Association Mapping of Quantitative Trait Loci for Grain Size in Introgression Line Derived from Oryza rufipogon

doi:10.1016/j.rsci.2020.04.007

Abstract

Abstract:

Grain size is one of the critical agronomic traits governing grain yield and quality in rice. However, the underlying genetic mechanisms that control grain size in rice are poorly understood. We used an introgression line derived from Zhonghui 8015 and Oryza rufipogon Griff. This introgression line was evaluated under two different environmental conditions to dissect the quantitative trait loci controlling grain size. Genome-wide association study (GWAS) was performed using 28 193 SNPs through a general linear model, and 56 significant SNPs on different loci associated with the 4 grain size traits were detected. Cloned genes including GS3 and qGL3 showed substantial effects on grain length and size. Seven new stable loci were identified with pleiotropic effects on grain size. Haplotype, gene expression analyses, combined gene-based associations, and functional annotations permitted the shortlisting of important dominant genes including GS3 and qGL3.

Key words: grain size, association mapping, Oryza sativa, Oryza rufipogon, quantitative trait locus, genome-wide association analysis

Hussain Kashif, Yingxing Zhang, Anley Workie, Riaz Aamir, Abbas Adil, Hasanuzzaman Rani Md., Hong Wang, Xihong Shen, Liyong Cao, Shihua Cheng. Association Mapping of Quantitative Trait Loci for Grain Size in Introgression Line Derived from Oryza rufipogon[J]. Rice Science, 2020, 27(3): 246-254.

Figures/Tables 12

Fig. 1. Schematic presentation of material construction for association mapping.

Table 1 Distributions of markers on 12 chromosomes.

Chromosome	No. of markers	Size (Mb)	Spacing (kb)
1	1007	43.2	15.5
2	757	35.8	15.8
3	742	36.4	17.4
4	876	35.4	18.1
5	625	29.7	17.1
6	811	31.2	16.3
7	632	29.6	18.7
8	458	28.3	15.3
9	397	22.9	16.8
10	211	23.2	15.3
11	301	29.0	15.5
12	256	27.5	17.7

Fig. 2. Phenotypic comparisons of two parents for grain size related traits. A and D, Grain length (GL) of PB100 (Upper) and ZH8015 (Lower). B and E, Grain width (GW) of PB100 (Upper) and ZH8015 (Lower). C, Panicles of PB100 (Left) and ZH8015 (Right). F, 1000-grain weight (TGW). G, Grain length and width ratio (GLWR).Data are Mean ± SE (n = 3). The asterisks represent statistical significance between ZH8015 and PB100, determined by the Student’s t-test (**, P < 0.01).

Table 2 Phenotypic variations for grain traits across two environments.

Trait	Environment	Mean ± SE	Range	H²_B (%)	G × E
GL (mm)	Lingshui	9.46 ± 0.04	8.60-10.40	56.3	***
	Hangzhou	9.61 ± 0.04	8.80-10.40	56.3	***
GW (mm)	Lingshui	3.42 ± 0.01	3.10-3.70	53.6	***
	Hangzhou	3.17 ± 0.02	2.90-3.80
GLWR	Lingshui	2.77 ± 0.01	2.50-3.30	49.2	***
	Hangzhou	3.05 ± 0.02	2.50-3.50	49.2	***
TGW (g)	Lingshui	36.40 ± 0.24	30.20-42.20	49.9	***
	Hangzhou	32.84 ± 0.22	26.90-40.30	49.9	***

Supplemental Fig. 1. Frequency distribution and variation of grain shape related traits in introgression lines.GL, Grain length; GW, Grain width; GLWR, Grain length and grain width ratio; TGW, 1000-grain weight.Brown arrow indicate parent PB100 (small grains) and blue arrow indicate ZH8015 (Large grain).

Fig. 3. Correlation analyses of four grain size traits.GL, Grain length; GW, Grain width; GLWR, Grain length-width ratio; TGW, 1000-grain weight. Top panel represent Lingshui-2017 and lower panel represent Hangzhou-2018. *, ** and *** represent the coefficient is signiﬁcant at the 0.05, 0.01 and 0.001 levels, respectively.

Table 3 Fifty-six SNPs on rice chromosome 3 significantly associated with grain size in GWAS analyses.

Trait	Position	Allele	MAF	P value	R²(%)	Trait	Position	Allele	MAF	P value	R²(%)
GL	16 733 441	A/G	0.33	1.56E-15	45.74	GLWR	9 189 010	T/C	0.40	2.83E-11	44.24
	16 734 121	A/G	0.33	3.29E-15			9 272 680	A/C	0.40	6.07E-11
	9 342 017	A/T	0.40	9.57E-15			25 042 825	T/C	0.40	6.07E-11
	9 228 066	T/G	0.39	1.52E-14			9 252 104	A/C	0.39	3.10E-10
	9 181 502	T/C	0.40	1.85E-14			25 043 314	A/G	0.27	8.60E-10
	9 529 975	A/C	0.39	2.85E-14			9 672 698	A/G	0.40	1.68E-09
	9 252 104	A/C	0.39	3.51E-13			9 270 291	T/C	0.39	6.81E-09
	9 254 860	C/G	0.40	1.04E-12			9 731 269	A/T	0.43	9.23E-09
	9 260 075	T/G	0.40	1.04E-12			9 776 599	T/C	0.34	6.67E-05
	9 189 010	T/C	0.40	1.06E-12		TGW	9 252 104	A/C	0.39	9.50E-10	63.00
	9 372 896	T/C	0.40	4.11E-12			16 733 441	A/G	0.39	2.17E-09
	9 260 953	T/C	0.39	5.16E-12			16 734 121	A/G	0.09	2.39E-09
	25 043 314	A/G	0.27	1.57E-11			9 529 975	A/C	0.39	7.19E-09
	9 272 680	A/G	0.40	7.26E-11			9 342 017	A/T	0.40	3.40E-08
	25 042 825	T/C	0.40	9.73E-11			9 181 502	T/C	0.40	4.17E-08
	9 731 269	A/T	0.30	9.73E-11			9 228 066	T/G	0.39	4.18E-08
	9 270 029	T/C	0.43	1.15E-09			9 189 010	T/C	0.40	1.87E-07
	9 776 599	T/C	0.39	3.97E-09			9 254 860	C/G	0.40	3.54E-07
	9 342 017	A/T	0.30	1.71E-05			9 260 075	T/G	0.40	3.54E-07
GLWR	16 733 441	A/G	0.09	9.73E-11	44.24		9 260 953	T/C	0.39	4.85E-07
	16 734 121	A/G	0.39	2.44E-13			9 372 896	T/C	0.40	1.87E-06
	9 228 066	T/G	0.39	9.03E-13			25 043 314	A/G	0.27	3.68E-06
	9 181 502	T/C	0.40	9.30E-13			9 731 269	A/T	0.43	6.47E-06
	9 254 860	C/G	0.40	1.03E-12			9 672 698	A/G	0.40	1.02E-05
	9 260 075	A/C	0.40	1.03E-12			9 272 680	A/G	0.40	2.30E-05
	9 529 975	A/C	0.39	8.18E-12			25 042 825	T/C	0.40	2.30E-05
	9 372 896	T/C	0.40	8.72E-12			9 270 291	T/C	0.39	5.80E-05
	9 260 953	T/C	0.39	1.07E-11

Table 3 Fifty-six SNPs on rice chromosome 3 significantly associated with grain size in GWAS analyses.

Trait	Position	Allele	MAF	P value	R²(%)	Trait	Position	Allele	MAF	P value	R²(%)
GL	16 733 441	A/G	0.33	1.56E-15	45.74	GLWR	9 189 010	T/C	0.40	2.83E-11	44.24
	16 734 121	A/G	0.33	3.29E-15			9 272 680	A/C	0.40	6.07E-11
	9 342 017	A/T	0.40	9.57E-15			25 042 825	T/C	0.40	6.07E-11
	9 228 066	T/G	0.39	1.52E-14			9 252 104	A/C	0.39	3.10E-10
	9 181 502	T/C	0.40	1.85E-14			25 043 314	A/G	0.27	8.60E-10
	9 529 975	A/C	0.39	2.85E-14			9 672 698	A/G	0.40	1.68E-09
	9 252 104	A/C	0.39	3.51E-13			9 270 291	T/C	0.39	6.81E-09
	9 254 860	C/G	0.40	1.04E-12			9 731 269	A/T	0.43	9.23E-09
	9 260 075	T/G	0.40	1.04E-12			9 776 599	T/C	0.34	6.67E-05
	9 189 010	T/C	0.40	1.06E-12		TGW	9 252 104	A/C	0.39	9.50E-10	63.00
	9 372 896	T/C	0.40	4.11E-12			16 733 441	A/G	0.39	2.17E-09
	9 260 953	T/C	0.39	5.16E-12			16 734 121	A/G	0.09	2.39E-09
	25 043 314	A/G	0.27	1.57E-11			9 529 975	A/C	0.39	7.19E-09
	9 272 680	A/G	0.40	7.26E-11			9 342 017	A/T	0.40	3.40E-08
	25 042 825	T/C	0.40	9.73E-11			9 181 502	T/C	0.40	4.17E-08
	9 731 269	A/T	0.30	9.73E-11			9 228 066	T/G	0.39	4.18E-08
	9 270 029	T/C	0.43	1.15E-09			9 189 010	T/C	0.40	1.87E-07
	9 776 599	T/C	0.39	3.97E-09			9 254 860	C/G	0.40	3.54E-07
	9 342 017	A/T	0.30	1.71E-05			9 260 075	T/G	0.40	3.54E-07
GLWR	16 733 441	A/G	0.09	9.73E-11	44.24		9 260 953	T/C	0.39	4.85E-07
	16 734 121	A/G	0.39	2.44E-13			9 372 896	T/C	0.40	1.87E-06
	9 228 066	T/G	0.39	9.03E-13			25 043 314	A/G	0.27	3.68E-06
	9 181 502	T/C	0.40	9.30E-13			9 731 269	A/T	0.43	6.47E-06
	9 254 860	C/G	0.40	1.03E-12			9 672 698	A/G	0.40	1.02E-05
	9 260 075	A/C	0.40	1.03E-12			9 272 680	A/G	0.40	2.30E-05
	9 529 975	A/C	0.39	8.18E-12			25 042 825	T/C	0.40	2.30E-05
	9 372 896	T/C	0.40	8.72E-12			9 270 291	T/C	0.39	5.80E-05
	9 260 953	T/C	0.39	1.07E-11

Supplemental Fig. 2. Manhattan plot (a) and Quantile-quantile plots (b) of four-grain shape related traits GL, Grain length; GW, Grain width; GLWR, Grain length-width ratio; TGW, Thousand grain weight.

Fig. 4. Summary of signiﬁcant trait-marker associations for grain size traits across genomic regions.

Table 4 Candidate genes/QTLs for grain length on chromosome 3.

Candidate gene	SNP site	P value
Os03g0407400	16 733 441	1.56E-15
Os03g0407400	16 734 121	3.29E-15
Os03g0646900	25 042 825	9.73E-11
Os03g0623700	25 043 314	1.71E-05

Fig. 5. Comparison and analyses of genomic DNA sequence of GS3.White boxes represent 5'- and 3'-UTR; black boxes indicate exons. Dark lines between exons represent introns. Translation start codon ATG and translation stop codon TGA indicated with the symbol of a star. C→A substitution shown in the second exon where C allele derived from small parent PB100 produce small grains and A allele derived from ZH8015 produce large grains, 3 bp TCC deletion found in all small grains indicated in exon 5.

Fig. 6. Comparative expression pattern of GS3.WT, Wild type; V1, Changbai 25; V2, Zhejing 88; V3, Wuyunjing 27; V4, Zhejing 22. Comparative expression pattern of GS3 in introgression line (IL) and four CRISPR/Cas9-induced GS3 mutants using real-time RT-PCR analyses of young panicles at 3 cm, 5 cm, 8 cm and 10 cm in length.

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