Natural Variation of OsNF-YC10 and Its Correlation with Grain Width in Rice

doi:10.16819/j.1001-7216.2025.240713

Abstract

Abstract:

【Objective】 Grain width is an important determinant of grain type and grain weight, which can affect rice yield and appearance quality. So far, a large number of genes have been found to affect grain width, but only a limited number of genes have breeding value because mutations of other genes often have negative collateral effects. Hence, it is of great significance to explore new grain width genes and uncover natural variations that have no negative effects on quality and yield. DR610 is a small-grain indica rice and Hagengdao 7 (HG7) is a large-grain japonica rice. 【Method】 In this study, a QTL-seq was performed using the extreme grain weight individuals of the DR610×HG7 F₂ population. Through the combined analysis of QTL-seq results and known genes affecting grain type, we found that a nonsense mutation of OsNF-YC10 in DR610 is likely to be the underlying genetic basis of its narrow grain. To determine the correlation between OsNF-YC10 variation and grain width, we analyzed the natural variation of OsNF-YC10 in more than 4000 rice germplasm accessions and its correlation with grain width. 【Result】 A total of 10 major OsNF-YC10 haplotypes were identified with HG7(Hap1) being the predominant haplotype (1464/1509) in japonica rice (1464 of 1509 accessions). The Hap5 (represented by DR610) contains a C(Hap^C)→T(Hap^T) nonsense mutation at nucleotide site 1048. The grain width of rice varieties with Hap^C (3.036 mm, n=1596) was significantly wider than that of Hap^T (2.938 mm, n= 309), indicating that this natural variation significantly affected the grain width. Further analysis showed that Hap1 and Hap5 are significantly differentiated between japonica and indica, with the latter mainly distributed in the low latitude regions. A molecular marker to distinguish C→T variants was developed using four tetra-primer ARMARMS-PCR, which can be used for marker-assisted selection of grain type. 【Conclusion】 In summary, this study determined the natural variation of OsNF-YC10, which is significantly correlated with the grain width. The finding is of importance to providing an important reference for breeding by design of grain type and weight in rice.

Key words: grain size, grain width, 1000-grain weight, QTL-seq, genotyping, tetra-primer ARMS-PCR

摘要：

【目的】水稻谷粒宽度是粒型和粒重的重要决定因子，可影响水稻产量和稻米外观品质。迄今已发现大量基因突变可影响粒宽，但因突变存在其他显著负效应，真正具有育种价值的基因不多，挖掘新的粒宽基因特别是对品质和产量没有负效应的自然变异具有重要意义。【方法】通过小粒型籼稻(DR610)和大粒型粳稻(哈粳稻7号，HG7)的F₂群体极端粒重个体的QTL-seq和已知影响粒型基因的联合分析，发现DR610的OsNF-YC10存在一个无义突变，这可能是其谷粒比HG7窄的原因。为探明OsNF-YC10变异与水稻粒宽的关系，我们对4000余份水稻种质中该基因的自然变异类型及其与粒宽的相关性进行分析。【结果】OsNF-YC10存在10种主要的单倍型，其中，以HG7为代表的单倍型(Hap1)为粳稻中的高频单倍型(1464/1509)；以DR610为代表的单倍型(Hap5)在第1048位核苷酸发生了一个C(Hap^C)→T(Hap^T)的无义突变，Hap^C型品种的粒宽(3.036 mm，n=1596)显著大于Hap^T型品种的粒宽(2.938 mm，n=309)，表明该基因的自然变异显著影响水稻粒宽。进一步分析发现，Hap1和Hap5单倍型存在明显的籼粳分化，后者主要分布在低纬度地区。最后，利用四引物ARMS-PCR，开发了一个区分C→T变异的分子标记，可用于粒型的分子标记辅助选择。【结论】本研究确定了OsNF-YC10的自然变异及其单倍型与粒宽的相关性，为粒宽的设计育种提供了重要参考。

关键词: 粒型, 粒宽, 千粒重, QTL-seq, 基因分型, 四引物 ARMS-PCR

CHEN Jiale, YU Qingtao, ZHENG Chenfan, WANG Qing, TAN Yuanyuan, CHEN Baicui, LI Chengxin, JIANG Meng, SHU Qingyao. Natural Variation of OsNF-YC10 and Its Correlation with Grain Width in Rice[J]. Chinese Journal OF Rice Science, 2025, 39(4): 552-562.

陈嘉乐, 于清涛, 郑琛凡, 汪庆, 谭瑗瑗, 陈百翠, 李承欣, 蒋萌, 舒庆尧. 水稻OsNF-YC10自然变异及其与谷粒宽度的相关性[J]. 中国水稻科学, 2025, 39(4): 552-562.

Figures/Tables 10

Table 1. PCR primers used in the present study

引物类型 Type of primer	引物序列(5’ to 3’) Primer sequence (5’ to 3’)	等位基因 Allele	产物长度 Size (bp)
扩增OsNF-YC10全长部分
Forward primer	CTGTGGGACTGTAAGATCATCTATG		1466
Reverse primer	CTTGTTGCCATTACTGGTGCTTGG		1466
Tetra-primer ARMS-PCR
Forward outer primer	TAGCCCTGCGGTTCTTGCAAGTATGA
Reverse outer primer	AATCCTGGTGTCCGAAACATCATCCT
Forward inner primer	TGCTGCTACAAAGGTTTTcAGTGcCt	T	241
Reverse inner primer	GTTTCTTTCTCATGTTGTTCTTCaTg	C	361

Fig. 1. Grain traits of HG7, DR610 and their F1 A and B, Grain phenotype, bar = 1 cm; C-F, Grain length, grain width, grain length-to-width ratio, and 1000-grain weight. Values are mean ± SD (n = 3). Different letters indicate significant differences at P < 0.05(analyzed by one-way ANOVA followed by Tukey’s multiple comparison test).

Fig. 2. Frequency distribution of DR610×HG7 F2 plants with different 1000-grain weight

Table 2. Statistics of whole-genome sequencing results of DR610, HG7 and their F2 large grain mixing pool (LGMP)

样品名称 Sample	总读序数 Total reads	比对率 Alignment (%)	基因组覆盖率 Genome coverage (%)	测序深度 Average depth (×)
HG7	85 448 290	90.70	97.47	30
DR610	113 535 356	98.54	92.73	43
LGMP	99 534 914	96.99	99.00	37

Table 3. Genomic regions and number of annotated genes, in which four QTLs of grain weight were mapped using QTL-seq of large grain F2 plants of DR610×HG7

染色体 Chr	开始 Start	结束 End	大小 Size (Mb)	基因数目 Number of genes
1	12 931 694	13 481 021	0.55	28
1	13 645 077	13 688 772	0.04	0
1	13 705 131	13 820 507	0.12	8
3	23 567 245	24 695 746	1.13	86
Total	-	-	1.84	122

Fig. 3. QTL-seq for rice grain QTL mapping and candidate gene identification The SNP-index plot (A) and InDel-index plot (B) revealed by QTL-seq, the red arrows indicate the candidate regions of grain QTLs. (C) The reported grain shape genes located on chromosomes 1 and 3. Genes highlighted in red indicated coding sequence(CDS) variation between DR610 and HG7. The gene (OsNF-YC10) marked with yellow background coincided with the QTL regions on panels A and B.

Fig. 4. Number of plants with different OsNF-YC10 genotypes (based on the 13789514 SNP) having extreme large (A) and extreme small grains (B) in the DR610×HG7 F2 population, and grain width of F2 plants with different OsNF-YC10 genotypes (C)

Fig. 5. Haplotype analysis of OsNF-YC10 in rice germplasm collections A, Pairwise r2 values (a measure of linkage disequilibrium) among all polymorphic sites in OsNF-YC10 coding sequence (CDS). The darkness of the color of each box corresponds to the r2 value, as shown in the legend. B, Haplotypes (Hap) of the OsNF-YC10 CDS region among 4726 rice accessions. C, Haplotype network analysis of OsNF-YC10. D, Grain width comparison of different OsNF-YC10 haplotypes from about 2000 rice germplasm accessions. Different letters indicate significant differences at P < 0.05(analyzed by one-way ANOVA followed by Tukey’s multiple comparison test).

Fig. 6. Global distribution of rice accessions of two major OsNF-YC10 haplotypes A, Geographical distribution of rice accessions with different OsNF-YC10 haplotypes. B and C, Box plot showing the distribution of rice accessions according to longitude (B) and latitude (C), respectively. ** indicates significant differences at P < 0.01(by Mann-Whitney U test).

Fig. 7. Genotyping of the SNP at nucleotide position 1048 of OsNF-YC10 using a tetra-primer ARMS-PCR

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