CN113789407B - SNP molecular marker combination for cyperus esculentus genotyping and application thereof - Google Patents

SNP molecular marker combination for cyperus esculentus genotyping and application thereof Download PDF

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CN113789407B
CN113789407B CN202111215856.7A CN202111215856A CN113789407B CN 113789407 B CN113789407 B CN 113789407B CN 202111215856 A CN202111215856 A CN 202111215856A CN 113789407 B CN113789407 B CN 113789407B
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杨向东
牛陆
杨春明
任伟
杨静
张原宇
邢国杰
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Jilin Academy of Agricultural Sciences
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Abstract

The invention relates to the fields of molecular biology and molecular plant breeding, in particular to SNP molecular marker combination for cyperus esculentus genotyping and application thereof. The molecular marker is at least one of 1292 SNP markers, and the information is shown in Table 1. The SNP molecular marker combination provided by the invention can be used for carrying out genotype identification on the Cyperus esculentus germplasm resources and varieties, can also be used for genetic background analysis, agronomic character association analysis and whole genome selective breeding of Cyperus esculentus breeding materials, and has good application prospects.

Description

SNP molecular marker combination for cyperus esculentus genotyping and application thereof
Technical Field
The invention relates to the fields of molecular biology and molecular plant breeding, in particular to SNP molecular marker combination for cyperus esculentus genotyping and application thereof.
Background
The Cyperus esculentus (Cyperus esculentus L.) is a novel oil crop with high ecological value, dual-purpose grain and oil feed and high competitive power. At present, most of the domestic cyperus esculentus is variety resources introduced from abroad in the sixth and seventies of the last century. The original genetic basis of the cyperus esculentus cannot accurately trace the source due to wide introduction and breeding, so that the germplasm resources are deficient and chaotic, and the healthy and rapid development of the cyperus esculentus industry in China is severely restricted. The identification of the chufa germplasm resources in the current production is mainly carried out by the phenotype differences of tuber shape, size, color and the like, the accuracy and the reliability are poor, and the accurate identification and the purity analysis are difficult to carry out. At the same time, the appearance of crops is easily influenced by environmental factors, and identification errors are often caused in the actual operation process. The similarity and the source tracing of the morphological characteristics of the cyperus esculentus are unclear, and great difficulty is caused to accurately identify and reasonably utilize the cyperus esculentus resources, in particular to the seed selection of the cyperus esculentus variety, the protection of the variety right and the like. Further clearing the current situation of the cyperus esculentus resources in China, accelerating the utilization of the cyperus esculentus germplasm resources and breeding of excellent varieties, and becoming a key problem to be solved in the development of the cyperus esculentus industry in China.
Unlike traditional morphological or biochemical marker identification methods, molecular marker technology (such as RAPD, RFLP, SSR, ALFP, SRAP, SNP) can essentially reflect the differences of biological individuals, has the advantages of small environmental impact, high detection specificity, high stability and accuracy and the like, and is widely applied to researches in the fields of biological diversity analysis, germplasm resource or variety classification, genetic map construction, molecular marker assisted selection breeding, comparative genomics and the like. Since the whole genome sequence of the cyperus esculentus is not published at present, the existing research is mainly to detect by using random-designed RAPD or SRAP primers, the specificity and stability of amplified fragments are poor, and the integration of data among different molecular markers is difficult, so that the method has not been widely applied. Compared with other molecular markers, the SNP marker (Single Nucleotide Polymorphisms, single nucleotide polymorphism) has the advantages of complete genome coverage, high throughput, site specificity, co-dominant inheritance, low false detection rate, easy data integration and the like, and part of the markers are related to functional genes or plant phenotypes, so that the SNP marker is taken as one of the preferred DNA fingerprint marking technologies by the international new variety protection alliance. Particularly, with the rapid development of the sequencing technology, the high-throughput and low-cost sequencing technology provides powerful technical support for detecting SNP markers at the whole genome level. No scheme for identifying the germplasm resources of the cyperus esculentus by adopting a SNP (single nucleotide polymorphism) marking method is found in the prior art.
Disclosure of Invention
The invention aims to provide a SNP molecular marker combination for cyperus esculentus genotyping and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an SNP molecular marker combination for cyperus esculentus genotyping, wherein the molecular marker combination at least comprises one of 1292 SNP markers, and the 1292 SNP markers are shown in table 1:
table 1 1292 SNP markers
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Preferably, the SNP molecular marker set comprises at least 2 SNP markers of 1292.
Preferably, the SNP molecular marker set comprises 1292 SNP markers.
The invention also provides application of the SNP molecular marker combination in identification of the germplasm resources and variety genotypes of the cyperus esculentus.
The invention also provides application of the SNP molecular marker combination in construction of the cyperus esculentus DNA fingerprint.
Compared with the prior art, the SNP molecular marker combination has the following advantages:
(1) Compared with the prior art, 1292 cyperus esculentus SNP markers provided by the invention have the advantages of high coverage rate and abundant polymorphism.
(2) Compared with the prior art, the invention has the advantages of high detection flux, low detection cost and the like.
(3) The domestic cyperus esculentus germplasm resources and varieties are deficient, and the existing resources are not clear in source tracing. The SNP marker or the combination provided by the invention can realize high-throughput detection of the cyperus esculentus germplasm resources and varieties, can be widely applied to the identification of the cyperus esculentus germplasm resources and varieties, the genetic background analysis of breeding materials, the selection of molecular markers or the selection breeding of whole genome, and the like, and has a good application prospect.
Drawings
FIG. 1 shows the result of cluster analysis of SNP fingerprints obtained by sequencing 42 parts of Cyperus esculentus by using Super-GBS technology in example 2 of the present invention.
FIG. 2 shows the SNP detection of 42 parts of Cyperus esculentus by one-generation sequencing in example 3 of the present invention.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, the examples were conducted under conventional experimental conditions or under conditions recommended by the manufacturer's instructions.
The 42 parts of Cyperus esculentus germplasm resource information described in the examples of the present invention are shown in Table 2.
TABLE 2 Cyperus esculentus germplasm resource information 42 parts
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EXAMPLE 1 Cyperus esculentus SNP marker development
And (3) carrying out de novo sequencing on the chufa JYD-02 by using a PacBIO sequence II single-molecule real-time sequencing system to obtain HiFi ready. Genome assembly was performed on HiFi reads using the Hifiasm software (version 0.15.2) to obtain an assembled book of 306,075,1840 bp. Using this assembly as a reference genomic sequence, 42 Cyperus esculentus resources described in Table 2 were subjected to simplified genomic sequencing (Super-GBS, illumina Hiseq Xten, PE 150) to obtain sequencing samples. The average sequencing depth was 39.3×, coverage ranged from 3.79% to 4.99%. Based on the results of the comparison of the sequencing samples to the reference genome, preliminary SNP and INDEL results were obtained. The obtained SNP and INDEL typing results were filtered using VCFtools software. The filtration conditions were as follows: (1) a Reads support number (DP) of not less than 4; (2) knockout of sites with MAF less than 0.01; (3) Sites with deletion rates higher than 20% for SNP or INDEL typing are deleted. A total of 87398 SNP sites and 5009 InDel sites were obtained.
Based on the obtained SNP loci, genotype conversion is carried out on all SNP loci by utilizing UgbS-Flex analysis flow. The lowest sequencing depth was set at 8,H/C/D identification threshold at 4 and the missing data threshold at 30%. Selecting SNP with the least deletion information from the detected co-isolation SNP markers as the representative of the group of markers, and co-screening to obtain 1292 SNP. The 1292 cyperus esculentus SNP marker information is shown in table 1.
Example 2 application of 1292 SNPs in Cyperus esculentus genomic DNA fingerprint analysis
The genotype of 42 parts of Cyperus esculentus resources of Table 2 was examined according to 1292 Cyperus esculentus SNP molecular marker combinations obtained in example 1 in combination with the Super-GBS technique. The specific method comprises the following steps:
(1) The reference genomic sequence obtained in example 1 was used as the reference genomic sequence of this example 2;
(2) Constructing a cyperus esculentus genome DNA sequencing library by using the Super-GBS technology, and sequencing to obtain clear Reads;
(3) Comparing the clear Reads with a reference genome sequence by using bowtie2 software, and detecting SNP or Indel by using GATK software to obtain a cyperus esculentus SNP locus;
(4) Based on the SNP molecular marker combination, the UgbS-Flex is utilized to carry out genotype conversion on the SNP locus of the cyperus esculentus, so as to obtain the DNA fingerprint of the cyperus esculentus.
The extraction method of the genome DNA adopts a CTAB method;
42 parts of the cyperus esculentus fingerprint in table 2 were constructed by this method, and the results are shown in table 3.
1 to 1292 in the SNP numbers in Table 3 represent CeSNP0001 to CeSNP1292, respectively.
The 42 parts of the Cyperus esculentus germplasm resources were subjected to clustering analysis, and the results are shown in FIG. 1. FIG. 1 shows that domestic Cyperus esculentus resources can be roughly classified into 3 categories, wherein 2 parts of germplasm resources (JYD-35 and JYD-36) introduced from African karst were relatively similar; is derived from Spanish (JYD-41), karma (JYD-34) and domestic partial resources (such as JYD-14, etc.) which are similar in genetics; while Russian (JYD-42), mary (JYD-33) and most domestic sources (e.g., JYD-23, etc.) are more similar. This result is more consistent with the phenotypic characterization result. The 1292 screened cyperus esculentus SNP markers are suitable for establishing the cyperus esculentus fingerprint spectrum, are convenient for comparing the genetic relationship between different resources or varieties, and are suitable for accurately identifying different cyperus esculentus germplasm resources and varieties.
TABLE 3 fingerprint of 42 Cyperus esculentus
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Example 3 core SNP for identifying Cyperus esculentus germplasm resources and uses
Based on the cyperus esculentus SNP markers obtained in example 1, the duty ratio and the segregation ratio of homozygotes therein were calculated, and the loci with high homozygote duty ratio and uniform segregation ratio were selected as core SNP loci. The invention screens 73 core SNP markers from 1292 SNP markers, and specific molecular marker information is shown in Table 4. And designing PCR detection primers or KASP primers by using the core SNP sequence information, and carrying out PCR amplification on the genomic DNA of the cyperus esculentus. And analyzing SNP locus variation conditions of different cyperus esculentus resources by adopting a first generation sequencing (Sanger method) or fluorescence detection.
The SNP numbers described in Table 4 correspond to the SNP numbers in the primers of Table 5.
SNP locus detection using one generation of sequencing includes the following steps.
Based on the SNP marker information of 73 cyperus esculentus cores, detection primers are designed, and specific primer sequences are shown in Table 5.
TABLE 5 primer sequences for Cyperus esculentus core SNP detection
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TABLE 4 73 core SNPs
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The CTAB method is used for extracting the genomic DNA of the cyperus esculentus. The concentration of the extracted cyperus esculentus genome DNA is diluted to 50 ng/. Mu.L to be used as a PCR amplification template.
PCR amplification and electrophoresis detection, wherein a PCR amplification system (25 uL) comprises: 10 XPCR buffer 2.5uL,10mmol/L dNTPs 0.5uL,5U/uL Taq enzyme 0.5uL, sample DNA 1.0uL, upstream and downstream primer mix (10 umol/L each) 1.0uL, ddH 2 O19.5 uL. The PCR amplification procedure was 95℃for 5min;94 ℃ for 30s,60 ℃ for 30s and 72 ℃ for 45s, and 35 cycles are total; and at 72℃for 10min. The PCR products were detected by 1.5% agarose gel electrophoresis.
And SNP detection, namely sequencing the PCR product of the electrophoresis detection result display band, and analyzing the SNP locus variation condition according to the sequencing result. The results are shown in FIG. 2.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (3)

1. A SNP molecular marker composition for cyperus esculentus genotyping, characterized in that said molecular marker composition comprises 1292 SNP markers, said 1292 SNP markers are shown in the following table:
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2. the use of the SNP molecular marker composition of claim 1 in the identification of cyperus esculentus germplasm resources and variety genotypes.
3. The use of the SNP molecular marker composition of claim 1 in constructing a cyperus esculentus DNA fingerprint.
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油莎豆SRAP 指纹图谱构建及遗传多样性分析;赵永国等;植物遗传资源学报;第14卷(第2期);222-225 *
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