CN112593005A - 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 on brassica napus and application thereof, the invention firstly identifies the leaf rolling locus on brassica napus in a 6126244bp-6833084bp region of a chromosome of brassica napus A02 and simultaneously discovers co-dominant/dominant molecular markers BnA02V099, BnA02INDEL1 and BnA02V573 closely linked with the leaf rolling locus, the invention also discloses a primer pair for amplifying the molecular markers BnA02V099, BnA02INDEL1 and BnA02V573, and the molecular markers are F36V 099, BnA INDEL1 and BnA V5732And F2:3The 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 single dominant site-controlled rolling leaf mutant Bndcl1 was obtained by EMS mutagenesis by the breeder, the control site was mapped to chromosome C05 by linkage mapping, and the mechanism of rolling leaf formation was analyzed using an itraQ-based comparative proteome (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 a 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 to different segments on the A05 chromosome.
Although breeders have made some positive researches on the inheritance of leaf rolling traits and breeding, related breeding works have not made a breakthrough, and new major sites and molecular markers thereof for controlling the leaf rolling traits of rape need to be continuously developed to provide a basis for the breeding works. The close linkage molecular marker of the new main effective site of rape leaf roll is 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 achieve the aim, the invention provides a leaf rolling locus BnUC3 of Brassica napus, wherein the BnUC3 locus is 6126244bp-7833204bp on the chromosome of Brassica napus A02. 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 closely linked molecular markers BnA02V099, BnA02V573 and BnA02INDEL1, wherein the molecular marker BnA02V099 is positioned on 6274194bp-6174384bp on a chromosome of the brassica napus A02; the molecular marker BnA02V573 is located on chromosome 6907939bp-6908155bp of Brassica napus A02; the molecular marker BnA02INDEL1 is located on the chromosome 6406298bp-6407901bp of Brassica napus A02; the nucleotide sequences of the 3 closely linked molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.
The invention also provides a primer pair of molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 for amplifying the leaf rolling position BnUC3 of the brassica napus, 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 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 are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.
The invention also provides application of the molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 of the leaf rolling locus BnUC3 on the brassica napus in detection of brassica napus varieties or germplasm containing the leaf rolling locus BnUC3 on the brassica napus.
The invention also provides application of the molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 of the leaf rolling locus BnUC3 on the brassica napus in breeding varieties or germplasm of the leaf rolling rape.
The invention also provides a method for breeding a rape variety with the molecular marker or the primer pair, wherein the primer pair corresponding to BnA02V099, BnA02V573 and BnA02INDEL1 is adopted to amplify rape genome DNA, 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 locus is indicated, and the rape is predicted to be the rape leaf rolling. 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 F1Two of F1Backcrossing the first generation of the BC with recurrent parent NJAU-M3756 and Zhongshui 11 respectively to obtain the BC1Group, F1Selfing to obtain F2Group, F2(NJAU-M3756 XNJAU-M1295) population derivation to obtain 2F2:3And (4) a group.
(2)F1All exhibit intermediate phenotype, for F2And F2:3The generation group counts phenotypes and is subjected to chi-square test, which shows that the ratio of the leaf rolling character, the intermediate character and the flat leaf character accords with the Mendelian segregation ratio of 1:2:1, backcross generations of the hybridization combination show the same genetic rule, and the genetic control of the leaf rolling character by a single dominant gene is demonstrated.
(3) BC obtained by chip detection1(ZS11 × (ZS11 × NJAU-M1295)) 37 individuals in the population were assigned SNP markersType results, together with their phenotypic data, were subjected to mapping analysis, and the BnUC3 locus controlling the leaf rolling trait in Brassica napus was mapped to the 6126244bp-7833204bp segment on chromosome A02. 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 the 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) F produced by using "NJAU-M3756 XNJAU-M12952Population, identifying the genetic banding pattern of the newly designed molecular marker.
(6) According to F2And F2:3The phenotype data and the molecular marker data of 584 individuals in the population are total, closely linked codominant/dominant molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 are found, and the genetic banding pattern is 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 a region of 6126244bp-7833204bp of a chromosome of the brassica napus A02, and the codominant/dominant molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 which are closely linked with the rolling leaf locus are simultaneously discovered;
2) at F2And F2:3In the population, the leaf rolling character of the cabbage type rape is very obviously related to the 3 molecular markers, F2And F2:3The genotype and the phenotype of a single plant are consistent, so that 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 part is an intermediate character rape phenotype map, and the right side is an upper rolling leaf rape phenotype map;
FIG. 2: molecular marker BnA02V099 was genotyped on a portion of the individual strains, where lanes 1 and 11 are homozygous leaf-rolled banding; 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: molecular marker BnA02V573 is used for genotyping part of the individual, wherein lanes 1, 2, 3, 6, 8, 10, 11 and 12 are homozygous leaf-curl or homozygous leaf-curl banding; lanes 4, 5, 7 and 9 are homozygous flat leaf banding;
FIG. 4: molecular marker BnA02INDEL1 genotyping a portion of the individual, lanes 1, 4, 7 and 11 are homozygous leaf-rolled banding patterns; lanes 3, 6, 8, 9 and 12 are homozygous closed leaf band types, and lanes 2, 5 and 10 are homozygous flat leaf band types.
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 F1，F1Backcrossing with Zhongshui 11 to obtain BC1Group, F1Selfing to obtain F2And (4) a group.
(2) Brassica napus BC1Group phenotype assay
For the above BC1And (4) carrying out phenotypic observation and agronomic character investigation on the single plants in the single plant population.
(3) Construction of genetic maps
Selecting BC137 individual leaf DNA samples from the population were used for SNP marker data acquisition. The SNP chip has 52157 sites in total, but the BC is1DNA 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 are subjected to positioning analysis together, the BnUC3 locus controlling the leaf rolling character of the cabbage type rape is positioned to the A02 chromosome, and the positioning interval is positioned in a segment of 6126244bp-7833204 bp.
Example 2: obtaining of molecular marker closely linked to leaf rolling site on cabbage type rape
(1) Molecular marker development
The research utilizes the SNP chip technology to position the upper rolling leaf locus in the 6126244bp-7833204bp section of the chromosome of rape A02, downloads the reference genome sequence (http:// www. genoscope. cns. fr/branched/data /) of the rape variety Darmor, utilizes SSR hunter 1.3 software to search the 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 an SSR BnA02V + 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 number of molecular markers were designed within the BnaA02:6126244bp-7833204bp segment 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 of NJAU-M3756 and NJAU-M1295 hybridization.
(2) Molecular marker identification
Extraction of F by CTAB method2(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 (1mmol/L), 5. mu.l of Mix, and 4. mu.l of ddH2And O. PCR reaction procedure: denaturation at 95 deg.C for 5 min; followed by 35 cycles of denaturation at 95 ℃ for 30s, annealing at Tm for 30s, and extension at 72 ℃ for 30 s; 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. Films were scanned for analysis in a BIO-RAD visadoc3.0(Bio-RAD, USA) imaging system.
(3) Results and analysis
Among the designed multiple molecular markers, BnA02V099, BnA02V573 and BnA02INDEL1 molecular markers have 99% of phenotype consistency, and the 3 molecular markers are considered to be closely linked with the leaf rolling locus on the brassica napus. At F2The 3 closely linked molecular markers in the population have 2 or 3 banding patterns, BnA02V099 is a codominant marker,the sizes of the bands are 191bp and 206bp respectively, and the bands have two bands simultaneously, a single plant with the 191bp band is a homozygous rolling leaf single plant, a single plant with the 206bp band is a homozygous flat leaf rape, and a single plant with the 191bp and 206bp bands is a rolling leaf heterozygote; BnA02V573 is a dominant marker, the sizes of the bands are 217bp and no band, the single plant with 217bp band is homozygous curled leaf single plant or curled leaf heterozygote, and the single plant without band is flat leaf rape. BnA02INDEL1 is co-dominant marker, the bands are 1604bp, 198bp and have two bands at the same time, the single plant with 198bp band is homozygous rolled leaf single plant, the single plant with 1604bp band is flat leaf rape, and the single plant with 1604bp and 198bp bands is rolled leaf heterozygote. 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
The method is used for verifying the rape parent NJAU-M1295 (the allelic band of BnA02V099 is 191bp, the allelic band of BnA02V573 is 217bp, the allelic band of BnA02INDEL1 is 198bp) and the parent NJAU-M3756 (the allelic band of BnA02V099 is 206bp, the allelic band of BnA02V573 is not present, and the allelic band of BnA02INDEL1 is 1604 bp).
(2) Population expansion detection and marker analysis
Hybridizing the two parents to obtain F1Growing into an F after planting1Generation individual plant, selfing, fruiting and harvesting to obtain F2Generation seed grown to F comprising segregation trait2The population, in which individual plants are selected, selfed to give F2:3Separating the population, and determining the agronomic characters and phenotypes of individual plants of the population.
Respectively extracting F by CTAB method2And F2:3Genomic 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 (1mmol/L), 5. mu.l Mix, and 4. mu.l ddH 2O. PCR reaction procedure: denaturation at 95 ℃ for 5min; followed by 35 cycles of denaturation at 95 ℃ for 30s, annealing at Tm for 30s, and extension at 72 ℃ for 30 s; 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. Films were scanned for analysis in a BIO-RAD visadoc3.0(Bio-RAD, USA) imaging system. The types of bands in the parents were analyzed for BnA02V099, BnA02INDEL1, and BnA02V 573.
(3) Results and analysis
Progeny F of a cross combination between NJAU-M1295 and NJAU-M37562And F2:3In the detection, the molecular markers BnA02V099, BnA02INDEL1 and BnA02V573 are found to have 99% of phenotype consistency. The results show that molecular markers BnA02V099, BnA02INDEL1 and BnA02V573 are used for predicting the better prediction effect of leaf rolling on 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 (7)
1. The cabbage type rape upper leaf rolling site BnUC3 is characterized in that the BnUC3 site is located on the chromosome 6126244bp-7833204bp of the cabbage type rape A02.
2. The molecular marker of the rolling leaf locus BnUC3 on the brassica napus is characterized by comprising 3 closely linked molecular markers BnA02V099, BnA02V573 and BnA02INDEL1, wherein the molecular marker BnA02V099 is positioned on 6274194bp-6174384bp on the chromosome of the brassica napus A02; the molecular marker BnA02V573 is located on chromosome 6907939bp-6908155bp of Brassica napus A02; the molecular marker BnA02INDEL1 is located on the chromosome 6406298bp-6407901bp of Brassica napus A02; the nucleotide sequences of the 3 closely linked molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.
3. Primer pairs of molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 for amplifying leaf rolling sites BnUC3 of the brassica napus are characterized in that the primer pairs for amplifying the molecular markers BnA02V099 are 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 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 are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.
4. Application of molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 of a cabbage type rape rolling leaf site BnUC3 in detection of cabbage type rape varieties or germplasm containing a cabbage type rape rolling leaf site BnUC 3.
5. Application of molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 of leaf rolling locus BnUC3 on cabbage type rape in breeding of leaf rolling rape varieties or germplasm.
6. A method for selectively breeding a rape variety with a rolling leaf by using the molecular marker of claim 2 or the primer pair of claim 3, wherein the genomic DNA of rape is amplified by using the primer pairs corresponding to BnA02V099, BnA02V573 and BnA02INDEL1, and whether the rape variety is homozygous is judged by judging whether the rape is of the rolling leaf phenotype or not based on the obtained amplified fragments.
7. The screening method of the molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 of the rolling leaf site BnUC3 on the brassica napus is characterized by comprising the following steps:
respectively hybridizing the mutant strain NJAU-M1295 of the upper rolling leaf with excellent germplasm resources NJAU-M3756 and Zhongshuang 11 of a normal leaf to obtain a hybrid F1Two of F1Backcrossing the first generation of the BC with recurrent parent NJAU-M3756 and Zhongshui 11 respectively to obtain the BC1Group, F1Selfing to obtain F2Group, F2(NJAU-M3756 XNJAU-M1295) population derivation to obtain 2F2:3A population;
F1all exhibit intermediate phenotypes, for BC1、F2And F2:3Counting phenotypes of generation groups, carrying out chi-square test, and judging whether the genetic transmission of the leaf rolling character is controlled by a single dominant gene;
BC obtained by chip detection1(ZS11 x (ZS11 XNJAU-M1295)) in the population, carrying out positioning analysis together with phenotype data on SNP marker typing results of a plurality of individuals, positioning the SNP marker typing results and the phenotype data, controlling a BnUC3 locus of leaf rolling characters on the cabbage type rape to be positioned in a 6126244bp-7833204bp segment on an A02 chromosome, wherein no polymorphism exists in SNP marker loci in a positioning interval, and the population used for later marker development and positioning is changed into a progeny of NJAU-M3756 and NJAU-M1295 hybridization;
designing a molecular marker by utilizing the SSR information of the chromosome 6126244bp-7833204bp of rape A02, and designing the molecular marker based on the INDEL locus obtained by sequencing and comparing the genome sequence;
f produced by using "NJAU-M3756 XNJAU-M12952Identifying the genetic banding pattern of the newly designed molecular marker;
according to F2And F2:3Group phenotype data and molecular marker data, closely linked co-dominant/dominant molecular markers BnA02V099, BnA02V573 and BnA02INDEL1 were found, and their genetic banding patterns were clearly visible.
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| CN116622874A (en) * | 2022-10-21 | 2023-08-22 | 南京农业大学 | Molecular marker of low-temperature male sterile locus of brassica napus, primer pair and application of molecular marker |
| 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 |
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| CN116716425A (en) * | 2023-01-16 | 2023-09-08 | 南京农业大学 | Molecular marker of drooping character site of cabbage type rape leaf-rolling/pod 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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