CN106591470B - One set of chloroplast SNP and INDEL molecular marker combination for corn maternal traceability - Google Patents
One set of chloroplast SNP and INDEL molecular marker combination for corn maternal traceability Download PDFInfo
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Abstract
The invention relates to a set of chloroplast SNP and INDEL molecular marker combinations for corn maternal traceability. The invention develops a set of corn chloroplast genome polymorphic site combinations based on high-quality re-sequencing data, wherein the polymorphic sites are CPM001-CPM100 and comprise 96 SNP sites and 4 IDNEL sites. The 100 chloroplast loci can be applied to the construction of a corn chloroplast DNA fingerprint database, parent source tracing and positive and negative cross identification. The application of chloroplast polymorphic sites expands the range of available marker sites of corn 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.
Description
Technical Field
The invention belongs to the technical field of crop molecular biology, and particularly relates to a set of chloroplast SNP and INDEL molecular marker combination for corn maternal traceability.
Background
Chloroplasts are organelles of green plants that perform photosynthesis and possess an intact set of genomes known as the chloroplast genome. The chloroplast genome structure is well conserved, DNA is generally a double-stranded circular molecule, and the chloroplast genome size in higher plants is generally 120-160 kb. Double-stranded circular DNA consists of 4 basic parts, namely a large single copy region (LSC), a small single copy region (SSC), and two Inverted Repeats (IRs).
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 genes are maternal inheritance, gene exchange and fusion among different individuals rarely occur, and the 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 primers (Wanfengge, etc., 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.
With the development of high-throughput sequencing technology and the maturity of genomic bioinformatics analysis technology, research on chloroplast genomes is effectively promoted. It is possible to excavate Single Nucleotide Polymorphism (SNP), insertion deletion polymorphism (INDEL) and simple repeat sequence (SSR) molecular marker sites from the chloroplast genome level. The marker locus and the molecular identification application related to the development of the corn chloroplast genome are not reported.
Disclosure of Invention
The invention aims to provide a set of chloroplast SNP and INDEL molecular marker combinations suitable for the traceability of a corn maternal line.
In order to realize the purpose of the invention, 170 parts of maize inbred line materials with wide sources, rich phenotypes and genotypes and strong representativeness are collected, chloroplast genomes of corresponding materials are sequenced and nucleotide polymorphisms of the chloroplast genomes of the corresponding materials are compared, and maize chloroplast genome polymorphic sites are developed. The invention provides a set of chloroplast SNP and INDEL molecular marker combinations for corn maternal traceability, the number of the SNP markers is CPM001-CPM096, the number of the INDEL markers is CPM097-CPM100, and the information is shown in Table 1:
TABLE 1100 chloroplast loci combination information
The physical location of the molecular marker was determined based on the chloroplast genome sequence of maize variety B73, which has a Version number Version 3 of the chloroplast genome of maize variety B73.
The invention also provides application of the molecular marker combination in constructing a corn variety chloroplast DNA fingerprint database. The method comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) taking the DNA extracted in the step 1) as a template, respectively designing primers based on the KASP detection platform technology according to the SNP and INDEL molecular marker combination, and carrying out PCR amplification;
3) the PCR product was analyzed using a fluorescence detector.
PCR amplification System: a total volume of 1. mu.L was added to the PCR plate 1.5. mu.L, oven dried, 0.5. mu.L deionized water, 0.5. mu.L mastermix (LGC Co., UK).
The PCR amplification procedure was: 15min at 94 ℃; 94 ℃ for 20s, 62 ℃ for 1min, 10 cycles (0.8 ℃ reduction per cycle); circulating at 94 deg.C for 20s and 55 deg.C for 1min for 26 times; storing at 15 ℃.
The invention also provides application of the molecular marker combination in maternal traceability analysis of a corn sample.
The invention also provides application in positive and negative cross identification of corn samples.
The invention also provides application of the molecular marker combination in corn molecular marker-assisted breeding.
The invention also provides application in preparing the corn chloroplast genome chip.
The invention also provides application of the molecular marker combination in genotype identification of maize filial generation.
The invention has the following advantages:
the invention develops a set of corn chloroplast genome polymorphic site combination based on high-quality re-sequencing data, and compared with nuclear genome sites, the corn chloroplast genome polymorphic site combination is more suitable for construction of a corn chloroplast DNA fingerprint database, parent source tracing and positive and negative cross identification. The application of chloroplast polymorphic sites expands the range of available marker sites of corn 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.
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FIG. 1 is a technical scheme for the development and evaluation of 100 maize chloroplast SNPs and INDEL loci in example 1 of the present 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.
Example 1 obtaining of maize chloroplast genome polymorphic site combinations
Selecting a sample: 170 parts of widely representative maize inbred lines were selected for whole genome sequencing. The 170 samples comprise corn types such as common corn, waxy corn, sweet corn, cracked corn and the like; including all heterosis groups in China, Tang Si Jiang, Luda honggu, Reid, Lanka, modified Reid, modified Lanka, P group and local varieties.
Sample preparation: the total DNA of 170 maize samples was extracted by CTAB method and RNA was removed. And respectively detecting the quality of the extracted DNA by using an ultraviolet spectrophotometer and agarose electrophoresis, and finding that the extracted DNA meets the sequencing related requirements. Agarose electrophoresis showed that the DNA band was single and not degraded; detecting A260/280 by an ultraviolet spectrophotometer to be between 1.8 and 2.0 (DNA has no protein pollution and low RNA content); a260/230 is between 1.8 and 2.0 (the content of DNA salt ions is low); the DNA concentration is greater than 1000 ng/. mu.L.
High throughput sequencing of total DNA: breaking the DNA of 170 parts of corn sample and PCR product by using ultrasound, cutting gel and recovering 400-and 600-bp DNA fragment, and utilizing
The library construction kit constructs a library with the size of 500bp, a sequencing platform of Hiseq4000PE150 is used for sequencing, the sequencing depth is 5 times, and about 10GB data are obtained on average for each sample.
High-throughput sequencing data processing, chloroplast genome splicing: high throughput sequencing data were stitched independently using two software, SPAdes (Bankevich et al, 2012) and soaldenovo 2(Luo et al, 2012), respectively. For each software spliced contig, the contig of the chloroplast genome was screened using the Blast program (Altschul et al, 1997); the selected chloroplast genome contigs were assembled using Sequencher. All reads maps were then aligned onto the spliced chloroplast genome sequence using geneous 8.1(Kearse et al, 2012) to verify whether the spliced contig sequence was correct.
Chloroplast genome annotation, polymorphic site determination: chloroplast genome annotation was performed using dodma (dual organic university geno Me antanotator) (Wyman et al, 2004) and BLASTX and BLASTN searches were used to identify the location of the encoding gene. 170 maize chloroplast genomes were aligned using MAFFT software (Katoh and Standard, 2013) and then manually adjusted using Se-al software. The principle of the inverted alignment occurring within the sequence is to pull it apart so as not to cause erroneous data polymorphisms. Utilizing DnaSP 5.0 to count variation sites and sequence polymorphism (Librado and Rozas,2009) in a chloroplast genome to obtain 100 polymorphic site combinations, wherein the physical positions and flanking sequences of the 100 sites are determined based on the chloroplast genome sequence of a maize variety B73, the 100 sites are CPM001-CPM100, and the specific information is shown in Table 1.
A technical scheme for the development and evaluation of 100 maize chloroplast SNPs and INDEL loci of the invention is shown in FIG. 1.
The key points of the technology of the invention are as follows: (1) the analysis of the whole genome sequencing data of corn is difficult and key point to process the whole genome sequencing data because the genome of corn is large and complex. The method comprises the steps of evaluating the quality of original data, independently splicing by using SPAdes software and SOAPdenovo2 software to obtain contig spliced with high quality, wherein sequence splicing is a key point, and parameters are set relatively rigorously. (2) The difficulty and the key point of the invention are the separation of the corn chloroplast genome data and the separation of the chloroplast data from the total DNA data. Since the chloroplast genome sequence is relatively conserved and the sequencing quality and length are sufficient, in the present invention chloroplast genome data is obtained by a splicing scheme, in combination with alignment with the maize chloroplast genome. The method mainly comprises the steps of screening contigs of chloroplast genomes by using a Blast program, assembling the contigs of the chloroplast genomes by using Sequencher software, and comparing and verifying the assembled sequences with maize chloroplast reference genomes (maize variety B73, Version 3) to ensure accurate and reliable chloroplast genome sequences. (3) Chloroplast polymorphic site determination is the final objective and result 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 abundant phenotypes and genotypes are selected, based on 170 spliced chloroplast genome sequences, DnaSP 5.0 is utilized to count variation sites and sequence polymorphisms, and finally chloroplast polymorphic sites are determined, wherein different genetic background materials and high-quality gene sequences are important factors for obtaining accurate and reliable chloroplast polymorphic sites.
Example 2 construction of maize variety chloroplast DNA fingerprint database Using polymorphic site combinations
Extracting the DNA of the corn variety: the general CTAB method is adopted to respectively extract the total DNA of all corn varieties for constructing the chloroplast DNA fingerprint database, and the DNA is diluted to form working solution with the concentration of 20 ng/mu L.
Designing and synthesizing 100 polymorphic site primers: the polymorphic sites provided by the embodiment are SNP and INDEL markers of biallelic genes, so that 100 sites can be used for designing primers based on KASP detection platform technology, and the primers are common primers and do not contain fluorescence.
PCR amplification System: a total volume of 1. mu.L was added to the PCR plate 1.5. mu.L, oven dried, 0.5. mu.L deionized water, 0.5. mu.L mastermix (LGC Co., UK).
The PCR amplification procedure was: 15min at 94 ℃; 94 ℃ for 20s, 62 ℃ for 1min, 10 cycles (0.8 ℃ reduction per cycle); circulating at 94 deg.C for 20s and 55 deg.C for 1min for 26 times; storing at 15 ℃.
Chloroplast DNA fingerprint data: the amplification products were scanned for fluorescence signals using a BMG Pherastar (LGC, UK) instrument to obtain raw data. The raw data was imported into Kraken software (LGC, england) for analysis to obtain fingerprint data for each data point.
Example 3 maternal traceability analysis Using maize chloroplast genome polymorphic sites
The corn hybrid A to be identified is used for extracting DNA, PCR amplification is carried out by utilizing the chloroplast marker provided by the invention, and fingerprint data are obtained by scanning a fluorescent signal. The specific procedure is the same as in example 2. Comparing the DNA fingerprint data of the sample A (Jingke 968) with a chloroplast genome fingerprint database of a known maize inbred line variety, determining that 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 (Jingke 724).
Example 4 identification of maize samples for reciprocal crossing using maize chloroplast genome polymorphic sites
The specific method is the same as the example 2, DNA is extracted from the hybrid sample C (Zhengdan 958) to be identified for positive and negative cross, PCR amplification and fluorescent signal scanning are carried out by utilizing the chloroplast marker provided by the invention to obtain fingerprint data. And (3) comparing the fingerprint data of the chloroplast marker of the sample to be detected with the fingerprint data of the female parent (sample D, Zheng 58) and the male parent (sample E, Chang 7-2) of the sample C to determine the chloroplast fingerprint data. 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 chloroplast fingerprint data of the corn samples referred to in this invention are shown in table 2.
TABLE 2 chloroplast fingerprint data of corn samples
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference to the literature
1.Altschul SF,Madden TL,Schaffer AA,Zhang J,Zhang Z,Miller W,LipmanDJ.1997.Gapped BLAST and PSI-BLAST:a new generation of protein databasesearch programs.Nucleic Acids Res 25:3389-3402.
2.Bankevich A,Nurk S,Antipov D,Gurevich AA,Dvorkin M,Kulikov AS,LesinVM,Nikolenko SI,Pham S,Prjibelski AD et al.2012.SPAdes:a new genome assemblyalgorithm and its applications to single-cell sequencing.J Comput Biol19:455-477.
3.Darriba D,Taboada GL,Doallo R,Posada D.2012.jModelTest 2:moremodels,new heuristics and parallel computing.Nat Methods9:772-772.
4.Katoh K,Standley DM.2013.MAFFT multiple sequence alignment softwareversion 7:improvements in performance and usability.Mol Biol Evol30:772-780.
5.Kearse M,Moir R,Wilson A,Stones-Havas S,Cheung M,Sturrock S,BuxtonS,Cooper A,Markowitz S,Duran C et al.2012.Geneious Basic:an integrated andextendable desktop software platform for the organization and analysis ofsequence data.Bioinformatics 28:1647-1649.
6.Librado P,Rozas J.2009.DnaSP v5:a software for comprehensiveanalysis of DNA polymorphism data.Bioinformatics 25:1451-1452.
7.Luo R,Liu B,Xie Y,Li Z,Huang W,Yuan J,He G,Chen Y,Pan Q,Liu Y etal.2012.SOAPdenovo2:an empirically improved memory-efficient short-read denovo assembler.Gigascience1:18.
8.Ronquist F,Teslenko M,van der Mark P,Ayres DL,Darling A,Hohna S,Larget B,Liu L,Suchard MA,Huelsenbeck JP.2012.MrBayes 3.2:efficient Bayesianphylogenetic inference and model choice across a large model space.Syst Biol61:539-542.
9.Santorum JM,Darriba D,Taboada GL,Posada D.2014.jmodeltest.org:selection of nucleotide substitution models on the cloud.Bioinformatics30:1310-1311.
10.Wyman SK,Jansen RK,Boore JL.2004.Automatic annotation oforganellar genomes with DOGMA.Bioinformatics20:3252-3255.
11.Ganal MW,Durstewitz G,Polley A,Be′rard A,Buckler ES,Charcosset A,Clarke JD,Graner EM,Hansen M,Joets J,Paslier MC,McMullen MD,Montalent P,RoseM,Schǒn CC,Sun Q,Walter H,Martin OC,FalqueM.2011.A large maize(Zea mays L.)SNP genotyping array:development and germplasm genotyping,and genetic mappingto compare with the B73reference genome.PLoS One 6:e28334.
12.Tian HL,Wang FG,Zhao JR,Yi HM,Wang L,Wang R,Yang Y,SongW.2015.Development of maizeSNP3072,a high-throughput compatible SNP array,forDNA fingerprinting identification of Chinese maize varieties.MolecularBreeding 35:136,DOI 10.1007/s11032-015-0335-0.
13. Wangfeng grids, Yihongmei, Zhao longevity, Liuping, Zhang Xinming, Tianhongli, Yuan yuan stem-up, 2014, a corn variety identification technical specification SSR marking method, the agricultural industry standard of the people's republic of China.
Claims (9)
1. The set of chloroplast SNP and INDEL molecular marker combinations for the maternal tracing of corn is characterized in that the SNP markers are numbered CPM001-CPM096 respectively, and the INDEL markers are numbered CPM097-CPM100 respectively, and the information is as follows:
2. the molecular marker combination of claim 1, wherein the physical location of the molecular marker is determined based on the chloroplast genome sequence of maize variety B73, which has a Version number Version 3 of the chloroplast genome of maize variety B73.
3. Use of the combination of molecular markers of claim 1 or 2 in the construction of a database of DNA fingerprints of chloroplast of maize varieties.
4. Use according to claim 3, characterized in that it comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) taking the DNA extracted in the step 1) as a template, respectively designing primers based on the KASP detection platform technology according to the SNP and INDEL molecular marker combination, and carrying out PCR amplification;
3) the PCR product was analyzed using a fluorescence detector.
5. Use of the combination of molecular markers of claim 1 or 2 in maternal traceability analysis of maize samples.
6. Use of a combination of molecular markers according to claim 1 or 2 in the identification of a reciprocal cross-section of a maize sample.
7. Use of the molecular marker combination of claim 1 or 2 in maize molecular marker assisted breeding.
8. Use of the combination of molecular markers of claim 1 or 2 in the preparation of a maize chloroplast genome chip.
9. Use of the combination of molecular markers of claim 1 or 2 for genotyping progeny of a maize cross.
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| CN107090504B (en) * | 2017-05-03 | 2020-05-15 | 袁隆平农业高科技股份有限公司 | SNP molecular marker combination for corn genotyping and application thereof |
| CN108060261B (en) * | 2018-01-30 | 2021-01-15 | 袁隆平农业高科技股份有限公司 | Method for capturing and sequencing corn SNP marker combination and application thereof |
| CN108411020B (en) * | 2018-02-06 | 2021-10-01 | 北京市农林科学院 | Corn chloroplast InDel molecular marker suitable for capillary electrophoresis detection platform |
| CN108486266B (en) * | 2018-02-06 | 2022-06-24 | 北京市农林科学院 | Molecular marker of corn chloroplast genome and application of molecular marker in variety identification |
| CN108441572B (en) * | 2018-02-06 | 2022-06-24 | 北京市农林科学院 | Method for identifying maize chloroplast cytoplasm type based on KASP technology |
| CN108192991B (en) * | 2018-02-06 | 2021-12-21 | 北京市农林科学院 | Identification of maize male sterility S-type cytoplasmic types using chloroplast markers |
| CN108220473B (en) * | 2018-02-06 | 2021-10-01 | 北京市农林科学院 | Identification of maize S-type cytoplasmic male sterile material by using chloroplast InDel marker |
| CN108486263A (en) * | 2018-02-06 | 2018-09-04 | 北京市农林科学院 | Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker |
| CN108486264B (en) * | 2018-02-06 | 2022-08-09 | 北京市农林科学院 | Identification of maize male sterility type C cytoplasm type by chloroplast marker |
| CN108486265B (en) * | 2018-02-06 | 2022-06-24 | 北京市农林科学院 | Method for identifying type of male sterile cytoplasm of corn based on KASP technology |
| CN108416189B (en) * | 2018-02-08 | 2022-02-08 | 北京市农林科学院 | Crop variety heterosis mode identification method based on molecular marker technology |
| CN108676906B (en) * | 2018-05-31 | 2021-08-24 | 北京市农林科学院 | SSR locus of corn chloroplast genome and application of SSR locus in variety identification |
| CN110964845B (en) * | 2020-01-03 | 2022-06-07 | 江苏省农业科学院 | Method for tracing hybrid source of corn pollination and InDel molecular marker |
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