CN108676906B - SSR locus of corn chloroplast genome and application of SSR locus in variety identification - Google Patents
SSR locus of corn chloroplast genome and application of SSR locus in variety identification Download PDFInfo
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Abstract
The invention provides an SSR locus of a corn chloroplast genome and application thereof in variety identification. According to the invention, the accuracy and high efficiency of the chloroplast polymorphic sites are ensured through representative sample selection, high-quality sequencing data and accurate data analysis, corn materials with wide sources and rich phenotypes and genotypes are selected, the spliced corn material chloroplast genome sequence is used for searching SSR, and 13 chloroplast SSR polymorphic sites are finally determined by using MISA software. The use of these SSR sites allows: (1) constructing a chloroplast DNA-SSR fingerprint database of a corn variety; (2) performing maternal traceability analysis on the corn sample; (3) and (4) carrying out positive and negative cross identification on the corn sample. The chloroplast SSR locus can expand the range of available marker loci of corn on the genome level, and provides a new thought and method for the research of corn variety, germplasm resource identification, genetic relationship evaluation, cytoplasm hereditary property and the like.
Description
Technical Field
The invention belongs to the technical field of crop molecular biology, and particularly relates to a corn chloroplast SSR molecular marker and application thereof.
Background
Chloroplasts are organelles of green plants that perform photosynthesis and possess an intact set of genomes known as the chloroplast genome. Chloroplast genome information is widely applied to research and application of plant variety, germplasm resource identification, genetic relationship evaluation, system evolution, cytoplasm genetic characteristics and the like due to the following advantages. (1) The chloroplast genome is small and relatively conserved, and the complete sequence is easily obtained; (2) chloroplast genome is maternal inheritance, gene exchange and fusion between different individuals rarely occur, and all genes of chloroplast have good colinearity; (3) the chloroplast genome is single copy genes except for the inverted repeat region, and the paralogous gene interference hardly exists; (4) the evolution speed difference of chloroplast coding regions and non-coding regions is obvious, and some high mutation regions exist, so that the problem of the following classification units can be solved.
In the research and application of corn varieties, breeding materials and germplasm resource identification, the genetic information of the nuclear genome of corn is adopted at present. For example, the SSR marking method for identifying corn varieties adopts 40 pairs of SSR (simple sequence repeat) primers (Wangfeng et al, 2014); chip maizeSNP3072 (tianan et al, 2015) suitable for corn DNA fingerprinting; commercial corn chip product maizensnp 50K (Ganal et al, 2011); and GBS based high throughput sequencing technology, simplified genome sequencing methods, and the like. The existing corn sample molecular identification method is based on the corn cell nuclear genome sequence information, and plant cells have cytoplasmic inheritance, namely chloroplast and mitochondrial genome information besides nuclear inheritance. And the cytoplasmic genome information, particularly the chloroplast genome information, has the advantages, and is more suitable for identification of maternal traceability and the like of corn samples. The existing maize genome polymorphic site set is not recorded with SSR polymorphic sites developed aiming at chloroplast genomes.
Disclosure of Invention
The invention aims to provide a set of chloroplast SSR molecular markers suitable for the traceability of a corn maternal line.
In order to achieve the purpose of the invention, 170 parts of maize inbred line materials which are wide in source, rich in phenotype and genotype and strong in representativeness are collected, and the SSR locus combination of the maize chloroplast genome is developed based on high-quality re-sequencing data and the application of the SSR locus combination in maize sample identification is explained. The general concepts and steps are as follows. (1) And selecting 170 parts of corn representative test materials, wherein the types of the corn representative test materials comprise all heterosis groups in China, and three types of samples of sweet glutinous, local varieties and CMS sterility. (2) High concentration and high quality of total DNA preparation. (3) Based on the high-throughput sequencing of a second-generation sequencing platform, the size of a constructed library is 500bp, PE140 is obtained, and the sequencing depth is 5 times. (4) Whole genome sequence data processing, chloroplast genome splicing, independent splicing by using two software, screening contigs belonging to chloroplast genome by using BLAST program based on a maize B73 chloroplast genome sequence, assembling and verifying sequence accuracy. (5) And (3) chloroplast genome annotation, polymorphic site determination and annotation of the chloroplast genome by using DOGMA software. (6) 170 parts of material chloroplast genome sequences are compared, MISA (http:// pgrc. ipk-gatersleen. de/MISA /) software is utilized to search SSR sites, the specific screening indexes are that the repetition times of single bases are higher than 10, the repetition times of two bases are higher than 8, the repetition times of three bases are higher than 5, the repetition times of four bases are higher than 4, the repetition times of five bases are higher than 3, the repetition times of six bases are higher than 3, and 33 SSR sites are obtained. (7) Primers are designed on two sides of the 33 SSR loci, verification and analysis are carried out on the basis of a fluorescence capillary electrophoresis platform, analysis is carried out according to indexes such as whether amplification is successful, whether amplification is a single peak or whether amplification is polymorphic, and finally evaluation is carried out to determine 13 chloroplast SSR polymorphic loci.
The physical positions of the 13 SSR polymorphic sites are determined based on comparison of chloroplast genome sequences of a corn variety B73, the 13 SSR polymorphic sites are numbered from CPMSSRP-01 to CPMSSRP-13, and specific site information is shown in Table 1. The technical scheme for the development and evaluation of 13 maize chloroplast SSR loci of the invention is shown in figure 1.
TABLE 113 chloroplast SSR polymorphic site information
The 13 chloroplast SSR loci provided by the invention are single-base length polymorphism, so that the set of locus combination can realize the acquisition of genotyping data on a fluorescence capillary electrophoresis platform. Designing primers based on SSR locus flanking sequences, wherein a fluorescent group is marked at the 5' end of one primer; preparing PCR reaction system and adding DNA, primer, dNTP and MgCl2Taq enzyme, Buffer; operating a reaction program; detecting the amplification product on a fluorescent capillary electrophoresis system; and (3) collecting original data by using capillary electrophoresis system matched software, and introducing SSR Analyzer software to analyze the original data to obtain genotype data in a fragment length format.
The invention also provides a method for constructing the corn chloroplast genome SSR-DNA fingerprint database. The method specifically comprises the following steps: (1) extracting the total genome DNA of a variety for constructing a fingerprint database; (2) a chloroplast genome DNA fingerprint database is obtained by utilizing a set of 13 SSR loci provided by the invention based on a fluorescence capillary electrophoresis platform.
The invention also provides a method for maternal traceability analysis of the corn sample. The 13 polymorphic sites provided by the invention are developed based on a corn chloroplast genome and are strict maternal genetic markers, so that the polymorphic sites can be applied to maternal traceability analysis of a corn sample. The application is based on the construction of a known maize inbred line variety chloroplast genome SSR-DNA fingerprint database.
The invention also provides a method for identifying the positive and negative cross of the corn hybrid. Maize hybrids are typically produced from two inbred parents, and the parents typically belong to different heterotic model groups. Analysis based on 170 maize samples showed that except for the minor heterotic patterns like reed/lanka, both the female and male parents of the major heterotic pattern could be identified by chloroplast markers. Therefore, seeds produced by the same hybridization combination in a positive and negative cross mode can be identified by extracting total DNA and utilizing chloroplast marker loci. Compared with the traditional mode of extracting the pericarp DNA and the endosperm DNA, the method is simple and easy to implement.
The invention provides application of the SSR molecular marker in maize molecular marker-assisted breeding.
The invention provides application of the SSR molecular marker in preparation of a corn genome chip.
The invention provides application of the SSR molecular marker in identification of maize germplasm resources.
The invention provides application of the SSR molecular marker in corn cytoplasm genetic research.
Any of the applications described above, comprising the steps of:
1) extracting DNA of a corn sample to be detected;
2) performing PCR amplification on the SSR molecular marker by using the DNA extracted in the step 1) as a template;
3) and detecting the PCR product by adopting a fluorescent capillary electrophoresis system.
In the step 2) of the application, 13 SSR loci are respectively and independently subjected to PCR amplification; since the amplification procedure is the same, it can be performed 1 time in different wells on one plate. The PCR reaction system is 20 μ L, and comprises 4 μ L DNA, 0.25 μmol/L primer, 0.15 μmol/L dNTP, 2.5mmol/L MgCl21 unit Taq enzyme (Genacea, USA), 1 XPCR Buffer. The PCR reaction program is 94 ℃ for 5min, 94 ℃ for 40sec, 60 ℃ for 35s, 72 ℃ for 45s, and 35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.
Electrophoresis and fingerprint data acquisition: the PCR products were electrophoresed on a capillary fluorescence electrophoresis system AB3730XL DNA analyzer (Applied Biosystems, USA), and the PCR products, formamide, and an internal standard (GeneScan. TM. -500LIZ, Applied Biosystems, USA) were added to each well of a 96-well electrophoresis plate. And (3) running the mixed sample on a PCR (polymerase chain reaction) instrument at 95 ℃ for 5min, taking out the denatured electrophoresis product, centrifuging at 1000rpm/min for 1min, and performing electrophoresis on an AB3730XL DNA analyzer. And collecting the original data by using Date Collection Ver.1.0 software matched with the electrophoresis apparatus, and importing the original data into data analysis software to obtain the genotype data in a fragment length format.
The key points of the technology of the invention are as follows:
(1) and (3) analyzing the whole genome sequencing data of the corn. Because the maize genome is large and complex, processing whole genome sequencing data is the first difficult point to encounter and the key point. The method for analyzing the whole genome sequencing data of the corn comprises the following steps: and evaluating the quality of original data, independently splicing by using SPAdes software and SOAPdenovo2 software to obtain contig of high-quality splicing, wherein sequence splicing is a key point, and the parameter setting is relatively strict.
(2) And (4) separating the corn chloroplast genome data. The difficulty and key point of the present invention is to isolate chloroplast data from total DNA data. Since the chloroplast genome sequence is relatively conserved and of sufficient sequencing quality and length, chloroplast genome data is obtained in the present invention by a splicing protocol, coupled with alignment with the maize chloroplast genome. The corn chloroplast genome data acquisition main steps are as follows: screening contigs of chloroplast genomes by using a Blast program, assembling the chloroplast genome contigs by using Sequencher software, and comparing, verifying and confirming the assembled sequence with a corn chloroplast reference genome (corn variety B73, Version 3) to provide guarantee for obtaining accurate and reliable chloroplast genome sequences.
(3) The determination of chloroplast SSR polymorphic sites is key data and results of the invention. The accuracy and the high efficiency of obtaining the chloroplast polymorphic sites are ensured through representative sample selection, high-quality sequencing data and accurate data analysis. 170 parts of materials with wide sources and rich phenotypes and genotypes are selected, the SSR sequences are searched by MISA software based on the spliced 170 chloroplast genome sequences, and the chloroplast SSR polymorphic sites are finally determined, wherein different genetic background materials and high-quality gene sequences are important factors for obtaining accurate and reliable chloroplast polymorphic sites.
The invention develops a set of maize chloroplast genome polymorphic SSR loci based on high-quality re-sequencing data aiming at chloroplast genome characteristics, and compared with nuclear genome loci, the maize chloroplast genome polymorphic SSR loci are more suitable for construction of a maize chloroplast DNA fingerprint database, parent source tracing and positive and negative cross identification. By applying chloroplast SSR polymorphic sites and primers thereof, the range of available marker sites of corn can be expanded on the genome level; provides a new idea and method for the research of maize variety and germplasm resource identification, genetic relationship evaluation, cytoplasm genetic characteristics and the like.
Drawings
FIG. 1 is a technical roadmap for the development and evaluation of 13 maize chloroplast SSR loci in example 1 of the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions. Those skilled in the art will appreciate that the details of the present invention not described in detail herein are well within the skill of those in the art.
Example 1 construction of maize variety chloroplast DNA-SSR fingerprint database Using the SSR polymorphic site combinations provided by the invention
Extracting the DNA of the corn variety: each sample was seeded and irradiated to form green shoots. The DNA extraction adopts a mode of extracting DNA by mixed strains, 30 single-strain green leaves are mixed, and the specific steps of DNA extraction are carried out according to the corn DNA molecular identification standard (Wanfengge et al, 2014). The DNA was diluted to give a working solution at a concentration of 20 ng/. mu.L.
Designing and synthesizing a primer: the 13 SSR loci (table 1) provided by the invention are single-base length polymorphism, so that the loci can be used for designing primers based on a fluorescence capillary electrophoresis platform. Each site was designed with a pair of primers based on its flanking sequence, wherein the 5' end of one primer was labeled with a fluorescent group.
And (3) PCR amplification: the PCR reaction system was 20. mu.L, and included 4. mu.L of DNA, 0.25. mu. mol/L of primers, 0.15. mu. mol/L of dNTP, 2.5mmol/L of MgCl2, 1 unit of Taq enzyme (Genacea, USA), and 1 XPCR Buffer. The PCR reaction program is 94 ℃ for 5min, 94 ℃ for 40sec, 60 ℃ for 35s, 72 ℃ for 45s, and 35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.
Electrophoresis and fingerprint data acquisition: the PCR products were electrophoresed on a capillary fluorescence electrophoresis system AB3730XL DNA analyzer (Applied Biosystems, USA), and the PCR products, formamide, and an internal standard (GeneScan. TM. -500LIZ, Applied Biosystems, USA) were added to each well of a 96-well electrophoresis plate. And (3) running the mixed sample on a PCR (polymerase chain reaction) instrument at 95 ℃ for 5min, taking out the denatured electrophoresis product, centrifuging at 1000rpm/min for 1min, and performing electrophoresis on an AB3730XL DNA analyzer. And collecting the original data by using Date Collection Ver.1.0 software matched with the electrophoresis apparatus, and introducing the original data into SSR Analyzer software for analysis to obtain genotype data in a fragment length format.
Example 2 maternal traceability analysis was performed using the maize chloroplast genome SSR polymorphic sites provided by the invention
And extracting DNA from green leaves formed by seed sprouting to obtain the corn hybrid A to be identified, and performing PCR amplification and fluorescence capillary electrophoresis by using the 13 SSR loci provided by the invention to obtain fingerprint data. The specific procedure is the same as in example 1. Based on the comparison of the SSR fingerprint data of the sample A (Jingke 968) and a known corn inbred line variety chloroplast SSR marker fingerprint database (referred to from China corn standard DNA fingerprint database to foreign platform, the website is www.maizedna.org), determining that the chloroplast fingerprint information of the sample to be detected is the same as that of the inbred line B (Jingke 724), and presuming that the female parent of the hybrid A (Jingke 968) is B (Jing 724).
Example 3 identification of maize sample reciprocal crossing by using maize chloroplast genome SSR polymorphic site provided by the invention
To identify the reciprocal hybrid sample C (Zhengdan 958), DNA was extracted from the green leaves of the seed seedlings. Based on DNA fingerprint data of the male and female parents of Zhengdan 958 in the known corn inbred line variety chloroplast SSR marker fingerprint database, polymorphic sites in the male and female parents of the Zhengdan 958 to be detected are selected from 13 polymorphic SSR sites for PCR amplification, and the fingerprint of the sample C is obtained after the fluorescence electrophoresis data analysis, and the specific method is the same as the embodiment 1. And comparing the fingerprint data of the sample C to be detected with the fingerprint data of the female parent (sample D, Zheng 58) and the male parent (sample E, Chang 7-2). If the fingerprint data of the C sample and the fingerprint data of the D sample, namely the female parent, are the same, the C sample and the D sample are orthogonal; and if the fingerprint data of the sample C is the same as that of the sample E, namely the male parent, performing backcross.
The fingerprint data of the maize variety 13 related to the chloroplast SSR polymorphic sites are shown in Table 2.
TABLE 2 fingerprint data of maize variety 13 against chloroplast polymorphic primers
| Site numbering | Multiple state type | Jingke 968(A) | Jing 724(B) | Zhengdan 958(C) | Zheng 58(D) | Chang 7-2(E) |
| CPMSSRP-01 | (A)12 | (A)11 | (A)11 | (A)11 | (A)11 | (A)12 |
| CPMSSRP-02 | (A)11 | (A)11 | (A)11 | (A)11 | (A)11 | (A)11 |
| CPMSSRP-03 | (T)15 | (T)14 | (T)14 | (T)14 | (T)14 | (T)15 |
| CPMSSRP-04 | (A)12ctc(T)10 | (A)11ctc(T)9 | (A)11ctc(T)9 | (A)11ctc(T)9 | (A)11ctc(T)9 | (A)11ctc(T)9 |
| CPMSSRP-05 | (T)11 | (T)10 | (T)10 | (T)10 | (T)10 | (T)10 |
| CPMSSRP-06 | (T)15 | (T)14 | (T)14 | (T)14 | (T)14 | (T)14 |
| CPMSSRP-07 | (G)14 | (G)12 | (G)12 | (G)12 | (G)12 | (G)9 |
| CPMSSRP-08 | (A)6g(A)11 | (A)6g(A)9 | (A)6g(A)9 | (A)6g(A)9 | (A)6g(A)9 | (A)5g(A)11 |
| CPMSSRP-09 | (T)9 | (T)8 | (T)8 | (T)8 | (T)8 | (T)9 |
| CPMSSRP-10 | (A)4(T)10 | (A)4(T)8 | (A)4(T)8 | (A)4(T)8 | (A)4(T)8 | (A)4(T)8 |
| CPMSSRP-11 | (T)13 | (T)10 | (T)10 | (T)10 | (T)10 | (T)11 |
| CPMSSRP-12 | (A)8 | (A)8 | (A)8 | (A)8 | (A)8 | (A)8 |
| CPMSSRP-13 | (T)10 | (T)9 | (T)9 | (T)9 | (T)9 | (T)10 |
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Claims (9)
1. The SSR molecular marker of the corn chloroplast genome is characterized in that the molecular marker is a combination of the following 13 SSR molecular markers, and the information of the 13 SSR molecular markers is as follows: the Version of B73 is Version 3,
2. the use of the SSR molecular marker of claim 1 in the construction of a corn variety chloroplast DNA fingerprint database.
3. Use of the SSR molecular marker of claim 1 in maternal traceability analysis of corn samples.
4. Use of the SSR molecular marker of claim 1 in the identification of positive and negative crossovers in a corn sample.
5. Use of the SSR molecular marker of claim 1 in maize molecular marker assisted breeding.
6. Use of the SSR molecular marker of claim 1 in the preparation of a corn genome chip.
7. The use of the SSR molecular marker of claim 1 in the identification of maize germplasm resources.
8. Use of the SSR molecular marker of claim 1 in the study of maize cytoplast.
9. Use according to any one of claims 2 to 8, characterized in that it comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) performing PCR amplification on the SSR molecular marker by using the DNA extracted in the step 1) as a template;
3) and detecting the PCR product by adopting a capillary electrophoresis system, and collecting and analyzing data to obtain genotype data.
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| CN106520961A (en) * | 2016-11-16 | 2017-03-22 | 江汉大学 | Corn microsatellite marker locus development method and length detection method of microsatellite marker in microsatellite marker locus |
| CN106591470A (en) * | 2017-01-11 | 2017-04-26 | 北京市农林科学院 | Set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize |
| CN107190053A (en) * | 2017-03-13 | 2017-09-22 | 北京林业大学 | The combination of cypress microsatellite molecular marker, primer screening method and its application |
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| CN106520961A (en) * | 2016-11-16 | 2017-03-22 | 江汉大学 | Corn microsatellite marker locus development method and length detection method of microsatellite marker in microsatellite marker locus |
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