Crops ›› 2022, Vol. 38 ›› Issue (4): 14-21.doi: 10.16035/j.issn.1001-7283.2022.04.003

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Identification and Fine Mapping of Sugary Endosperm Mutant m5788 in Rice (Oryza sativa L.)

Zheng Siyi1(), Yang Ye1, Song Yuanhui1, Hua Qin1, Lin Quanxiang1, Zhang Haitao1, Cheng Zhijun2()   

  1. 1College of Agriculture, Anhui Agricultural University, Hefei 230036, Anhui, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2021-10-06 Revised:2021-12-08 Online:2022-08-15 Published:2022-08-22
  • Contact: Cheng Zhijun E-mail:2363085742@qq.com;chengzhijun@caas.cn

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Abstract:

Starch synthesis in endosperm development determines the appearance and flavor quality of rice grains. m5788 is a shriveled grain sweet embryo mutant derived from the tissue culture progeny of Japonica var. Zhonghua 11. The 1000-grain weight and the number of grains per panicle decreased significantly. Due to the blocking of starch synthesis, the level of soluble sugar increased significantly. Genetic analysis revealed that m5788 was determined by a pair of recessive nuclear genes. Linkage analysis localized the candidate gene to the 110kb region flanked by indel markers of Z8-25.8 and Z8-25.9 on chromosome 8 in 569 F2 mutant individuals. In this region there was a putative isoamylase encoding the gene LOC_Os08g40930, which was 82.2% homologous to maize Sugary 1 and was involved in the DBE pathway of the starch debranching enzyme ISA1. The sequencing results showed that no genomic sequence change occurs in LOC_Os08g40930 between wild type and m5788. However, the qRT-PCR analysis showed that the decrease in the expression level of the gene LOC_Os08g40930 in the mutant m5788 was presented together with the expression level of the coding-gene of pullulanase-type in same DBE pathway. All results suggest that isa1 carried by m5788 is a novel allele.

Key words: Rice, Endosperm development, Fine mapping, Starch synthesis

Table 1

New developed polymorphic Indel and SNP primers used for fine mapping"

标记Marker 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
Z8-25.6 CTCGTGCGCGCGCACCGTTGG GCAGATGGATGGGACGGAGT
Z8-25.7 TTTGGGGCTGGAGCACCTTG CATCATAGAATGTGCTGA
Z8-28.8 TTTTTCTTCTTCTCATGCCTT TTTAGCTTCTCTTCCATTCCA
Z8-25.9 GAAGAGAGCTGATTCAGGAGAGAGC ATAGTTAGGCAGCAACAGCAACG
Z8-26.2 ATGATGGTCCATAAGAGTCG CAGTGTTCAAGATGGTCATTG

Table 2

Primers used for qRT-PCR"

引物Primer 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
ISA1 GTACTTCGATGTCTCCAATGTCG ATACTCTCCTCGGCTTATCACTG
PUL CCTAAAGCTTACTACCCATGCAT ACATGTCCTTGTCTCCACTTCTT

Fig.1

Agronomic traits comparison between wild type and the mutant m5788 (a) Plant at maturity stage; (b) Plant height components (left: wild type, right: m5788); (c) Grain appearance (Zhonghua 11: two circles outside; m5788: two circles inside); (d) Grain width; (e) I2-KI dyeing"

Table 3

Comparison of agronomic traits between wild type and the mutant m5788"

农艺性状
Agronomic trait		 野生型
Wild type		 突变体
Mutant
株高Plant height (cm) 117.0±2.8 100.2±2.7**
分蘖数Tiller number 9.5±2.1 12.6±1.9*
有效穗数Effective tiller number 9.3±1.7 13.1±2.7**
穗长Panicle length (cm) 24.2±1.3 20.6±1.0**
一次枝梗数Number of primary branches 14.0±1.8 12.9±1.1
二次枝梗数Number of secondary branches 45.8±4.0 43.0±6.6
穗粒数Grain number per panicle 278.0±45.8 184.0±12.3*
结实率Seed setting (%) 97.0±0.0 95.0±2.0
千粒重1000-grain weight (g) 29.6±1.95 15.6±0.87**

Table 4

Comparison of quality index between wild type and the mutant %"

品质指标Quality index 野生型Wild type 突变体Mutant
水分Moisture 9.99±0.0011 9.89±0.0004
蛋白质Protein 5.67±0.0005 7.58±0.0012**
直链淀粉Amylose 19.26±0.0035 19.62±0.0008*
脂肪酸Fatty acid 14.67±0.0113 16.19±0.0092**
胶稠度Gel consistency 50.63±0.0073 35.97±0.0227**

Fig.2

Comparison of soluble sugar content between wild type and mutant at different stages “**”represents extremely significantly different at P < 0.01"

Fig.3

Schematic diagram of map-based cloning on the candidate gene M5788 (a) rough mapping of the gene M5788 flanked by two markers Z8-24 and Z8-26.2, (b) M5788 was finely mapped to a 110kb interval delimited by two markers of Z8-25.8 and Z8-25.9, (c) the structure of target gene LOC_Os08g40930"

Table 5

Gene annotations in the mapping interval"

基因Gene 基因注释Gene annotation
LOC_Os08g40830 含有pumilio结构域的蛋白PPD1
LOC_Os08g40890 与精胺合酶有关
LOC_Os08g40940 表达蛋白
LOC_Os08g40840 胞外复合亚基EXO70
LOC_Os08g40910 表达蛋白
LOC_Os08g40950 表达蛋白
LOC_Os08g40850 线粒体载体蛋白
LOC_Os08g40919 表达蛋白
LOC_Os08g40860 RNA假尿苷合酶
LOC_Os08g40930 α淀粉酶,含有蛋白质的催化结构域
LOC_Os08g40990 受体蛋白激酶1
LOC_Os08g40870 五肽重复蛋白
LOC_Os08g40880 含有蛋白质的RNA识别基序
LOC_Os08g40900 生长素响应因子

Fig.4

Phylogenetic tree of LOC_Os08g40930 homologs GRMZM2G138060, GRMZM2G150796, and GRMZM2G090905 are maize genes, Bradi3g40410, Bradi4g32707, and Bradi2g26170 are wheat genes, and AT2G39930 and AT1G03310 are Arabidopsis genes, LOC_Os05g32710 and LOC_Os09g29404 are rice genes"

Fig.5

Comparison of expression levels of OsISA1 and OsPUL in wild type and mutant"

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