CN114836569A - KASP molecular marker of flowering Chinese cabbage branch trait major QTL and application thereof - Google Patents
KASP molecular marker of flowering Chinese cabbage branch trait major QTL and application thereof Download PDFInfo
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Abstract
The invention discloses a KASP molecular marker of a main effect QTL of a vegetable heart branch number and application thereof, wherein the main effect QTL is positioned on an A07 chromosome of a vegetable heart and is named as qBrTL 1; the qBrTL1 localization interval is 34.24cM between KASP markers A0716 and A0717, which can account for 20.81% of phenotypic variation with an additive effect of 1.52; the qBrTL1 comprises two linked KASP molecular markers of A0716 and A0717, wherein the A0716 is developed according to the 28007027bp presentation polymorphism of the A07 chromosome, the base of the A0716 is A or G, the A0717 is developed according to the 28031606bp presentation polymorphism of the A07 chromosome, and the base of the A0717 is T or G. The two KASP molecular markers can realize high-throughput and rapid auxiliary selection of the flowering characteristics of the flowering cabbage, improve the selection efficiency, accelerate the breeding process and provide a new genetic marker for molecular breeding of the multi-branched flowering cabbage.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a KASP molecular marker of a flowering characteristic major QTL of flowering cabbage and application thereof.
Background
The flowering cabbage (Brassica campestris L.ssp. Chinese var. utilis Tsen et Lee), also known as a flowering cabbage, is a biennial herb (Zhang Hua, etc., 2010; Huangxiu, etc., 2017) in Brassica brassicae species of cruciferae with the flowering cabbage as an edible organ, is native to the Guangdong, is a special vegetable (Chenhan Cai, etc., 2021) in southern areas of China, and has a wide cultivation area. The main inflorescence growth advantage of the common flowering cabbage variety is remarkable, only 1 flowering bolt is usually harvested in production, the yield is lower than that of the characteristic variety Zengcheng flowering cabbage in Guangdong region, the Zengcheng flowering cabbage can form about 15 multi-side branches at the stem base part close to the ground, the multi-side branches can be harvested for multiple times after one-time planting, the side branches are used as commodity organs, and the number of the side branches can be used as the standard for measuring the yield of the flowering cabbage (Liu le Tung et al, 1998), so that the method has important significance in breeding the multi-branch flowering cabbage variety.
The branch number of the cabbage heart is quantitative, the forming mechanism of the branch number is very complex, and the branch number is easily influenced by the environment, so that the traditional conventional breeding method based on phenotype selection has poor selection effect on the complex quantitative character, long breeding period, low selection efficiency and prolonged breeding period, and the requirement of current cabbage heart breeding cannot be completely met. The combination of molecular marker technology and quantitative genetics can decompose complex quantitative traits into single Quantitative Trait Loci (QTL), and then research multiple genes controlling quantitative traits like the research of quality traits, and the QTL positioning can be used for determining the genetic loci and positions influencing the quantitative traits. Molecular marker assisted breeding is a new means for effectively combining molecular genetics and traditional phenotype selection, and directly utilizes molecular markers closely linked or coseparated with target character genes to screen target regions and whole genomes of individuals so as to achieve the purposes of improving the target character selection efficiency and shortening the breeding period. The key of the molecular marker assisted selective breeding technology is to identify DNA molecular markers closely linked with important agronomic traits. At present, KASP is successfully applied to genotyping by constructing a genetic map to mine genes related to traits. The KASP marker is not limited by the base type and site, and can be used for detecting indels and SNPs. The fine positioning of candidate genes can be realized by using KASP markers in combination with phenotype data through QTL mapping software.
The cultivation of multi-branch Chinese flowering cabbage variety is an effective way for improving the yield and economic benefit of Chinese flowering cabbage. The currently identified genes for controlling the branch number of the flowering cabbage are few, and the requirements of molecular breeding cannot be met. Therefore, the discovery of the major QTL of the flowering characteristic of the flowering cabbage and the development of the molecular marker closely linked with the major QTL have important significance for the molecular marker-assisted selective breeding of the multi-branch flowering cabbage variety.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the problems occurring in the prior art.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions:
the KASP molecular marker primer sequence of the KASP molecular marker of A0716 is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
The KASP molecular marker primer sequence of A0717 is shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
The invention also aims to provide the application of the KASP molecular marker primer group of the flowering cabbage character major QTL in multi-branch flowering cabbage breeding. The invention can be applied to the early identification and screening of the vegetable heart branch character, improves the selection efficiency and provides a new genetic marker for multi-branch vegetable heart molecular breeding.
In order to achieve the purpose, the invention adopts the following technical measures:
the KASP molecular marker of the flowering Chinese cabbage character major QTL is obtained by the following method:
(1) using common less-branched rape heart DH line 'CX 010' and multi-branched Zengcheng rape heart DH line 'CX 020' as parent, hybridizing to generate F 1 Generation by selfing to produce F 2 And (4) separating the populations.
(2) All F 2 The growth conditions of the plants of the generation segregation population are completely consistent, and in the period with the most obvious branching phenotype, the two parent materials and the F are subjected to 2 Statistics of the number of branches were performed for each individual of the generation segregating population. The specific method comprises the following steps: the standard for investigating the number of branches was that the number of branches within 5cm of the stem base was the stem base branch.
(3)F 2 The branch number of the plants in the population is separated and shows a trend of normal distribution, which indicates that the flowering characteristic of the cabbage heart is quantitative. From F 2 Selecting 50 plants with extreme branching phenotype and middle branching phenotype from segregating population, and extracting 'CX 010' and 'CX 020' and F by using novel plant genome DNA extraction kit (Tiangen, Beijing, China) 2 Total DNA of plant leaves.
(4) Constructing 3 mixed pools of multi-branch phenotypes (12-15 branches), few-branch phenotypes (1-3 branches) and intermediate-branch phenotypes (7-8 branches), performing Graded pool-Seq sequencing on the three mixed pools and two parents, breaking a DNA sample into fragments with the length of 350bp by a fragmentation kit, and constructing a library. The sequencing platform was NovaSeq 6000(Illumina) and the sequencing mode was PE 150.
(5) The original data are subjected to quality control, reads are compared to a Chinese cabbage V3.0 reference genome (http:// branched. org/read/datasets/pub/genome/Brassica _ rapa/V3.0/) by using BWA, the comparison result is subjected to SAMTOOLS to remove repetition, the mutation site is analyzed according to the analysis process of GATK to obtain SNP and InDel information, and the structure annotation is carried out on the mutation site by using SNPeff.
(6) And after Ridit test and analysis, carrying out noise reduction on the data, and counting the proportion of the sites with the significant p value in the total sites of the sliding window area, thereby judging the area significantly associated with the characters. The noise reduction parameter is a window size of 0.2Mb, and the threshold value of the p value is 10 -8 . And finally, positioning an interval of 28.0-28.9 Mb region of the chromosome A07 as a candidate QTL interval, and positioning the gene for regulating the branch number on the chromosome A07.
(7) And uniformly selecting SNP sites with no other variation of 100bp before and after the selection in the candidate interval according to the SNP information, and then designing a primer to convert the SNP sites into KASP markers. By detecting parent and part F 2 And screening the genotype of the individual plant of the population to obtain a KASP marker with good genotyping, and using the KASP marker for constructing a genetic linkage map.
(8) Analysis of F Using specific KASP markers 2 The genotype of each individual in the population was analyzed using QTL IciMapping software by combining genotype and phenotype data. Based on the technical measures, a QTL locus qBrTL1 for controlling the branching number of the cabbage heart is finally obtained, and the main QTL is positioned on the A07 chromosome of the cabbage heart. The qBrTL1 includes two linked KASP molecular markers A0716 and A0717, the LOD peak (10.41) is located at 27cM chromosome 7, the contribution rate is 20.81%, and the additive effect and dominant effect are 1.52 and 2.37 respectively. The physical positions of the polymorphic base marked with A0716 and the polymorphic base marked with A0717 are at 28007027bp and 28031606bp on A07 chromosome, respectively, and the distance between the two is about 24.6 kb.
Compared with the prior art, the invention has the beneficial effects that: the KASP molecular marker of the flowering cabbage main effect QTL and the application thereof are obtained by developing the marker closely linked with the flowering cabbage main effect QTL, and the QTL positioned by the KASP molecular marker has obvious and stable genetic effect and can meet the requirement of molecular breeding.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail with reference to the accompanying drawings and detailed embodiments, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise. Wherein:
FIG. 1 is a morphological diagram of two parents of the present invention. ('CX 010' is branched in such a way that 1 to 3 axillary buds are produced but only one effective branch is formed for harvesting. 'CX 020' is branched in such a way that 12 to 15 branches are produced in the vicinity of rosette leaves.)
FIG. 2 is a statistical chart of the number of branches of two parents of the present invention.
FIG. 3 is F 2 Distribution map of branch number of the group plants. (the abscissa represents the branch frequency distribution, and the ordinate represents the number of individuals.
FIG. 4 is a primary map of the identification of the gene for the branching number of flowering cabbage using the GradedPool-Seq method. (the abscissa is the position coordinate of the chromosome, the unit is 100kb, the ordinate is the ratio of the point with the p value lower than the threshold value in the window to the total number of the points in the window, the higher the position of the point is, the stronger the association degree with the character is, the final location of 28.0-28.9 Mb region of the A07 chromosome, namely the interval of the highest peak is a candidate QTL interval, and the gene for regulating the branch number is located on the A07 chromosome).
FIG. 5 is a mapping diagram of the major QTL (qBrTL1) of the flowering Chinese cabbage trait in the present invention.
FIG. 6 shows A0716 pairs of F in an embodiment of the present invention 2 Genotype identification result map of the population. (individuals having the same genotype as that of the less branched flowering cabbage 'CX 010' and having GG genotypes showing less branching; individuals having the same genotype as that of the more branched flowering cabbage 'CX 020' and having AA genotypes showing more branching; individuals having a heterozygous genotype and having GA genotypes showing intermediate branching.)
FIG. 7 shows A0717 pairs of F in an embodiment of the present invention 2 Genotype identification result map of the population. (individuals of the same genotype as that of the less branched flowering cabbage 'CX 010' with TT showing less branching; individuals of the same genotype as that of the more branched flowering cabbage 'CX 020' with GG showing more branching; individuals of the heterozygous genotype with TG showing intermediate branching。)
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and it will be apparent to those of ordinary skill in the art that the present invention may be practiced without departing from the spirit and scope of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Next, the present invention will be described in detail with reference to the drawings, wherein for convenience of illustration, the cross-sectional view of the device structure is not enlarged partially according to the general scale, and the drawings are only examples, which should not limit the scope of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in the actual fabrication.
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
Construction and property survey of the Chinese flowering cabbage branch number segregation population:
in this example, ordinary less-branched cardiac DH line ` CX010 ` (FIG. 1a) and multi-branched Zengcheng cardiac DH line ` CX020 ` (FIG. 1b) were used as parents, and the difference in the number of branches between the two was significant (FIG. 2), and F was constructed by crossing and selfing 2 Isolating the population. Pairing two parent materials and F during the period of the most obvious flowering phenotype of the cabbage heart 2 Statistics of the number of branches were performed for each individual of the progeny segregating population. The specific method comprises the following steps: the standard for investigating the number of branches was that the number of branches within 5cm of the stem base was the stem base branch.
F 2 The number of branches of the plants in the population is separated and shows a trend of normal distribution, which indicates that the flowering characteristic of the cabbage heart is quantitative (figure 3).
Parent and F 2 Extracting total DNA of the colony leaves:
(1) adding liquid nitrogen into fresh leaves, grinding into powder, adding 400 μ L buffer solution LP1 and 6 μ L RNase A, shaking for 1min, and standing at room temperature for 10 min; adding 130 μ L buffer solution LP2, mixing well, and shaking for 1 min; centrifuging at 12000rpm for 5min, and transferring the supernatant into a 1.5mL centrifuge tube;
(2) adding LP3 buffer solution with the volume of 1.5 times, shaking and mixing uniformly for 15s, wherein flocculent precipitates appear in a centrifugal tube; transferring the solution into an adsorption column CB3, centrifuging at 12000rpm for 1min, and pouring off waste liquid; adding 600 μ L of rinsing solution PW into the adsorption column, centrifuging at 12000rpm for 1min, pouring off waste liquid, and repeating twice; centrifuging at 12000rpm for 2min, pouring out waste liquid, and placing the adsorption column at room temperature until the residual rinsing liquid is dried;
(3) transferring the adsorption column into a new 1.5mL centrifuge tube, suspending, dripping 50 μ L of elution buffer TE, standing at room temperature for 5min, centrifuging at 12000rpm for 2min, detecting the concentration of the genomic DNA by using an enzyme-labeling instrument, and storing in a refrigerator at-30 ℃.
Preliminary positioning of a QTL candidate interval of flowering Chinese cabbage:
(1) constructing 3 mixed pools of multi-branch phenotypes (12-15 branches), few-branch phenotypes (1-3 branches) and intermediate-branch phenotypes (7-8 branches), performing Graded pool-Seq sequencing on the three mixed pools and two parents, breaking a DNA sample into fragments with the length of 350bp by a fragmentation kit, and constructing a library. The sequencing platform was NovaSeq 6000(Illumina) and the sequencing mode was PE 150.
(2) The original data are subjected to quality control, reads are compared to a Chinese cabbage V3.0 reference genome (http:// branched. org/read/datasets/pub/genome/Brassica _ rapa/V3.0/) by using BWA, the comparison result is subjected to SAMTOOLS to remove repetition, the mutation site is analyzed according to the analysis process of GATK to obtain SNP and InDel information, and the structure annotation is carried out on the mutation site by using SNPeff.
(3) And after Ridit test and analysis, carrying out noise reduction on the data, and counting the proportion of the sites with the significant p value in the total sites of the sliding window area, thereby judging the area significantly associated with the characters. The noise reduction parameter is a window size of 0.2Mb, and the threshold value of the p value is 10 -8 . And finally, positioning an interval of 28.0-28.9 Mb region of the A07 chromosome as a candidate QTL interval, and positioning genes for regulating and controlling branching on the A07 chromosome (figure 4).
And uniformly selecting SNP sites with no other variation before and after 100bp selection in the candidate interval according to the SNP information, and then designing a Primer by utilizing Primer 5 software to convert the SNP sites into KASP markers.
(1) KASP label of a 0716: forward primers (5 ' -3 ') SEQ ID NO.1(GAAGGTGACCAAGTTCATGCTCCAACACAACCAGCCCTAT) and SEQ ID NO.2(GAAGGTCGGAGTCAACGGATTCCAACACAACCAGCCCTAC) were ligated to the 5 ' ends with FAM and VIC linker (adaptor) sequences, respectively, as their KASP Forward primers to detect polymorphisms in the major QBR TL1 close-linked marker, the single nucleotide marker (SNP) of A0716, respectively. The sequence of the common reverse primer (Reveser primer,5 '-3') is SEQ ID NO.3 (AACGTATTAAATATTGGAATTGAA).
(2) KASP label of a 0717: forward primers (5 ' -3 ') SEQ ID No.4(GAAGGTGACCAAGTTCATGCTGATGTGCTTATTTATGTCCAC) and SEQ ID No.5(GAAGGTCGGAGTCAACGGATTGATGTGCTTATTTATGTCCAA) were ligated to the 5 ' ends with FAM and VIC linker (adaptor) sequences, respectively, as their KASP Forward primers to detect polymorphisms in the major QBR TL1 close-linked marker, the single nucleotide marker (SNP) of A0716, respectively. The sequence of the common Reverse primer (Reverse primer,5 '-3') is SEQ ID NO.6 (AAAACAACAAAAACATCCCCGCTCCG).
Genetic map construction and QTL mapping:
PCR reaction (1.6. mu.L): DNA 0.8. mu.L, 2 XKASP Master mix + Assay 0.8. mu.L.
PCR reaction procedure: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, extension at 61-55 ℃ for 60s, and reduction of 0.6 ℃ per cycle for 10 cycles; denaturation at 94 ℃ for 20s and elongation at 55 ℃ for 60s for 26 cycles.
Genotyping based on KASP technique using IntelliQube genotyping detection platform (LGC), and F is judged from fluorescence signal of amplification product 2 The genotype of the plant.
Using QTL IciMapping 4.2 software to F 2 Linkage analysis of the population KASP marker genotype and phenotype data to construct a genetic linkage map of molecular markers near the target site on chromosome A07.
Based on the genetic map, F 2 Genotype data and branch phenotype data for a populationQTL detection is carried out by using QTL IciMapping 4.2 software, and a main QTL locus is detected (table 1, figure 5).
TABLE 1 basic information of major QTL sites for branching on chromosome A07
The KASP molecular marker of the flowering cabbage main effect QTL and the application thereof are obtained by developing the close linkage marker of the flowering cabbage main effect QTL, and the KASP molecular marker primer can identify the flowering cabbage genotype of filial generations and can meet the requirement of molecular breeding.
Sequence listing
Sequence listing
While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the disclosed embodiments of the invention may be used in any combination, provided that no structural conflict exists, and the combinations are not exhaustively described in this specification merely for the sake of brevity and resource conservation. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (2)
1. KASP molecular marker of flowering Chinese cabbage character major QTL and application thereof are characterized in that: the primer group consists of a KASP molecular marker primer of A0716 and a KASP molecular marker primer of A0717:
the KASP molecular marker primer sequence of A0716 is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;
the KASP molecular marker primer sequence of A0717 is shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
2. The application of the molecular marker primer group of the flowering cabbage character major QTL in claim 1 in multi-branch flowering cabbage breeding.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117344045A (en) * | 2023-09-13 | 2024-01-05 | 广东省农业科学院蔬菜研究所 | Cabbage core KASP molecular marker developed based on whole genome resequencing and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102144560A (en) * | 2011-02-22 | 2011-08-10 | 沈阳农业大学 | Method and application method for obtaining novel germ plasm of brassica A genome vegetable |
| CN109182592A (en) * | 2018-11-08 | 2019-01-11 | 中国农业科学院油料作物研究所 | Chain SNP marker and application with rape multi-branched character main effect QTL site |
| CN114164294A (en) * | 2021-12-08 | 2022-03-11 | 沈阳农业大学 | SNP site related to green-keeping property of Chinese cabbage and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102144560A (en) * | 2011-02-22 | 2011-08-10 | 沈阳农业大学 | Method and application method for obtaining novel germ plasm of brassica A genome vegetable |
| CN109182592A (en) * | 2018-11-08 | 2019-01-11 | 中国农业科学院油料作物研究所 | Chain SNP marker and application with rape multi-branched character main effect QTL site |
| CN114164294A (en) * | 2021-12-08 | 2022-03-11 | 沈阳农业大学 | SNP site related to green-keeping property of Chinese cabbage and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117344045A (en) * | 2023-09-13 | 2024-01-05 | 广东省农业科学院蔬菜研究所 | Cabbage core KASP molecular marker developed based on whole genome resequencing and application thereof |
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