CN113789407A - 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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CN113789407A
CN113789407A CN202111215856.7A CN202111215856A CN113789407A CN 113789407 A CN113789407 A CN 113789407A CN 202111215856 A CN202111215856 A CN 202111215856A CN 113789407 A CN113789407 A CN 113789407A
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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 a 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 genotyping of cyperus esculentus germplasm resources and varieties, can also be used for genetic background analysis, agronomic trait association analysis and whole genome selective breeding of cyperus esculentus breeding materials, and has good application prospect.

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 a SNP molecular marker combination for cyperus esculentus genotyping and application thereof.
Background
Cyperus esculentus L is a novel oil crop with high ecological value, dual purposes of grain and oil feeding and extremely high competitiveness. At present, the cyperus esculentus planted at home is a variety resource introduced from abroad in the sixth and seventies of the last century. Due to wide introduction and breeding, the original genetic basis of the cyperus esculentus cannot be traced accurately, germplasm resources are deficient and disordered, and the healthy and rapid development of the cyperus esculentus industry in China is severely restricted. The identification of the germplasm resources of the cyperus esculentus in the current production is mainly realized by differentiating the differences of the tuber shapes, sizes, colors and other phenotypes, the accuracy and the reliability are poor, and the accurate identification and the purity analysis are difficult to carry out. Meanwhile, the appearance and the character of the crops are easily influenced by environmental factors, and identification errors are often caused in the actual operation process. The similarity and the source tracing of the form characteristics of the cyperus esculentus are unclear, and great difficulty is caused for accurate identification and reasonable utilization of the cyperus esculentus resources, particularly for seed selection and variety right protection of the cyperus esculentus varieties. Further clarifying the current situation of domestic cyperus esculentus resources, accelerating the utilization of the cyperus esculentus germplasm resources and breeding of excellent varieties become key problems which are urgently needed to be solved in the development of the cyperus esculentus industry in China.
Different from the traditional morphological or biochemical marker identification method, the molecular marker technology (such as RAPD, RFLP, SSR, ALFP, SRAP, SNP and the like) can reflect the difference of biological individuals essentially, has the advantages of small environmental influence, strong detection specificity, stability and accuracy and the like, and is widely applied to the research in the fields of biodiversity analysis, germplasm resource or variety classification, genetic map construction, molecular marker-assisted selective breeding, comparative genomics and the like. Because the whole genome sequence of the cyperus esculentus is not published at present, the existing research mainly utilizes random-designed RAPD or SRAP primers for detection, the specificity and stability of amplified fragments are poor, and data integration among different molecular markers is difficult, so that the cyperus esculentus is not widely applied. Compared with other molecular markers, the SNP marker (Single Nucleotide Polymorphisms) has the advantages of whole genome coverage, high flux, site specificity, co-dominant inheritance, low false detection rate, easy data integration and the like, and part of the marker is related to a functional gene or a plant phenotype, and is taken as one of the first-choice DNA fingerprint marking technologies by the International New-species protection Union. Particularly, with the rapid development of sequencing technology, the high-throughput and low-cost sequencing technology provides powerful technical support for the detection of SNP markers at the whole genome level. The prior art does not find a scheme for identifying the germplasm resources of the cyperus esculentus by adopting an SNP (single nucleotide polymorphism) marking method.
Disclosure of Invention
The invention aims to provide an 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, which at least comprises one of 1292 SNP markers, wherein the 1292 SNP markers are shown in Table 1:
TABLE 11292 SNP markers
Figure BDA0003310588250000011
Figure BDA0003310588250000021
Figure BDA0003310588250000031
Figure BDA0003310588250000041
Figure BDA0003310588250000051
Figure BDA0003310588250000061
Figure BDA0003310588250000071
Figure BDA0003310588250000081
Figure BDA0003310588250000091
Figure BDA0003310588250000101
Figure BDA0003310588250000111
Figure BDA0003310588250000121
Figure BDA0003310588250000131
Figure BDA0003310588250000141
Figure BDA0003310588250000151
Figure BDA0003310588250000161
Figure BDA0003310588250000171
Figure BDA0003310588250000181
Figure BDA0003310588250000191
Figure BDA0003310588250000201
Figure BDA0003310588250000211
Figure BDA0003310588250000221
Figure BDA0003310588250000231
Figure BDA0003310588250000241
Figure BDA0003310588250000251
Figure BDA0003310588250000261
Figure BDA0003310588250000271
Figure BDA0003310588250000281
Figure BDA0003310588250000291
Figure BDA0003310588250000301
Figure BDA0003310588250000311
Figure BDA0003310588250000321
Figure BDA0003310588250000331
Figure BDA0003310588250000341
Figure BDA0003310588250000351
Figure BDA0003310588250000361
Figure BDA0003310588250000371
Figure BDA0003310588250000381
Figure BDA0003310588250000391
Figure BDA0003310588250000401
Figure BDA0003310588250000411
Figure BDA0003310588250000421
Figure BDA0003310588250000431
Figure BDA0003310588250000441
Figure BDA0003310588250000451
Figure BDA0003310588250000461
Figure BDA0003310588250000471
Figure BDA0003310588250000481
Figure BDA0003310588250000491
Figure BDA0003310588250000501
Figure BDA0003310588250000511
Figure BDA0003310588250000521
Figure BDA0003310588250000531
Figure BDA0003310588250000541
Figure BDA0003310588250000551
Figure BDA0003310588250000561
Figure BDA0003310588250000571
Figure BDA0003310588250000581
Figure BDA0003310588250000591
Figure BDA0003310588250000601
Figure BDA0003310588250000611
Figure BDA0003310588250000621
Figure BDA0003310588250000631
Figure BDA0003310588250000641
Figure BDA0003310588250000651
Figure BDA0003310588250000661
Figure BDA0003310588250000671
Figure BDA0003310588250000681
Figure BDA0003310588250000691
Preferably, the SNP molecular marker combination at least comprises 2 of 1292 SNP markers.
Preferably, the SNP molecular marker combination comprises 1292 SNP markers.
The invention also provides application of the SNP molecular marker combination in identification of germplasm resources and variety genotypes of cyperus esculentus.
The invention also provides application of the SNP molecular marker combination in constructing the DNA fingerprint of the cyperus esculentus.
Compared with the prior art, the SNP molecular marker combination has the following advantages:
(1) compared with the prior art, the 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 method has the advantages of high detection flux, low detection cost and the like.
(3) The germplasm resources and varieties of the cyperus esculentus in China are deficient, and the existing resources are not clear to trace. The SNP marker or the combination provided by the invention can realize high-throughput detection on the germplasm resources and varieties of the cyperus esculentus, can be widely applied to identification of the germplasm resources and varieties of the cyperus esculentus, genetic background analysis of breeding materials, molecular marker selection or whole genome selective breeding and the like, and has good application prospect.
Drawings
FIG. 1 shows the result of cluster analysis of SNP fingerprints obtained by sequencing 42 Cyperus esculentus by Super-GBS technique in example 2 of the present invention.
FIG. 2 shows the result of SNP detection of 42 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 carried out according to conventional experimental conditions or according to the manufacturer's instructions.
The germplasm resource information of 42 cyperus esculentus in the example of the invention is shown in table 2.
TABLE 242 parts of Cyperus esculentus germplasm resource information
Figure BDA0003310588250000692
Figure BDA0003310588250000701
Example 1 Cyperus SNP marker development
And (3) performing de novo sequencing on the cyperus esculentus JYD-02 by using a PacBio queue II single-molecule real-time sequencing system to obtain HiFi reads. HiFi reads were genome-assembled using hifiasm software (version 0.15.2) to obtain 306,075,184bp assemblages. The set of samples was used as a reference genome sequence to perform simplified genome sequencing (Super-GBS, Illumina Hiseq Xten, PE150) on 42 cyperus esculentus resources described in table 2 to obtain a sequencing sample. The average sequencing depth is 39.3 x, and the coverage range is 3.79-4.99%. Preliminary SNP and INDEL results were obtained based on the alignment of the sequenced sample to the reference genome. The obtained SNP and INDEL typing results were filtered using VCFtools software. The filtration conditions were as follows: (1) (ii) Reads support number (DP) is not less than 4; (2) eliminating sites with MAF less than 0.01; (3) the SNP or INDEL typing deletion rate is higher than 20%. 87398 SNP sites and 5009 InDel sites are obtained in total.
And (3) carrying out genotype conversion on all the SNP sites by utilizing a UGbS-Flex analysis process based on the obtained SNP sites. The lowest sequencing depth was set to 8, the H/C/D identification threshold was 4, and the missing data threshold was 30%. And selecting SNP with the least deletion information from the detected co-segregation SNP markers as a representative of the group of markers, and co-screening to obtain 1292 SNP. The 1292 chufa SNP marker information is shown in table 1.
Example application of 21292 SNPs in Cyperus esculentus genomic DNA fingerprinting analysis
The genotype of 42 cyperus esculentus resources of table 2 was examined according to 1292 cyperus esculentus SNP molecular marker combinations obtained in example 1, in combination with 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 in example 2;
(2) constructing a Cyperus esculentus genome DNA sequencing library by using a Super-GBS technology, and sequencing to obtain Clean Reads;
(3) comparing Clean Reads with a reference genome sequence by utilizing bowtie2 software, and carrying out SNP or Indel detection on a comparison result by utilizing GATK software to obtain a Cyperus esculentus SNP site;
(4) based on the SNP molecular marker combination, UGbS-Flex is utilized to carry out genotype conversion on the SNP locus of the cyperus esculentus, and the DNA fingerprint of the cyperus esculentus is obtained.
The extraction method of the genome DNA adopts a CTAB method;
42 cyperus esculentus fingerprints in table 2 were constructed using this method, and the results are shown in table 3.
1 to 1292 of the SNP numbers in Table 3 represent CeSNP0001 to CeSNP1292, respectively.
42 portions of cyperus esculentus germplasm resources were subjected to cluster analysis, and the results are shown in fig. 1. FIG. 1 shows that domestic Cyperus esculentus resources can be roughly classified into 3 types, wherein 2 germplasm resources (JYD-35 and JYD-36) introduced from African Kemylon are relatively similar; is derived from Spanish (JYD-41), karilon (JYD-34) and part of domestic resources (such as JYD-14 and the like) which are relatively similar genetically; the sources of Russia (JYD-42), Mary (JYD-33) and most domestic resources (such as JYD-23) are more similar. This result is more consistent with the results of phenotypic identification. The 1292 chufa SNP markers screened by the invention are suitable for establishing chufa fingerprint spectra, are convenient for comparing the genetic relationship among different resources or varieties, and are suitable for accurately identifying the germplasm resources and varieties of different chufa.
TABLE 342 parts of Cyperus esculentus fingerprint
Figure BDA0003310588250000721
Figure BDA0003310588250000731
Figure BDA0003310588250000741
Figure BDA0003310588250000751
Figure BDA0003310588250000761
Figure BDA0003310588250000771
Figure BDA0003310588250000781
Figure BDA0003310588250000791
Figure BDA0003310588250000801
Figure BDA0003310588250000811
Figure BDA0003310588250000821
Figure BDA0003310588250000831
Figure BDA0003310588250000841
Figure BDA0003310588250000851
Figure BDA0003310588250000861
Figure BDA0003310588250000871
Figure BDA0003310588250000881
Figure BDA0003310588250000891
Figure BDA0003310588250000901
Figure BDA0003310588250000911
Figure BDA0003310588250000921
Figure BDA0003310588250000931
Figure BDA0003310588250000941
Figure BDA0003310588250000951
Figure BDA0003310588250000961
Figure BDA0003310588250000971
Figure BDA0003310588250000981
Figure BDA0003310588250000991
Figure BDA0003310588250001001
Figure BDA0003310588250001011
Figure BDA0003310588250001021
Figure BDA0003310588250001031
Figure BDA0003310588250001041
Figure BDA0003310588250001051
Figure BDA0003310588250001061
Figure BDA0003310588250001071
Figure BDA0003310588250001081
Figure BDA0003310588250001091
Figure BDA0003310588250001101
Figure BDA0003310588250001111
Figure BDA0003310588250001121
Figure BDA0003310588250001131
Figure BDA0003310588250001141
Figure BDA0003310588250001151
Figure BDA0003310588250001161
Figure BDA0003310588250001171
Figure BDA0003310588250001181
Figure BDA0003310588250001191
Figure BDA0003310588250001201
Figure BDA0003310588250001211
Figure BDA0003310588250001221
Figure BDA0003310588250001231
Figure BDA0003310588250001241
Figure BDA0003310588250001251
Figure BDA0003310588250001261
Figure BDA0003310588250001271
Figure BDA0003310588250001281
Figure BDA0003310588250001291
Figure BDA0003310588250001301
Figure BDA0003310588250001311
Figure BDA0003310588250001321
Figure BDA0003310588250001331
Figure BDA0003310588250001341
Figure BDA0003310588250001351
Figure BDA0003310588250001361
Figure BDA0003310588250001371
Figure BDA0003310588250001381
Figure BDA0003310588250001391
Figure BDA0003310588250001401
Figure BDA0003310588250001411
Figure BDA0003310588250001421
Figure BDA0003310588250001431
Figure BDA0003310588250001441
Figure BDA0003310588250001451
Figure BDA0003310588250001461
Figure BDA0003310588250001471
Figure BDA0003310588250001481
Figure BDA0003310588250001491
Figure BDA0003310588250001501
Figure BDA0003310588250001511
Figure BDA0003310588250001521
Figure BDA0003310588250001531
Figure BDA0003310588250001541
Figure BDA0003310588250001551
Figure BDA0003310588250001561
Figure BDA0003310588250001571
Figure BDA0003310588250001581
Figure BDA0003310588250001591
Figure BDA0003310588250001601
Figure BDA0003310588250001611
Figure BDA0003310588250001621
Figure BDA0003310588250001631
Figure BDA0003310588250001641
Figure BDA0003310588250001651
Figure BDA0003310588250001661
Figure BDA0003310588250001671
Example 3 core SNP for identifying Cyperus esculentus germplasm resources and application
Based on the cyperus esculentus SNP markers obtained in example 1, the homozygote occupancy ratio and the segregation ratio were calculated, and sites with high homozygote occupancy ratio and uniform segregation ratio were selected as core SNP sites. 73 core SNP markers are screened from 1292 SNPs, and the specific molecular marker information is shown in Table 4. And designing a PCR detection primer or a KASP primer by using the core SNP sequence information, and carrying out PCR amplification on the Cyperus esculentus genome DNA. And analyzing the variation condition of the SNP sites of different cyperus esculentus resources by adopting a generation sequencing (Sanger method) or fluorescence detection.
The SNP numbers indicated in Table 4 correspond to the SNP numbers indicated in the primers of Table 5.
The detection of SNP sites by means of one-generation sequencing comprises the following steps.
Based on 73 cyperus esculentus core SNP marker information, detection primers are designed, and specific primer sequences are shown in Table 5.
TABLE 5 primer sequences for Cyperus esculentus core SNP detection
Figure BDA0003310588250001681
Figure BDA0003310588250001691
TABLE 473 core SNPs
Figure BDA0003310588250001701
Figure BDA0003310588250001711
Figure BDA0003310588250001721
Figure BDA0003310588250001731
Figure BDA0003310588250001741
Figure BDA0003310588250001751
And (3) extracting the Cyperus esculentus genome DNA by using a CTAB method. The extracted Cyperus esculentus genome DNA was diluted to 50 ng/. mu.L concentration and used as a PCR amplification template.
PCR amplification and electrophoresis detection, wherein a PCR amplification system (25uL) comprises: 2.5uL of 10 XPCR buffer, 0.5uL of 10mmol/L dNTPs, 0.5uL of 5U/uL Taq enzyme, 1.0uL of sample DNA, 1.0uL of upstream and downstream primer mixture (10 umol/L each), ddH2O19.5 uL. The PCR amplification program is that the temperature is 95 ℃ for 5 min; 35 cycles of 94 ℃ for 30s,60 ℃ for 30s and 72 ℃ for 45 s; 10min at 72 ℃. The PCR product was detected by electrophoresis on a 1.5% agarose gel.
And (3) SNP detection, namely sequencing the PCR product of the electrophoresis detection result display band, and analyzing the variation condition of the SNP locus according to the sequencing result. The results are shown in FIG. 2.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (5)

1. An SNP molecular marker set for genotyping of cyperus esculentus, which comprises at least one of 1292 SNP markers, wherein the 1292 SNP markers are shown in the following table:
Figure FDA0003310588240000011
Figure FDA0003310588240000021
Figure FDA0003310588240000031
Figure FDA0003310588240000041
Figure FDA0003310588240000051
Figure FDA0003310588240000061
Figure FDA0003310588240000071
Figure FDA0003310588240000081
Figure FDA0003310588240000091
Figure FDA0003310588240000101
Figure FDA0003310588240000111
Figure FDA0003310588240000121
Figure FDA0003310588240000131
Figure FDA0003310588240000141
Figure FDA0003310588240000151
Figure FDA0003310588240000161
Figure FDA0003310588240000171
Figure FDA0003310588240000181
Figure FDA0003310588240000191
Figure FDA0003310588240000201
Figure FDA0003310588240000211
Figure FDA0003310588240000221
Figure FDA0003310588240000231
Figure FDA0003310588240000241
Figure FDA0003310588240000251
Figure FDA0003310588240000261
Figure FDA0003310588240000271
Figure FDA0003310588240000281
Figure FDA0003310588240000291
Figure FDA0003310588240000301
Figure FDA0003310588240000311
Figure FDA0003310588240000321
Figure FDA0003310588240000331
Figure FDA0003310588240000341
Figure FDA0003310588240000351
Figure FDA0003310588240000361
Figure FDA0003310588240000371
Figure FDA0003310588240000381
Figure FDA0003310588240000391
Figure FDA0003310588240000401
Figure FDA0003310588240000411
Figure FDA0003310588240000421
Figure FDA0003310588240000431
Figure FDA0003310588240000441
Figure FDA0003310588240000451
Figure FDA0003310588240000461
Figure FDA0003310588240000471
Figure FDA0003310588240000481
Figure FDA0003310588240000491
Figure FDA0003310588240000501
Figure FDA0003310588240000511
Figure FDA0003310588240000521
Figure FDA0003310588240000531
Figure FDA0003310588240000541
Figure FDA0003310588240000551
Figure FDA0003310588240000561
Figure FDA0003310588240000571
Figure FDA0003310588240000581
Figure FDA0003310588240000591
Figure FDA0003310588240000601
Figure FDA0003310588240000611
Figure FDA0003310588240000621
Figure FDA0003310588240000631
Figure FDA0003310588240000641
Figure FDA0003310588240000651
Figure FDA0003310588240000661
Figure FDA0003310588240000671
Figure FDA0003310588240000681
Figure FDA0003310588240000691
2. the SNP molecular marker set according to claim 1, comprising at least 2 out of 1292 SNP markers.
3. The SNP molecular marker set according to claim 2, comprising 1292 SNP markers.
4. The use of the SNP molecular marker combination according to any one of claims 1 to 3 for identification of germplasm resources and variety genotypes of cyperus esculentus.
5. The use of the SNP molecular marker combination according to any one of claims 1 to 3 for constructing a DNA fingerprint of cyperus esculentus.
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CN116411121A (en) * 2023-04-10 2023-07-11 河南省农业科学院经济作物研究所 Kit for amplifying primers and simultaneously identifying multiple grain types and seed coat traits of cyperus esculentus

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