Targeted Identification of Rice Grain-Associated Gene Allelic Variation through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy

doi:10.21203/rs.3.rs-1457542/v1

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Research Article

Targeted Identification of Rice Grain-Associated Gene Allelic Variation through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy

https://doi.org/10.21203/rs.3.rs-1457542/v1

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Background: The mining of excellent allelic variation and the creation of new crop germplasm are the basis for improving breeding efficiency.

Results: In this study, a total of 3872 heavy ion-irradiated M2 generation rice seeds and individual leaves were collected, and 484 mixed samples were obtained at a seed-to-leaf ratio of 8:1. Targeted sequencing technology was used to detect the major grain length genes GS3 and grain length. The allelic variation in the broad major gene GW5 and the phenotype data were measured, and 12 mixed samples containing potential mutations and 15 SNPs were obtained; 96 individual plants in the 12 mixed samples were then subjected to Sanger sequencing, combined with the phenotypic data obtained 13 key mutants and their corresponding SNPs; the protein structure and function analysis of the key mutants was carried out, and 6 allelic variants that led to the change in grain shape were mined, as well as the corresponding mutants, including long-grain mutants GS3-2 and GS3-7, short-grain mutants GS3-3 and GS3-5, wide-grain mutant GW5-1 and narrow-grain mutant GW5-4; sequencing identified new grain length gene allelic variants GS3-G1, GS3-G2 and GS3-G3, which provided new functional gene loci and a material basis for molecular breeding and genotype mutation and phenotype analysis, particularly in the context of mutational associations.

Conclusion: Based on the above studies, we propose a method for targeted identification of allelic variation in rice grain type genes by combining targeted sequencing of mixed samples and whole genome sequencing. This method can sequence the target fragments of mixed samples, and the detection cost is low; furthermore, the identification cycle based on present method is short due to screening process is on the mutation offspring and the recombination genetic population is not necessary. The principle of present method is not limited to the grain type phenotype and can also be used for the directional detection and identification of other target traits and allele genes.

Rice grain type

Heavy-ion irradiation

Allelic variation

Targeted sequencing

Whole genome sequencing

Rice (Oryza sativa L.) is one of the three major food crops and is a staple food for nearly half of the world's population (Miura et al., 2010). Excellent germplasm resources are the basis for improving the breeding efficiency of new rice varieties (Zhao et al., 2011). Various physical or chemical mutagenic factors can induce changes in biological genetic material, resulting in new allelic variations and species. As a new radiation mutagenesis method, heavy ion mutagenesis has unique advantages, such as a high mutation rate, wide mutation spectrum, fast mutation stability, and stable and reliable mutagenesis, and this method is simple and easy to implement (Qu Ying et al., 2007; Hase et al., 2012). This method can induce heritable variation in plant genomes in contemporary times. The resulting mutants are important materials for functional genomics research. In recent years, this technique has provided a new way to create germplasm and plays an important role in plant breeding (Yang et al., 2019; Li et al., 2019; Oka-Kira et al., 2005; Ishikawa et al., 2012;).

Mining of allelic variations is the key to creating new germplasm for plants and animals. The rice Wx gene is the main gene that controls amylose synthesis, and the discovery and utilization of its allelic variation is an important way to analyze rice quality variation and is also an important basis for rice quality improvement. Currently, the Wx gene has been discovered and identified, and multiple important allelic variants, including Wx^lv, Wx^a, Wx^b, Wxⁱⁿ, Wx^mp, Wx^op, and wx (Zhang et al., 2019), have also been identified. GS3 is a major QTL controlling grain length in rice, and the protein it encodes negatively regulates grain length (Fan et al., 2006). Mao et al. (Hailiang et al., 2010) identified the four alleles GS3:GS3-1, GS3-2, GS3-3, and GS3-4, in which GS3-3 has an SNP mutation in exon 2, resulting in a long grain; GS3-4 has a 1-bp deletion at 357 bp, resulting in a decrease in long grain length. GW5 is a major QTL that controls grain width in rice. The 1212 bp deletion in wide-grain varieties regulates the expression of GW5 and then the size of the grain. Using CRISPR technology to delete approximately 5 kb downstream of GW5 can increase the grain width and size. Moreover, grain weight can increase yield (Jiafan et al., 2017).

For the screening and identification of allelic variants, in classical forward genetics studies, complex mapping populations and linkage maps are usually constructed, and genetic markers are used for gene linkage analysis (Serquen et al., 1997; Zhang et al., 2012; Fazio et al., 2003). The targeted induced local lesions in genomes (TILLING) technique is a reverse genetics technique developed in the 1990s. This technique is based on chemical mutagenesis materials and combines chemical mutagenesis technology with PCR screening technology and high-throughput detection methods. Linked together, a high-throughput and rapid detection of point mutations in target gene regions has formed a technical system (Henikoff et al., 2003) that has been applied in a variety of plants and promoted mutagenesis and breeding development (Boualem et al., 2014; Anai, 2012; Ochiai et al., 2011; Chen et al., 2012; Minoia et al., 2010). MutMap is a forward genetic gene mapping strategy and genetic analysis method developed based on whole genome sequencing (WGS) (Abe et al., 2012). The MutMap method also includes a variety of developments and extensions, such as MutMap + and MutMap-Gap. These methods do not require the establishment of cumbersome progeny mapping groups and do not rely on genetic hybridization and any linkage information. The identification process of variant loci has been successfully applied to the study of multiple gene mappings in different species (Hiroki et al., 2013; Rym et al., 2017; Takagi et al., 2015; Takagi et al., 2013). In recent years, targeted sequencing technology has been widely used. Targeted sequencing is GenoPlexs based on multiplex PCR and GenoBaits based on liquid-phase probe capture, which can detect multiple SNPs within a single amplicon, greatly improving intratarget variation and detection efficiency. This technique has the characteristics of high marker flexibility and high detection efficiency and can be widely used in biological evolution, genetic map construction, gene location cloning, marker trait association detection, allelic variation detection, etc. (Shen et al., 2021; Guo et al., 2019; Lu et al., 2019; Li et al., 2020; Du et al., 2019; Yang et al., 2019).

In this study, 3872 12C6 + radiation mutagenized mutant materials for the second generation were identified by mixed sample targeted sequencing technology, and the mutant mixed samples and SNPs related to the granulotype genes GS3 and GW5 were mined and then selected by Sanger sequencing. Mutant individual plants, combined with phenotype and protein function analysis, were utilized to further select key mutant individual plants and conduct WGS to analyze the relationship between mutant phenotype and genotype, discover new allelic variations, and establish a system. An efficient and accurate method for directional identification of allelic variation in rice grain type genes through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy, abbreviated as MTWA, was developed.

Grain Type Phenotype Investigation

The wild-type (WT) material used in this study was Huahang No. 31 (Fig. 2a). A total of 3872 seeds of the M2 generation per plant were harvested. The grain length was between 8 and 10.22 mm, the average length was 9.28 mm, and the coefficient of variation was 1.89%. The grain length was 9.31 mm (Fig. 2c). The grain width was between 1.54 and 2.87 mm, with an average width of 2.02 mm, and the WT grain width was 2.03 mm, with a coefficient of variation of 0.56% (Fig. 2d). Both the grain length and grain width conformed to a normal distribution and had a wide variation range. Compared with the WT, there were many materials with large grain type differences, indicating that there were several potential grain type mutations (Fig. 2b)

Table 1

Grain type phenotype survey
trait	Min	Max	Average	Standard deviation	Coefficient of variation/%
Length (mm)	8.00	10.22	9.28	0.18	1.89
Width (mm)	1.54	2.87	2.02	0.01	0.56

Targeted Sequencing of Mixed Samples to Screen a Mixed Pool of Potential Mutations

A total of 484 mixed samples and 1 WT sample were obtained by mixing 3872 individual DNAs in equal amounts at a ratio of 8:1. The detected fragments were the grain length major gene GS3 (Fig. 3a) and the grain width major gene GW5 (Fig. 3b). After evaluating the target segment location information, a set of primers that could cover the entire target segment (Supplementary Table S1) was designed to perform targeted sequencing on 485 pooled samples (Fig. 3c). After the sequencing was completed, the vaf of the SNP site was first analyzed and calculated (the calculation method is shown in Fig. S 1), and the WT sample was used as a reference to screen for mutation sites: 1) When the parent is a pure genotype at this site, the offspring and the parent are at this site; 2) when the parent is a heterozygous genotype at this locus and the mutation frequency difference between the offspring and the parent at this locus is greater than or equal to 1/16, the locus shall be retained (site retention).

According to the above screening method, a total of 179 mutation sites were obtained, all of which were homozygous mutations, of which 110 sites were in the GS3 interval and 69 were in the GW5 interval. The total mutation frequency in the GS3 interval was calculated to be 4.05× 10 − 5, and the total mutation frequency in the GW5 interval was 9.02×10 − 5 (total mutation frequency = mutation base number/gene fragment length). Among the 179 mutation sites, 63.57% were located in the intron region, and 30% were located in the exon region (Fig. 3d). We retained only the nonsynonymous and nonsense mutations located in the exon region that could cause amino acid changes. At the same time, reliable sites with relatively high reads were screened, and a total of 15 SNPs were obtained (Table 2), of which 14 were nonsynonymous mutations and 1 was a nonsense mutation, including 11 GS3 interval loci and 4 GW5 interval locus points (Fig. 3e), for a total of 12 mixed samples.

Table 2

Targeted sequencing mutation site information
SNP number	Mixed sample number	Gene	Location	Ref	Mut	Function type
SNP-1	4-101	GS3	16732992	G	C	nonsynonymous SNV
SNP-2	4-101	GS3	16734441	G	T	stopgain
SNP-3	5–6	GS3	16734009	G	C	nonsynonymous SNV
SNP-4	6–23	GS3	16731920	C	A	nonsynonymous SNV
SNP-5	6–44	GS3	16729753	A	T	nonsynonymous SNV
SNP-6	6–44	GS3	16729861	G	T	nonsynonymous SNV
SNP-7	7-112	GS3	16735064	G	C	nonsynonymous SNV
SNP-8	7–41	GS3	16729815	C	T	nonsynonymous SNV
SNP-9	7–78	GS3	16729903	G	A	nonsynonymous SNV
SNP-10	7–78	GS3	16729863	C	A	nonsynonymous SNV
SNP-11	9 − 5	GS3	16729886	C	A	nonsynonymous SNV
SNP-12	5–35	GW5	5365411	A	G	nonsynonymous SNV
SNP-13	7–82	GW5	5366479	C	G	nonsynonymous SNV
SNP-14	10–14	GW5	5366520	T	G	nonsynonymous SNV
SNP-15	10–51	GW5	5366501	A	G	nonsynonymous SNV

Screening of Individual Mutant Plants and Identification of Their Authenticity

To further screen out the mutant individual plants from the mixed samples, we isolated individual plants in the 12 mixed samples, which contained a total of 96 individual plant materials, and added 200 bp before and after the 15 SNPs to amplify the SNP-containing strains. The 96 individual plant materials of the fragment were subjected to Sanger sequencing and compared with the results of targeted sequencing to determine the target mutant individual plant. A total of 13 loci were consistent with the targeted sequencing results, among which the Sanger sequencing results of SNP-5 and SNP-6 were different from the targeted sequencing results (Fig. 4a), so the mutants at these two loci were excluded and targeted sequencing. The concordance rate with Sanger sequencing was 86.67%, and a total of 13 mutants were screened. The complete 15 SNP results are shown in Fig. S2.

To verify the authenticity of the selected mutants, we identified the authenticity of the selected 13 single-plant materials according to the technical regulations for the identification of rice varieties (SSR marking method) issued by the Ministry of Agriculture and designed a total of 10 pairs of SSR markers. The agarose gel electrophoresis detection results of the 13 mutant individual plants were consistent with the WT, indicating that they were all true mutations (Fig. 4b).

Phenotypic Verification and Protein Function Analysis of Individual Mutant Plants

After verification, the corresponding grain type and phenotype data of the real variant individual plants were found according to the number. Only 6 of the 13 individual plants showed significant changes in grain type, and the grain types of the remaining 7 individual plants were the same as those of the control. There was no significant difference in the ratios between samples (Table 3). According to the screening results of targeted sequencing, 13 SNPs are nonsynonymous and nonsense mutations, which theoretically lead to amino acid changes, while some SNPs do not cause significant changes in phenotype, presumably not changing the function of a protein or structural or other genetic mutations.

Table 3

Amino acid mutation and phenotypic information of single mutant plants
Mutant number	Single plant number	Amino acid mutation	Grain length (mm)	Grain width (mm)
WT	WT		9.31	2.03
GS3-1	7-112-2-4	A5G	9.21	2.12
GS3-2	4-101-1-3	C55X	10.22**	2.03
GS3-3	5-6-1-4	C135W	8.06**	2.04
GS3-4	4-101-2-2	S141C	9.41	2.17
GS3-5	6-23-1-4	C173F	8.51**	1.9
GS3-6	7-78-1-3	G167C	9.33	1.94
GS3-7	9-5-1-1	C190F	9.97**	1.86
GS3-8	7-78-2-3	S184F	9.33	2.09
GS3-9	7-41-1-4	R200H	9.30	1.9
GW5-1	5-35-1-2	V411G	9.53	2.29**
GW5-2	7-82-1-4	A405G	9.42	2.02
GW5-3	10-14-2-1	K397E	9.46	2.03
GW5-4	10-51-2-3	D97G	9.40	1.84**
: represents significant variation at p < 0.05; *: represents highly significant variation at p < 0.01

We screened a total of 9 grain length mutants, of which 2 grain lengths were significantly longer and 2 grain lengths were significantly shorter than WT grains (Fig. 5a). Nine SNPs related to GS3 were identified, including 8 nonsynonymous mutations and 1 nonsense mutation (GS3-2), in which GS3-1 was located in exon 1 and GS3-2 was located in exon 2. The remaining seven mutations were located in exon 5 (Fig. 5b). The mutation position of GS3-1 is relatively advanced, and it is not located in the functional structural region and has no effect on the structure and function of the protein, so the grain length does not change significantly (Fig. 5b). GS3-2 is located in the OSR domain, the 55th amino acid is mutated to a stop codon, the OSR domain is deleted, and the protein structure and function are severely affected (Fig. 5d), resulting in a significant increase in grain length. Both GS3-3 and GS3-4 are located in the TNFR domain of Cys-rich mutants, and the grain length of GS3-3 is significantly reduced. Protein structural analysis showed that the mutation of amino acid No. 135 leads to two additional β sheets in the secondary structure of the protein. and presumably resulted in impaired TNFR domain function (Fig. 5d), whereas the GS3-4 grain length was not significantly altered. GS3-5, GS3-6, GS3-7, GS3-8 and GS3-9 are all located in the Cys-rich VWFC domain, among which only the grain length of GS3-5 is significantly reduced, and the mutation of GS3-5 may lead to the impaired structure of the VWFC domain function, but there is no significant difference in protein structure compared with WT plants (Fig. S3). The GS3-6, GS3-8 and GS3-9 phenotypic results were similar to those of GS3-4; functional domain amino acid point mutation occurred, but the phenotype did not change significantly. It was speculated that the mutation of these 4 amino acids may not affect the function of the protein or that other gene mutations have an impact on the phenotype; however, the grain length of GS3-7. In contrast, the functional site analysis of its protein showed that the mutation of amino acid 183 of GS3-7 was located in the ligand binding site of the protein (Fig. 5c). After the mutation, the function of the protein was affected, so the particle shape changed. However, GS3-4, GS3-5, GS3-6, GS3-8 and GS3-9 showed no significant difference when compared to the WT in terms of protein structure and function (Fig. S3).

We screened 4 mutants with grain width, of which 1 grain width increased, 1 grain width decreased, and the remaining two grain widths had no significant changes (Fig. 6a). The four identified GW5-related SNPs were all nonsynonymous mutations, of which GW5-4 was located in exon 1, and the other three were located in exon 2 (Fig. 6b). GW5-1, GW5-2 and GW5-3 are all located in the calmodulin-binding domain; the difference is that only the granule width of GW5-1 is significantly wider than that of other mutants, and the other two mutants have no obvious change in phenotype. We predicted the protein structure and function of mutants and found that the protein structures of GW5-1 and GW5-2 were more similar to each other than to the WT, while the protein structure of GW5-3 had no obvious change (Fig. 6c). The phenotypes of GW5-1 and GW5-2 of the same domain differ, presumably due to interference from other genes. The GW5-4 mutation position is relatively forward, not located in the functional structural region, and has no effect on the structure and function of the protein (Fig. 6b), but the grain width is significantly narrowed, and the protein structure is relatively concentrated (Fig. 6c).

WGS of Key Mutants Identifies New SNPs Affecting the GS3 Mutation Effect

To explore the reasons for the contradiction between genotype mutation and phenotype mutation and to clarify whether allelic variation in other grain length-related genes had an impact on the phenotype, we used the mutant GS3-5. For GS3-7, DNA was extracted, and equal amounts of DNA were mixed to construct mixed sample GS3-M1; mutants GS3-4, GS3-6, GS3-8, and GS3-9 with genotype mutation but no change in phenotype were utilized to construct mixed sample GS3-M2; and WT plants were utilized to prepared the WT sample. A total of 3 samples were subjected to WGS.

A total of 2,084,534 SNPs and 336,039 InDels were obtained by sequencing GS3-M1, and 2,116,343 SNPs and 341,777 InDels were obtained from GS3-M2. After screening, three new allelic variants related to grain length were finally obtained (Table 4) (Fig. 7a). GS3-G1 is located in the second exon of OsNST1, which mutates serine No. 65 to threonine. At present, there are few reports on this gene, and its protein structure cannot be predicted. Mutants exhibit reduced cell wall cellulose content and structural changes, resulting in reduced mechanical strength and abnormal plant development, such as dwarf plants and smaller seed size (Song et al., 2011). GS3-G2 is a variant located in the first exon of OsMAPK6 that mutates the aspartic acid at No. 131 to arginine, which affects only one of its functional domains. Inhibition of OsMPK6 expression can make rice panicles denser and grains smaller, and mutation of this gene can significantly reduce grain length, grain width and thousand-grain weight (Guo et al., 2018). GS3-G3 is located in the second exon of RAE2, resulting in a frameshift insertion mutation at amino acid 99 and impaired function of the cysteine-rich region of the encoded protein EPFL1. The number of kernels decreased, the kernels became longer, and the proportion of awned kernels increased (Jin et al., 2016) (Fig. 7b).

Table 4

Whole-genome mutation site information
Number	Chr	Location	Ref	Alt	Structure gene	Function type
GS3-G1	2	24236917	C	T	Os02g0614100	nonsynonymous SNV
GS3-G2	6	2812755	T	C	Os06g0154500	nonsynonymous SNV
GS3-G3	8	23999540	-	C	Os08g0485500	frameshift insertion

Reliability of MTWA to Identify Mutants and Allelic Variants

Screening mutants and identifying allelic variants are important foundations for innovative germplasm materials and functional genomics research (Guo Longbiao et al., 2006). To efficiently utilize germplasm resources, the most fundamental way is to excavate new alleles and purposefully aggregate or transfer them in conventional breeding or molecular breeding and then combine them with molecular design to achieve the purpose of improving breeding efficiency. In this study, the seeds of Huahang 31 were irradiated with 12C6+, and the M2 generation population containing 3872 individual plants was obtained. The variation coefficient of grain length was 1.89%, and the variation coefficient of grain width was 0.56%, indicating that there are many potential mutations in grain type. body. In this study, a new method for allelic variation and mutant identification, MTWA, was proposed, which used targeted sequencing technology to initially identify mixed sample materials. After screening, a total of 15 SNPs and 12 mixed sample materials were obtained, and Sanger sequencing was performed. The key mutants in the mixed samples were screened out and verified for authenticity. The consensus rate between targeted sequencing and Sanger sequencing was 86.67%, proving the feasibility of the method. A total of 13 mutants and 15 SNPs were screened out. Analysis of the phenotype of the mutant individual plant, the protein functional structural analysis of the mutation site, and WGS of the key mutants were conducted. Mining new grain length-related allelic variations, analyzing the connection between genotype mutations and phenotype mutations, and establishing a set of systematic, efficient and accurate new methods for allelic variation identification were also conducted. At the same time, a batch of mutation sites and mutant materials with breeding value were screened and identified.

Advantages of MTWA

Traditional forward genetic identification methods usually require the construction of complex mapping populations and linkage maps and gene linkage analysis using genetic markers (Wei-Wei et al., 2012; Felix et al., 1997; G. et al., 2003) but can only map mutation sites to a large range of chromosomal regions (Hazen et al., 2005; Samuel et al., 2005), and fine mapping of mutant genes is expensive and time consuming (Korbinian et al., 2009; Abe et al., 2012). MTWA can be utilized to analyze the M2 generation without the need to construct a complex genetic population, which greatly shortens the detection time. This technique is also a directional mutant identification method. TILLING technology uses CEL I enzyme to digest PCR amplification products and detects and selects mutants by capillary electrophoresis (Yan Zhihui et al., 2014), but the process is relatively complicated; however, the TILLING technique can detect gene fragments. There are certain length requirements; usually, the length of the target gene fragment is less than 1.5 kb, and the high-resolution melting curve (HRM) detection region is only 150-500 bp (Sikora et al., 2011). On the other hand, MTWA directly performs second-generation sequencing on the target fragment to determine the mutation sites. The MTWA process is relatively simple, and the sequencing results are more accurate than those of the TILLING method. At the same time, multiple mutation sites can be detected in a single amplicon, and there is no restriction on the fragment length. The detection efficiency is higher and has wider applicability than the TILLING method. Compared to MutMap and its derived methods, mutants in MTWA do not require backcrossing of parental lines, thus greatly reducing time and effort (Allen et al., 2013; Abe et al., 2012); if the genome of the target crop is large and complex, MutMap will have the problems of high sequencing costs, large datasets, and difficult comparison and analysis, especially in allopolyploid species with high genome heterozygosity, and highly homologous sequences and subtypes of genomes could also be detected (Li et al., 2015; Ling et al., 2013; Michael et al., 2018; The, 2014). As a mutant and allelic variant directional identification method, MTWA only needs to sequence the target fragments of mixed samples in the early stage, and the number of WGS samples of key mutants in the later stage is small, so the costs of sequencing and the data analysis are greatly saved. These mutants can be detected in the M2 band, and the detection duration of this method is short and not limited to the particle type phenotype, allowing for capturing and detecting any target fragment with high flexibility.

The Breeding Significance of Mining New Allelic Variations

A total of 13 grain type-related SNPs were identified in this study, of which 6 SNPs had an impact on phenotypic changes, which can be used to develop molecular markers; 7 SNPs that did not affect phenotypes can also provide theories for follow-up research via nonsense allelic variation studies. At the same time, we screened a batch of germplasm materials with obvious differences in grain shape from the mutagenized progeny, such as the long-grain mutants GS3-2 and GS3-7, short-grain mutants GS3-3 and GS3-5, wide-grain mutant somatic GW5-1 and narrow-grain mutant GW5-4. Among these mutants, the genotype mutation of GS3-2 contributed 9.77% to the grain length, which was consistent with the mutation position of the mutant obtained by Hailiang (2010), indicating that the allelic mutation was reliable and the mutation mechanism was clear; GW5-4 mutation led to a 13.43% reduction in grain width and was the site with the largest variation in grain shape among all mutations. These two sites can be used as key SNPs to develop molecular markers and provide new functional genes for molecular breeding. locus, serving breeding practice.

GS3 encodes a transmembrane protein consisting of 232 amino acids. The protein product consists of three conserved domains: OSR, MR and Cys-rich domains. The Cys-rich domain includes two regions, TNFR/NGFR and VWFC. The OSR domain plays an important role as a negative regulator, and loss of the OSR structure and function results in the formation of long grains. In GS3-2 identified in this study, the 55th amino acid of the OSR domain was mutated to a stop codon, and the OSR domain was deleted. The protein structure and function were severely affected, resulting in a significant increase in grain length. This mutation is also similar to that of previous studies. The results of the study of GS3-5 are the same as those of previous studies (Hailiang et al., 2010); moreover, the mutation of GS3-5 may lead to the impaired function of the VWFC domain, which is also consistent with previous conclusions (Hailiang et al., 2010), but the protein structure is not obvious. GS3-7 also resulted in impaired VWFC domain function but increased grain length, contrary to previous conclusions (Hailiang et al., 2010). Additionally, in the mutation of GW5, GW5-1 and GW5-2 are in the same domain, but the phenotypes are different, while GW5-4, which is not located in the functional domain, has a significantly reduced grain width. Therefore, we speculate that the mutants with phenotypic variation identified in this study that do not match the genotypic variation may have other grain-related variants that have an impact on the phenotype.

Analysis of the Effect of New Grain Length-related SNPs on the Effect of GS3 Mutation

Among the allelic variants identified in this study, there are many contradictions between loci and phenotypic traits, such as GS3-4, GS3-5, and GS3-7. To explore whether there are new GL gene variants for expression, we found new allelic mutations in GS3-G1, GS3-G2 and GS3-G3 by WGS. The protein structure of GS3-5 was not significantly different from that of WT, but the grain length was significantly reduced, it is speculated to be due to the effect of the mutation of GS3-G1 or GS3-G2 locus. The GS3-G1 mutation will lead to a significant reduction in grain size, and the 1000-grain weight is reduced to 50% of WT (Zhang et al., 2011). GS3-G2 site will result in a 20% reduction in grain length (Guo et al., 2018), so the presence of GS3-G1 or GS3-G2 sites ultimately results in a reduction in grain length in GS3-5; GS3-7 The mutation type of GS3-G3 will theoretically lead to a decrease in grain length, but the actual grain length increases by 7.09%, while the mutation at the GS3-G3 site will lead to an increase of about 8% in grain length (Jin., 2016). The phenotypic mutation effect is greater than that of GS3-7, so the final response is the grain length in the phenotype. An increase in GS3-4, GS3-6, GS3-8, and GS3-9 genotype mutations will theoretically lead to reduced grain length, but the actual grain length of these mutants was not significantly different from that of the WT plants, presumably due to the mutation of GS3-G3. The increase in grain length counteracted the phenotypic mutation effect of the original GS3 mutant, so the result reflected in grain length was no significant change in phenotype.

Material Handling and Mutant Phenotype Investigation

The WT material used in this experiment was Huahang 31 material. The dry seeds of rice were irradiated by the high-energy ion radioactive beam 12C⁶⁺ provided by the baryon radiation device HIRFL in Lanzhou China Institute of Modern Physics. The irradiation energy was 80.55 MeV/u, the dose was 80 Gy, and the dose rate was 60 Gy/min. At the same time, the unirradiated seeds were prepared as the control, the mutagenized generation was M1, and the main ear of M1 was mixed to obtain M2. The seeds of the M2 generation mutant materials were harvested according to individual plants, dried and stored in a freezer at -20°C. The grain shape pictures were collected by the scanner, and then the pictures were analyzed by SmartGRAIN to obtain the phenotypic data of grain length and grain width.The obtained grain type data were statistically analyzed by SPSS19, and statistical graphs were drawn by GraphPad Prism7.

DNA Extraction and Targeted Sequencing

Single leaves of M2 generation plants were taken, and DNA was extracted by the CTAB method. The extracted DNA samples were subjected to agarose gel electrophoresis and Qubit quality inspection. DNA samples that passed the quality inspection were stored at -80°C for subsequent experiments. Quality DNA was utilized to prepare mixed samples at a seed-to-leaf ratio of 8:1, and the mixed samples were analyzed by targeted sequencing technology. The detected fragments were the main gene GS3 of rice grain length and the main gene of grain width GW5. Firstly, specific amplification primers were designed for multiple SNP loci to be tested, nonspecific amplification were suppressed in the first round of PCR, and targeted primers were enabled to achieve highly uniform amplification in one tube of PCR, thereby enriching the target fragments in large quantities. Subsequently, in the second round of PCR, sequencing adapters and library barcodes were added to finally obtain the library required for sequencing. Finally, the marker genotypes of the target loci were revealed by massively parallel sequencing.

Sanger Sequencing

In order to amplify the fragments with the target SNP, we added 200bp before and after each of the 15 SNPs and used Primer5 design 15 pairs of primers(Supplementary Table S2), and performed PCR amplification on the individual material corresponding to each SNP to obtain Sanger sequencing was performed after the target fragment, and the sequencing data was compared with the wild type by SnapGene, and the peak map was drawn.

Identification of Mutants Authenticity

In order to verify the authenticity of the selected mutants, we identified the authenticity of the 13 single-plant materials screened according to the technical regulations for the identification of rice varieties (SSR marking method) issued by the Ministry of Agriculture and Rural Affairs of People’s Republic of China, and selected 10 pairs of SSR markers and designed the corresponding primers through Prime5 software, then the PCR productions were detected by 1% agarose gel electrophoresis (Supplementary Table S3).

Sequencing Data Filtering and SNP Acquisition

Fastp software was used to filter the raw reads to remove the adapter sequence (adapter); when the N content contained in the sequencing read exceeded 10% of the length of the read, the paired reads needed to be removed. When the number of bases of quality (Q≤20) exceeded 40% of the length of the read, the paired reads needed to be removed. Variation detection was performed using GATK software. When the coverage depth of a sample at a certain SNP site was less than 5×, the sequencing depth of the sample at this site was insufficient. To ensure the accuracy of genotyping results, the site is treated as a deletion. In the parental sample, if vaf (variant allele frequency) ≥ 0.8 or ≤ 0.2, the locus was a pure-sum mutation, and if the mutation frequency was between 0.2 and 0.8, the SNP locus was a heterozygous mutation.

Protein Structure and Function Analysis

For the amino acid mutations corresponding to the identified SNPs, the functional site of the protein was predicted by I-TASSER (https://zhanggroup.org/I-TASSER/), and then the functional site of the protein was predicted by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/) to predict the three-dimensional structural model of proteins. PyMOL was used to draw protein functional sites and three-dimensional structure maps and perform annotation and comparison.

WGS

The DNA of the key mutants was extracted by the CTAB method. After passing the quality inspection, the genomic DNA was randomly broken into short DNA fragments with enzymes and then blunt-end repaired. dA tails were ligated to both ends of the DNA fragments, and sequencing adapters were ligated. The DNA fragments with adapters were purified by AMPure XP magnetic beads, and fragments in the range of 300-400 bp were selected for PCR amplification. The constructed library was purified, checked against a library, and sequenced on a HiSeq X10 PE150 instrument.

Ethics approval and consent to participate

This study complied with the ethical standards of China where this research was performed.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no conflict of interest.

Funding

This work was supported by the Research and Development Plan for Key Areas in Guangdong Province (No.2018B020206002) and the Special Rural Revitalization Funds of Guangdong Province (2021KJ382)

Authors' contributions

KS and TG designed the experiment and wrote the manuscript. KS, DDL, AYX, QW and SSJ conducted the experiments and performed data analysis. JFW, GLY, DHZ, CHH, HW and ZQC drafted proposals and corrected the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would thank American Journal Experts for their valuable language service.

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No competing interests reported.

FigureS1.tif
Additional file 1: Fig. S1 Vaf calculation method, green bases are mutation bases, red bases are reference genome bases, and the ratio of green bases to all bases is the vaf.
FigureS2.tif
Additional file 2: Fig. S2 The Sanger sequencing results of the mutation sites of the 15 mutant individual plants, in which the results of SNP-5 and SNP-6 are different from the targeted sequencing results, these two mutant individual plants are excluded, and the rest are the same as the targeted sequencing results, which are true variation.
FigureS3.tif
Additional file 3: Fig. S3 Protein structures of 9 GS3 mutants, the red part is the difference from WT.
TableS1.xlsx
Additional file 4: Table S1 Targeted sequencing primer information.
TableS2.xlsx
Additional file 5: Table S2 Primers for amplifying fragments of the target site.
TableS3.xlsx
Additional file 6: Table S3 Primers for Authenticity Verification of Mutant Individuals.
TableS4.xlsx
Additional file 7: Table S4 Grain type datas.
TableS5.xlsx
Additional file 8: Table S5 Mutation mixed sample information screened by targeted sequencing.
TableS6.xls
Additional file 9: Table S6 Mutation site information obtained by targeted sequencing.
TableS7.xls
Additional file 10: Table S7 Mutation site information screened by whole genome sequencing.

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Targeted Identification of Rice Grain-Associated Gene Allelic Variation through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy

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