CN108486264B - Identification of maize male sterility type C cytoplasm type by chloroplast marker - Google Patents
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Abstract
The invention provides a method for identifying the type of male sterile C cytoplasm of corn by utilizing a chloroplast marker, wherein the chloroplast marker is developed based on a chloroplast genome and has 11 molecular markers in total, including 9 SNPs and 2 InDel markers. The molecular marker combination can be used for identifying germplasm materials of C-type male sterile cytoplasm and normal cytoplasm of corn, can be used for identifying hybrid seeds of C-type sterile seed production and conventional seed production, and can also be used for identifying purity of C-type sterile materials. The molecular marker combination provided by the invention provides identification technical support for corn C-type cytoplasmic sterility seed production and sterile material breeding, and provides powerful guarantee for production of corn three-line matched seeds; the range of available marker loci of the corn is expanded on the genome level, and a new thought and method are provided for the research of cytoplasmic hereditary property and the like of the corn.
Description
Technical Field
The invention belongs to the technical field of crop molecular biology, and particularly relates to identification of a corn male sterility C-type cytoplasm type by using a chloroplast marker.
Background
Corn is the crop which can realize the utilization of heterosis at the earliest time, and the breeding and utilization of hybrid seeds are praised as a revolution in crop production. The corn hybrid seed production method has two methods of conventional method and male sterility, and the male sterility is used for preparing the corn hybrid seed, so that not only is a large amount of labor saved and the cost reduced, but also the purity of the hybrid seed can be improved and the yield can be improved.
Maize Male Sterility (MS) refers to the phenomenon that maize stamens develop abnormally to produce non-functional pollen, while pistils develop normally to pollinate and fruit. Maize male sterility can be broadly divided into Cytoplasmic Male Sterility (CMS) and nuclear male sterility (GMS). Cytoplasmic Male Sterility (CMS) is a nuclear-cytoplasmic interactive male sterile line commonly controlled by nuclear and cytoplasmic genes, easily realizes the matching of a sterile line, a maintainer line and a restorer line, and is a main male sterile type used in corn breeding and fine variety production. To date, more than 200 CMS materials from different cytoplasm sources have been discovered, and the CMS materials are divided into three types, namely S type, C type and T type according to the special effect reaction of male flower fertility restoration, and the male flower fertility reaction and cytoplasm effect of different types of materials are different.
The method adopts CMS seed production to solve three problems, namely identification of the type of the cytoplasm of the first male sterility, identification of the purity of the second male sterility material and identification of the hybrid prepared by the sterile line. Therefore, it is very important to obtain a set of site combinations suitable for identifying CMS materials. 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 high mutation regions exist, so that the problems of the following classification units can be solved. As CMS is closely related to cytoplasm, the identification of the sterile cytoplasm type by adopting the marker locus on the chloroplast genome is a stable, reliable and innovative method. Development of chloroplast SNP or InDel marker loci suitable for CMS material identification, simultaneous use of multiple locus combinations, can enhance identification reliability and reduce uncertainty.
The method for identifying the types of the male sterile cytoplasm of the corn is reported to have the methods of field fertility observation, cytological difference comparison, fertility restoration specificity identification, marker locus PCR amplification and the like. The first three methods have long identification period and limited range of identification materials; the PCR-based method does not report a method for identifying the type of the male sterile cytoplasm of the corn by developing SNP or InDel locus aiming at chloroplast genome at present, and can not realize high-throughput and automatic identification. The development of marker loci and methods for CMS type identification in maize based on the chloroplast genome has not been reported. Therefore, it is necessary to develop a set of chloroplast molecular marker loci suitable for the maize male sterility type C cytoplasm type by using high-quality re-sequencing data.
Disclosure of Invention
The invention aims to provide a method for identifying the type C cytoplasm of male sterility of corn by using a chloroplast marker.
Another objective of the invention is to provide a combination of SNP and INDEL molecular markers for identifying maize male sterility type C cytoplasm types.
In order to realize the purpose of the invention, 170 parts of maize inbred line materials (including three CMS type materials) with wide sources, rich phenotypes and genotypes and strong representativeness are collected to obtain a chloroplast genome sequence, nucleotide polymorphisms are compared, and SNP and InDel loci for identifying maize male sterility C type cytoplasm types are developed. The method mainly comprises the following steps: (1) selecting 170 parts of corn representative test materials, wherein the types comprise all heterosis groups in China, sweet and glutinous, local varieties, CMS sterile types and other samples. (2) High concentration, high quality 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 materials are compared with each other, and 100 SNP/InDel chloroplast genome variation sites are screened out. (7) Determining the specific loci for identifying the types of the C-type sterile cytoplasm of the corn, analyzing Fst values (genetic differentiation coefficients) among different groups, wherein the Fst values are group specific loci if the Fst values are more than 0.9, and analyzing the C-type sterile cytoplasm material of the corn to obtain 11 specific loci which comprise 9 SNP loci and 2 InDel loci. Specific information on the loci is shown in Table 1, and the physical location of the loci was determined based on alignment of chloroplast genome sequences of maize variety B73. A technical scheme developed for the 11 chloroplast loci of the invention suitable for identifying maize male sterile type C cytoplasmic types is shown in FIG. 1.
The invention provides molecular markers for identifying corn male sterility C-type cytoplasm, which comprise any one or more of the following 11 molecular markers, wherein the 11 molecular markers comprise 9 SNP molecular markers and 2 InDel molecular markers, and the information of the 11 molecular markers is shown in Table 1.
TABLE 1
As the 11 SNP/InDel sites provided by the invention are all two-state markers, the molecular marker or any molecular marker combination thereof can realize the acquisition of genotyping data based on a KASP platform. The specific scheme is that according to KASP technical requirements, a primer is designed aiming at the locus provided by the invention, and the primer is a common primer and does not contain a fluorescent group; purchasing a PCR amplification system MasterMix matched with the KASP technology; preparing a reaction system, and adding DNA, a primer and MasterMix; operating a reaction program; scanning the fluorescence signal in situ; and (4) analyzing the data to obtain genotype data.
Except the molecular marker with the site number of CPMIDP09, other molecular markers are obtained by amplifying the following primers respectively: CPMSNP18, SEQ ID NO. 23-25; CPMSNP38, SEQ ID NO. 26-28; CPMSNP39, SEQ ID NO. 29-31; CPMSNP44, SEQ ID NO. 32-34; CPMSNP48, SEQ ID NO. 35-37; CPMSNP57, SEQ ID NO. 38-40; CPMSNP59, SEQ ID NO. 41-43; CPMSNP72, SEQ ID NO. 44-46; CPMSNP81, SEQ ID NO. 47-49; CPMIDP10, SEQ ID NO. 50-52.
Further, the invention provides a specific primer combination for detecting the molecular marker, which is specifically any one of the following groups: CPMSNP18, SEQ ID NO. 23-25; CPMSNP38, SEQ ID NO. 26-28; CPMSNP39, SEQ ID NO. 29-31; CPMSNP44, SEQ ID NO. 32-34; CPMSNP48, SEQ ID NO. 35-37; CPMSNP57, SEQ ID NO. 38-40; CPMSNP59, SEQ ID NO. 41-43; CPMSNP72, SEQ ID NO. 44-46; CPMSNP81, SEQ ID NO. 47-49; CPMIDP10, SEQ ID NO. 50-52.
Although the embodiment of the invention does not provide amplification primers of CPMIDP09 locus, a person skilled in the art can design suitable specific primers aiming at the locus numbered CPMIDP09 and can identify the type of corn type C male sterile cytoplasm based on the locus information of CPMIDP09 given in Table 1, so that the molecular marker with the locus numbered CPMIDP09 and the application of the molecular marker in identifying the type C male sterile cytoplasm of corn belong to the protection scope of the invention.
Further, the invention provides application of the molecular marker or the specific primer pair in identifying the type C cytoplasm of the male sterility of the corn.
The invention provides application of the molecular marker or the specific primer pair in identifying hybrid seeds prepared from a corn C-type cytoplasmic male sterile line.
The invention provides application of the molecular marker or the specific primer pair in maize molecular marker assisted breeding.
The invention provides application of the molecular marker or the specific primer pair in corn material identification.
The application specifically comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) respectively designing primers based on KASP detection platform technology according to the molecular markers by using the DNA extracted in the step 1) as a template, and performing PCR amplification;
3) the PCR product was analyzed using a fluorescence detector.
Wherein, the PCR amplification procedure in the step 2) is as follows: 15min at 94 ℃; cycling 10 times at 94 ℃ for 20s and 61 ℃ for 1min, and reducing the temperature by 0.6 ℃ in each cycle; circulating for 30 times at 94 ℃ for 20s and 58 ℃ for 1 min.
In the step 3), if the PCR product is the same as the allele described in Table 1, the individual is a C-type sterile individual, and if the PCR product is the other allele, the individual does not belong to the C-type sterile individual.
The invention provides application of the molecular marker in identifying the purity of the C-type cytoplasmic male sterile corn material.
In the specific process of identifying the purity of the C-type cytoplasmic male sterile maize material, the method comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) using the DNA extracted in the step 1) as a template, respectively designing primers based on KASP detection platform technology according to the molecular marker of the invention, and carrying out PCR amplification;
3) analyzing the PCR product by a fluorescence detector;
4) if the PCR product is the same as the allele described in the table 1, the PCR product is a C-type sterile single plant, and if the PCR product is the other allele, the PCR product does not belong to the C-type sterile single plant; purity calculation is carried out on each site, and the calculation method comprises the following steps: (number of sterile individuals of type C/total number of individuals detected). times.100%.
The key points of the invention are as follows:
(1) isolation of maize chloroplast genome data: the difficulty and the key point of the invention are that chloroplast genome data are separated from whole genome 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 protocol, in combination with alignment with the maize chloroplast reference 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, AGPv3) to confirm so as to provide guarantee for obtaining accurate and reliable chloroplast genome sequences.
(2) Digging of chloroplast mutation sites: 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.
(3) And (3) determining the identification site of the C-type sterile cytoplasm type of the corn: through analyzing the variation locus of chloroplast genome of all types of maize inbred lines (including T, C, S three types of CMS materials), screening out the specific locus of the C-type sterile material, wherein the specific locus shows that the genotype of the C-type sterile material is different from that of all other types of inbred line materials and belongs to the specific locus of the C-type sterile material.
The invention has the following advantages: in view of the characteristics of chloroplast genomes, the invention develops a group of corn chloroplast genome SNP molecular markers and InDel molecular markers and the combination of the molecular markers based on high-quality re-sequencing data, can be used for identifying germplasm materials of corn C-type male sterile cytoplasm and normal cytoplasm, can be used for identifying hybrid seeds of C-type sterile seed production and conventional seed production, and can also be used for identifying the purity of the C-type sterile material. The molecular marker provided by the invention provides identification technical support for corn C-type cytoplasmic male sterile seed production and sterile material breeding, and provides powerful guarantee for production of corn three-line matched seeds.
Drawings
FIG. 1 is a technical scheme obtained at 11 chloroplast loci suitable for identifying maize male sterile type C cytoplasmic types in example 1 of the invention.
Fig. 2 is an analysis result diagram of the invention for identifying whether the maize inbred line or hybrid is C-sterile by 10 pairs of primers, except for the molecular marker whose locus number is CPMIDP09, the primers of other 10 molecular markers can identify whether the maize inbred line or hybrid is male-sterile C-cytoplasmic type.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. 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 acquisition of molecular markers for maize chloroplast genome associated with maize male sterile C-type cytoplasmic types
(1) Selecting a sample: 170 parts of maize inbred lines with extensive representatives 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; comprises all heterosis groups in China, four heads of Tang, red bone, Reid, Lanka, improved Reid, improved Lanka, P group and local variety, and T, C, S cytoplasm sterility type materials.
(2) Sample preparation: seeds of 170 corn samples were allowed to germinate in the incubator for 5 days, with no light conditions for the first 3 days and light conditions for the next 2 days (i.e., sufficient light was given after emergence). 30 green shoot leaves were selected from each sample and mixed, and ground thoroughly under liquid nitrogen. Total DNA was extracted by CTAB method and RNA was removed. Respectively detecting the quality of the extracted DNA by using an ultraviolet spectrophotometer and agarose electrophoresis, wherein the agarose electrophoresis shows that the DNA has single band and is 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.
(3) 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, and a sequencing platform of Hiseq 4000PE150 is utilized for sequencingThe sequencing depth was 5-fold, and about 10GB of data was obtained per sample on average.
(4) 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, screening out contigs of the chloroplast genome using Blast program (Altschul et al, 1997); the selected chloroplast genome contigs were assembled using Sequencher. All reads maps were then applied to the spliced chloroplast genome sequence using geneous 8.1(Kearse et al, 2012) to verify that the spliced contig sequence was correct.
(5) Chloroplast genome annotation, polymorphic site determination: chloroplast genome annotation was performed using dodma (dual organic island 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. The DnaSP 5.0 is used for counting variation sites and sequence polymorphism in two chloroplast genomes (Librado and Rozas,2009) and 100 SNP/INDEL polymorphic sites are developed and obtained.
(6) Determination of the cytoplasmic type site suitable for identifying maize type C sterility: based on 100 SNP/INDEL polymorphic genotype data of 170 parts of corn representative materials, we-Fst-pop function in VCFtools software is utilized to analyze Fst values among different groups, namely genetic differentiation coefficients among different groups, and the Fst value is larger than 0.9, so that the site is considered as a group specific site. 11 specific sites including 9 SNP sites and 2 IDNEL sites are obtained by analyzing the C-type sterile type material of the corn.
The technical scheme developed for the 11 chloroplast loci used in the present invention to identify maize male sterile type C cytoplasmic types is shown in figure 1. Finally, 11 molecular markers for identifying the corn male sterility C-type cytoplasm are obtained through screening, wherein the molecular markers comprise 9 SNP molecular markers and 2 InDel molecular markers, and the information of the molecular markers is shown in Table 1.
Besides the molecular marker with the site number of CPMIDP09, other molecular markers are obtained by amplifying the following primers respectively: CPMSNP18, SEQ ID NO. 23-25; CPMSNP38, SEQ ID NO. 26-28; CPMSNP39, SEQ ID NO. 29-31; CPMSNP44, SEQ ID NO. 32-34; CPMSNP48, SEQ ID NO. 35-37; CPMSNP57, SEQ ID NO. 38-40; CPMSNP59, SEQ ID NO. 41-43; CPMSNP72, SEQ ID NO. 44-46; CPMSNP81, SEQ ID NO. 47-49; CPMIDP10, SEQ ID NO. 50-52.
Example 2 identification of maize C-type cytoplasmic male sterile inbred line by using the molecular marker obtained by the present invention
Identifying the C-type cytoplasmic sterile inbred line sample from 96 maize inbred line samples by the following specific method.
Extracting DNA of a maize inbred line sample to be identified: and randomly selecting 50 seeds from each sample, sprouting, and giving sufficient light to form green seedlings. The DNA extraction adopts a mode of mixed plant extraction DNA, green leaves of 30 individual plants are randomly selected from 50 individual plants in each sample and mixed, and the specific steps of DNA extraction are carried out according to the corn DNA molecular identification standard (Wanfengge et al, 2014, corn variety identification technical specification SSR marking method, the agricultural industry standard of the people's republic of China). The DNA was diluted to give a working solution at a concentration of 20 ng/. mu.L.
Designing and synthesizing primers of 11C-type sterile specific sites: the polymorphic sites provided by the invention are SNP and InDel markers of biallelic genes, so that primers can be designed for 11 sites based on a KASP technical platform, and the synthetic primer is a common primer without fluorescence.
And (3) PCR amplification: from the 10 pairs of primers determined in example 1, amplification was performed for each pair of primers. The PCR reaction system was 1. mu.L, and included 1.5. mu.L of total DNA (oven dried), 0.5. mu.L of KASP ROX standard reaction mix (Kbiosciences, Herts UK), 0.014. mu.L of primer mixture, and 0.5. mu.L of deionized water. The PCR reaction program is 94 ℃ for 15 min; circulating for 10 times, adopting falling PCR mode, at 94 deg.C for 20s, at 61 deg.C for 1min, (at 61 deg.C falling to 55 deg.C, each circulation reducing by 0.6 deg.C); circulating for 30 times at 94 ℃ for 20sec and 58 ℃ for 1 min.
Fingerprint data acquisition: the amplification product was scanned for fluorescence signal using a BMG Pheastar (LGC, Middlesex, UK) instrument to obtain raw data. Raw data is imported into Kraken software (LGC, Middlesex, UK) to be analyzed to obtain fingerprint data of each data point of each corn variety, and the fingerprint data is imported into a data management system.
And (4) judging a result: the determination is made based on the alleles of each locus listed in table 1 in the maize type C sterile cytoplasmic types, which are C sterile material if they are the same as the alleles in the table and not C sterile material if they are another allele. The result shows that primers designed by other molecular markers except the molecular marker with the site number of CPMIDP09 determined in example 1 have good specificity, and can respectively detect the specificity of the targeted sites, thereby completing the identification work of the maize C-type cytoplasmic sterile inbred line.
The analysis result chart of whether the maize inbred line is C-sterile or not by using the 10 pairs of primers provided by the invention is shown in figure 2. Besides the molecular marker with the site number of CPMIDP09, other 10 molecular marker primers can identify whether the maize inbred line or hybrid is male sterile C-type cytoplasm type. In the results typing map for each pair of primers in FIG. 2, each data point is the result of typing for each sample in each pair of primers; the data points near the origin are negative control samples, and part of the rest data points are close to the X axis and the other part of the data points are close to the Y axis, which respectively represent two homozygous genotype results displayed by each pair of primers; the genotype data read by software is respectively checked with the information of each pair of primers in the table 1, if the genotype data is consistent with the allele of the maize C-type cytoplasmic male sterility sample, the C-type sterility is determined, and the other type is non-C-type sterility.
Example 3 identification of maize hybrid for C-type cytoplasmic sterility by Using the molecular marker obtained in the present invention
Identifying whether 96 different packaging bags or Jingke 968 from different sources are hybrid seeds produced by C-type cytoplasmic male sterile seed production, and the specific method is as follows.
And (3) extracting DNA of a corn hybrid sample to be identified: and randomly selecting 50 seeds from each packaging bag, sprouting, and giving sufficient light to form green seedlings. The DNA extraction adopts a mode of extracting DNA from mixed plants, and green leaves of 30 individual plants are randomly selected from 50 individual plants in each sample to form 96 samples to be detected. The specific steps of DNA extraction are carried out according to the identification standard of the corn DNA molecules (Wanfengge et al, 2014, corn variety identification technical specification SSR marking method, and agricultural industry standard of the people's republic of China), and the DNA is diluted to form working solution with the concentration of 20 ng/mu L.
Primer design synthesis, PCR amplification, fingerprint data acquisition methods see example 2.
And (4) judging a result: based on the allele of each locus listed in the table 1 in the corn C-type sterile cytoplasm type, the determination is carried out, if a sample to be tested is the same as the allele in the table, the hybrid generated by the C-type sterile mode is generated, and if the sample is the other allele, the hybrid does not belong to the hybrid generated by the C-type sterile mode.
Example 4 identification of the purity of type C sterile samples Using the loci obtained according to the invention
The purity of the C-type sterile sample seeds of 1-package corn, namely the content of the C-type sterile seeds, is identified by the following specific method.
And (3) extracting DNA of a sample to be identified: randomly selecting 120 seeds from a sample package to be identified, sprouting, and giving sufficient light to form green seedlings. The DNA extraction adopts a single-plant extraction mode, DNA of 100 single-plant green leaves is randomly extracted, and the specific steps of DNA extraction are carried out according to the corn DNA molecular identification standard (Wanfengge et al, 2014, corn variety identification technical specification SSR marking method, and the agricultural industry standard of the people's republic of China). The DNA was diluted to give a working solution at a concentration of 20 ng/. mu.L.
1 or more sites are randomly selected from the 11C-type sterility specific sites determined in the example 1, and primers are designed. And (3) PCR amplification: from the 10 sets of primers identified in example 1, each set of primers was subjected to PCR amplification. The PCR reaction system was 1. mu.L, and included 1.5. mu.L of total DNA (oven dried), 0.5. mu.L of KASP ROX standard reaction mix (Kbiosciences, Herts UK), 0.014. mu.L of KASP Primer mix mixture, and 0.5. mu.L of deionized water. The PCR reaction program is 94 ℃ for 15 min; circulating for 10 times, adopting falling PCR mode, at 94 deg.C for 20s, at 61 deg.C for 1min, (at 61 deg.C falling to 55 deg.C, each circulation reducing by 0.6 deg.C); cycling 30 times at 94 ℃ for 20sec and 58 ℃ for 1 min.
Fingerprint data acquisition: the amplification product was scanned for fluorescence signal using a BMG Pheastar (LGC, Middlesex, UK) instrument to obtain raw data. Raw data is imported into Kraken software (LGC, Middlesex, UK) for analysis to obtain fingerprint data of each data point of each individual plant, and the fingerprint data is imported into a data management system.
And (4) judging a result: the determination is made based on the alleles of each locus listed in table 1 in the maize type C sterile cytoplasmic type, which is a type C sterile individual if the allele is the same as the allele in the table, and which is not a type C sterile individual if the allele is another allele. Purity calculation is carried out on each locus by multiplying (the number of C-type sterile single plants/the total detected single plant number) by 100%.
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.
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<213> Artificial Sequence (Artificial Sequence)
<400> 11
gcgtttcgat gaatgagcct atggtaatgc ttttatctct attctatggc gcaatcgacc 60
<210> 12
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcgagtctat aaacaagtac taaataagga aaagaaaact atactaaagg aaacataaga 60
<210> 13
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cccgggacgc gaagtagtag gattggttct cataattatc acataatttt caaaaaaaaa 60
<210> 14
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gaatttgtcg aaattttttt tcttgttgaa taatgccaaa tcaaaaaaaa tatccaaaaa 60
<210> 15
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ttttttaatt atagttattc ctatgcgaga gatagaattc ttcgtgacat gacgaaaatt 60
<210> 16
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cccctttttg aattcttttt tagtatatga agcaaaaaga aagaaaagat ggataaggat 60
<210> 17
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aaaagcttct ttttcgaaag attacccctg tctttgttta tgcttcggat tggaacaaat 60
<210> 18
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
actctaattc gcccacgcct gcgaatcagt cgacattttg tacaaatttt acgaacggaa 60
<210> 19
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gattggattt gcaccaaagg aaaccataaa ttccatatac catagaaatc ttaggataga 60
g 61
<210> 20
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cttctctatc ctattcattg gtaccgatca tggatacttc aaaaatttta ttatttgttt 60
g 61
<210> 21
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gattggattt gcaccaaagg aaaccataaa ttccatatac catagaaatc ttaggataga 60
g 61
<210> 22
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ctctatccta ttcattggta ccgatcatgg atacttcaaa aattttatta tttgtttgaa 60
c 61
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gtgctcgttt agtgttcaga cca 23
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gtgctcgttt agtgttcaga ccc 23
<210> 25
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cttagtttag tgcgagatgc ccacat 26
<210> 26
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
attctaaaat cattctttag aaagccacac 30
<210> 27
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ctaaaatcat tctttagaaa gccacat 27
<210> 28
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ggccaagtca ggttagatct atatcttta 29
<210> 29
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
atgggaactc aaagatatcg aagagta 27
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gggaactcaa agatatcgaa gagtc 25
<210> 31
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
caaccaatca ctcttttatt ccatcctttt 30
<210> 32
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gcctatacta ctattctatg gataaagct 29
<210> 33
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cctatactac tattctatgg ataaagcg 28
<210> 34
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tcgctcacta attgatcttt acggtgttt 29
<210> 35
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
atcctcgtcc gattaatcca ctttta 26
<210> 36
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atcctcgtcc gattaatcca cttttt 26
<210> 37
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ccttcaattc attgttttcg agatctttta 30
<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tatttagtac ttgtttatag actcgac 27
<210> 39
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ccttatttag tacttgttta tagactcgat 30
<210> 40
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
aatgctttta tctctattct atggcgcaat 30
<210> 41
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
attcaacaag aaaaaaaatt tcgacaaatt cc 32
<210> 42
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
attcaacaag aaaaaaaatt tcgacaaatt ct 32
<210> 43
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gcgaagtagt aggattggtt ctcataatt 29
<210> 44
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tcatatacta aaaaagaatt caaaaagggg a 31
<210> 45
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
catatactaa aaaagaattc aaaaaggggg 30
<210> 46
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gagatagaat tcttcgtgac atgacgaaa 29
<210> 47
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
aggcgtgggc gaattagagt c 21
<210> 48
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
caggcgtggg cgaattagag tt 22
<210> 49
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gtctttgttt atgcttcgga ttggaacaa 29
<210> 50
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cggtaccaat gaataggata gags 24
<210> 51
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
atcggtacca atgaatagga tagaga 26
<210> 52
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ccaaaggaaa ccataaattc catataccat 30
Claims (5)
1. Any one of the following uses of a molecularly-labeled combined detection reagent:
(1) the application of the compound in identifying the type C cytoplasm of the male sterility of the corn,
(2) the application in identifying the hybrid prepared by the corn C-type cytoplasmic male sterile line,
(3) the application in the molecular marker assisted breeding of the C-type cytoplasmic male sterile maize,
(4) application in identifying the purity of the C-type cytoplasmic male sterile corn material,
the molecular marker combination comprises the following 11 molecular markers, wherein the 11 molecular markers are 9 SNP molecular markers and 2 InDel molecular markers, and the information of the 11 molecular markers is as follows:
2. the use according to claim 1, wherein the molecular markers other than the molecular marker with the site number CPMIDP09 are amplified by the following primers: CPMSNP18, SEQ ID NO. 23-25; CPMSNP38, SEQ ID NO. 26-28; CPMSNP39, SEQ ID NO. 29-31; CPMSNP44, SEQ ID NO. 32-34; CPMSNP48, SEQ ID NO. 35-37; CPMSNP57, SEQ ID NO. 38-40; CPMSNP59, SEQ ID NO. 41-43; CPMSNP72, SEQ ID NO. 44-46; CPMSNP81, SEQ ID NO. 47-49; CPMIDP10, SEQ ID NO. 50-52.
3. Use according to any of claims 1-2, characterized in that it comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) respectively designing primers based on KASP detection platform technology according to the 11 molecular markers by using the DNA extracted in the step 1) as a template, and performing PCR amplification;
3) the PCR product was analyzed using a fluorescence detector.
4. The use of claim 3, wherein the PCR amplification procedure of step 2) is: 15min at 94 ℃; cycling 10 times at 94 ℃ for 20s and 61 ℃ for 1min, and reducing the temperature by 0.6 ℃ in each cycle; circulating for 30 times at 94 ℃ for 20s and 58 ℃ for 1 min.
5. The use according to claim 1, comprising the steps of:
1) extracting DNA of a corn sample to be detected;
2) respectively designing primers based on KASP detection platform technology according to the 11 molecular markers by using the DNA extracted in the step 1) as a template, and performing PCR amplification;
3) analyzing the PCR product by a fluorescence detector;
4) if the PCR product is the same as the allele, the PCR product is a C-type sterile single plant, and if the PCR product is the other allele, the PCR product does not belong to the C-type sterile single plant; purity calculation is carried out on each site, and the calculation method comprises the following steps: (number of sterile individuals of type C/total number of individuals detected). times.100%.
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