CN116676410A - SNP molecular marker combination for constructing sugarcane DNA fingerprint and application - Google Patents
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Abstract
The invention relates to the technical field of sugarcane molecular biology, and particularly discloses SNP molecular marker combinations for constructing sugarcane DNA fingerprint patterns and application thereof. The SNP molecular marker combination for constructing the sugarcane DNA fingerprint consists of 128 SNP molecular markers, has high specificity and resolution, and can reveal the true level utilization of sugarcane genetic diversity; the DNA fingerprint library is established by utilizing the SNP molecular marker combination, DNA fingerprint information of different sugarcane varieties can be correctly reflected, variety identification can be carried out on the sugarcane, the detection result is accurate and efficient, 245 sugarcane germplasm resource varieties are effectively distinguished for the first time, the identification capability is strong, and technical support is provided for the protection of sugarcane germplasm resources.
Description
Technical Field
The invention belongs to the technical field of sugarcane molecular biology, and particularly relates to SNP molecular marker combination for constructing a sugarcane DNA fingerprint and application thereof.
Background
Sugarcane is the first large sugar crop in the world and is widely distributed in more than one hundred countries, and the global planting area exceeds more than one thousand, nine hundred thousand hectares. The sugarcane germplasm resources are rich, and the sugarcane varieties are many. Early, sugarcane variety (line) identification relies on observing agronomic characters such as leaf bud appearance, stem diameter and the like, so that the sugarcane variety (line) identification is obviously influenced by cultivation conditions and habitat. In addition, most sugarcane varieties (lines) adopt a hybridization and backcross breeding mode, backbone parent germplasm resources are narrow, offspring genetic variation is tiny, similarity is higher and higher, and the offspring genetic variation is difficult to distinguish only by morphological difference. Therefore, the efficient and accurate variety identification has important significance for the cultivation and popularization of new varieties of sugarcane, the protection of intellectual property rights and the like.
The DNA fingerprint can reflect the biological individual difference essentially, has high individual specificity and stable reliability, and improves the effective means for realizing accurate and rapid variety identification. Currently, RFLP, RAPD, AFLP, ISSR, SSR, SNP and other common molecular markers for constructing DNA fingerprints are used, and the genetic diversity analysis of 13 Saccharum and Saccharum materials from the United states and Australia by utilizing RFLP molecular fingerprints is reported in the literature, but the application of the RFLP markers is greatly hindered by the use of radioactive isotopes, and the RFLP markers are low in reliability. There are also reports of research on 68 parts of sugar cane AFLP sugar cane fingerprints from Australia, france, america and Philippines, and the research results show that the AFLP markers have the characteristics of high polymorphism and good reliability. However, AFLP technology requires high DNA quality and purity, requires steps such as digestion, ligation, pre-amplification, selective amplification, and the like, is cumbersome in process, shows polymorphism of amplified fragment length, has difficulty in distinguishing DNA fragments with the same length but different sequences, and therefore has no large-scale application. SNP is used as a third generation molecular marker, has the advantages of high density, strong stability and the like, and has been widely used for identifying crop varieties. Compared with other crops, the sugarcane molecular fingerprint based on the SNP mark typing technology has few reports, the SNP loci are utilized to identify the germplasm of the sugarcane, and the sugarcane DNA fingerprint database based on the core SNP loci is constructed, so that the method has important significance in the aspects of high-efficiency identification of sugarcane varieties, cultivation of new varieties of the sugarcane and the like.
Disclosure of Invention
The invention aims to provide SNP molecular marker combinations and applications for constructing sugarcane DNA fingerprint patterns.
In order to achieve the above purpose, the invention provides an SNP molecular marker combination for constructing a sugarcane DNA fingerprint, wherein the SNP molecular marker combination consists of 128 SNP molecular markers, the numbers of the SNP molecular markers are GWHbEII-001 to GWHbEII-128, and specific information is shown in table 1.
TABLE 1 sugarcane 128 SNP molecular marker information
On the other hand, the invention also provides application of the SNP molecular marker combination in any one of the following aspects:
(1) The method is used for constructing sugarcane DNA fingerprint;
(2) Is used for sugarcane genotyping;
(3) The method is used for identifying the germplasm resources and varieties of the sugarcane;
(4) The method is used for breeding sugarcane germplasm resources;
(5) The method is used for sugarcane molecular marker assisted breeding.
On the other hand, the invention also provides a method for constructing the DNA fingerprint of the sugarcane germplasm resource or variety, which comprises the following steps:
s1, extracting DNA of a sugarcane germplasm resource or variety for constructing a DNA fingerprint, and detecting genotypes of 128 sites according to claim 1;
s2, combining and arranging genotypes of each sugarcane germplasm resource or variety at the 128 SNP loci according to the locus numbering sequence to form a specific sequence, wherein the specific sequence is a DNA fingerprint of the sugarcane germplasm resource or variety.
Preferably, in the method for constructing the DNA fingerprint of the sugarcane germplasm resource or variety, the specific sequence obtained in the step S2 is encoded, and a two-dimensional code or a bar code which can be identified by a terminal of the device is constructed, so that the sugarcane germplasm resource or variety is intelligently identified and managed.
On the other hand, the invention also provides a sugarcane variety DNA fingerprint library constructed by the construction method.
On the other hand, the invention also provides a method for identifying sugarcane varieties, which comprises the steps of extracting DNA of a sugarcane germplasm sample to be detected, detecting 128 SNP loci combined by the SNP molecular markers in the genome DNA of the sample to be detected, determining genotypes of the SNP molecular markers according to detection results of the SNP loci, and comparing the genotypes with the constructed DNA fingerprint library in the claim 5 to identify sugarcane germplasm.
Compared with the prior art, the invention has the following beneficial effects:
the SNP molecular marker combination for constructing the sugarcane DNA fingerprint consists of 128 SNP molecular markers, has high specificity and resolution, and can reveal the true level utilization of sugarcane genetic diversity; the DNA fingerprint library is established by utilizing the SNP molecular marker combination, DNA fingerprint information of different sugarcane varieties can be correctly reflected, variety identification can be carried out on the sugarcane, the detection result is accurate and efficient, 245 sugarcane germplasm resource varieties are effectively distinguished for the first time, the identification capability is strong, and technical support is provided for the protection of sugarcane germplasm resources.
Drawings
FIG. 1 is a cluster map of 248 parts of sugarcane germplasm resources according to the invention.
FIG. 2 shows MAF and PIC maps of 1343 SNP sites in example 1 according to the invention.
FIG. 3 is a map of heterozygosity of 1343 SNP sites in example 1 of the invention.
FIG. 4 is a graph showing genetic diversity of germplasm resources in example 1 of the present invention.
FIG. 5 shows the genotyping results for sugarcane material in example 2 of the present invention: wherein each row represents a SNP genotype, each column represents a single material, and yellow, green, blue and purple represent nucleotides C/C, A/A, T/T and G/G, respectively. The missing data are shown in gray and the heterozygous sites in white.
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments.
Example 1 SNP loci for construction of sugarcane DNA finger print
The invention uses 248 sugarcane germplasm resource samples, the germplasm resource information is shown in table 2, a CTAB method is adopted to extract genome DNA from sugarcane leaf tissues, complete genome simplified genome sequencing is completed in Dio biotechnology (Guangzhou) limited company, and 128 SNP loci are obtained based on sequencing data. The 248 parts of sugarcane germplasm are from the places of China, the United states, india, brazil and the like, and the cluster diagram of 248 parts of sugarcane germplasm resources is shown in FIG. 1.
The SNP locus screening method comprises the following steps:
(1) Filtering the mutation sites by using Plink to obtain 2717029 SNP+indels marks, deleting Indels and sites with deletion rate of more than 20% or MAF <5%, and remaining 122188 SNPs;
(2) The PIC <0.35 site was deleted, leaving 42318 sites;
(3) SNPs with p-value <0.01 tested by the hadamard-hilbert equalization (HWE) were deleted, leaving 3413 SNPs;
(4) Removing one of two sites with higher LD value by Plink1.9 according to condition-index-paper 50100.2, and remaining 1594 sites;
(5) The SNP loci of other mutations of 100bp before and after the SNP are removed, the SNP loci on 80 chromosomes are reserved, and 1343 SNPs remain. MAF and PIC values of 1343 SNP loci vary from 0.22 to 0.50 and from 0.35 to 0.50 (see FIG. 2), the average values are 0.31 and 0.40, respectively, the observed heterozygosity of 1343 variable SNP loci varies from 0.29 to 0.59 (see FIG. 3), and averages to 0.45, and genetic diversity of germplasm resources was also evaluated, and found to be as low as 0.35, as high as 0.51, and average to be 0.40 (see FIG. 4). The MAF, PIC and the observed heterozygosity value are utilized to further screen SNP loci which are distributed on 80 chromosomes, have no genotype data deletion and are mainly homozygote variation loci, and are detected in as many individuals as possible, so that 248 SNP loci are finally obtained;
(6) 128 SNPs were further screened to obtain by allowing 1 SNP to exist in the 10M range, and specific information is shown in Table 1.
Example 2 genotyping sugarcane germplasm resources and constructing finger print
Constructing a fingerprint spectrum: extracting leaf sample DNA of sugarcane germplasm resources for constructing DNA fingerprint, and obtaining genotypes of 128 SNP loci of sugarcane germplasm resources samples by simplifying genome sequencing (GBS method) at Basodiao biotechnology (Guangzhou) limited company; according to the genotyping result, the genotypes of 128 SNP loci of each sugarcane germplasm resource are combined and arranged according to the sequence of locus numbers to form a specific sequence, wherein the specific sequence is the DNA fingerprint of the sugarcane germplasm resource or variety, and the specific sequence is specifically shown in Table 2. The typing results of 248 test materials are shown in FIG. 5, in which each row represents one SNP genotype, each column represents one material, and the colors yellow, green, blue and purple represent nucleotides C/C, A/A, T/T and G/G, respectively, the missing data are shown in gray, and the heterozygous sites are shown in white. The genotype data (specific sequence) of 248 parts of core SNP of the sugarcane germplasm is encoded by using online software Caolaoerweima (http:// cli. Im /), a two-dimensional bar code is constructed for each part of sugarcane material, specifically, the table is shown in table 2, the "-" in the table indicates that the SNP locus is missing in the material, the effective SNP data ratio in the sample is more than or equal to 0.80 and is an effective sample, wherein the ineffective SNP data ratio of S12, S141 and S234 is more than 20%, and is an ineffective sample, and 245 parts of sugarcane germplasm resources can be distinguished in 248 parts of sugarcane germplasm resources.
TABLE 2 sugarcane germplasm resources and finger print
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Example 3
The identification method of the sugarcane varieties comprises the steps of extracting DNA of the sugarcane germplasm to be detected, detecting genotypes of 128 SNP loci of genome DNA of the sugarcane germplasm to be detected by adopting a simplified gene sequencing method, and comparing the genotypes with a sugarcane DNA fingerprint library to identify the sugarcane germplasm. Blind DNA identification was performed on a sample containing ROC22 with a DNA fingerprint of ROC 22. In blind sample identification, 10 sugarcane samples (No. 2 is ROC 22) are randomly extracted, genotypes of 128 SNP loci are detected on the 10 sugarcane samples by adopting simplified genome sequencing, and the result shows that the 128 core SNP loci of the No. 2 sample have 126 coincidences, the coincidence rate is more than 98 percent, two ineffective loci are arranged in the ROC22, the effective loci of the No. 2 sample are consistent with the effective loci of the ROC22, the approximation coefficient is 100 percent (the number of the effective loci of the sample to be detected is the same as that of the standard sample and the number of the effective loci of the standard sample is the same as that of the standard sample), and the No. 2 sample is judged to be an approximate or suspected identical variety of the ROC 22.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (8)
1. The SNP molecular marker combination for constructing the sugarcane DNA fingerprint is characterized by comprising 128 SNP molecular markers, wherein the number of the SNP molecular marker combination is GWHEIII-001-GWHEIII-128, specific information is shown in table 1, the marker GWHEIII-001 is positioned at 52692696 of chromosome 1 of the sugarcane genome, the nucleotide is T or C, and the rest markers are similar.
2. The use of the SNP molecular marker combination of claim 1 in construction of sugarcane DNA fingerprint.
3. The use of the SNP molecular marker combination of claim 1 in sugarcane genotyping or sugarcane germplasm resource and variety identification.
4. The use of the SNP molecular marker combination of claim 1 in sugarcane germplasm resources or sugarcane molecular marker assisted breeding.
5. A method for constructing a DNA fingerprint of a sugarcane germplasm resource or variety, comprising the steps of:
s1, extracting DNA of a sugarcane germplasm resource or variety for constructing a DNA fingerprint, and detecting genotypes of 128 sites according to claim 1;
s2, combining and arranging genotypes of each sugarcane germplasm resource or variety at the 128 SNP loci according to the locus numbering sequence to form a specific sequence, wherein the specific sequence is a DNA fingerprint of the sugarcane germplasm resource or variety.
6. The method for constructing a DNA fingerprint of a sugarcane germplasm resource or variety according to claim 5, wherein the specific sequence obtained in the step S2 is encoded, a two-dimensional code or a bar code which can be identified by a terminal of a construction device is constructed, and the sugarcane germplasm resource or variety is intelligently identified and managed.
7. A DNA fingerprint of a sugar cane variety constructed using the construction method of claim 5 or 6.
8. The identification method of the sugarcane varieties is characterized by comprising the steps of extracting DNA of sugarcane germplasm to be detected, detecting genotypes of 128 SNP loci combined by the SNP molecular markers in the genome DNA of the sugarcane germplasm to be detected, determining the genotypes of the SNP molecular markers according to detection results of the SNP loci, and comparing the genotypes with the constructed DNA fingerprint library in claim 5 to identify the sugarcane germplasm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105474956A (en) * | 2015-12-04 | 2016-04-13 | 扶绥县科学技术局 | High-yield planting method for sugarcane |
| CN113881794A (en) * | 2021-09-02 | 2022-01-04 | 广东省科学院南繁种业研究所 | Group of molecular markers obviously related to sugarcane leaf included angle and application thereof |
| CN115820923A (en) * | 2022-12-21 | 2023-03-21 | 海南大学 | Molecular marker combination for constructing sugarcane DNA fingerprint and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105474956A (en) * | 2015-12-04 | 2016-04-13 | 扶绥县科学技术局 | High-yield planting method for sugarcane |
| CN113881794A (en) * | 2021-09-02 | 2022-01-04 | 广东省科学院南繁种业研究所 | Group of molecular markers obviously related to sugarcane leaf included angle and application thereof |
| CN115820923A (en) * | 2022-12-21 | 2023-03-21 | 海南大学 | Molecular marker combination for constructing sugarcane DNA fingerprint and application thereof |
Non-Patent Citations (1)
| Title |
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| 黄玉新等: "甘蔗遗传连锁图谱构建及QTL 定位研究进展", 《分子植物育种》, 23 February 2022 (2022-02-23), pages 1 - 23 * |
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