An SNP molecular marker for identifying sesame plant height and its application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to an SNP molecular marker for identifying the plant height of sesame and its application. The present invention discloses an SNP molecular marker that can be used for identifying the plant height of sesame. Through the molecular marker and method provided by the present invention, the seed DNA can be directly detected, without being restricted by factors such as time and environment, saving the time and investment for identifying the plant height of sesame, and accelerating the breeding efficiency of sesame varieties suitable for timely harvesting.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an SNP molecular marker for identifying sesame plant height and its application. Background Art

[0002] Sesame is a healthy and characteristic oil crop widely planted in the world, with an oil content as high as 55%, and is known as the "queen of oilseeds". China has a sesame planting history of more than 2,200 years and is still an important sesame producer and consumer in the world, making important contributions to the development of the global sesame industry (Wang Ruiyuan, China has made important contributions to the development of the global sesame industry. China Oils and Fats, 2019, 44(12): 1-2).

[0003] Plant height is an important agronomic trait of sesame. Traditional sesame varieties are mostly tall and large, which affects the improvement of yield and the implementation of mechanization. The plant height of sesame is controlled by one pair (or two pairs of major genes) + multiple genes or multiple genes, and the initial capsule height of the main stem and the length of the fruit axis of the main stem are the main factors affecting plant height. Zhu Xiaofeng et al. used sesame recombinant inbred lines as experimental materials to construct a genetic map containing 344 pairs of primers, and used different software to conduct QTL mapping on plant height data over many years and at multiple locations.

[0004] SNP (Single nucleotide polymorphism) is the most ideal molecular marker with the largest number, the widest distribution, the best polymorphism and the construction of high-density genetic maps in the genome. High-throughput sequencing technology is the main method for high-quality SNP development, and with the reduction of sequencing costs, SNPs have gradually become a new generation of widely used molecular markers. Wei et al. sequenced 705 sesame materials and identified 5,407,981 SNPs, and associated 12 high-quality SNP markers related to plant height. Wang et al. mapped 41 QTL loci related to plant height, providing more molecular genetics knowledge for the study of sesame plant type. However, plant height is a complex quantitative trait, and existing molecular markers are difficult to provide technical support for the breeding of new sesame varieties suitable for mechanical harvesting. Summary of the Invention

[0005] To solve the above problems, the present invention provides an SNP molecular marker for identifying sesame plant height and its application.

[0006] The present invention provides an SNP molecular marker related to the sesame plant height trait. The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1; the mutation site of the SNP molecule is located on the gene at 20,818,590 bp of sesame LG8 chromosome, and the mutation site corresponds to T / C at 20,818,590 bp of LG8 chromosome.

[0007] The present invention provides the application of the above SNP molecular markers in the identification of sesame plant height.

[0008] The present invention also provides a method for identifying the sesame plant height trait, which is characterized in that the SNP molecular marker shown in SEQ ID NO.1 is used for identification. When it is C at 20818590 bp of the sesame LG8 chromosome, the corresponding sample is a tall plant; when it is T at 20818590 bp of the sesame LG8 chromosome, the corresponding sample is a medium or low plant.

[0009] The present invention also provides a primer set for identifying the sesame plant height trait, which is a PCR primer set for amplifying the above SNP molecular marker.

[0010] The present invention also provides a kit for identifying the sesame plant height trait, which includes a PCR primer set for amplifying the above SNP molecular marker.

[0011] The present invention also provides a method for identifying the sesame plant height trait, using the above primer set to amplify the sesame genome.

[0012] The beneficial effect of the present invention is that the SNP molecular marker provided by the present invention can be used to efficiently and accurately identify and screen the sesame plant height. Compared with the prior art, the method provided by the present invention can directly detect the seed DNA, is not restricted by factors such as time and environment, saves the time and investment for identifying the sesame plant height, and accelerates the breeding efficiency of sesame varieties suitable for timely harvesting. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Analysis of the significance of plant height differences among groups in the validation population, where A is the group with the same base type as Jihangzhi 1; H is the heterozygous group; B is the group with the same base type as Jizhi DW01. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. Creation of research materials and field design

[0015] Using Jihangzhi 1 as the female parent and Jizhi DW01 as the male parent to construct a hybrid combination. The hybridization work was completed in Shijiazhuang, Hebei in the summer of 2021. The F1 was planted in Sanya, Hainan for generation addition in the winter of the same year, self-crossed and the F2 was harvested. In the summer of 2022, it was planted in Shijiazhuang, Hebei, with a total of 409 individual plants, and conventional field management was carried out.

[0016] 2. DNA extraction

[0017] Using the CTAB method to extract the DNA of the parents and F2 individual plants, using agarose gel electrophoresis to detect the integrity of the DNA, and using NanoDrop 2000 to measure the DNA concentration.

[0018] 3. BSA-seq

[0019] Based on the plant height phenotypic data of the parents and the F2 population, two DNA bulks with extreme plant heights were constructed. For each of the two parents, 10 plants were selected to take leaves, and for the offspring, 30 tall / dwarf extreme plants were selected to take leaves. After DNA extraction and qualification testing, the DNA of individual plants with extreme plant heights was mixed in equal amounts respectively to construct two DNA bulks of extreme pools. Resequencing of the parental and pool DNA was completed through the Illumina HiSeq platform. The sequencing depth of the parental DNA was 10×, and the sequencing depth of the offspring pool DNA was 30×.

[0020] 4. Develop SNP and InDel markers

[0021] Filter the raw data according to the data analysis method of LI et al. (High-density genetic linkage map construction by F2 populations and QTL analysis of early-maturity traits in upland cotton (Gossypium hirsutum L.). Plos One, 2017, 12(8): e0182918.), and align the high-quality sequences to the sesame reference genome (Wang L H, Yu S, Tong C B, Zhao Y Z, Liu Y, Song C, Zhang Y X, Zhang X D, Wang Y, Hua W, Li D H, Li D, Li F, Yu J Y, Xu C Y, Han X L, Huang S M, Tai S H, Wang J Y, Xu X, Li Y R, Liu S Y, Varshney R K, Wang J, Zhang X R. Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis[J]. Genome Biology, 2014.15(2): 1-13.) using the BWA (0.7.17) software (Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009, 25(14): 1754-1760.). Identify SNP and InDel markers using the GATK (4.0.11.0) software (The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research, 2010, 20: 1297-1303.).

[0022] 5. Chromosome segment analysis related to plant height

[0023] First, filter out the following low-quality SNPs / InDels: SNPs / InDels with multiple genotypes, SNPs / InDels with a read support of <4, and SNPs / InDels with consistent genotypes between the bulk pools. Finally, obtain high-quality SNPs / InDels.

[0024] Association analysis was performed by two methods, ED (Euclidean Distance) and SNP-index (Hill J T, Demarest B L, Bisgrove B W, Gorsi B, Su Y C, Yost H J. MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq[J]. Genome Research, 2013, 23(4): 687-697.). When performing the ED method analysis, the cube of the original ED was taken as the association value in this patent to eliminate background noise. Theoretically, there are significant differences only in the loci related to the target trait among the pooled samples. Therefore, the larger the ED value or ΔSNP-index of the target locus, the closer the association between this locus and the target trait, and the ED values and ΔSNP-index of other loci tend to be 0. The intersection of the results obtained by the two algorithms was selected as the candidate region. Finally, a chromosomal region significantly associated with plant height was found at 20,680,000 bp - 20,890,000 bp on chromosome NC_026152.1 (LG8). The base sequences of 50 bp upstream and downstream of the SNP at position 20,818,590 bp, where the parents are different in the middle of this region, are shown in Table 1. The base at this position in Jihangzhi 1 is C, and the base in Jizhi DW01 is T.

[0025] Table 1 SNP quantity and base positions at the target location

[0026]

[0027] 6. Validation and application of SNP markers

[0028] The F2 population containing 409 individual plants (numbered 1 - 409) was constructed with Jihangzhi 1 as the female parent and Jizhi DW01 as the male parent. The plant height (PH) of the population was investigated. After the genomic DNA was extracted by the CTAB method and passed the detection, the DNA sequences of the target loci of the individual plants in the F2 population were detected by the Genotyping by sequencing (GBS) method published by ELSHIRE et al. (Arobust, simple genotyping-by-sequencing (GBS) approach for high diversityspecies. PloS one, 2011, 6(5): e19379.). The base types of each F2 individual plant at the target SNP position were detected using bioinformatics techniques and software. First, the 409 F2 individual plants were divided into two validation populations, I (1 - 200) and II (201 - 409), according to the numbers. Secondly, according to the genotypes of the parental SNPs, the two validation populations were respectively divided into 3 groups, namely group I-A and II-A with the same base type as Jihangzhi 1, group I-B and II-B with the same base type as Jizhi DW01, and heterozygous groups I-H and II-H. According to the grouping results, the average plant height and the significance of the difference between different groups were calculated.

[0029] The results of the significance analysis of the differences found that in the two validation populations, the group (A) with the same base type as Jihangzhi 1 had the highest plant height, the heterozygous group (H) had the medium plant height, and the group (B) with the same base type as Jizhi DW01 had the lowest plant height. The differences in plant height between groups reached an extremely significant level ( Figure 1 ).

[0030] This proves that the discovered SNP can very significantly distinguish the plant height of sesame and can be used for the auxiliary identification of plant height in the breeding process of new sesame varieties.

Claims

1. A method for identifying the plant height of sesame using SNP molecular markers, characterized in that, The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the base polymorphism at the 51st position of the nucleotide sequence is C or T; the identification method is to identify the sesame genome. When the 51st position of the SNP molecular marker is C, the corresponding sample is a tall plant; when the 51st position of the SNP molecular marker is T, the corresponding sample is a medium or short plant.

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