Molecular markers of genes related to subaleurone thickness in sorghum and their application

Abstract

The present invention relates to a molecular marker of a gene related to the thickness of the subaleurone layer of sorghum and its application. The present invention mines a SNP site significantly related to the thickness of the subaleurone layer through a whole genome association analysis method, and then develops a molecular marker SA1. The base 1296 of the amplified product of the molecular marker in materials with thin subaleurone layers is C, and the base 1296 of the amplified product in materials with thick subaleurone layers is T. The marker can significantly affect the thickness of the subaleurone layer of sorghum, but has no significant effect on the grain size. Therefore, the present invention provides a new technical means for screening sorghum materials with high nutritional quality and enrichment.

Description

Technical Field

[0001] The invention relates to a molecular marker of a sorghum subaleurone thickness related gene and application thereof, and belongs to the field of sorghum nutritional quality improvement and crop molecular biology design breeding. Background Art

[0002] The endosperm of cereal grains is composed of the outermost layer of aleurone cells and the innermost layer of starchy endosperm cells. The aleurone layer mainly accumulates proteins, lipids and phenolic substances, while the starchy endosperm mainly accumulates starch. During the maturity stage of cereal grains, the accumulation rate of endosperm starch is asynchronous. According to the amount of nutrient accumulation, the different cells formed in the starchy endosperm can be divided into three categories: subaleurone cells, prismatic cells and central cells (Laser microdissection-based gene expression analysis in the aleurone layer and starchy endosperm of developing rice caryopses in the early storage phase. Rice. 8: 57; Ishimaru T, et al. (2015) Spatial distribution of functional components in the starchy endosperm of wheat grains. J. Cereal Sci. 91: 102869; Shewry PR, et al. (2019) Protein accumulation in aleurone cells, sub-aleurone cells and the center starchendosperm of cereals. Plant Cell Rep.33:1607-1615; Zheng Y, et al. (2014)). In cereal seeds, the aleurone layer and subaleurone layer are the main storage sites for proteins. Morphological analysis found that compared with other types of cells in the starchy endosperm, subaleurone cells contain higher protein content and lower starch content. Microscopic observation also showed that only a small part of starch granules were distributed in the protein-rich subaleurone cells; these results indicate that subaleurone cells may have different origins from other cell types in the starchy endosperm; in addition, semi-thin section staining can more intuitively show that the subaleurone layer is rich in proteins, such as Figure 1As shown, in fact, previous studies have shown that the subaleurone content of wheat flour averages 54%; therefore, the subaleurone content is directly related to the nutritional value of flour (Variation inprotein composition within the endosperm of hard wheat. Cereal Chem. 46: 293-300; Kent N, et al. (1969) Subaleuron endosperm cells of high protein content. Cereal Chem. 43: 585-601; Kent N. (1966) Starch synthesis in the cerealendosperm. Curr. Opin. Plant Biol. 6: 215-222; James MG, et al. (2003) Spatialdistribution of functional components in the starchy endosperm of wheatgrains. J. Cereal Sci. 91: 102869; Shewry PR, et al. (2019)).

[0003] With the development of sequencing technology and the reduction of costs, more and more plant genomes have been analyzed with increasing accuracy. Based on the whole genome genotype, association analysis with specific phenotypes has become a method for quickly obtaining candidate sites. Association analysis has significant advantages such as saving time and cost, wide breadth and high accuracy, and has gradually become a powerful tool for studying plant quantitative traits (Genetic association mapping and genome organization of maize. Current Opinion in Biotechnology, 17: 155-160; Yu J, et al. (2006)). Summary of the invention

[0004] One of the purposes of the present invention is to provide a molecular marker of a gene related to the thickness of the sorghum subaleurone layer, which can be used for the screening or identification of germplasm resources and provide a new technical means for screening sorghum varieties with high nutritional quality and enrichment.

[0005] Another object of the present invention is to provide the application of molecular markers of genes related to the thickness of sorghum subaleurone layer.

[0006] A molecular marker of a gene related to the thickness of the sorghum subaleurone layer, wherein the molecular marker is a SNP site obtained by PCR amplification using a primer pair and a sorghum V3.1 genome as a template, and the sequence of the primer pair is as follows:

[0007] SEQ ID NO.1: 5'CCATGCATGTGTGTAAATATTCGA-3',

[0008] SEQ ID NO. 2: 5'ACTGACTGACGAGTATACTGCA-3'.

[0009] The base 1296 of the amplified product of the marker in the material with a thin subaleurone layer is C, and the base 1296 of the amplified product in the material with a thick subaleurone layer is T.

[0010] The related gene is located on chromosome 6 of sorghum.

[0011] A primer pair for amplifying molecular markers of genes related to sorghum subaleurone thickness, wherein the forward primer sequence is SEQ ID NO.1, and the reverse primer sequence is SEQ ID NO.2.

[0012] Application of molecular markers of genes related to sorghum subaleurone thickness in the screening or identification of germplasm resources.

[0013] The application is to use a primer pair to obtain a SNP site by PCR amplification using the sorghum genome as a template. The genotype of the material with thick subaleurone layer is T at the 1296th position, and the genotype of the material with thin subaleurone layer is C at the 1296th position. The sequence of the primer pair is as follows:

[0014] SEQ ID NO.1: 5'CCATGCATGTGTGTAAATATTCGA-3',

[0015] SEQ ID NO. 2: 5'ACTGACTGACGAGTATACTGCA-3'.

[0016] The present invention uses a combination of association analysis and linkage analysis to dig out SNP sites significantly associated with subaleurone thickness, and then develops a molecular marker. The marker can significantly correlate with sorghum subaleurone thickness, but has no significant effect on grain size. Therefore, the invention provides a new technical means for screening sorghum varieties with high nutritional quality enrichment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Semi-thin sections of mature sorghum seeds show the distribution of nutrients in the starchy endosperm. A: Coomassie Brilliant Blue staining to observe the distribution of protein; B: Schiff reagent staining to observe the distribution of starch;

[0018] Figure 2 For genome-wide association analysis, the significant locus 6:47313665 and candidate segment association analysis were located.

[0019] Figure 3 Haplotype analysis of the SbSTP10 polymorphic site.

[0020] Hap 1 and 2 are two haplotypes. Least significant difference method was used for multiple comparisons. If there was a significant difference between the two groups at the p<0.05 level, different lowercase letters were used to mark them. If there were the same letters, it meant that there was no significant difference between the two groups. DETAILED DESCRIPTION

[0021] The present invention is achieved through the following technical solutions:

[0022] A molecular marker for a gene related to sorghum subaleurone thickness (sequence number: Sobic.006G102800), the related gene is located on chromosome 6 of sorghum, and its phenotypic contribution rate to subaleurone thickness is 16.2% in 253 sorghum inbred line varieties collected from all over the world. The present invention uses the advantages of whole genome association analysis, uses a wide range of variant germplasm resources to determine candidate genes and functional sites through association analysis, and then develops molecular markers.

[0023] The molecular marker linked to the gene is SA1, and the primer pair for amplifying the molecular marker of the gene related to the subaleurone thickness of sorghum, wherein the SA1 forward primer sequence is SEQ ID NO.1, and the reverse primer sequence is SEQ ID NO.2. The molecular marker amplification product in the material with thick subaleurone layer is T, indicating that the material has the subaleurone layer thick allele and exhibits the subaleurone layer thick phenotype; the amplification product in the material with thin subaleurone layer is C, indicating that the material has the subaleurone layer thin allele and exhibits the subaleurone layer thin phenotype.

[0024] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description of the specific embodiments. However, the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0025] Example 1 Acquisition of molecular markers

[0026] Genome-wide association analysis: In the single nucleotide polymorphism (SNP) analysis of 253 sorghum accessions, 99 accessions were derived from our previous study (Zhang et al., 2018b), and the remaining 67 sorghum varieties were analyzed according to the previous resequencing and SNP detection process. In brief, sequencing was performed on the Illumina Hiseq X 10 platform with a paired-end sequencing and an average depth of ~5.67×. Low-quality reads were first strictly filtered, and the filtered reads were aligned to the BTx623 (v3.1) reference genome, and duplicate reads were filtered out. Then, GATK (Genome Analysis Toolkit, version 3.2-2) was used to detect SNP sites and insertion and deletion sites (InDels). In addition, the further filtering criteria for SNP markers used in genome-wide association study (GWAS) are as follows: 1. Delete the long sequence fragments (Scaffolds) assembled from the overlapping groups generated in the splicing stage; 2. Remove SNP sites with a missing rate greater than 20%; 3. Retain SNP sites with a minor allele frequency (MAF) less than 5%. Results A total of 4478843 high-quality SNP sites were identified in 253 different sorghum germplasm resources. Only SNP_6:47313665 (6:47313665) was significantly associated with leaf number (p=4.4E-08), which could explain 16.2% of the phenotypic variation ( Figure 2 (A). According to the reference genome annotation of sorghum inbred line Btx623, this SNP is located in the promoter region of Sobic.006G102800 gene. The gene function is annotated as MFS superfamily-STP sugar transporter, so we call it SbSTP10. Candidate segment association analysis was performed on the SNP sites and InDel sites in the 50kb region upstream and downstream of this site. The results showed that the significant association site "6:47313665" appeared, and the threshold reached 6.89E -09 ( Figure 2 (middle B).

[0027] Linkage disequilibrium analysis: The software used for linkage disequilibrium (LD) analysis of significant loci in association analysis was Haploview 4.2 (Barrett et al., 2005). The candidate gene SbSTP10 found by LD analysis was then analyzed for sequence variation by Sanger sequencing. The primers used were designed based on the BTx623 reference genome. SAL analysis was performed with SNP_6:47313665 as the center, and the analysis showed that there were 5 genes in this range, all of which were highly linked ( Figure 2 Middle C).

[0028] Haplotype analysis: Resequencing data revealed a total of 305 polymorphic sites (217 SNPs and 88 InDels) within 3Kb upstream and downstream of SbSTP10. Candidate segment association analysis was performed using SNP sites and InDel sites within 50kb upstream and downstream of the site. The results showed a significant association site "6:47313665" with a threshold of 6.89E -09 This polymorphic site can form two haplotypes, among which haplotype 2 (Hap2) has the thickest subaleurone layer and is the optimal haplotype ( Figure 3 ).

[0029] Example 2 Screening of Sorghum Germplasm Resources with Thick Subaleurone Layer

[0030] Molecular marker development: The sorghum inbred line Btx623 was used as the reference sequence. Primer 3 was used to design the primer pair with pleiotropy 6:47313665. The amplified fragment length was 3200 bp. The specific primer pair sequences are as follows:

[0031] Upstream primer: 5'CCATGCATGTGTGTAAATATTCGA-3',

[0032] Downstream primer: 5'ACTGACTGACGAGTATACTGCA-3'.

[0033] The molecular marker SA1 of the present invention was used to screen the sub-aleurone thickness of 253 collected sorghum germplasms: 253 germplasms were planted in a large field in Beijing, leaves were collected in the seedling stage to extract DNA, and extended sequencing was performed using the SA1 primer pair, and it was found that 6:47313665 was 92 T genotype materials and 161 C genotype materials. After the plants matured, the grains were collected to count the sub-aleurone thickness. Through statistics, it was found that the average sub-aleurone thickness of the 92 materials with genotype T was 0.34, while the average sub-aleurone thickness of the materials with genotype C was 0.21. It can be seen that the materials with genotype T all showed a phenotype of thick sub-aleurone layer, while the materials with genotype C showed a phenotype of thin sub-aleurone layer ( Figure 3). This marker can be used for the screening of subaleurone sorghum germplasm. Sequence Listing <110> Institute of Botany, Chinese Academy of Sciences <120> Molecular markers of genes related to subaleurone thickness in sorghum and their application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <400> 1 ccatgcatgt gtgtaaatat tcga 24 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <400> 2 actgactgac gagtatactg ca 22

Claims

1. Application of molecular markers in the screening or identification of germplasm resources of sorghum submud layer thickness related traits; characterized in that: The primer pair was used to obtain a molecular marker containing a SNP site by PCR amplification with the sorghum V3.1 genome as a template. The genotype of the material with thin sub-aleurone layer thickness was C at the 1296th position of the molecular marker, and the genotype of the material with thick sub-aleurone layer thickness was T at the 1296th position of the molecular marker. The primer pair sequence is as follows: SEQ ID NO.1: 5'CCATGCATGTGTGTAAATATTCGA-3', SEQ ID NO. 2: 5'ACTGACTGACGAGTATACTGCA-3'.

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