdao 1 in <i>sbe3-rs</i> coding for high resistant starch; and C Because the point mutation resulted in the loss of a restriction enzyme site <i>Spe</i>I, <i>sbe3-rs</i> of the mutant was not digested with <i>Spe</i>I, while <i>SBE3</i> of the wild type was (M: marker DL2000; Lane 1–2: wild type before and after digestion; and lane 3–4: Jiangtangdao 1 before and after digestion).</p

Primers used for sequencing the putative gene <i>SBE3</i> (LOC_Os02g32660).

<p>Primers used for sequencing the putative gene <i>SBE3</i> (LOC_Os02g32660).</p

Genotypes of four recombinant F₃ individuals for putative resistant starch <b><i>sbe3-rs</i></b> flanked by RM6611 and RM13366: A – ‘Miyang 23’, B – ‘Jiangtangdao 1’ and H – heterozygous.

<p>Genotypes of four recombinant F₃ individuals for putative resistant starch <b><i>sbe3-rs</i></b> flanked by RM6611 and RM13366: A – ‘Miyang 23’, B – ‘Jiangtangdao 1’ and H – heterozygous.</p

Schematic representation of the region on rice chromosome 2 harboring <i>sbe3-rs</i>.

<p>A: High-resolution genetic map built with eight SSRs and a STS markers: numbers between the markers indicate genetic distance (cM), and the number of recombinants was among extreme low RS content plants. B: Physical maps between SSR marker RM8254 and RM 13313. C: Recombinant genotypes and their corresponding phenotypes. +, indicates RS content segregating in F₄ line; − indicates no segregation in F₄ line. <>\raster(85%)="rg2"<>: Genotypes between two flanking markers are heterozygotes; □: Break point of the recombination; <>\raster(85%)="rg1"<>: Miyang 23 genotype and ▪: Jiangtangdao 1 genotype.</p

Physical location and domain structure of SBE3 gene.

<p>A: Diagram of genomic interval within the target region on chromosome 2 of Nipponbare. B: Conserved domain of predicted candidate gene <i>SBE3</i> (LOC_Os02g32660). E_Set superfamily: early set domain associated with the catalytic domain of sugar utilizing enzymes; AmyAc_Family superfamily: alpha amylase catalytic domain; Alpha-amylase_C: alpha amylase, C-terminal all-beta domain; and ▴: Mutation site.</p

Genetic linkage map of rice resistant starch on chromosome 2 for <i>sbe3-rs</i>, genetic distance (Kosambi, centiMorgan) and SSR marker name on the left and right of the chromosome, respectively and highlighted region for the estimated position of <i>seb3-rs</i>.

<p>Genetic linkage map of rice resistant starch on chromosome 2 for <i>sbe3-rs</i>, genetic distance (Kosambi, centiMorgan) and SSR marker name on the left and right of the chromosome, respectively and highlighted region for the estimated position of <i>seb3-rs</i>.</p

Verification of the CAPS marker using a part of F₂ individuals.

<p>Verification of the CAPS marker using a part of F₂ individuals.</p