Phenotypic Analysis and Genetic Mapping of a Rice Floury Endosperm Mutant

doi:10.13560/j.cnki.biotech.bull.1985.2025-0670

Abstract

Abstract:

Objective Rice floury endosperm mutants, which show an opaque endosperm phenotype due to abnormal starch or protein synthesis and accumulation, serve as ideal genetic materials for studying endosperm development and its regulatory networks. This study is aimed to conduct grain phenotypic analysis and mutant gene mapping of the floury endosperm mutant M97. Method The starch granule morphology, starch chain length distribution and physicochemical properties of mutant M97 were analyzed using scanning electron microscopy, fluorescence-assisted capillary electrophoresis, X-ray powder diffractometer, and rapid viscosity analyzer. Additionally, the mutated gene of M97 was identified via the MutMap method combined with SNP/Indel filtering. Result The whole cross-section of Kitaake grain was transparent, and the central area of M97 endosperm showed floury and opaque, and the periphery remained transparent. The grain length, width and thickness of M97 brown rice were significantly lower than those of the wild type, resulting in a decrease in its 100-brown rice weight. Observation via scanning electron microscopy showed that the starch granules in the central area of M97 endosperm were loose with large gaps, while those in the peripheral region were similar to those of the wild type, showing tightly arranged and regular polygonal starch granules. Compared with the wild type, the total starch content of M97 decreased significantly, the soluble sugar content increased significantly, and the apparent amylose content decreased significantly. Both M97 and wild-type starch belonged to A-type crystals, but the relative crystallinity of M97 significantly increased. The starch of M97 showed increased swelling power, decreased water solubility, higher peak viscosity and breakdown values, and lower hot paste viscosity and final viscosity. The mutant gene was mapped to chromosome 2 by the MutMap method. SNP filtering identified a T-to-C substitution in the third exon of the BT1 gene, leading to the substitution of isoleucine (Ile) at position 322 with threonine (Thr). Moreover, the KASP marker verified that this mutation site co-segregated with the floury endosperm phenotype. Homologous protein sequence analysis of BT1 showed that isoleucine was highly conserved across species. Conclusion This study identified a new allelic mutant of the BT1 gene and investigated the effects of a single amino acid substitution on rice endosperm morphology and starch properties, laying a foundation for further study on the mechanism of BT1 participating in regulating rice starch synthesis and providing insights for rice quality improvement.

Key words: rice, floury endosperm, BT1, starch characteristics, endosperm development

WANG Cheng-cheng, HUANG Tian-yu, ZHANG Qing, SHI Lai-quan, FANG Hui-min, ZHANG Long. Phenotypic Analysis and Genetic Mapping of a Rice Floury Endosperm Mutant[J]. Biotechnology Bulletin, 2026, 42(2): 169-177.

Figures/Tables 6

Fig. 1 Appearance phenotype and components of brown riceA, B: Brown rice. C, D: Cross-section of brown rice. E-H: Quantification of brown rice length (E), width (F), thickness (G), 100-brown rice weight (H). I: Total starch content. J: Soluble sugar content. Scale bars, 1 mm. The data were analyzed by the t-test, *P<0.05, **P<0.01, the same below

Fig. 2 Morphologies of starch granulesA-F: Scanning electron microscope photograph of starch granules. Scale bars, 10 μm

Fig. 3 Determination of starch chain length distribution and functional propertiesA: Iodine absorbance spectra. B: Chain length distribution of amylopectin. C: X-ray diffraction patterns of starches. D: RVA patterns of the starch. RC: Relative crystallinity

Table 1 Swelling power, water solubility and pasting parameters of starches

Genotype	Watering solubility	Swelling power	RVA parameters of the starch （mPa/s）
Genotype	Watering solubility	Swelling power	PV	HV	BV	FV	SV
WT	6.2±0.1	17.2±1.0	2 480±9	2 393±9	87±18	2 863±2	470±7
M97	5.5±0.1**	24.5±1.3**	2 568±53	2 205±44*	363±9**	2 668±41*	463±3

Fig. 4 Mapping of the mutated gene in M97A: Flowchart of MutMap method for locating mutant gene. B: SNP/Indel-Index filtering principle of candidate sites. C. Candidate gene structure. The 965th base with T is replaced by C. D: Sequence chromatograms of the mutation site. The 322nd amino acid Ile is replased with Thr, and the mutation site is marked with a red box. E: Co-separation analysis was performed using KASP marker

Fig. 5 Amino acid sequence alignment of BT1 protein mutation region in different species

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