CN112593005B - Molecular marker of leaf rolling site on brassica napus and application thereof - Google Patents
Molecular marker of leaf rolling site on brassica napus and application thereof Download PDFInfo
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Abstract
The invention relates to the field of plant breeding, in particular to a molecular marker of a leaf rolling locus of a cabbage type rape and application thereof, the invention firstly identifies the leaf rolling locus of the cabbage type rape controlled in a 6126244bp-6833084bp region of a cabbage type rape A02 chromosome, and simultaneously discovers codominant/dominant molecular markers BnA02V099, bnA02INDEL1 and BnA02V573 which are closely linked with the leaf rolling locus, the invention also discloses primer pairs for amplifying the molecular markers BnA02V099, bnA02INDEL1 and BnA02V573, and the molecular markers are F02V 099, bnA02INDEL1 and BnA02V573 2 And F 2:3 The expression of the population makes the population have very important value in the leaf rolling breeding and detection of the brassica napus.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a molecular marker of leaf rolling sites on brassica napus and application thereof.
Background
Brassica napus is one of the most important oil crops in the world, and can be used as feed, industrial production raw materials and the like while providing edible oil for human beings (Allender et al, 2010). Optimizing the plant type of the cabbage type rape and improving the photosynthetic efficiency of the cabbage type rape are important ways for improving the yield, and the cultivation of the rape variety with high photosynthetic efficiency plant type needs proper rape germplasm resources and develops corresponding molecular markers.
The leaf type regulation mechanism of rape is relatively complex, and most of rape is controlled by multiple genes. Through artificial EMS mutagenesis and natural mutation, a plurality of short-stalk mutants are obtained and subjected to genetic research. The breeder obtained a single dominant site-controlled rolling leaf mutant, bndcl1, by EMS mutagenesis, mapped the control site on the C05 chromosome by linkage mapping, and analyzed the mechanism of rolling leaf formation using iTRAQ-based comparative proteomics (Wang et al, 2016 chen et al, 2018. "dwarf source No. 1" is a dwarf variety introduced by the farm college of Jiangsu province from Pacific seeds, australia, which also exhibits a subtenon phenotype during the seedling stage (Puhuimin et al, 1995). Qugaping et al obtained an upper leaf curl rape Bnucl1 by EMS mutagenesis of No.9 in brassica napus variety (Qugaping et al, 2014). Yang et al (2019) and Huang et al (2020) mapped the control sites of the rolling leaf mutant Bnuc1 and Bnuc2 on Brassica napus in different segments on the A05 chromosome.
Although breeders make some positive researches on the heredity and breeding of the leaf rolling traits, related breeding works have not made a breakthrough, and new major loci and molecular markers thereof for controlling the leaf rolling traits of the rapes need to be continuously developed to provide a basis for the breeding works. The closely linked molecular markers of the new major locus of rape leaf roll are developed, and the selection efficiency can be improved in the separation generation. Therefore, the development and application of molecular markers closely linked with the leaf rolling sites of rape are key technologies for applying the leaf rolling of rape to breeding.
Disclosure of Invention
The invention aims to provide a molecular marker of a leaf rolling locus on a cabbage type rape and application thereof, finds a new locus for controlling the leaf rolling character on the cabbage type rape, develops a molecular marker technology closely linked with the locus, and can be used for detecting the leaf rolling rape and breeding a variety of the leaf rolling rape.
In order to realize the aim, the invention provides a leaf rolling locus BnUC3 of the Brassica napus, wherein the BnUC3 locus is 6126244bp-7833204bp on the chromosome A02 of the Brassica napus. The reference genomic sequence (Brassica napus A02 chromosomal sequence) is found in the NCBI database (GenBank: LT 220456.1).
The invention also provides a molecular marker of a leaf rolling locus BnUC3 on the brassica napus, which comprises 3 molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 which are closely linked, wherein the molecular marker BnA02V099 is positioned on a chromosome 6274194bp-6174384bp of the brassica napus A02; the molecular marker BnA02V573 is positioned on a brassica napus A02 chromosome 6907939bp-6908155bp; the molecular marker BnA02INDEL1 is positioned on a cabbage type rape A02 chromosome 6406298bp-6407901bp; the nucleotide sequences of the 3 closely linked molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
The invention also provides a primer pair of molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 for amplifying the leaf rolling position BnUC3 of the cabbage type rape, wherein the primer pair for amplifying the molecular marker BnA02V099 is Seq V099-F and Seq V099-R, and the sequences of the primer pairs are respectively shown as SEQ ID No.4 and SEQ ID No. 5; the primer pair for amplifying the molecular marker BnA02V573 is Seq V573-F and Seq V573-R, and the sequences of the primer pair are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7; the primer pair for amplifying the molecular marker BnA02INDEL1 is Seq INDEL1-F and Seq INDEL1-R, and the sequences of the primer pair are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.
The invention also provides application of molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 of the cabbage type rape rolling leaf site BnUC3 in detection of cabbage type rape varieties or germplasm containing the cabbage type rape rolling leaf site BnUC 3.
The invention also provides application of molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 of the leaf rolling locus BnUC3 of the brassica napus in breeding of leaf rolling rape varieties or germplasm.
The invention also provides a method for breeding a cabbage rape variety by using the molecular marker or the primer pair, wherein the primer pair corresponding to BnA02V099, bnA02V573 and BnA02INDEL1 is adopted to amplify the genome DNA of the rape, and after electrophoresis of an amplification product on 40% polyacrylamide gel or 1% agarose gel, if an amplification fragment of NJAU-M1295 is obtained, the existence of the cabbage rape leaf rolling site is indicated, and the rape is predicted to be the cabbage rape. If the amplified fragment of the parent NJAU-M3756 is obtained, the existence of the normal leaf locus of the brassica napus is indicated, and the brassica napus is predicted to be the normal flat leaf rape.
The molecular marker linked with the leaf rolling site on the cabbage type rape is screened by the following steps:
(1) A rolling leaf mutant strain is found in the breeding process, and a stable rolling leaf material NJAU-M1295 is obtained through continuous selfing; respectively hybridizing NJAU-M1295 with excellent germplasm resources NJAU-M3756 and Zhongshuang 11 of normal leaves to obtain a hybrid F 1 Two of F 1 Backcrossing respectively with recurrent parent NJAU-M3756 and Zhongshu 11 to obtain BC 1 Group, F 1 Selfing to obtain F 2 Group, F 2 (NJAU-M3756 XNJAU-M1295) population derivation to obtain 2F 2:3 And (4) a group.
(2)F 1 All exhibit intermediate phenotypes, for F 2 And F 2:3 The generation population counts phenotypes and is subjected to chi-square test, which shows that the proportions of the leaf rolling character, the intermediate character and the flat leaf character accord with a Mendelian segregation ratio of 1.
(3) BC obtained by chip detection 1 (ZS 11 x (ZS 11 XNJAU-M1295)) in the group, the SNP marker typing results of 37 individuals are subjected to mapping analysis together with the phenotype data, and the BnUC3 locus controlling the leaf rolling character of the brassica napus is mapped to a 6126244bp-7833204bp segment on the A02 chromosome. SNP marker sites in the positioning interval are all free of polymorphism, and the population change of subsequent marker development and positioning is the offspring of NJAU-M3756 and NJAU-M1295 hybridization.
(4) The SSR information of rape A02 chromosome 6126244bp-7833204bp is utilized to design a molecular marker. Molecular markers were designed using INDEL sites obtained by genomic sequence sequencing and alignment.
(5) Using "NJAU-M3756 XNJAU-M1295' produced F 2 Population, identifying the genetic banding pattern of the newly designed molecular marker.
(6) According to F 2 And F 2:3 The phenotype data and the molecular marker data of 584 individuals in the population are total, and the closely linked codominant/dominant molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 are found, and the genetic banding patterns of the markers are clearly visible.
Compared with the prior art, the invention has the beneficial effects that:
1) The locus for controlling the rolling leaves of the brassica napus is firstly identified in the 6126244bp-7833204bp region of the brassica napus A02 chromosome, and the codominant/dominant molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 which are closely linked with the rolling leaf locus are simultaneously found;
2) At F 2 And F 2:3 In the population, the leaf rolling character of the cabbage type rape is very obviously related to the 3 molecular markers, F 2 And F 2:3 The genotype and the phenotype of a single plant are consistent, so the 3 molecular markers have great application prospects in the auxiliary selective breeding of the leaf-rolling rape in the future;
3) The invention can find the pure leaf-rolling rape in the cabbage type rape and is greatly helpful for breeding the leaf-rolling plant type.
Drawings
FIG. 1: rolling leaf rape surface type graph in seedling stage; the left side is a rape phenotype map of normal flat leaves, the middle is a rape phenotype map with intermediate characters, and the right side is an upper rolling leaf rape phenotype map;
FIG. 2: the molecular marker BnA02V099 is used for genotyping part of single-plant genes, wherein lanes 1 and 11 are homozygous leaf curl band types; 2. 5, 6, 7, 8, 9, 10 and 12 are of hybrid upper leaf curl belt type; lanes 3 and 4 are homozygous flat leaf banding;
FIG. 3: the molecular marker BnA02V573 is used for genotyping part of single strains, wherein lanes 1, 2, 3, 6, 8, 10, 11 and 12 are hybrid rolling leaves or homozygous rolling leaf bands; lanes 4, 5, 7 and 9 are homozygous flat leaf banding;
FIG. 4 is a schematic view of: the molecular marker BnA02INDEL1 is used for genotyping part of single-plant genes, wherein lanes 1, 4, 7 and 11 are homozygous leaf curl band types; lanes 3, 6, 8, 9 and 12 are heterozygous, homozygous, flat leaf band, lanes 2, 5 and 10.
Detailed Description
The invention is further described in the following with reference to the accompanying drawings. The experimental methods in the following implementation methods are all conventional methods, and the experimental materials are all conventional biochemical reagents.
Example 1: obtaining leaf rolling sites on brassica napus
(1) Genetic population construction
Hybridizing two cabbage type rape materials NJAU-M1295 with the double 11 of the cabbage type rape varieties to obtain a hybrid F 1 ，F 1 Backcrossing with Zhongshui 11 to obtain BC 1 Group, F 1 Selfing to obtain F 2 And (4) a group.
(2) Brassica napus BC 1 Group phenotype assay
For the above BC 1 And (4) carrying out phenotypic observation and agronomic character investigation on the single plants in the single plant population.
(3) Construction of genetic maps
Selecting BC 1 37 individual leaf DNA samples from the population were used for data acquisition of SNP markers. The SNP chip has 52157 sites in total, but the BC is 1 DNA samples of the population are not polymorphic at all sites and correlation analysis is performed after removal of the null markers.
(4) Results and analysis
The phenotype data of 37 DNA samples used for the SNP chip and the SNP typing data thereof are positioned and analyzed together, the BnUC3 locus controlling the leaf rolling character of the cabbage type rape is positioned to an A02 chromosome, and the positioning interval is positioned in a 6126244bp-7833204bp section.
Example 2: obtaining of closely linked molecular markers of leaf rolling sites on brassica napus
(1) Molecular marker development
The research utilizes an SNP chip technology to position the upper rolling leaf locus in a 6126244bp-7833204bp section of a rape A02 chromosome, downloads a reference genome sequence (http:// www. Genoscope. Cns. Fr/branched/data /) of a rape variety Darmor, utilizes SSR hunter 1.3 software to search for an SSR locus, 150bp design primers are respectively added at the upstream and the downstream of the SSR locus, and the SSR marker locus is named as a 'BnA 02V + SSR locus'. The INDEL locus discovered by sequencing the genome sequence of the important gene in the positioning interval is used for developing a molecular marker; a plurality of molecular markers are designed in a BnaA02:6126244bp-7833204bp segment by using Primer Premier 5.0 software. SNP marker sites in the positioning interval are all free of polymorphism, and the population used for the subsequent marker screening is changed into offspring obtained by crossing NJAU-M3756 and NJAU-M1295.
(2) Molecular marker identification
Extraction of F by CTAB method 2 (NJAU-M3756 XNJAU-M1295) genomic DNA of Brassica napus leaves of the population, PCR reaction System (10. Mu.l) containing 0.5. Mu.l of DNA template, 0.25. Mu.l each of upstream and downstream primers (1 mmol/L), 5. Mu.l of Mix, and 4. Mu.l of ddH 2 And (O). PCR reaction procedure: denaturation at 95 deg.C for 5min; followed by 35 cycles of denaturation at 95 ℃ for 30s, tm value annealing for 30s, and extension at 72 ℃ for 30s; extending for 10min at 72 ℃; finally, storing at 4 ℃. The PCR amplification product was electrophoresed with 40% polyacrylamide gel or 1% agarose. The films were scanned for analysis on a BIO-RAD visadoc3.0 (Bio-RAD, USA) imaging system.
(3) Results and analysis
Among the designed multiple molecular markers, the molecular markers BnA02V099, bnA02V573 and BnA02INDEL1 have the phenotype consistency of 99 percent, and the 3 molecular markers are considered to be closely linked with the leaf rolling locus on the cabbage type rape. At F 2 The 3 closely linked molecular markers in the population have 2 or 3 banding patterns, bnA02V099 is a codominant marker, the sizes of the banding patterns are 191bp and 206bp respectively, and the banding patterns have two banding patterns simultaneously, a single plant with the 191bp banding pattern is a homozygous rolling leaf single plant, a single plant with the 206bp banding pattern is a homozygous flat leaf rape, and a single plant with the 191bp and 206bp banding patterns is a rolling leaf heterozygote; bnA02V573 is a dominant marker, the sizes of bands are 217bp and no band, a single plant with the band of 217bp is a homozygous curled leaf single plant or a curled leaf heterozygote, and a single plant without the band type is flat leaf rape. The BnA02INDEL1 is a co-dominant marker, the size of the bands is 1604bp,198bp and has two bands simultaneously, the single plant with 198bp bands is a homozygous rolling leaf single plant and has a homozygous rolling leaf single plantThe single strain with 1604bp band is flat leaf rape, and the hybrid with 1604bp and 198bp bands is rolling leaf. The upstream primer sequences of the 3 closely linked molecular markers are Seq V099-F, seq V573-F and Seq INDEL1-F, respectively, and the downstream primer sequences are Seq V099-R, seq V573-R and Seq INDEL1-R, respectively.
Example 3: application of closely linked molecular markers in selection of brassica napus
(1) Parental genome amplification detection
Used for verifying that the rape parent of the leaf curl is 'NJAU-M1295' (the BnA02V099 allelic band is 191bp, the BnA02V573 allelic band is 217bp, the BnA02INDEL1 allelic band is 198 bp), the parent 'NJAU-M3756' (the BnA02V099 allelic band is 206bp, the BnA02V573 is no allelic band; the BnA02INDEL1 allelic band is 1604 bp.
(2) Population expansion detection and marker analysis
Hybridizing the above two parents to obtain F 1 Growing into an F after planting 1 Generation individual plant, selfing and fruiting to obtain F 2 Generation seed grown to F comprising segregation trait 2 The population, in which individual plants are selected, selfed to give F 2:3 Separating the population, and determining the agronomic characters and phenotypes of individual plants of the population.
Respectively extracting F by CTAB method 2 And F 2:3 Genomic DNA of leaves of individual plants of the population. PCR reaction (10. Mu.l) containing 0.5. Mu.l of DNA template, 0.25. Mu.l each of upstream and downstream primers (1 mmol/L), 5. Mu.l of Mix, and 4. Mu.l of ddH2O. PCR reaction procedure: denaturation at 95 deg.C for 5min; followed by 35 cycles of denaturation at 95 ℃ for 30s, tm annealing for 30s, and extension at 72 ℃ for 30s; extending for 10min at 72 ℃; finally, the mixture is stored at 4 ℃. The PCR amplification product was electrophoresed with 40% polyacrylamide gel or 1% agarose gel. The films were scanned for analysis on a BIO-RAD visadoc3.0 (Bio-RAD, USA) imaging system. Band types of BnA02V099, bnA02INDEL1 and BnA02V573 in parents were analyzed.
(3) Results and analysis
Progeny F of a cross combination between NJAU-M1295 and NJAU-M3756 2 And F 2:3 In the detection, bnA02V099, bnA02INDEL1 and BnA02 are foundThe V573 molecular marker has 99% consistency with the phenotype. The results show that the molecular markers BnA02V099, bnA02INDEL1 and BnA02V573 have better prediction effect on the leaf rolling of rape.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
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Molecular marker of leaf rolling locus on brassica napus and application thereof
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Claims (5)
1. Leaf rolling site of cabbage type rapeBnUC3Closely linked molecular markers, wherein the nucleotide sequences of the molecular markers BnA02V099 and BnA02V573, bnA02V099 and BnA02V573 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. For amplifying the leaf rolling site of Brassica napus according to claim 1BnUC3The primer pair of the closely linked molecular markers BnA02V099 and BnA02V573 is characterized in that the primer pair for amplifying the molecular marker BnA02V099 is Seq V099-F and Seq V099-R, and the sequences of the primer pair are respectively shown as SEQ ID No.4 and SEQ ID No. 5; the primer pair for amplifying the molecular marker BnA02V573 is Seq V573-F and Seq V573-R, and the sequences are shown as SEQ ID NO.6 and SEQ ID NO.7 respectively.
3. The primer pair of claim 2, wherein the primer pair is a leaf rolling site on Brassica napusBnUC3The application in detecting cabbage type rape varieties or germplasm.
4. Use of the primer pair of claim 2 for breeding a brassica oleracea variety or germplasm.
5. A method for selectively breeding a rape variety with a leaf curl by using the primer pair as claimed in claim 2, characterized in that the genomic DNA of rape is amplified by using the primer pairs corresponding to BnA02V099 and BnA02V573, and whether the rape is of a leaf curl phenotype or not is judged according to the obtained amplified fragment, and whether the rape is a homozygous strain or not can be judged.
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| CN116622874B (en) * | 2022-10-21 | 2024-04-12 | 南京农业大学 | Molecular marker of low-temperature male sterile locus of brassica napus, primer pair and application of molecular marker |
| CN115948599B (en) * | 2022-12-29 | 2023-09-08 | 中国农业科学院油料作物研究所 | InDel molecular marker of RAD17 gene, detection primer and application thereof |
| CN116716425B (en) * | 2023-01-16 | 2023-11-28 | 南京农业大学 | Molecular marker of drooping character site of cabbage type rape leaf-rolling/pod and application thereof |
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| CN108239647A (en) * | 2017-11-29 | 2018-07-03 | 华中农业大学 | A kind of gene, molecular labeling and application for controlling rape plant type |
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| CN108239647A (en) * | 2017-11-29 | 2018-07-03 | 华中农业大学 | A kind of gene, molecular labeling and application for controlling rape plant type |
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