CN115074459A - Method for developing specific fingerprint spectrum of tobacco hybrid - Google Patents

Method for developing specific fingerprint spectrum of tobacco hybrid Download PDF

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CN115074459A
CN115074459A CN202210824391.3A CN202210824391A CN115074459A CN 115074459 A CN115074459 A CN 115074459A CN 202210824391 A CN202210824391 A CN 202210824391A CN 115074459 A CN115074459 A CN 115074459A
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任民
贾振国
罗成刚
张玉
陈志强
潘旭浩
佟英
耿锐梅
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Qingzhou Tobacco Research Institute of China National Tobacco Corp of Institute of Tobacco Research of CAAS
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Abstract

A method for developing a specific fingerprint of a tobacco hybrid comprises the steps of taking massive SNP loci of the whole genome of the tobacco hybrid as a data base, obtaining an SNP variant genome through gene sequencing, and screening out the specific SNP variant genome of the tobacco hybrid by taking large-scale tobacco germplasm resources as control data; the method disclosed by the invention has the advantages that the basic data quantity and the data quantity of the control population are far higher than those of the prior art, the accuracy of the obtained fingerprint is higher, the genetic specificity of the tobacco hybrid is more truly disclosed, and the target variety can be more effectively identified. The invention explores variety specific sites with credibility from the scale of whole genome and large-scale germplasm resource groups, thereby solving the problems of the existing SSR molecular marker fingerprint technology, lack of specific sites of target varieties, limited resolution of fingerprints and the like.

Description

Method for developing specific fingerprint spectrum of tobacco hybrid
Technical Field
The invention relates to the technical field of biology, in particular to a method for developing a specific fingerprint spectrum of a tobacco hybrid.
Background
At present, the development of the tobacco germplasm resource fingerprint spectrum is mainly carried out by utilizing molecular markers, and SSR (Simple Sequence Repeat) molecular markers are mostly applied at present. The SSR molecular marker has the advantages of high polymorphism, co-dominant inheritance, good repeatability and the like. The construction of the tobacco fingerprint by using the SSR molecular markers generally requires that from 400-600 pairs of SSR markers, dozens to two hundred varieties or germplasm resources are used as control groups, 40-60 pairs of SSR markers with high polymorphism are screened and used as selection markers to construct the molecular fingerprint of a target strain, variety or germplasm resource group. The SSR molecular marker is widely used for developing a fingerprint spectrum of a tobacco germplasm resource material and a pure line bred variety, and can be used for identifying the pure line bred variety of the tobacco.
The existing SSR molecular marker detection technology has the following main problems due to the difficulty in improving the automation degree and realizing high flux: (1) in the development process of the SSR molecular marker fingerprint, the number of candidate markers and candidate markers is small, and the representativeness of the markers is low; (2) in consideration of controlling the number of markers, only marker sites with higher polymorphism can be selected, and the markers have better universality but lack specificity; (3) the number of the control population (or the reference population) is limited, the population number is usually dozens to one or two hundred, even less dozens, the resolution of the SSR molecular marker fingerprint constructed by the small-scale population is inevitably influenced, and the probability of false positive in the process of variety identification is relatively high; (4) when the SSR fingerprint is used, the workload is large, the labor consumption is high, and the detection cost is relatively high.
From the application direction, currently, SSR molecular markers are utilized, so that more universal fingerprints are developed, and fewer fingerprints are identified aiming at the variety specificity of important varieties or breeding materials. In addition, the current fingerprint spectrum mainly aims at the pure line cultivated varieties and is rare for hybrid varieties.
Disclosure of Invention
The invention aims to provide a method for developing a specific fingerprint of a tobacco hybrid, thereby solving the problems in the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for developing a specific fingerprint of a tobacco hybrid comprises the following steps:
s1, extracting the DNA of the tobacco hybrid of the specific fingerprint spectrum to be constructed, and recording the DNA as V F And extracting the DNA of the male parent and the female parent of the tobacco hybrid and recording as V P1 And V P2 ；
S2, pair V F 、V P1 And V P2 Performing genome sequencing to obtain corresponding SNP typing information, and recording SNP locus information of SNP typing as SNP A ；
S3 SNP A Screening the genome corresponding to the SNP locus, and screening out the genome at V P1 And V P2 Is homozygous in, whereas in V F The SNP site corresponding to the heterozygous genome in (1) is marked as SNP C ；
S4, setting the interval threshold between the physical positions of the genome of two SNP sites, and recording the interval threshold as T dist Screening out that the distance between the physical positions of the genomes of the two SNP loci is less than or equal to T dist Said SNP site of (1) and denoted as SNP D ；
S5, calculating SNP by using SNP variation genome of tobacco germplasm resource population as a control group D The polymorphism index value of the SNP site in (1);
s6, setting polymorphism threshold value of said polymorphism index value, and recording it as T Pi Screening and filtering the SNP locus by using the polymorphism threshold value to screen out a polymorphism index value less than or equal to T Pi The SNP site of (1) is denoted as SNP E ；
S7、SNP E The SNP locus in the tobacco hybrid is the specific fingerprint map locus of the tobacco hybrid, and the genome of the corresponding SNP locus is the specific fingerprint map of the tobacco hybrid.
Preferably, step S3 includes the following steps:
s31 SNP A Screening the genome corresponding to the SNP locus, and screening out the genome at V F 、V P1 And V P2 The SNP sites corresponding to the genomes present in all of them are denoted as SNPs B ；
S32 SNP B Screening the genome corresponding to the SNP locus, and screening out the genome at V P1 And V P2 Is homozygous in, whereas in V F The SNP site corresponding to the heterozygous genome in (1) is marked as SNP C 。
Preferably, the method of genome sequencing comprises simplified genome sequencing and whole genome sequencing.
Preferably, the calculation formula of the polymorphism index value is as follows:
Figure BDA0003743471580000021
wherein, pi i Indicates the polymorphism index value of the ith SNP site, and x indicates the distance between the physical position of the genome of the SNP site and the replication origin of the gene. j represents SNP C Or SNP D The sequence number of the SNP site(s) in (1) is from near to far from the replication origin of the gene, i ═ j +1, pi ═ j j Indicates the polymorphism index value of the jth SNP site.
Preferably, the simplified genomic Sequencing comprises Genotyping-by-Sequencing and regeneration-site associated DNA Sequencing.
Preferably, the number of samples in the tobacco germplasm resource population as the control group in step S5 is 1000 or more.
The beneficial effects of the invention are: the invention discloses a method for developing a specific fingerprint map of a tobacco hybrid, which comprises the steps of taking massive SNP loci of a whole genome of the tobacco hybrid as a data base, carrying out gene sequencing to obtain an SNP variant genome, and screening out the specific SNP variant genome of the tobacco hybrid by taking large-scale tobacco germplasm resources as contrast data; the method disclosed by the invention has the advantages that the basic data quantity and the data quantity of the control population are far higher than those of the prior art, the accuracy of the obtained fingerprint is higher, the genetic specificity of the tobacco hybrid is more truly disclosed, and the target variety can be more effectively identified. The specific fingerprint obtained by the invention has high robustness, and part of the specific fingerprint can be used as the specific identification characteristic of the tobacco hybrid in practical application.
Drawings
FIG. 1 is a flow chart of the development of a specific fingerprint of a hybrid tobacco;
FIG. 2 is a distribution diagram of the specificity fingerprint of Zhongchuan 208 in the genome.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
A method for developing a specific fingerprint of a tobacco hybrid comprises the steps of screening out specific fingerprint SNP sites of the hybrid by taking a large-scale tobacco germplasm resource group as a control group on the basis of obtaining the whole genome SNP variation of the hybrid and parents of the hybrid, and constructing the specific fingerprint of the tobacco hybrid;
as shown in fig. 1, the method comprises the following steps:
s1, extracting the microstructureDNA of tobacco hybrid by establishing specific fingerprint spectrum and recording as V F DNA of the male and female parent thereof, designated V P1 And V P2 ；
S2, pair V F 、V P1 And V P2 Carrying out genome sequencing to obtain SNP typing information of the genome, and recording SNP locus information of SNP typing as SNP A ；
S3 SNP A Screening the genome corresponding to the SNP locus, and screening out the genome at V F 、V P1 And V P2 The SNP sites corresponding to the genomes present in all of them are denoted as SNPs B ；
S4 SNP B Screening the genome corresponding to the SNP locus, and screening out the genome at V P1 And V P2 Is homozygous in, whereas in V F The SNP site corresponding to the heterozygous genome in (1) is marked as SNP C 。
S5, setting the threshold value of the interval between the physical positions of the genome of two SNP sites, denoted as Td ist Passing said interval threshold T dist Screening out the distance between the physical positions of the two SNP variant genomes is less than or equal to T dist And marking the SNP site of the selected SNP variation genome as SNP D
S6, calculating SNP by using SNP variant genome of large-scale tobacco germplasm resource population as a control group and using computer software vcftools D The polymorphism index value of the SNP site in (1),
the calculation formula of the polymorphism index value is as follows:
Figure BDA0003743471580000041
wherein, pi i Indicates the polymorphism index value of the ith SNP site, and x indicates the distance between the physical position of the genome of the SNP site and the replication origin of the gene. j represents SNP C Or SNP D The sequence number of the SNP site(s) in (1) is from near to far from the replication origin of the gene, i ═ j +1, pi ═ j j Denotes the firstPolymorphism index values of j SNP sites;
s7, setting polymorphism threshold value of said polymorphism index value, and recording it as T Pi SNP of C Or SNP D The SNP sites in the SNP site are sorted according to the sequence of the corresponding polymorphism index values from low to high, screening and filtering are carried out by utilizing the polymorphism threshold value, and the screened polymorphism index value is less than or equal to T Pi The SNP site of (1) is denoted as SNP E ；
S8、SNP E The SNP locus in the tobacco hybrid is the specific fingerprint map locus of the tobacco hybrid, and the corresponding SNP variant genome is the specific fingerprint map of the tobacco hybrid.
The genome sequencing in the above step S2 includes whole genome sequencing and simplified genome sequencing; such simplified genomic Sequencing includes, but is not limited to, Genotyping-by-Sequencing and Restriction-site associated DNA Sequencing.
Examples
The embodiment develops the specific fingerprint spectrum of the boat 208 in the flue-cured tobacco hybrid, and specifically comprises the following steps:
s1, wherein the parents of Zhongchuan 208 comprise MS Zhongyan 300 and a strain T136, and GBS simplified genome sequencing is carried out on DNAs of Zhongchuan 208, MS Zhongyan 300 and the strain T136;
s2, obtaining 42,456 SNP sites that Zhongchuan 208 and the parents thereof are not deleted, and screening 428 SNP sites that the Zhongchuan 208 parents are homozygous and differentiated, and the Zhongchuan 208 is heterozygous;
s3, filtering the 1428 SNP sites which are initially screened according to a standard that the interval is more than 50Kb, and removing the SNP sites which are too close to each other to obtain 520 SNP sites which meet the standard;
s4, using DNA of 5000 more tobacco germplasm resources as a control group, carrying out GBS simplified genome sequencing, using sequencing data of the control group to calculate the polymorphism indexes of 520 SNP sites obtained in the step S3, and setting the polymorphism threshold of the SNP sites to be 0.2; 275 SNP sites <0.2 after polymorphism threshold screening; the specific fingerprint map information of 275 SNP sites is shown in Table 1, and the distribution of the specific fingerprint maps in the genome of tobacco hybrid is shown in FIG. 2.
Table 1: zhongchuan 208 variety specific fingerprint spectrum and partial information of SNP (single nucleotide polymorphism) sites thereof
Figure BDA0003743471580000051
Figure BDA0003743471580000061
In the actual use process, the problem that partial fingerprint map site information of a sample to be detected is lost possibly occurs, so that the detection test of the fingerprint map robustness is developed. Randomly removing a certain number of SNP sites from the 275 screened fingerprint SNP sites, and checking whether the sample to be detected can still be judged to be Zhongchuan 208; 4 number gradients of 50, 100, 150 and 200 fingerprint map sites are set, each number gradient is repeated for 5 times, and corresponding number of site information is randomly deleted at each time. The test result shows that even if the deletion rate of 275 fingerprint loci is as high as 72.7%, the detection effect of the fingerprint is still not affected by inputting only 75 fingerprint information, and whether the sample to be detected is Zhongchuan 208 or not can be accurately judged.
By adopting the technical scheme disclosed by the invention, the following beneficial effects are obtained:
the invention discloses a method for developing a specific fingerprint map of a tobacco hybrid, which comprises the steps of taking massive SNP loci of a whole genome of the tobacco hybrid as a data base, carrying out gene sequencing to obtain an SNP variant genome, and screening out the specific SNP variant genome of the tobacco hybrid by taking large-scale tobacco germplasm resources as contrast data; the method disclosed by the invention has the advantages that the basic data quantity and the data quantity of the control population are far higher than those of the prior art, the accuracy of the obtained fingerprint is higher, the genetic specificity of the tobacco hybrid is more truly disclosed, and the target variety can be more effectively identified. The specific fingerprint obtained by the invention has high robustness, and part of the specific fingerprint can be used as the specific identification characteristic of the tobacco hybrid in practical application.
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 improvements can be made without departing from the principle of the present invention, and such modifications and improvements should also be considered within the scope of the present invention.

Claims (5)

1. A method for developing a specific fingerprint of a tobacco hybrid is characterized by comprising the following steps:
s1, extracting the DNA of the tobacco hybrid of the specific fingerprint spectrum to be constructed, and recording the DNA as V F And extracting the DNA of the male parent and the female parent of the tobacco hybrid and recording as V P1 And V P2 ；
S2, pair V F 、V P1 And V P2 Performing genome sequencing to obtain corresponding SNP typing information, and recording SNP locus information of SNP typing as SNP A ；
S3 SNP A Screening the genome corresponding to the SNP locus, and screening out the genome at V P1 And V P2 Is homozygous in, whereas in V F The SNP site corresponding to the heterozygous genome in (1) is marked as SNP C ；
S4, setting the interval threshold between the physical positions of the genome of two SNP sites, and recording the interval threshold as T dist At SNP C The distance between the physical positions of the genome of any two SNP sites is screened to be more than or equal to T dist The SNP site of (1) is described as SNP D ；
S5, calculating SNP by using SNP variation genome of tobacco germplasm resource population as a control group D The polymorphism index value of the SNP site in (1);
s6, setting polymorphism threshold value of said polymorphism index value, and recording it as T Pi Screening and filtering the SNP locus by using the polymorphism threshold value to screen out a polymorphism index value less than or equal to T Pi The SNP site of (1) is denoted as SNP E ；
S7、SNP E SN in (1)The P site is the specific fingerprint map site of the tobacco hybrid, and the genome of the corresponding SNP site is the specific fingerprint map of the tobacco hybrid.
2. The method for developing a fingerprint specific to a hybrid tobacco plant according to claim 1, wherein the step S3 comprises the steps of:
s31 SNP A Screening the genome corresponding to the SNP locus, and screening out the genome at V F 、V P1 And V P2 The SNP sites corresponding to the genomes present in all of them are denoted as SNPs B ；
S32 SNP B Screening the genome corresponding to the SNP locus, and screening out the genome at V P1 And V P2 Is homozygous in, whereas in V F The SNP site corresponding to the heterozygous genome in (1) is marked as SNP C 。
3. The method for developing a specific fingerprint of a tobacco hybrid according to claim 1, wherein said method for genome sequencing comprises simplified genome sequencing and whole genome sequencing.
4. The method of claim 1, wherein the simplified genomic Sequencing comprises Genotyping-by-Sequencing and Restriction-site assisted DNA Sequencing.
5. The method for developing a specific fingerprint of a hybrid tobacco plant according to claim 1, wherein the number of samples in said population of tobacco germplasm resources used as a control group in step S5 is 1000 or more.
CN202210824391.3A 2022-07-13 2022-07-13 Method for developing specific fingerprint spectrum of tobacco hybrid Pending CN115074459A (en)

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