CN108220402B - Method for identifying pedigree relationship between Chinese cabbage germplasm and variety - Google Patents
Method for identifying pedigree relationship between Chinese cabbage germplasm and variety Download PDFInfo
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Abstract
The invention discloses an identification method of the genetic relationship between Chinese cabbage germplasm and variety pedigree. The invention converts the detection result of the co-dominant marker with simple band type into A, G, T, C four letters, effectively combines the sequence analysis of conventional nucleic acid diversity analysis software such as DNAMAN, clusterW, MEGA and the like with the assignment analysis of the diploid Chinese cabbage co-dominant marker, and solves the fussy problem that NTSYS can only recognize binary 0 and 1 data matrixes but can not interpret genotype data or the marker detection result needs to be converted into 0/1 matrixes or AA/BB letter matrixes according to the requirements of the software. The method of the invention is basically consistent with the NTSYS clustering result, but omits the preparation step of the marker file of the NTSYS clustering analysis software, has simple operation, and is suitable for analyzing the relationship between the hybrid and the pure line.
Description
Technical Field
The invention belongs to the technical field of plant breeding, particularly relates to a Chinese cabbage breeding technology, and particularly relates to a method for identifying germplasm and variety pedigree relationship of Chinese cabbages.
Background
The research on genetic diversity is an important basis for effective utilization and protection of plant germplasm resources, the genetic diversity of the plant germplasm resources is detected and researched, a mechanism for forming and maintaining the genetic diversity can be disclosed, and scientific basis and an implementation scheme are provided for the establishment of efficient utilization and reasonable protection strategies of the plant germplasm resources.
The tools for plant germplasm genetic diversity research mainly comprise biochemical markers such as enzyme marker and DNA-based molecular markers (Zhanghong, etc., research on 61 national barley genetic diversity by applying SSR marker, plant genetic resources bulletin 2008,9(1): 15-19; GarrisAJ, TaiTH, CoburnJ, et, genetic structure and diversity in Oryza sativa, Genetics, 2005, 69: 1631-. Among them, especially, with the development of molecular biology, various molecular markers developed based on sequence differences of genetic materials, i.e., DNA, are widely used for genetic relationship identification of crop varieties or resources. The molecular marker tools adopted by the genetic diversity research mainly comprise dominant and codominant, and the codominant marker with wide distribution and good repeatability is particularly suitable for the used molecular markers, such as SSR, InDels, SNP and the like. Simple Sequence Repeats (SSRs), also called microsatellite DNA (microsatellite DNA), are DNA sequences consisting of 1-6 nucleotide tandem repeats, such as (CA) n, (ATG) n, (TAGG) n and the like, have short length, are widely distributed at different positions of a genome, cause polymorphism of each locus due to different numbers of repeated elements and the number of element bases, and can be divided into two types of genome SSRs and EST-s. The marker of insertion/deletion polymorphism (InDels) is characterized in that the DNA sequence at the same site generates insertion/deletion of nucleotide fragments among different individuals, specific primers are designed according to sequences at two sides of a target site for PCR amplification, the length polymorphism of an amplified fragment is an InDels marker, the InDel marker is developed according to insertion/deletion difference between homologous sequences, and the InDel marker has certain difficulty for development of crops with unknown genome sequences, is a co-dominant marker, and has the advantages of better stability, abundant polymorphism and the like.
In the study of genetic diversity, two different types of molecular markers, dominant and co-dominant, produce different data types, dominant molecular markers produce binary data (which can be labeled with 1/0), and co-dominant molecular markers produce genotype data, and the different data forms bring various difficulties to the analysis and study of genetic diversity. For co-dominant molecular markers such as SSR, InDels and the like, for a long time, when researchers use the markers to identify the genetic relationship of crop germplasm, specific software such as NTSYS, Popgene and the like is generally adopted, and a marker detection result needs to be converted into an 0/1 matrix or an AA/BB letter matrix according to the requirements of the software, and then the result can be obtained through complicated calculation. However, for example, when NTSYS cluster analysis software is used, the marker file preparation step is complicated, and NTSYS can analyze only the relationship between pure lines, and is not suitable for analyzing the relationship between hybrids and pure lines.
Disclosure of Invention
Aiming at the problems in the prior art, the invention develops a method for identifying the genetic relationship between the germplasm and the variety of Chinese cabbage, converts the detection results of a series of co-dominant markers of the same sample into a 'marker sequence' consisting of A, C, G, T four base letters by a co-dominant marker assignment method, in particular to an InDels marker assignment method, and analyzes and compares the marker detection results of different samples by using nucleic acid diversity analysis software by combining conventional nucleic acid diversity analysis software such as DNAMAN, clusterW, MEGA and the like to draw a homologous relationship tree, so that the genetic relationship among the materials can be visually seen.
Specifically, the invention relates to the following technical scheme:
the invention discloses a method for identifying the genetic relationship between Chinese cabbage germplasm and variety pedigree, which comprises the following steps:
(1) extracting the genomic DNA of a sample to be detected;
(2) selection and screening criteria for co-dominant markers: designing primers according to the codominant marker for amplification and PAGE detection, selecting a primer combination with polymorphism and good specificity, and performing PCR amplification and capillary electrophoresis detection;
(3) and (3) tag assignment: arranging the mark detection results of all samples to be detected in sequence, wherein the sequence of marks in a batch of samples is consistent; if the size of the target fragment of the single marker in all detection samples is larger than four, the marker is eliminated; the detection results of the single polymorphism markers in all samples are sequentially assigned as A, G, T, C according to the target fragments from large to small; the detection result assignment of a single marker in a particular sample consists of two letters; for the condition that the selected marker in the detection sample is completely deleted, the proportion of the deleted sample is more than sixty percent of the detection sample, the marker is eliminated, and the deleted band is expressed by NN;
(4) and (3) outputting the identification result of the germplasm and variety pedigree relationship: assigning the marks to generate a DNA sequence consisting of A/T/G/C letters, analyzing and comparing the mark detection results of different samples to be detected by using nucleic acid diversity analysis software, drawing a homologous relation tree, and obtaining the genetic relationship among the samples to be detected.
The invention identifies the germplasm and variety pedigree relationship of Chinese cabbage, the Chinese cabbage is diploid crop, the genotype data generated by co-dominant molecular markers (such as SSR, InDels and the like) has diversity, most of the co-dominant molecular markers such as SSR/InDels have effective allele factors within 4 (Zezuke and the like, the development and the universal analysis of Simple Sequence Repeat (SSR) and insertion/deletion (InDel) markers of the Chinese cabbage, agricultural biotechnology reports 2012,20(12): 1398-1406, the technical contents of the article are incorporated into the application), because DNA sequence comparison software such as DNMAN can intuitively provide homologous relation tree files of all sequences according to the similarity of a plurality of DNA sequences, the invention effectively combines the sequence analysis of conventional nucleic acid diversity analysis software such as DNAMMAN, clusterW, diploid and the like with assignment analysis of the co-dominant markers of the Chinese cabbage, the method solves the problem that NTSYS can only recognize binary 0 and 1 data matrixes but can not interpret genotype data or NTSYS, Popgene and the like need to convert a marker detection result into an 0/1 matrix or an AA/BB letter matrix according to the requirements of software.
Specifically, in the step (1) of the identification method, the method known in the prior art can be adopted for extracting the genomic DNA of the sample to be detected, for example, the method for extracting the genomic DNA of the Chinese cabbage sample refers to Liu bolt peaches and the like (journal of agricultural biotechnology, 2014,22(7): 853-861).
In a preferred embodiment of the present invention, the co-dominant marker of the present invention is one of SSR, InDels, and SNP.
In a preferred embodiment of the invention, the co-dominant marker is an InDels marker. The InDels marker selected in the step (2) can be publicly reported in the prior art, such as Zezuke and the like, the development and the universality analysis of Chinese cabbage Simple Sequence Repeat (SSR) and insertion/deletion (InDel) markers, agricultural biotechnology reports 2012,20(12): 1398-1406, or newly developed markers, such as Zhang Zhi and the like, agricultural biotechnology reports 2016,24(4): 510-518, the contents of which are incorporated into the application.
For example, in marker development, a Chinese cabbage sample is subjected to whole genome re-sequencing, sites with deletion difference of more than 5bp are inserted into sequencing data through screening, primers are designed in two side conserved regions, and PCR amplification and PAGE detection are carried out.
In the selection and screening standard of the specific InDels codominant marker, a primer combination which has difference in InDels markers in a selected sample and can amplify only a single band is selected for PCR amplification and capillary electrophoresis detection.
Preferably, the material used for development and marking is the inbred line 06-247 of Jianchun, a hybrid variety of Japanese cabbage, and the inbred line He102 of Henan Bibao, a local variety of cabbage.
In a specific embodiment, the assignment of the detection result of a single marker in a specific sample consists of two letters, wherein the homozygous type is AA, GG, TT and CC, the heterozygous type is AG, AT, AC, GT, GC and TC or CT, CG, CA, TG, TA and GA, and the assignment criteria in a batch of samples are consistent.
Specifically, the sequence of the heterozygote assignments in a batch of samples, or the assignment of a relatively large fragment followed by the assignment of a relatively small fragment, may be AG, AT, AC, GT, GC, TC, or vice versa, i.e., the assignment of a relatively small fragment followed by the assignment of a relatively large fragment, i.e., CT, CG, CA, TG, TA, GA, follows a sequence in a batch of samples.
In a specific embodiment, the nucleic acid diversity analysis software is selected from one or more of DNAMAN, clusterW and MEGA.
In the specific embodiment, DNAMAN and MEGA6.0 software are adopted to identify the genetic relationship of the hybrid so as to identify the genetic relationship between the newly marketed hybrid and the sold variety; and the MEGA6.0 is adopted to carry out UPGMA cluster analysis on the core germplasm and the hybrid, so that the pedigree relationship of the hybrid is analyzed, and the parental source of a certain hybrid is easily judged.
In a preferred embodiment, the identification method of the invention can be used for identifying the genetic relationship between Chinese cabbage hybrids.
In a preferred embodiment, the identification method is used for identifying molecular markers of the collected hybrid resources, judging the genetic relationship between the hybrid resources and the existing germplasm, and searching materials with relatively close genetic relationship for backcross transformation so as to purposefully perform germplasm innovation and avoid blindness.
The invention achieves the following beneficial effects:
compared with the cluster analysis software NYSIS, popgene and the like, the DNA comparison software DNMAN, MEGA and the like has higher popularity among molecular biology researchers, so that the detection result of the InDels co-dominant marker with simple band type is converted into A, G, T, C four letters, the four letters sequentially represent the bands from large to small, because the Chinese cabbage is a diploid crop, the detection result of one marker consists of two letters, namely homozygous type AA, GG, TT or CC, the sequence of heterozygous type in a batch of samples is that a relatively large fragment is preceded and a relatively small fragment is followed, namely AG, AT, AC, GT, GC and TC are possible, or vice versa, that a relatively small fragment is preceded and a relatively large fragment is followed, namely CT, CG, CA, TG, TA and GA are followed in a batch of samples. In addition, the selected marker may be completely deleted in a part of the test sample, and if the proportion of the deleted sample exceeds more than sixty percent of the test sample, the marker is eliminated, and the deleted band is represented by NN when the percentage is less than sixty percent. The detection results of a group of marks are arranged in a batch of samples according to a certain sequence, and all the detection results of the marks of a certain material are a DNA sequence, so that the results can be easily compared by using DNAMAN and MEGA software, and visual comparison results are output and basically consistent with NTSYS clustering results through comparison. The assignment method omits a marker file preparation step of NTSYS cluster analysis software, is simple to operate, and is suitable for analyzing the relationship between the hybrid and the pure line, unlike the method in which NTSYS only analyzes the relationship between the pure lines, so that the assignment method is convenient and practical.
Drawings
Screening of the markers of FIG. 1
FIG. 240 DNAMAN5.0 analysis results of hybrids
FIG. 340 MEGA6.0 analysis results of hybrids
FIG. 4127 MEGA6.0 analysis results of resources
FIG. 5127 NTSYS2.0 clustering results of resources
FIG. 640 genetic relationships between hybrids and 127 core resources
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
Materials and methods:
1.1 materials
The development marker is made of a material selected from a selfing separation line 06-247 of a Jianchun Chinese cabbage hybrid variety and a selfing separation line He102 of a Henan Bibao Chinese cabbage local variety. The detailed purchase of 40 Chinese cabbage hybrids from the market is shown in table 1, 127 parts of core resources are independently created for Chinese cabbage subject groups of vegetable and flower research institute of agricultural academy of sciences of Shandong province, and the detailed purchase is shown in table 2.
Table 140 hybrid information
Table 2127 germplasm resources information
1.2 extraction of genomic DNA from test Material
Refer to Liu Ji peach (journal of agricultural biotechnology 2014,22(7): 853-861).
1.3 development and screening criteria for InDels markers
The method for marking and developing Zhangzhigang and the like (agricultural biotechnology report, 2016,24(4): 510-518) comprises the steps of performing whole genome re-sequencing on 06-247 and He102, screening a site with deletion difference of more than 5bp in sequencing data, designing primers in conserved regions on two sides, and performing PCR amplification and PAGE detection. Selecting those primer combinations with polymorphism and good specificity, i.e. primer combinations which have difference in 06-247 and He102 and can amplify only single band respectively, and making PCR amplification and capillary electrophoresis detection on hybrid and core germplasm.
1.4 tag assignment method and assignment
All the marks to be detected are arranged according to a certain sequence, and the sequence of the marks in a batch of samples cannot be changed. Because the specificity of the selected marker is good, only 1-2 bands can be amplified in a single sample, the detection results of the single polymorphic marker in all materials are sequentially assigned to A, G, T, C according to the target fragments from large to small, and the marker is eliminated if the target fragments of the single marker in all detection samples are larger than four in size. Since celery cabbage is a diploid crop, the detection result of a single marker in a specific sample will consist of two letters, homozygous type is AA, GG, TT, CC, and heterozygous type assignment in a batch of samples follows either relatively large segment assignment first and relatively small segment assignment, i.e. AG, AT, AC, GT, GC, TC, or vice versa, i.e. relatively small segment assignment first and relatively large segment assignment later, i.e. CT, CG, CA, TG, TA, GA, follows one sequence in a batch of samples. In addition, the selected marker may be completely deleted from a part of the test sample, and if the proportion of the deleted sample exceeds more than sixty percent of the test sample, the marker is eliminated and the deletion band is represented by NN.
1.5 output of results
According to the above method, a sample is detected with X markers, which will result in a "DNA" sequence consisting of 2X letters, such sequence file being very well recognizable by nucleic acid diversity analysis software such as DNAMAN, clusterW, etc.
2 results
2.1 markers for selection
As shown in FIG. 1, the PAGE detection results in the screening of markers, wherein the numbers are marker numbers, each marker corresponds to two lanes, the left side uses 06-247 as a template, the right side uses He102 as a template, according to the detection results, markers without polymorphism (such as 9 # and 17 # and a dominant marker (such as 11 #) and with complex amplification bands (such as 3 # and 4 # and 6 # and the like) are eliminated, and only those markers which can amplify single polymorphism bands respectively in 06-247 and He102, such as 1 # and 16 # and 18 # are selected. Using this stringent screening method, co-selection 80 was performed for subsequent studies of markers numbered from X1 to X80.
2.2 identification of the hybrids
The results of genetic relationship identification of 40 hybrids using DNMAN and MEGA6.0 software are shown in FIGS. 2 and 3, and there are no reports on genetic relationship identification between hybrids in the existing literature. By the above-mentioned identification results, the genetic relationship between the newly marketed hybrid and the already marketed variety can be easily identified, and it is known that it is a substitute or upgraded version of that variety. By contrast with old varieties, molecular markers linked with improved traits can be screened and used for germplasm innovation.
2.3 identification of core germplasm
UPGMA clustering analysis of 127 core germplasm using MEGA6.0 is shown in fig. 4, which is substantially identical to the result of ntsys2.0 analysis (see fig. 5). There is no report in the literature about the analysis of genetic resource correlation by MEGA software.
2.4 identification of the phylogenetic relationship of hybrid lines
The source of the parent is judged by the unique mark of a hybrid, the genetic relationship between the hybrid and the germplasm needs to be analyzed, and the research on the relationship is not available in the prior literature. By adopting the assignment method of the invention, the pedigree relationship of the hybrid can be conveniently analyzed, for example, FIG. 6 shows the relationship between all 40 hybrids and 127 germplasms, from which the parental source of a certain hybrid can be easily judged.
Along with the popularization and the promotion of the hybrid, the farmyard varieties almost die, so that the collection of the hybrid becomes an important form for germplasm resource collection, the collected hybrid is subjected to field agronomic characters and comprehensive resistance identification, then self-bred to separate excellent strains, and the existing strains are improved through backcross transformation, so that the method is a main form for germplasm innovation of the existing Chinese cabbage. In order to avoid the homogeneity of parents, the collected resources need to be subjected to molecular marker identification in advance, the genetic relationship between the collected resources and the existing germplasm is judged, and materials with relatively close genetic relationship are found for backcross transformation, so that germplasm innovation is purposefully performed, and blindness is avoided.
Claims (10)
1. A method for identifying the genetic relationship between Chinese cabbage germplasm and variety pedigree is characterized by comprising the following steps:
(1) extracting the genomic DNA of a sample to be detected;
(2) selection and screening criteria for co-dominant markers: designing primers according to the codominant marker for amplification and PAGE detection, selecting a primer combination with polymorphism and good specificity, and performing PCR amplification and capillary electrophoresis detection;
(3) and (3) tag assignment: arranging all the marks to be detected in sequence, wherein the sequence of the marks in a batch of samples is consistent; if the size of the target fragment of the single marker in all detection samples is larger than four, the marker is eliminated; the detection results of the single polymorphism markers in all samples are sequentially assigned as A, G, T, C according to the target fragments from large to small; the detection result assignment of a single marker in a particular sample consists of two letters; for the condition that the selected marker in the detection sample is completely deleted, the proportion of the marker-deleted sample is more than sixty percent of the detection sample, the marker is eliminated, and the deletion band is expressed by NN;
(4) and (3) outputting the identification result of the germplasm and variety pedigree relationship: assigning a label to generate a DNA sequence consisting of A/T/G/C, analyzing and comparing the label detection results of different samples to be detected by using nucleic acid diversity analysis software, drawing a homologous relation tree, and obtaining the genetic relationship among the samples to be detected.
2. The method of claim 1, wherein the co-dominant marker is one of SSR, InDels, SNP.
3. The method of claim 1, wherein the co-dominant marker is an InDels marker.
4. The identification method of claim 3, wherein the Chinese cabbage sample is subjected to genome wide re-sequencing, sites with deletion difference of more than 5bp are inserted into the sequencing data by screening, primers are designed in conserved regions on two sides, and PCR amplification and PAGE detection are performed.
5. The method of claim 4, wherein the primer combinations that differ in InDels markers and are capable of amplifying only a single band are selected for PCR amplification and capillary electrophoresis detection.
6. The method of any one of claims 1 to 5, wherein in step (3) the assignment of a single label to the test result in a particular sample consists of two letters, homozygous for AA, GG, TT, CC and heterozygous for AG, AT, AC, GT, GC, TC or CT, CG, CA, TG, TA, GA, and wherein the assignment criteria in a batch of samples are identical.
7. The method of claim 6, wherein the heterozygote assignment is in the order of the values in a sample set, or the relatively large fragment assignment precedes the relatively small fragment assignment, i.e., AG, AT, AC, GT, GC, TC, or vice versa, i.e., the relatively small fragment assignment precedes the relatively large fragment assignment, i.e., CT, CG, CA, TG, TA, GA.
8. The method of any one of claims 1 to 5, wherein the nucleic acid diversity analysis software is selected from the group consisting of DNAMAN, clusterW, MEGA.
9. The identification method according to claim 8, wherein the genetic relationship identification of the hybrid is performed using DNAMAN and MEGA6.0 software to identify the genetic relationship between a newly marketed hybrid and a variety already sold; performing UPGMA clustering analysis on the core germplasm and the hybrid by adopting MEGA6.0, analyzing the pedigree relationship of the hybrid, and judging the parental source of a certain hybrid.
10. The method according to any one of claims 1 to 5, wherein the method is used for genetic relationship identification between Chinese cabbage hybrids.
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| CN109694919B (en) * | 2018-12-29 | 2022-04-12 | 山东省农业科学院蔬菜花卉研究所 | Four-type Chinese cabbage hybrid purity identification core primer and hybrid purity InDel molecular marker identification kit |
| CN109811075B (en) * | 2019-01-28 | 2020-11-13 | 北京市农林科学院 | Method for identifying authenticity of Chinese cabbage variety and special SNP primer combination thereof |
| CN111793708B (en) * | 2019-10-11 | 2021-04-27 | 北京市农林科学院 | Method for identifying authenticity of Chinese cabbage variety and special SSR primer combination thereof |
| CN111445953B (en) * | 2020-03-27 | 2022-04-26 | 武汉古奥基因科技有限公司 | Method for splitting tetraploid fish subgenome by using whole genome comparison |
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