CN116987815A - InDel molecular marker for identifying leaf margin cracks of cabbage type rape and application thereof - Google Patents

InDel molecular marker for identifying leaf margin cracks of cabbage type rape and application thereof Download PDF

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CN116987815A
CN116987815A CN202311103991.1A CN202311103991A CN116987815A CN 116987815 A CN116987815 A CN 116987815A CN 202311103991 A CN202311103991 A CN 202311103991A CN 116987815 A CN116987815 A CN 116987815A
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braa10
type rape
cabbage type
leaf margin
margin
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王玉刚
孙云霞
范楚川
胡利民
王利辉
王显
马港丽
何宇
孙义飞
姜国红
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Shenyang Agricultural University
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Abstract

The invention discloses an InDel molecular marker for identifying cabbage type rape leaf margin crack and application thereof, belonging to the technical field of molecular genetics. The InDel molecular marker for identifying the leaf margin defect of the cabbage type rape has a nucleotide sequence shown as SEQ ID NO. 1 and a primer nucleotide sequence shown as SEQ ID NO. 2-4, and the leaf margin defect of the cabbage type rape can be rapidly and accurately distinguished according to an electrophoresis band by extracting genome DNA of the leaf of the cabbage type rape, performing PCR amplification by using the primer and then performing agarose gel electrophoresis. The InDel molecular marker can be used for auxiliary selection in the cotyledon period, so that the breeding efficiency of the cabbage type rape is effectively improved, and the breeding process is accelerated.

Description

InDel molecular marker for identifying leaf margin cracks of cabbage type rape and application thereof
Technical Field
The invention belongs to the technical field of molecular genetics, and particularly relates to an InDel molecular marker for identifying leaf margin lacerations of cabbage type rape and application thereof.
Background
Cabbage type rape (Brassica rapa l.) belongs to Brassica of the cruciferae family and is an important oil crop. Leaf dehiscence is an important agronomic trait that has a large variation in brassica crops. Split leaves are a unique morphological feature of crop improvement. Compared with round leaves, the leaf cutting under the same genetic background is easier to promote the convection of air and optimize the canopy structure, so that crops can be more suitable for a high-density planting mode, and researches show that the leaf cutting can improve the stress resistance of plants, including drought resistance and cold resistance.
At present, a molecular marker of a cabbage type rape leaf crack gene is not obtained yet, and cannot be applied to molecular marker assisted selective breeding. InDel has the following advantages over SSR markers: the quantity is large, and the distribution is wide; the method is easy to parting and convenient to detect in a rapid and large scale, so that molecular markers are designed aiming at InDel differences existing between leaf cracks and all-material, seedlings can be rapidly identified in a cotyledon period, the breeding efficiency can be effectively improved, and the breeding process is accelerated.
Disclosure of Invention
In view of the above, the invention aims to provide an InDel molecular marker for identifying the leaf margin crack of the cabbage type rape and application thereof, and the InDel molecular marker developed by the invention can be used for rapidly and accurately identifying the leaf margin crack character of the cabbage type rape in a cotyledon period, and the genotype of the leaf margin crack of the cabbage type rape can be economically, efficiently and accurately distinguished by agarose gel electrophoresis, so that the breeding efficiency of the cabbage type rape is effectively improved, and the breeding process is accelerated.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the first aspect of the invention provides an InDel molecular marker for identifying the leaf margin cleavage character of cabbage type rape, which has a nucleotide sequence of AAGGAGCTTCGTCTGCAGCCA (SEQ ID NO: 1).
The second aspect of the present invention provides a PCR amplification primer set of the above InDel molecular markers, comprising:
BraA10-F1：GCTGTCGAAGGAGCTACG(SEQ ID NO:2)；
BraA10-F2：GCTGTCGAAGGAGCTTCT(SEQ ID NO:3)；
BraA10-R：GACCCACAATAATGCCTCTC(SEQ ID NO:4)。
the third aspect of the invention provides a method for identifying the leaf margin cracking character of cabbage type rape, which comprises the following steps:
(1) Extracting genome DNA of cabbage type rape leaves;
(2) The PCR amplification is carried out by taking cabbage type rape genomic DNA as a template, adopting the BraA10-F1 and the BraA10-F2 to be respectively paired with the BraA 10-R;
(3) Performing agarose gel electrophoresis on the PCR amplification product obtained in the step (2);
(4) When only the combination of the BraA10-F1 and the BraA10-R is used for obtaining an amplification product, the leaf margin of the cabbage type rape plant is a crack, and the leaf margin of the self-bred seed is a crack; when only the combination of the BraA10-F2 and the BraA10-R is used for obtaining an amplification product, the leaf margin of the cabbage type rape plant is the whole margin, and the leaf margin of the self-bred seed is the whole margin; when BraA10-F1 and BraA10-R are combined and BraA10-F2 and BraA10-R are combined and amplified into a strip, the leaf margin of the cabbage type rape plant is split, and the leaf margin of the self-bred seed is split and full margin 3: 1.
Based on the technical scheme, further, the CTAB method is utilized to extract the genome DNA of the rape leaves in the step (1).
Based on the technical scheme, further, the PCR amplification reaction system in the step (2) is as follows: 1. Mu.L of genomic DNA, 5. Mu.L of 2 XTaq Master Mix, 0.5. Mu.L of upstream primer BraA10-F1/BraA10-F2, 0.5. Mu.L of downstream primer BraA10-R, ddH was added 2 O to 10. Mu.L.
Based on the above technical scheme, in the step (2), the concentration of the genome DNA is 50-200 ng/. Mu.L, the concentration of the upstream primer BraA10-F1/BraA10-F2 is 5-20 mu M, and the concentration of the 0.5 mu L downstream primer BraA10-R is 5-20 mu M.
Based on the above technical scheme, further, the PCR amplification reaction procedure in step (2) is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 30s,30 cycles; extending at 72℃for 5min.
Based on the technical scheme, in the step (3), the concentration of agarose is 1%, the voltage is 200V during electrophoresis, the current is 100mA, and the electrophoresis time is 8-10 min.
The fourth aspect of the invention provides the application of the InDel molecular marker and the PCR amplification primer pair of the InDel molecular marker in identifying the leaf margin laceration or auxiliary selective breeding of the cabbage type rape.
Compared with the prior art, the invention has the following beneficial effects:
(1) The InDel molecular marker developed by the invention is a dominant marker, and can generate 2 specific bands to distinguish 3 genotypes.
(2) The InDel molecular marker developed by the invention can be used for identifying the leaf margin cracking phenotype in advance in the cotyledon stage of the cabbage type rape.
(3) The identification method provided by the invention has the advantages of simplicity, rapidness, accuracy, low cost and the like, and can be applied to auxiliary selection breeding of cabbage type rape with high flux.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
Fig. 1: cabbage type rape parent RcBr, parent NIL and donor parent 08a061 leaf margin laceration phenotype, wherein a: split leaf parental phenotype, B: circular leaf parent phenotype, C: a donor parent phenotype.
Fig. 2: the comparison result of the differential sequences of the parent RcCr and the parent NIL of the cabbage type rape, wherein RCO_gene_3C: v3.0 reference genomic sequence, P1: circular leaf parent, P2: split leaf parent.
Fig. 3: the agarose gel electrophoresis result of the circular leaf parent (P1) and the split leaf parent (P2) is amplified by using a PCR amplification primer pair of a cabbage type rape leaf margin split gene InDel molecular marker BraA10-F1/BraA 10-R.
Fig. 4: the agarose gel electrophoresis result of the circular leaf parent (P1) and the split leaf parent (P2) is amplified by using a PCR amplification primer pair of a cabbage type rape leaf margin split gene InDel molecular marker BraA10-F2/BraA 10-R.
Fig. 5: the leaf margin cleavage gene InDel of the cabbage type rape is respectively paired with the molecular markers BraA10-F1/BraA0-F2 of the BraA10-R, and the amplification results are obtained on individuals of the round leaf parent (P1) and the split leaf parent (P2) and F2; wherein A: when amplified with BraA0-F1/BraA10-R, B: amplification was performed with BraA0-F2/BraA 10-R.
Detailed Description
The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.
Example 1:
development of InDel molecular marker for identifying leaf margin crack of cabbage type rape
According to the results of positioning of the brassica napus leaf margin cracking genes, candidate gene analysis and the like in the earlier stage, the BrRCO (BraA 10g 03440.3C) gene is found to be a candidate gene for controlling brassica napus leaf margin cracking. By applying to circular leaf parent NIL RcBr Comparing with the sequencing result of split leaf parent RcCr clone, the method has the following advantages that RcBr In contrast, in the parent RcBr, there is an insertion of one base in the second exon of the BrRCO gene, the nucleotide sequence of which is
AAGGAGCTTCGTCTGCAGCCA (SEQ ID NO:1, as shown in FIG. 2).
Designing upstream and downstream primers at the mutation site and at both sides by using Primer 5.0 Primer design software, wherein the sequences of the primers are as follows:
BraA10-F1：GCTGTCGAAGGAGCTACG(SEQ ID NO:2)；
BraA10-F2：GCTGTCGAAGGAGCTTCT(SEQ ID NO:3)；
BraA10-R：GACCCACAATAATGCCTCTC(SEQ ID NO:4)。
example 2:
a method for identifying leaf margin cracks of cabbage type rape comprises the following specific steps:
(1) Extraction of cabbage type rape genome DNA
Extracting circular leaf parent NIL by CTAB method RcBr Genomic DNA in fresh young leaves of split leaf parent RcBr;
(2) PCR amplification was performed using the above-described cabbage type rape genomic DNA as a template and the primers of example 1;
PCR amplification System (10. Mu.L): 1. Mu.L of genomic DNA at a concentration of 100 ng/. Mu.L, 5. Mu.L of 2 XTaq Master Mix, 0.5. Mu.L of the upstream primer BraA10-F1/BraA10-F2 at a concentration of 10. Mu.M, 0.5. Mu.L of the downstream primer BraA10-R at a concentration of 10. Mu.M, ddH was added 2 O to 10. Mu.L.
The PCR amplification reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 30s,30 cycles; extending at 72 ℃ for 5min;
(3) Agarose gel electrophoresis detection of PCR amplification products:
adding 2 mu L of Loading Buffer into 10 mu L of PCR amplification product, centrifuging, and then, adding 6 mu L of Loading quantity into 1% agarose gel, wherein the voltage is 200V, the current is 100mA, and the electrophoresis time is 8-10 min. When the parent RcBr was amplified using the above primer pair, only the BraA10-F1 combination gave an amplified product (as shown in fig. 3), with a characteristic band of 264bp, nucleotide sequence as follows:
TAAAGGGAGACGGATGCTCTAAGATCCCATGATTTTTTTTTTTAAATGGTGCATA
TATATACCTCATCGTGCGTTGTCAAAAAAAGAAAATATATATATATATATACCTC
ATCGTGTAGCATTTGATTCTGTCTTGAAACCACGTCGTACTCTTTCCTTAGCGAG
TTGTACGACTCCTCGAGATGCTTCACCCTCCACCGTGCACGGCGGTTCTGGAACC
AAACAGCTACCTGACGTGGCTGCAGACGAAGCTCCTTCGACAGC (SEQ ID NO: 5); when the parent NIL is amplified using the above primer pair, only the BraA10-F2 combination gives an amplified product (as shown in fig. 4), with a characteristic band of 263bp, the nucleotide sequence being as follows:
TAAAGGGAGACGGATGCTCTAAGATCCCATGATTTTTTTTTTTAAATGGTGCATA
TATATACCTCATCGTGCGTTGTCAAAAAAAGAAAATATATATATATATATACCTC
ATCGTGTAGCATTTGATTCTGTCTTGAAACCACGTCGTACTCTTTCCTTAGCGAG
TTGTACGACTCCTCGAGATGCTTCACCCTCCACCGTGCACGGCGGTTCTGGAACC
AAACAGCTACCTGACGTGGCTGCAGAGAAGCTCCTTCGACAGC(SEQ ID NO:6)。
example 3:
the InDel molecular marker is applied to auxiliary selection of cabbage type rape leaf margin cleavage.
NIL was identified using the identification method of example 2 RcBr And RcCr produced F 2 The segregating population was validated, which contains 200 individuals, with known leaf margin split phenotypes for the inbred offspring. The results of partial electrophoresis are shown in FIG. 5.
When amplified with BraA0-F1/BraA10-R, no bands were designated A and the bands were designated C; when amplified with BraA0-F2/BraA10-R, no band was designated B and the band was designated C; wherein, when the amplification results of the two pairs of primers are respectively marked as A/C or B/C, the genotype of the single plant is homozygotic A or homozygotic B; when the amplification result of the two pairs of primers is marked as C/C, the genotype of the single plant is heterozygous "H".
TABLE 1 identification of leaf margin genotype and phenotype of F2 population of Brassica napus
As can be seen from Table 1, the lines with round leaves are all of genotype "A"; strains with phenotypes of split She Ju offspring not isolated, genotypes were all "B"; the phenotype was the occurrence of isolated lines of the offspring of split She Ju, all genotypes were "H". The results show that the accuracy of the InDel molecular marker for identifying the leaf margin cracking of the cabbage type rape is 100%. Therefore, the marker can be rapidly, accurately and high-flux applied to auxiliary selection breeding of cabbage type rape.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (9)

1. An InDel molecular marker for identifying the leaf margin cracking character of cabbage type rape is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. The InDel molecular tagged PCR amplification primer pair of claim 1, comprising: an upstream primer with a nucleotide sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 4.
3. A method for identifying the leaf margin cracking character of cabbage type rape, which is characterized by comprising the following steps:
(1) Extracting genome DNA of cabbage type rape leaves;
(2) Performing PCR amplification by using cabbage type rape genomic DNA as a template and adopting the BraA10-F1 and the BraA10-F2 as defined in claim 2 to pair with the BraA10-R respectively;
(3) Performing agarose gel electrophoresis on the PCR amplification product obtained in the step (2);
(4) When only the combination of the BraA10-F1 and the BraA10-R is used for obtaining an amplification product, the leaf margin of the cabbage type rape plant is a crack, and the leaf margin of the self-bred seed is a crack; when only the combination of the BraA10-F2 and the BraA10-R is used for obtaining an amplification product, the leaf margin of the cabbage type rape plant is the whole margin, and the leaf margin of the self-bred seed is the whole margin; when BraA10-F1 and BraA10-R are combined and BraA10-F2 and BraA10-R are combined and amplified into a strip, the leaf margin of the cabbage type rape plant is split, and the leaf margin of the self-bred seed is split and full margin 3: 1.
4. A method according to claim 3, wherein the genomic DNA of canola leaves is extracted in step (1) using the CTAB method.
5. The method according to claim 3, wherein the PCR amplification reaction system in the step (2) is: 1. Mu.L of genomic DNA, 5. Mu.L of 2 XTaq Master Mix, 0.5. Mu.L of upstream primer BraA10-F1/BraA10-F2, 0.5. Mu.L of downstream primer BraA10-R, ddH was added 2 O to 10. Mu.L.
6. The method according to claim 5, wherein the concentration of genomic DNA in step (2) is 50 to 200 ng/. Mu.L, the concentration of the upstream primer BraA10-F1/BraA10-F2 is 5 to 20. Mu.M, and the concentration of the downstream primer BraA10-R is 0.5. Mu.L is 5 to 20. Mu.M.
7. The method of claim 3, wherein the PCR amplification reaction procedure in step (2) is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 30s,30 cycles; extending at 72℃for 5min.
8. The method according to claim 3, wherein the agarose concentration in the step (3) is 1%, the voltage during electrophoresis is 200V, the current is 100mA, and the electrophoresis time is 8-10 min.
9. The use of the InDel molecular marker of claim 1, the PCR amplification primer pair of the InDel molecular marker of claim 2, for identifying brassica napus leaf margin laceration or assisted selection breeding.
CN202311103991.1A 2023-08-30 2023-08-30 InDel molecular marker for identifying leaf margin cracks of cabbage type rape and application thereof Pending CN116987815A (en)

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