CN111996280B - SNP marker co-separated from brassica napus dwarf compact trait and application thereof - Google Patents
SNP marker co-separated from brassica napus dwarf compact trait and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP marker co-separated from the dwarf stalk compact trait of Brassica napus, which is located at the coding region of a gene BnaA04T0200700ZS and corresponds to the position of 19965380bp of the A04 chromosome of a Brassica napus reference genome ZS11 (published in 2020), and the polymorphic site of the basic group is C or T. The invention also discloses a primer based on the five-primer amplification hindered mutation system technology and a primer based on the conventional PCR technology for detecting the SNP marker. By utilizing the SNP marker and the primer provided by the invention, the rape plant height and plant type can be accurately and efficiently predicted, a scientific method is provided for realizing early identification or screening of the rape plant height and plant type characters, and the progress of rape plant type breeding is accelerated.
Description
Technical Field
The present invention belongs to the field of biotechnology and plant molecular breeding technology. Specifically, the invention relates to an SNP marker co-separated from the compact trait of Brassica napus dwarf and application thereof.
Background
Rape is the first big oil crop in China, the annual planting area is about 1.1 hundred million acres, and rapeseed oil accounts for more than 55% of domestic edible vegetable oil. At present, the self-sufficiency rate of edible vegetable oil in China is about 35%, the dependence degree on the international market is high, and the development of rape production has important strategic significance on maintaining the national edible oil supply safety. The plant type plays a key role in crop breeding potential, and researches show that the rape with short stalks and compact plant type is more favorable for improving the photosynthetic efficiency and the fertilizer resistance of the population, is favorable for ventilation of the middle lower part of the rape, reduces plant diseases and insect pests, resists lodging, increases the harvest index and is favorable for mechanical production of the rape. The dwarf, lodging-resistant and plant-type compact variety is the development trend of the genetic improvement of the rape at home and abroad at present.
The plant height is the main constitutive character of the plant type, and the appropriate plant height can improve the lodging resistance of crops, which is a necessary prerequisite for obtaining high and stable yield of the crops. The green revolution of wheat and rice since the 60 s in the 20 th century is one of the most great achievements of crop breeding, and cultivated short-stalk varieties are widely planted all over the world, so that the yield is increased by nearly one time, and the basis of the successful revolution of the green revolution is the full utilization of short-stalk resources. Some dwarf resources are also currently found in oilseed rape, for example, Ds-1(17cm), Ds-2(19cm), Ds-3(69.7cm), dwarf No.1 (24cm), oilseed rape ED1(100cm), ED1(23cm), GRC1157(80cm), DW871(139.1cm), 99CDAM (85cm), NDF-1(70cm), Apl03(130cm), 9804(110cm), Bndwf1(80cm), Bndwf1/dcl1(50cm), etc. However, compared with crops such as rice, wheat and the like, the discovered and identified rape dwarf germplasm is few, the successful application of the rape dwarf germplasm in dwarf breeding is still few, and the dwarf breeding of the rape in China has not been remarkably developed.
Traditional breeding is a time-consuming and labor-intensive process and relies on the experience of breeders. Compared with the traditional breeding technology, the molecular marker technology can not be influenced by environmental factors and can quickly and accurately track the target gene. The molecular marker technology is combined with the traditional breeding technology, so that the genotype of the plant can be identified in the early stage, the breeding efficiency is obviously improved, and the breeding period is shortened. SNP (single nucleotide polymorphism) refers to DNA sequence polymorphism caused by single base variation on a genome DNA sequence, has the advantages of wide and uniform distribution, high genetic stability, high flux, quick detection, easy automation and standardized operation and the like, and is the most potential marker at present. Currently, there are more than 20 SNP genotyping methods, including restriction amplification polymorphism sequence method (CAPS), allele-specific PCR (AS-PCR), single-strand conformation polymorphism (SSCP), gene chip technology, competitive allele-specific PCR (KASP), sequencing method, etc. The fluorescent molecular marking technology of the five-primer amplification hindered mutation system (PARMS) has the characteristics of high flux, accuracy, time saving, convenience and low cost, and is a rapid genotyping technology developed in recent years.
Disclosure of Invention
The invention aims to: in the earlier stage research, an EMS mutagenesis technology is utilized, and a genetically stable dwarf stalk compact mutant DF110 (plant height 67.6cm) is obtained from mutagenesis progeny of a conventional rape Ningyou No. 18 (NY18, plant height 190cm), and has the advantages of short stalk, lodging resistance, compact plant type and the like. DF110 is hybridized with 5 high-quality and high-yield rape varieties, and the combined hybrid has the characteristics of moderate plant height, compact plant type, high yield and lodging resistance and the like, and meets the standard of the requirement of 'ideal plant type' of rape. The result of testing the yield of the hybrid seeds shows that the yield per mu is 266.6 to 339.6 kilograms, 31.2 to 41.3 percent of the yield of the hybrid seeds is higher than that of the hybrid seeds, and the yield is increased by more than 15 percent of the yield of the hybrid seeds popularized in a large area. Therefore, DF110 is an excellent germplasm resource for rape plant type breeding. The invention aims to solve the technical problem of providing the SNP marker which is co-separated from the compact trait of the brassica napus dwarf. DF110 has SNP marker co-separated with dwarf stalk compact character, and has important application prospect in rape plant type breeding.
The technical problem to be solved by the present invention is to provide primers for detecting the SNP markers.
The technical problem to be solved by the invention is to provide a kit for detecting the SNP marker.
The invention also aims to solve the technical problem of providing the application of the SNP marker, the arbitrary one or more groups of primers or the kit in identifying or screening the rape dwarf compact trait material.
The invention also solves the technical problem of providing the application of the SNP marker, the arbitrary group or several groups of primers or the kit in molecular marker-assisted selection breeding.
The technical problem to be solved by the invention is to provide a method for early predicting the plant height and plant type characters of the cabbage type rape.
The technical scheme is as follows: in order to solve the technical problems, the invention provides an SNP marker co-separated with the dwarf stalk compact trait of the Brassica napus, wherein the SNP marker is positioned at the coding region of a gene BnaA04T0200700ZS and corresponds to the position of 19965380bp of the A04 chromosome of a Brassica napus reference genome ZS11 (published in 1 month 2020), and the polymorphic site of the base is C or T.
Wherein when the genotype of the SNP marker position is CC, the probability that the cabbage type rape is high-stalk and loose is 100 percent; when the genotype of the SNP marker position is TT, the probability that the cabbage type rape is short stalk and compact plant type is 100 percent.
The invention also comprises a five-primer amplification hindered mutation system PCR amplification primer for detecting the SNP marker, wherein the five-primer amplification hindered mutation system PCR amplification partial primer (BnaA04-199) comprises:
allele primer 1: BnaA04-199-Fg is
5′-GAAGGTGACCAAGTTCATGCTGTTCTTGGTACTCCAACCCGAG-3′，
Allele primer 2: BnaA04-199-Fa is
5′-GAAGGTCGGAGTCAACGGATTGTTCTTGGTACTCCAACCCGAA-3′，
Site amplification reverse primer: BnaA04-199-R is
5′-GCGGGAATCTGAAGTCTGTGTAG-3′。
The accuracy of detection of the BnaA04-199 marker was 100%.
The five-primer amplification hindered mutation system of the SNP marker is shown by three primers BnaA04-199, and the other primer amplification systems are 2 xPARMS Master Mix (Gentides Biotech Co., Ltd.) of Wuhan market peptide Biotechnology Co., Ltd.
The present disclosure also includes conventional PCR amplification primers (BnaA04-199PCR) for detecting said SNP markers, said primers comprising:
site amplification forward primer BnaA04-199 pcr-F: 5'-TGATCTTTGGGGCCACAGAGTAT-3' the flow of the air in the air conditioner,
site amplification reverse primer BnaA04-199 pcr-R: 5'-AAGTTGAACAAGGCTGGTAGAGG-3' the flow of the air in the air conditioner,
allele primer BnaA04-199 pcr-Fg: 5'-GTTCTTGGTACTCCAACCCGAG-3', and the adhesive tape is used for adhering the film to a substrate,
allele primer BnaA04-199 pcr-Rt: 5'-GTTTGGATTCATGCATCGGATTTCTTT-3' are provided.
The detection accuracy of the BnaA04-199pcr marker is 100%.
The invention also comprises a kit for detecting the SNP marker, which comprises any one or more groups of primers.
The invention also comprises the application of the SNP marker, any one or more groups of primers or the kit in the breeding of the cabbage type rape.
The invention also comprises the application of the SNP marker, any one or more groups of primers or the kit in identifying or screening the rape dwarf compact trait materials.
The invention also comprises the application of the SNP marker, any one or more groups of primers or the kit in the auxiliary selective breeding of the molecular marker of the rape.
The invention also comprises a method for early predicting or detecting the plant height and plant type character of the cabbage type rape, the plant height and plant type character of the cabbage type rape to be detected are predicted by detecting the SNP marker, and the method specifically comprises the following steps:
(1) extracting rape genome DNA to be detected;
(2) taking the genomic DNA to be detected as a template, and carrying out PCR amplification reaction in a fluorescent quantitative PCR instrument by using the five-primer amplification hindered mutation system PCR amplification primers;
(3) analyzing a fluorescent signal of the PCR amplification product, wherein the green signal is the genotype of the high-stem and plant-type dispersed rape; the blue signal is the genotype of the dwarf and plant type compact rape; the red signal is the genotype of the heterozygous rape; grey signals are indeterminate genotypes.
The invention also comprises a method for early predicting or detecting the plant height and the plant type character of the brassica napus, which predicts the plant height and the plant type character of the brassica napus to be detected by detecting the SNP marker, and specifically comprises the following steps:
(1) extracting rape genome DNA to be detected;
(2) taking the genome as a template, and carrying out conventional PCR amplification reaction by using a conventional PCR amplification primer marked by SNP;
(3) detecting the PCR amplification product through agarose gel electrophoresis, if the amplification product has only a single 406bp band, the amplification product is consistent with the wild type genotype, and predicting the amplification product to be homozygous high-stem and plant type dispersed rape; if the amplified product has a single band of 272bp, the genotype is consistent with that of the mutant, and the rape is predicted to be homozygous short-stalk compact rape; if the amplified product has two bands of 406bp and 272bp at the same time, the amplified product is similar to F 1 The genotypes are consistent, and the hybrid intervarietal rape is predicted.
In addition, according to the embodiment of the present invention, the method for performing SNP marker detection on oilseed rape to be detected is not particularly limited. SNP detection can be achieved by using technologies such AS Cleaved Amplified Polymorphic Sequence (CAPS), allele-specific PCR (AS-PCR), Single Strand Conformation Polymorphism (SSCP), high resolution melting curve (HRM), gene chip (gene chip), competitive allele-specific PCR (KASP), competitive allele-specific PCR, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MAL-DI-TOF-MS), and sequencing by GBS.
Has the beneficial effects that: the core requirements of the ideal plant type of the rape are moderate plant height and compact plant type. Due to the shortage of excellent germplasm resources, the plant type breeding of the rape has not made breakthrough progress. Compared with the prior art, the DF110 is a non-transgenic dwarf, lodging-resistant and plant-type compact mutant. The yield of the hybrid seed formed by combining DF110 and 5 high-quality and high-yield rape varieties is increased by 31.2-41.3% compared with the yield of the hybrid seed of the rape variety which is promoted in a large area, and the hybrid seed has the characteristics of moderate plant height, compact plant type, high yield and lodging resistance and the like, so that the DF110 has important application value in the rape plant type breeding. By using a map-based cloning method, the dwarf compact control locus BnDwf.A4 is finely positioned in the 177.3Kb interval of the A04 chromosome, and an SNP marker co-separated from the dwarf compact trait is obtained in the finely positioned interval, so that the plant height and the plant type of the progeny can be predicted by 100%.
1. The SNP marker BnaA04-199 which is co-separated with the dwarf compact trait is obtained on the cabbage type rape A04 chromosome for the first time, and a primer BnaA04-199 based on the PARMS technology and a primer BnaA04-199PCR based on the conventional PCR and agarose gel electrophoresis are developed.
2. The two groups of SNP marker primers are codominant markers, and can distinguish different genotypes 100%. Wherein, BnaA04-199 is a fluorescent molecular marker, and the detection with high flux, high accuracy, automation and low cost can be realized based on PARMS technology. The BnaA04-199PCR primer has low experimental requirements, and the plant height and the plant type of the rape can be accurately predicted only by a conventional PCR instrument and agarose gel electrophoresis. Different primers can be selected according to specific actual requirements.
3. The method can be used for molecular marker-assisted selective breeding of the compact trait of the short stalk of the rape plant, can accurately predict the plant height and the plant type of the rape by SNP marker detection, provides scientific basis for early detection of the compact trait of the short stalk of the rape, and greatly improves the breeding efficiency of the plant type of the rape.
Drawings
FIG. 1, Brassica napus dwarf mutant DF110, wild type NY18 and F 1 (ii) a phenotype of (a);
NY18 is wild type, the plant height is 190cm, and the plant type is loose; DF110 is a mutant, the plant height is 67.6cm, and the plant type is compact; f 1 The plant height is 122cm, and the plant type is an intermediate type;
FIG. 2 field phenotype of hybrid of rape cultivar Nannong 9808 with DF 110;
the height of Nannong 9808 is 168cm, the plant type is dispersed, the maturity period is lodging, and the yield measurement result shows that the yield per mu is 240.3 kg; the hybrid seed composed of Nannong 9808 × DF110 has the plant height of 125cm, moderate plant height, compact plant type, no lodging during the whole growth period and good lodging resistance, and the yield measurement result shows that the yield per mu reaches 339.6 kg;
FIG. 3 shows SNP marker BnaA04-199 pairs (NY18 XDF 110) F 2 PARMS PCR detection results of 92 individuals of the population;
green signal, high stalk genotype; blue signal, dwarf genotype; red signal, heterozygous genotype; gray signal, blank control;
FIG. 4 shows SNP marker BnaA04-199 pairs (Holly. times. DF110) F 2 PARMS PCR assay results for 92 individuals of the population;
green signal, high stalk genotype; blue signal, dwarf genotype; red signal, heterozygous genotype; gray signal, blank control or indeterminate genotype;
FIG. 5 shows SNP markers BnaA04-199 pairs (Zhongshuang No. 11. times.DF 110) F 2 PARMS PCR detection results of 92 individuals of the population;
green signal, high stalk genotype; blue signal, dwarf genotype; red signal, heterozygous genotype; gray signal, blank control or indeterminate genotype;
FIG. 6 shows SNP marker BnaA04-199 pairs (17P44 XDF 110) F 2 PARMS PCR assay results for 92 individuals of the population;
green signal, high stalk genotype; blue signal, dwarf genotype; red signal, heterozygous genotype; gray signal, blank control or indeterminate genotype;
FIG. 7 shows that the SNP markers BnaA04-199 pairs (Nannong 9808 XDF 110) F 2 PARMS PCR detection results of 92 individuals of the population;
green signal, high-stalk genotype; blue signal, dwarf genotype; red signal, heterozygous genotype; gray signal, blank control or indeterminate genotype;
FIG. 8 shows that SNP marker BnaA04-199pcr pair (NY18 XDF 110) F 2 Detecting results of partial individual plants of the population;
n, the amplification band type of the high-stem parent NY18 only has a single band of 406 bp; f1, (NY18 XDF 110) F 1 The amplified band type of (2) has two bands of 406bp and 272bp at the same time; d, amplification banding pattern of the dwarf parent DF110 is only a single band of 272 bp; m, DL2000plus DNA Marker;
1-7，F 2 (ii) amplified banding patterns of high-stem individuals in the population; 8-14, F 2 (ii) amplified banding patterns of medium stem individuals in the population; 15-21, F 2 Amplified banding patterns of short stalk individuals in the population;
BnaA04-199pcr can be accurately distinguished (N)Y18×DF110)F 2 High, medium and low stem individual plants in the population.
FIG. 9 shows that SNP marker BnaA04-199pcr pair (Holly. times.DF 110) F 2 The detection results of part of the individual plants of the population;
h, the amplification banding pattern of the high-pole parent Holly is only a single band of 406 bp; f1, (Holly × DF110) F 1 The amplified band type of (2) has two bands of 406bp and 272bp at the same time; d, the amplification banding pattern of the dwarf parental DF110 only has a single band of 272 bp; m, DL2000plus DNA Marker;
1-7，F 2 (ii) amplified banding patterns of high-stem individuals in the population; 8-14, F 2 (ii) amplified banding patterns of mid-stem individuals in the population; 15-21, F 2 (ii) an amplified banding pattern of short stalk individuals in the population;
BnaA04-199pcr can accurately distinguish (Holly × DF110) F 2 High-stem, medium-stem and low-stem individuals in the population.
FIG. 10 shows the SNP marker BnaA04-199pcr pair (Zhongshuang No. 11. times. DF110) F 2 The detection results of part of the individual plants of the population;
z, double No. 11 amplification bands in the high-stem parent are single bands of 406 bp; f1, (Zhongshuang No. 11X DF110) F 1 The amplified band type of (2) has two bands of 406bp and 272bp simultaneously; d, the amplification banding pattern of the dwarf parental DF110 only has a single band of 272 bp; m, DL2000plus DNA Marker;
1-7，F 2 (ii) amplified banding patterns of high stem individuals in the population; 8-14, F 2 (ii) amplified banding patterns of mid-stem individuals in the population; 15-21, F 2 (ii) an amplified banding pattern of short stalk individuals in the population;
BnaA04-199pcr can accurately distinguish (Zhongshuang No. 11 × DF110) F 2 High, medium and low stem individual plants in the population.
Detailed Description
The methods used in the following examples are conventional methods unless otherwise specified, and various reagents used in the examples are commercially available. 2 XPARMS Master Mix (cat. No. E001) and conventional PCR Mix were purchased from Wuhan City peptide Biotechnology Co., Ltd (Gentides Biotech Co., Ltd.), and the detection of PARMS PCR markers was technically supported by Wuhan City peptide Biotechnology Co., Ltd. The cabbage type rape varieties NY18, Holly, 17P44 and Nannong 9808 used in the experiment are provided for the research institute of economic crops of agricultural academy of sciences of Jiangsu province, and the Zhongshuan No. 11 is provided for the research institute of oil crops of agricultural academy of sciences of China.
Example 1 compact rape dwarf mutant DF110 and breeding application value thereof
EMS is utilized to mutate conventional rape NY18, and after years of multi-generation screening, the mutant DF110 with short stalk, lodging resistance and compact plant type is obtained. DF110 is preserved in China general microbiological culture Collection center (CGMCC) at 8, 11 and 2020, with the preservation number of CGMCC NO.19968 and the preservation address of: the microbiological research institute of western road 1, 3, national academy of sciences, north-kyo, chaoyang, the postal code: 100101, classification name: rape (Brassica napus).
The dwarf compact control site BnDwf.A4 is finely positioned in the 19829507-20006796bp interval of A04 chromosome by using a map-based cloning method. In the interval, the base at the 19965380bp position of the mutant DF110 is mutated from C to T, and the SNP is detected in 5 different parent offspring to verify that the SNP is cosegregated with the dwarf compact trait.
In autumn of 2018, DF110, 5 high-quality and high-yield rape varieties (NY18, Zhongshui No. 11, Holly, 17P44 and Nannong 9808) and hybrid seeds thereof are planted in Lishu plant scientific base of agricultural academy of sciences of Jiangsu province, and 10 continuous single plants are selected in the mature period for agronomic character investigation (Table 1). The data show that compared with the high-stalk parent, the main axis silique number, the whole plant silique number and the single plant seed yield of all the hybrids are obviously increased, the plant height is obviously reduced, and the lodging resistance is obviously enhanced. DF110 crosses parents of different genetic backgrounds, F 1 The generations show the obvious advantages of moderate plant height, compact plant type, resistance to yield increase and suitability for mechanized production of rapes (figure 2), and show that the DF110 has important application value in rape plant type breeding.
TABLE 1
Example 2 Fine localization of the short stalk compact trait control site BnDwf. A4
F at NY18 XDF 110 2 In the population, 30 high-stalk extreme single plants and 30 short-stalk extreme single plants are respectively selected, two DNA mixing pools are constructed, and the two DNA mixing pools and two parents are subjected to genome re-sequencing. Using a BSA character positioning method, taking the genome of Brassica napus ZS11 as a reference genome, preliminarily positioning the dwarf locus of DF110 in a 15.3-20.7Mb interval of A04 chromosome, and naming the dwarf locus as BnDwf.A 4;
adopting a plug seedling method to grow seedlings, and adding (Zhongshuang No. 11 multiplied by DF110) F 2 The seeds are sown into 50-hole trays, each hole is sown with one full seed, and 140 trays are sown in total. The homozygous short-stalk compact single plant has a phenotype of leaf shrinkage at the seedling stage, so that the homozygous short-stalk compact single plant can be accurately distinguished at the seedling stage, but the heterozygous type and the wild type cannot be distinguished. And (3) selecting single plants with shriveled leaves at the seedling age of 35 days, transplanting the single plants to a field, and performing conventional field management for fine positioning of the BnDwfA4 and identification of the later-stage plant height and plant type phenotype. Selecting tender leaves, and successfully extracting DNA of 1641 dwarf compact single plants by using a CTAB method. According to the result of parental re-sequencing, screening SNP in the positioning region of BnDwfA4, extracting 200bp sequences at both sides of the SNP, submitting to Wuhan city scenery peptide Biotechnology Limited, and designing PARMS PCR primers. And verifying the designed PARMS PCR primers, screening out primers with good polymorphism, performing gene analysis on the 1641 short-stalk compact single plants, and screening out crossover single plants. The mapping shares 11 pairs of PARMS PCR primers, and the positions of the SNP corresponding to the A04 chromosome of the Brassica napus reference genome ZS11 (published in 2020) are respectively as follows: 15334596 bp, 17986575 bp, 18676247 bp, 19626111 bp, 19654675 bp, 19829507 bp, 19965380bp, 20006796bp, 20354811 bp, 20401024 bp and 20744288 bp. Combining the dwarf compact phenotype and the crossover individual genotype, finally, the BnDwfA4 is finely positioned between the two SNP markers of 19829507 bp and 20006796bp, and the corresponding physical distance is 177.3 Kb.
Example 3 development of SNP sites and their markers cosegregating with dwarf compact trait
According to the brassica napus reference genomeSNP-designed PARMS PCR primers at 19829507 bp and 20006796bp of A04 chromosome of ZS11 (published 2020) (middle double 11. times. DF110) F 2 Of 1641 short-stalk compact individuals of the population, 31 and 7 crossover individuals were screened, respectively, and bndwf.a4 was finally pinpointed between these two markers. Design of PARMS PCR primers based on the SNP at 19965380bp of chromosome A04 (BnaA04-199 marker), allele primer 1: BnaA04-199-Fg was 5'-GAAGGTGACCAAGTTCATGCTGTTCTTGGTACTCCAACCCGAG-3', allele primer 2: the nucleotide sequence of BnaA04-199-Fa is 5'-GAAGGTCGGAGTCAACGGATTGTTCTTGGTACTCCAACCCGAA-3', and the nucleotide sequence of the site amplification reverse primer BnaA04-199-R is 5'-GCGGGAATCTGAAGTCTGTGTAG-3'. And (3) screening crossover individuals on 1641 short-stalk individuals by using a BnaA04-199 marker, wherein the SNP is co-separated from the short-stalk compact character, and the crossover individuals are not screened. The BnaA04-199 marker is located in the coding region of BnaA04T0200700ZS gene, and the mutation from C to T at 19965380bp causes the mutation of the coded amino acid from glutamic acid to lysine. BnaA04T0200700ZS (AT2G30980) encodes brassinolide signal transduction gene BIL1, and the mutation of the gene has been proved to cause the dwarfing of plants in Arabidopsis thaliana and other plants, but the function of the gene in rape is not clear. To verify that the BnaA04-199 marker cosegregated with the dwarf compact trait, the marker was verified in different populations. F constructed in NY18, Zhongshuang No. 11, Holly, 17P44, Nannong 9808 and DF110 2 In the population, 92 single plants with different plant height types are randomly selected, DNA is extracted, a BnaA04-199 marker is used for genotyping, and the BnaA04-199 marker is found to be completely coseparated with the short stalk compact character in different populations by combining the investigation results of the plant height and plant type characters in the mature period, and the screening efficiency is 100%.
The PCR reaction system is 5 μ L: 2 × PARMS Master Mix (cat # E001): 2.5 mu L; allele primer 1: 0.1 mu L; allele primer 2: 0.1 mu L; site amplification reverse primer: 0.3 mu L; genomic DNA: 1 μ L (50 ng/. mu.L); ddH 2 O：1μL。
The PCR procedure was: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 deg.C for 20s, annealing at 65 deg.C for 1min (0.8 deg.C per cycle), and performing 10 cycles; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 1min, and 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C. Performing a PCR amplification reaction in Q6 (ABI); and carrying out genotyping according to the fluorescent signal by using SNP Decoder software.
The results of genotyping are shown in FIGS. 3-7. Wherein the green signal is consistent with the wild type genotype (CC) and is homozygous high-stem plant type dispersed rape; the blue signal is consistent with the mutant genotype (TT), and is homozygous dwarf and plant-type compact rape; red signal and F 1 The genotypes (CT) are consistent, and the rape is heterozygous medium type rape; grey signals are blank controls or indeterminate genotypes.
Example 4: developing conventional PCR (polymerase chain reaction) marker aiming at SNP (single nucleotide polymorphism) locus co-separated by short-stalk compact traits
The BnaA04-199 label based on the PARMS technology can realize high-throughput, high-precision, automatic and low-cost detection, has important application value in group detection of large samples, but has higher experimental requirements and needs a fluorescent quantitative PCR instrument or a fluorescent scanner. To meet the requirements of detection of small samples and to enable breeders with no laboratory conditions to use molecular marker assisted selection, we designed the BnaA04-199 marker as a conventional PCR primer BnaA04-199 PCR. The primer comprises four primer sequences, wherein, the BnaA04-199pcr site amplifies a forward primer: BnaA04-199-F is 5'-TGATCTTTGGGGCCACAGAGTAT-3', site amplification reverse primer: BnaA04-199-R is 5'-AAGTTGAACAAGGCTGGTAGAGG-3', allele primer 1: BnaA04-199-Fg was 5'-GTTCTTGGTACTCCAACCCGAG-3', allele primer 2: the BnaA04-199-Rt is 5'-GTTTGGATTCATGCATCGGATTTCTTT-3'. The primer is used for amplification on a common PCR instrument (such as an Eppendorf AG 22331 Hamburg PCR amplification instrument), and the amplified product is electrophoresed on 2.5 percent agarose gel, so that the genotypes of homozygous high-stem, homozygous low-stem and heterozygous intermediate rape can be accurately identified.
The PCR reaction system is 20 μ L: PCR mix (genetics Biotech co., Ltd.): 10 mu L of the solution; BnaA 04-199-F: 0.8 mu L; BnaA 04-199-R: 0.8 mu L; BnaA 04-199-Fg: 0.8 mu L; BnaA 04-199-Rt: 0.8 mu L; genomic DNA: 1 μ L (50 ng/. mu.L); ddH 2 O：5.8μL。
The PCR procedure was: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing and extension at 65 ℃ for 1min (0.8 ℃ per cycle), and 10 cycles; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 1min, and 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C. Carrying out PCR amplification reaction in an EppendorfAG 22331 Hamburg PCR amplification instrument; the PCR amplification product was detected by electrophoresis on a 2.5% agarose gel.
To verify the accuracy of BnaA04-199pcr, we constructed F with DF110 in NY18, Holly and Zhongshuang No. 11 2 In the population, 7 high-stem individuals, 7 intermediate-type individuals and 7 short-stem individuals, wild type, mutant and F thereof are randomly selected 1 PCR amplification and electrophoresis detection were performed using BnaA04-199 PCR. The electrophoresis results are shown in FIGS. 8-10, in which the wild type and the single high-stem plant have a single strip of 406 bp; DF110 and the short stalk single plant only have a single band of 272 bp; f 1 Has two bands of 406bp and 272bp simultaneously with the intermediate single strain. The detection result is completely consistent with the phenotype (the accuracy is 100 percent), and the BnaA04-199pcr can accurately perform genotyping on the rape plant height and plant type character, and has important application value in molecular marker-assisted selection breeding.
Sequence listing
<110> institute of oil crops, national institute of agricultural sciences, Jiangsu province institute of agricultural sciences
<120> SNP marker cosegregating with compact trait of brassica napus dwarf and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 43
<212> DNA
<213> allele primer 1: BnaA04-199-Fg (artificial sequence)
<400> 1
gaaggtgacc aagttcatgc tgttcttggt actccaaccc gag 43
<210> 2
<211> 43
<212> DNA
<213> allele primer 2: BnaA04-199-Fa (artificial sequence)
<400> 2
gaaggtcgga gtcaacggat tgttcttggt actccaaccc gaa 43
<210> 3
<211> 23
<212> DNA
<213> site amplification reverse primer BnaA04-199-R (artificial sequence)
<400> 3
gcgggaatct gaagtctgtg tag 23
<210> 4
<211> 23
<212> DNA
<213> BnaA04-199pcr-F(Artificial Sequence)
<400> 4
tgatctttgg ggccacagag tat 23
<210> 5
<211> 23
<212> DNA
<213> BnaA04-199pcr-R(Artificial Sequence)
<400> 5
aagttgaaca aggctggtag agg 23
<210> 6
<211> 22
<212> DNA
<213> BnaA04-199pcr-Fg(Artificial Sequence)
<400> 6
gttcttggta ctccaacccg ag 22
<210> 7
<211> 27
<212> DNA
<213> BnaA04-199pcr-Rt(Artificial Sequence)
<400> 7
gtttggattc atgcatcgga tttcttt 27
Claims (2)
1. The application of PARMS PCR amplification primers for detecting SNP markers in identifying or screening Brassica napus dwarf and compact plant trait materials is characterized in that the amplification primers comprise:
allele primer 1:
5'-GAAGGTGACCAAGTTCATGCTGTTCTTGGTACTCCAACCCGAG-3'，
allele primer 2:
5'-GAAGGTCGGAGTCAACGGATTGTTCTTGGTACTCCAACCCGAA-3'，
site amplification reverse primer:
5'-GCGGGAATCTGAAGTCTGTGTAG-3'；
when the genotype of the SNP marker is CC, the cabbage type rape is high-stalk and loose type; when the genotype of the SNP marker is TT, the cabbage type rape is dwarf and compact; the brassica napus is a filial generation taking a mutant DF110 as a parent, the mutant DF110 is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) in 8-11-month 2020, and the preservation number is CGMCC NO. 19968.
2. A method for early predicting or detecting the plant height and plant type character of Brassica napus is characterized by comprising the following steps:
(1) extracting rape genome DNA to be detected;
(2) carrying out PCR amplification reaction in a fluorescent quantitative PCR instrument by using the amplification primer of claim 1 by using the genomic DNA to be detected as a template;
(3) analyzing a fluorescent signal of the PCR amplification product, wherein the fluorescent signal is consistent with the genotype CC and is high-stem and plant-type dispersed rape; the fluorescent signal is consistent with genotype TT, and is short-stalk and plant-type compact rape;
the brassica napus is a filial generation taking a mutant DF110 as a parent, the mutant DF110 is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) in 2020, 8 and 11 months, and the preservation number is CGMCC NO. 19968.
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