Crops ›› 2023, Vol. 39 ›› Issue (4): 22-30.doi: 10.16035/j.issn.1001-7283.2023.04.004

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Phenotypic Analysis and Map-Based Cloning of Chalkiness and FlouryEndospermMutantcsein Rice (Oryza sativa L.)

Hua Qin1(), Lin Quanxiang1, Song Yuanhui1, Sun Jiameng1, Zhang Zupu2,3, Chen Qingquan1, Li Jincai1, Zhang Haitao1()   

  1. 1College of Agronomy, Anhui Agricultural University, Hefei230036, Anhui, China
    2Jiangsu Hongqi Seed Co., Ltd., Taizhou225311, Jiangsu, China
    3Jiangsu Province (Hongqi) Engineering Research Center for Rice and Wheat Seed Breeding, Taizhou225311, Jiangsu, China
  • Received:2022-03-09 Revised:2022-05-28 Online:2023-08-15 Published:2023-08-15

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Abstract:

It is helpful to clarify the genetic regulatory mechanism of starch and storage protein biosynthesis related to quality and promote rice quality breeding by using mutants related to rice quality. The chalkiness and shrunken endosperm mutantnamed cse was obtained by screening a mutant library of tissue culture of Zhonghua 11, and the phenotype identification and physicochemical properties analysis of grains were carried out. Analysis of genetic behavior of cse and map-based cloning of target gene by F2 population. qRT-PCR was used to analyze the spatiotemporal expression pattern of relatedgenes.Compared with the wild type, the mutant showed significantly different in agronomic traits and starch physicochemical characteristics. The mutant starch granules were irregularly spherical with large interspaces. Genetic analysis showed that the mutant trait was controlled by a pair of recessive nuclear genes. Map-based cloning revealed that cse is located on the long arm of chromosome 4 between Os4-14-8 and Os4-15 with a physical interval of 40kb. Sequencing results showed that only one candidate gene LOC_Os04g55230 encoding TPR protein had mutation in the mapping interval. The deletion of three bases GGC in the first exon led to the deletion of one glycine, and the substitution of one base G in the fourteenth exon for base A led to the mutation of arginine to lysine. The results of qRT-PCR showed that the expression level of LOC_Os04g55230 reached the maximum in endosperm 14 days after fertilization.The mutant LOC_Os04g55230was a new allele of the reported gene OsFLO2/OsCNY8, but the phenotype of the mutant was not completely same as the known OsFLO2 mutant. The cse grains had shrunken appearance, and the effective tiller number, storage protein and fatty acid content were significantly increased.

Key words: Rice(Oryza sativa L.), Floury endosperm, Starch granules, Physicochemical characteristic, Map-based cloning

Table 1

Information of primers"

名称Name 类型Type 正向引物序列（5′-3′）Forward primer sequence (5′-3′) 反向引物序列（5′-3′）Reverse primer sequence (5′-3′)
Os4-10 定位 ACCTTTTCTTGGCTTGAGGG GCTTTTGCTACTTTTGGGGG
Os4-11 定位 ACGTCCATGTCGGTGTACG CCACCACCTCTACTTCTTCAGTG
Os4-12 定位 ACGGAAACGTAGGTGGTCTG ACCCGCTAGCTCTTCGATCT
Os4-13 定位 TTGGTTGCTTCCTCCCATAC GGCATTGTACGACGGATCTC
Os4-14 定位 ACTGTGGAGTACAGGTCGGC GAAACGGAAACGAAACCCTC
Os4-15 定位 CCGCTACTAATAGCAGAGAG GGAGCTTTGTTCTTGCGAAC
Os4-10-13 定位 TCGAAGGACCTTGTGTGATTCC TAGCTATGTTTGCCGAATATTG
Os4-12-11 定位 CAGGTCAAAACTCCTCACGC TGCAGAGTGGCTTTCAATGG
Os4-13-16 定位 CTCGAAAGTATAGACAGACAGCT ACTACTCCTCCCTCTCGTGA
Os4-14-8 定位 GTATTATGCTAACGTCAGCATG CTAGGTGTCAGCATGGTACTG
Os4-15-5 定位 CACCGAATCACCTAAGAATAG CTGAATTCACTAATTCTGTCTC
Primer 1 实时荧光定量PCR CATTCTGAAGGATGAAGTGGTTC ACTCAGACATCGCTAGTACTCAG
Ubiquitin 实时荧光定量PCR GCCCAAGAAGAAGATCAAGAAC GACTTTTATGCTTCCGTTGTTATCT

Table 2

Comp arison of agronomic traits between the wild type and cse"

农艺性状
Agronomic trait		 野生型
Wild type		 突变体
cse
株高Plant height(cm) 110.20±1.88 101.00±1.06**
主茎穗长Main panicle length (cm) 23.84±0.34 21.94±0.56*
第1节间长
The first internode length (cm)		 43.31±0.85 41.53±0.40
第2节间长
The second internode length (cm)		 22.40±0.40 19.62±0.56
第3节间长
The third internode length(cm)		 13.64±0.75 9.61±1.05**
第4节间长
The fourth internode length (cm)		 7.50±0.95 4.51±0.46**
有效分蘖数Effective tiller number 10.02±0.63 14.83±0.43**
一次枝梗数Primary branch number 13.33±1.05 13.40±0.91
二次枝梗数Secondary branch number 43.57±3.33 31.25±2.32**
穗粒数Grain number per panicle 243.88±22.03 172.12±14.21**
粒长Grain length(mm) 7.83±0.04 7.30±0.07*
粒宽Grain width (mm) 3.70±0.10 3.37±0.04**
粒厚Grain thickness (mm) 2.33±0.09 2.03±0.05**
千粒重1000-grain weight (g) 28.95±0.05 18.28±0.10**

Fig.1

Phenotypic characterization of wild type and thecse (a) Plant morphology at heading stage; (b) Mature internodes of ZH11 (left) and cse (right); (c) Panicle morphology at ripening stage; (d) Grain size; (e) Mature grain appearance of ZH11 (outside) and cse (inside); (f) Cross-sections of mature grains. ZH11: Zhonghua 11"

Fig.2Dry

matter accumulation in wildtype and cse at different stages “**”indicates extremely significant difference at P< 0.01 level, the same below"

Fig.3Iodi

ne staining and SEM observation of mature endosperm between wild type and cse a, b: endosperm staining comparison between the wildtype and cse; c, d: SEM observation of mature endosperm of wildtype and cse"

Table 3Phys

icochemical properties comparison between the wild type and cse"

指标Index 野生型Wild type 突变体cse
蛋白质Protein (%) 9.31±0.18 11.29±0.21**
总淀粉Total starch (%) 83.70±0.33 73.90±0.28**
直链淀粉Amylose (%) 17.65±0.48 8.81±0.25**
可溶性糖Soluble sugar (%) 4.64±2.38 6.21±0.57**
脂肪酸Fatty acid (%) 14.13±0.77 25.96±0.38**
垩白度Chalkiness (%) 17.23±0.24 76.84±2.30**
碱消值Alkali spreading value 8.06±0.27 7.64±0.08**
胶稠度Gel consistency (mm) 58.03±0.35 64.52±0.30**

Table 4Ther

mal parameters of the wild type and cse"

材料
Material		 起始温度
T0 (℃)		 峰值
TP (℃)		 结束温度
TC (℃)		 焓变
?H (J/g)
野生型
Wild type		 66.60±0.65** 71.96±0.45** 76.09±0.29** 7.18±0.83**
cse 61.98±0.54 68.31±0.43 73.76±0.50 5.15±0.30

Fig.4

The swollen volume of wild type and cse starch in urea solutions of various concentrations (a) The swollen volume of wildtype and cse starch in urea solutions of various concentrations; (b) The swollen volume of wild-type and cse starch in urea solutions of various concentrations"

Table 5

Genetic analysis of mutant gene cse"

年份
Year		 调查总株数
Total number of
plants investigated		 野生型表型个数
Number of wild
type phenotypes		 突变体表型个数
Number of mutant
phenotypes		 χ2
2020 212 168 44 1.81
2021 743 567 176 0.68

Fig.5

Fine mapping of cse (a)-(d) The numbers beneath the bold lines represented the recombinants identified by the corresponding markers. The red arrow box is the target gene; (e) A schematic representation of the cse gene, the boxes represent the deletion and replacement of bases in exons"

Table 6

Anno tated genes in the fine mapping range"

开放阅读框ORF 基因名称Gene name 注释功能Putative function
1 LOC_Os04g55180 含有α/β折叠家族结构域的蛋白质
2 LOC_Os04g55190 含SPOC结构域的蛋白质
3 LOC_Os04g55200 涂层亚基
4 LOC_Os04g55210 氯通道蛋白
5 LOC_Os04g55220 含有C2结构域的蛋白质
6 LOC_Os04g55230 含有四肽重复结构域的蛋白质

Fig.6

Phylogenetic analysis of CSE (LOC_Os04g55230) and CSE-like proteins"

Fig.7

Wildtype expressionanalysisof CSE in different tissues and endospern of different growth periods"

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