CN116158344A - New method for obtaining TuMV-resistant tuber mustard-purple cabbage differential additional line through distant hybridization and application - Google Patents
New method for obtaining TuMV-resistant tuber mustard-purple cabbage differential additional line through distant hybridization and application Download PDFInfo
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Abstract
The invention discloses a novel method for obtaining tuber mustard-purple cabbage heterologous line resisting TuMV through distant hybridization and application thereof, aiming at solving the problem of deficiency of tuber mustard germplasm resources resisting TuMV, realizing improvement of tuber mustard germplasm and enriching brassica germplasm resources of cruciferae. The invention takes heterohexaploid obtained by distant hybridization of hot pickled mustard tuber and purple cabbage as a starting material, has the characteristics of huge plants, dark green leaf color, high TuMV resistance, high total glucosinolate content and the like, and obtains TuMV resistance hot pickled mustard tuber-purple cabbage monomer/disomic additional line group through 2 rounds of backcross with hot pickled mustard tuber and TuMV infection identification. The invention has the advantages that: by using the heterohexaploid as a bridge parent, a tuber mustard-purple cabbage heterotopy line population resisting TuMV is obtained through backcrossing, on one hand, a new disease-resistant germplasm of brassica is obtained, and on the other hand, a molecular basis is provided for precise directional breeding of the tuber mustard. The method has strong operability and important practical significance for innovation and utilization of the germplasm resources of the hot pickled mustard tuber.
Description
Technical Field
The invention relates to a novel method for obtaining tuber mustard-purple cabbage heterologous line resisting TuMV through distant hybridization, thereby obtaining disease-resistant novel germplasm of tuber mustard and achieving the purpose of enriching brassica vegetable germplasm resources.
Background
The preserved szechuan pickle (Brassica juncea) is a special vegetable in China and plays an important role in promoting agricultural production in China. At present, the breeding of disease-resistant varieties of hot pickled mustard tuber mainly adopts traditional breeding or transgenic breeding and other methods, but has certain limitations. The brassica of cruciferae contains abundant genetic resources, and the purple cabbage of the same genus has excellent characteristics of high TuMV resistance, high nutritional quality and the like. Through distant hybridization and artificial chromosome doubling technology, the hot pickled mustard tuber and purple cabbage heterohexaploid are successfully obtained. The heterohexaploid has the characteristics of huge plants, dark green leaf color, high TuMV resistance, high total glucosinolate content and the like, and is an excellent germplasm resource in brassica of cruciferae. However, due to the high collinearity of A1/C1, A2/C2, and the dose compensating effect of homologous chromosome complementation, the A genome chromosomes are continually lost during the process of heterohexaploidy selfing, making excellent germplasm resources difficult to preserve. And a different additional line is further established on the basis of distant hybridization, so that a new way is provided for the accurate utilization and preservation of the dominant character.
The foreign addition line refers to a line in which one or a pair of heterologous chromosomes is added to a chromosome set of a species, and the foreign addition line not only realizes the transfer of exogenous excellent genes, but also avoids the influence of undesirable genes on other heterologous chromosomes. Therefore, the creation of excellent foreign lines using distant hybrids to develop new varieties with high quality has become a hot spot for international research. An et al breeds a new variety 'WR49-1' of a monomer heterologous strain resistant to wheat powdery mildew by distant hybridization and chromosome engineering through the use of 'Elytrigia minor No. 6' and 'German White' cultivars of rye; tan et al have found that the monomeric heterologous line to which the B3 chromosome is added has excellent properties of a purple stem by synthesizing a cabbage-black mustard monomeric heterologous line using cabbage and black mustard.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel method for obtaining a tuber mustard-purple cabbage heterologous line resisting TuMV through distant hybridization, which takes a heterologous hexaploid obtained in the earlier stage as a bridge parent, introduces a purple cabbage chromosome into a tuber mustard genome, realizes improvement of tuber mustard germplasm and enriches brassica germplasm resources of cruciferae.
The invention aims at realizing the following technical scheme: a novel method for obtaining TuMV-resistant tuber mustard-purple cabbage differential attachment by distant hybridization, comprising the following steps:
(1) Backcrossing the obtained heterohexaploid with hot pickled mustard tuber by distant hybridization to obtain heteroploid (BC) 1 )。
(2) Backcrossing of heterologous Galla chinensis to obtain primary heterologous line population (BC) 2 ) Backcrossing the primary differential additional line population and hot pickled mustard tuber to obtain candidate differential additional line population (BC 3 )。
(3) Screening monomer or disomic additional system by using specific molecular marker.
(4) TuMV inoculation is carried out on the tuber mustard-purple cabbage monomer or the two-body different additional system, new materials of the tuber mustard-purple cabbage monomer or the two-body different additional system resisting TuMV are screened and applied.
Further, in the step (1), the hot pickled mustard tuber is a homozygous restorer line material.
Further, in the step (1), the heterologous hexaploid genome is AABBCC, AABB represents the mustard genome, CC represents the red cabbage genome, and the chromosome number is 2n=54.
Further, in the step (1), the heterohexaploid has the characteristics of large plants, dark green leaves, high TuMV resistance, high total glucosinolate content and the like.
Further, in the step (2), the heterologous Chinese gall has the characteristics of large plants, dark green leaves, purple red stems and the like.
Further, in the step (3), the specific molecular marker adopts an EST-SSR identification method, and the nucleotide sequence of the molecular marker primer pair aiming at the purple cabbage chromosome is as follows:
CC01 forward primer: 5'-CGTTGCCGATAGGGTTCC-3'; (SEQ ID NO. 1)
Reverse primer: 5'-GAGGGCGAGACACTGCTTCC-3' (SEQ ID NO. 2)
CC02 forward primer: 5'-AGTAGCCGCCTCATCTTCCTC-3' (SEQ ID NO. 3)
Reverse primer: 5'-TTCCCATTCCTTAACTTGCTTCTC-3' (SEQ ID NO. 4) CC03 forward primer: 5'-AGGCCTTATTCTTCATCC-3' (SEQ ID NO. 5)
Reverse primer: 5'-GTTTCCCTCTCACAATCC-3' (SEQ ID NO. 6)
CC04 forward primer: 5'-TTTTTGCTTGTGATTGTGTTCTTC-3' (SEQ ID NO. 7)
Reverse primer: 5'-GAGGGTTTTAGGTTCCATTGTTAG-3' (SEQ ID NO. 8) CC05 forward primer: 5'-GAGGAGGAAGAAGAGCAAGATGAT-3' (SEQ ID NO. 9)
Reverse primer: 5'-ACGACCTGCAGATTAAGAAACACA-3' (SEQ ID NO. 10) CC06 forward primer: 5'-GCGGGGACTCTACCTCTA-3' (SEQ ID NO. 11)
Reverse primer: 5'-AGCAGCTCAGCATACAAG-3' (SEQ ID NO. 12)
CC07 forward primer: 5'-AGCCTGGCCTGAGCAATCC-3' (SEQ ID NO. 13)
Reverse primer: 5'-ATCATCTTTTCAATCTTTCATCTG-3' (SEQ ID NO. 14) CC08 forward primer: 5'-CGAAGGAGCGGGGATAGC-3' (SEQ ID NO. 15)
Reverse primer: 5'-AAAAGGATAGGACGACGACAAAAT-3' (SEQ ID NO. 16) CC09 Forward primer: 5'-CTGGAAATGCAATCGACAAGACT-3' (SEQ ID NO. 17)
Reverse primer: 5'-TGGGAGGGTGATAAAAGCAAAAA-3' (SEQ ID NO. 18)
Further, in the step (4), tuMV disease resistance is identified for the candidate foreign line population by a tribological inoculation method.
Further, the TuMV disease resistant heterologous line is a heterologous line with the genome AABB+C01 or AABB+C01, C05 (both containing chromosome 1 and chromosome 5).
Further, in the step (4), the virus strain identified by TuMV disease resistance is TuMV-UK.
The novel method for obtaining TuMV-resistant tuber mustard-purple cabbage heterologous line through distant hybridization is applied to tuber mustard breeding.
The invention has the advantages that: by using the heterohexaploid as a bridge parent, a tuber mustard-purple cabbage heterotopy line population resisting TuMV is obtained through backcrossing, on one hand, a new disease-resistant germplasm of brassica is obtained, and on the other hand, a molecular basis is provided for precise directional breeding of the tuber mustard. The method has strong operability and important practical significance for innovation and utilization of the germplasm resources of the hot pickled mustard tuber.
Drawings
FIG. 1 is a diagram of a route created by a mustard tuber-purple cabbage differential line;
FIG. 2 is a diagram of a heterologous five-fold volume;
FIG. 3 is a population diagram of candidate hot pickled mustard tuber-purple cabbage alien addition lines;
FIG. 4 is a screening chart of EST-SSR molecular marker primer pairs; CC01-CC09 represent EST-SSRs markers specific for the chromosomes of cabbage 9, respectively; a. b and c respectively represent hot pickled mustard tuber, purple cabbage and heterohexaploid;
FIG. 5 is a diagram of the identification of candidate iso-additive lines using EST-SSR; a-e represent 5 tuber mustard-purple cabbage monomer differential lines; the line frames and the numbers represent the chromosome numbers of the additional purple cabbage;
Detailed Description
The invention is further illustrated below with reference to examples.
Example 1:
materials: the present example uses, as starting material, heterologous hexaploids obtained from hot pickled mustard tuber and red cabbage by distant hybridization and artificial chromosome doubling. The genome of the hot pickled mustard tuber and the heterohexaploid of the red cabbage is AABBCC, the AABB represents the genome of the hot pickled mustard tuber, the CC represents the genome of the red cabbage, and the chromosome number is 2n=54. The obtained heterohexaploid has the characteristics of large plant, dark green leaf color, high TuMV resistance, and high total glucosinolate content.
The method comprises the following steps:
step (1): heterologous Chinese gall preparation
Heterologous hexaploids (Li JX, rao LL, meng QF, ghani MA, chen LP.product of Brassica tri-genomic vegetable germplasm by hybridization between tuber mustard (Brassica juncea) and red tissue (B.oleracea), euphytica,2015,204 (2): 323-333) obtained by distant hybridization and artificial chromosome doubling are transplanted to seed bases after hardening, and hybridization pollination is performed in the morning at a suitable temperature. Firstly, wiping the instruments such as pointed forceps, a culture dish (sleeve) and the like required by pollination by using absorbent cotton dipped with 75% alcohol, then sterilizing hands until the alcohol volatilizes completely, picking preserved szechuan pickle pollen which is put in the culture dish after 2d bags are put in advance by using the pointed forceps, and covering the preserved szechuan pickle pollen to prevent other pollen from mixing. Selecting heterohexaploid inflorescences with good growth vigor, manually removing stamens, sterilizing the pointed forceps and gloves with 75% alcohol again, and after alcohol is volatilized completely, clamping the hot pickled mustard tuber pollen, smearing the heterohexaploid inflorescences on the heterohexaploid stigma until visible faint yellow pollen exists on the stigma, wherein 10 flower buds are reserved in each inflorescences. And bagging the inflorescences after pollination by using a sulfuric acid paper bag to avoid pollution of other pollens, and hanging a tag for marking after fixation. And replacing the net bag when the fruit pod begins to form. The heterologous Galla chinensis seeds can be harvested after 2-3 months (figure 1).
Step (2): cultivation and identification of tuber mustard-purple cabbage monomer/disomic additional line
And sowing and raising the heterologous Chinese gall, transplanting the heterologous Chinese gall to a seed reproduction base (figure 2) when the seeds reach 4 leaves and 1 heart, wherein the heterologous Chinese gall has the characteristics of huge plants, greener leaf color, mauve stem and the like. First round backcrossing with hot pickled mustard tuber to obtain primary different additional line population (BC 2 ) Backcrossing the primary differential additional line population and hot pickled mustard tuber to obtain candidate differential additional line population (BC 3 ) (FIG. 3). By utilizing EST-SSR molecular marker technology, identifying chromosome composition of candidate heterologous line groups, and respectively screening 9 pairs of specific primer pairs for purple cabbage chromosomes as shown in FIG. 4:
CC01 forward primer: 5'-CGTTGCCGATAGGGTTCC-3'; (SEQ ID NO. 1)
Reverse primer: 5'-GAGGGCGAGACACTGCTTCC-3' (SEQ ID NO. 2)
CC02 forward primer: 5'-AGTAGCCGCCTCATCTTCCTC-3' (SEQ ID NO. 3)
Reverse primer: 5'-TTCCCATTCCTTAACTTGCTTCTC-3' (SEQ ID NO. 4) CC03 forward primer: 5'-AGGCCTTATTCTTCATCC-3' (SEQ ID NO. 5)
Reverse primer: 5'-GTTTCCCTCTCACAATCC-3' (SEQ ID NO. 6)
CC04 forward primer: 5'-TTTTTGCTTGTGATTGTGTTCTTC-3' (SEQ ID NO. 7)
Reverse primer: 5'-GAGGGTTTTAGGTTCCATTGTTAG-3' (SEQ ID NO. 8) CC05 forward primer: 5'-GAGGAGGAAGAAGAGCAAGATGAT-3' (SEQ ID NO. 9)
Reverse primer: 5'-ACGACCTGCAGATTAAGAAACACA-3' (SEQ ID NO. 10) CC06 forward primer: 5'-GCGGGGACTCTACCTCTA-3' (SEQ ID NO. 11)
Reverse primer: 5'-AGCAGCTCAGCATACAAG-3' (SEQ ID NO. 12)
CC07 forward primer: 5'-AGCCTGGCCTGAGCAATCC-3' (SEQ ID NO. 13)
Reverse primer: 5'-ATCATCTTTTCAATCTTTCATCTG-3' (SEQ ID NO. 14) CC08 forward primer: 5'-CGAAGGAGCGGGGATAGC-3' (SEQ ID NO. 15)
Reverse primer: 5'-AAAAGGATAGGACGACGACAAAAT-3' (SEQ ID NO. 16) CC09 Forward primer: 5'-CTGGAAATGCAATCGACAAGACT-3' (SEQ ID NO. 17)
Reverse primer: 5'-TGGGAGGGTGATAAAAGCAAAAA-3' (SEQ ID NO. 18)
The PCR amplification method is as follows:
the PCR reaction system was 20. Mu.L, including 2X Rapid Taq Master Mix 10.0.0. Mu.L, 1.0. Mu.L of DNA template, 1.0. Mu.L of forward primer, 1.0. Mu.L of reverse primer, and ddH 2 O7.0. Mu.L. The PCR amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 52-58℃for 30s, extension at 72℃for 20s,34 cycles; extending at 72℃for 7min. Detection was performed by 1.8% agarose gel electrophoresis. The monomer/disomy addition lines account for 25.51% and 35.03%, respectively, and the results of partial chromosome composition identification are shown in FIG. 5.
Step (3) identification of TuMV-resistant tuber mustard-purple cabbage differential addition line
When the plant of the different additional line grows to 1 heart of 4 leaves, 2g of the disease leaves of the TuMV-UK plant is taken in a mortar, 20mL of PBS (pH=7.2) with 0.02M is added to be ground into inoculation liquid, 200 mu L of the inoculation liquid is sucked up to the 3 rd true leaves (from bottom to top) of the plant of the different additional line by a liquid-transfering device, a little 600 meshes of silicon carbide is sprayed on the leaf surfaces to rub along the leaf veins, only buffer solution is added to rub as a control group, and ddH is used after 15min 2 O washes blade surface residues. The inoculated experimental group and the control group are placed in the dark for 24 hours and then transferred into an illumination incubator for culture, the photoperiod is 16/8 hours, and the temperature is 28 ℃/22 ℃. And observing the disease condition of inoculated leaves and system leaves 3-10d after inoculation. The mortar, 0.02M PBS (pH=7.2), pipette tip, carborundum, ddH 2 O is sterilized at high temperature and high pressure. After 10d of TuMV infection, the tuber mustard-purple cabbage differential attachment (BcF) 2 ) Well BC 3 -22 (AABB+C01) and BC 3 -62 (AABB+C01, C05) system leaves with significantly reduced disease-sensitive conditions. Therefore, the TuMV-resistant tuber mustard-purple cabbage heterologous line population can be obtained by the method, on one hand, the brassica disease-resistant new germplasm is obtained, and on the other hand, a molecular basis is provided for precise directional breeding of the tuber mustard. The method has strong operability and important practical significance for innovation and utilization of the germplasm resources of the hot pickled mustard tuber.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary or exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (9)
1. A novel method for obtaining TuMV-resistant tuber mustard-purple cabbage differential attachment by distant hybridization, comprising the following steps:
(1) Backcrossing the heterohexaploid obtained by distant hybridization of hot pickled mustard tuber and purple cabbage with hot pickled mustard tuber to obtain heteroploid BC 1 ；
(2) Backcrossing of heterologous Chinese gall and hot pickled mustard tuber to obtain primary heterologous additional line group BC 2 Backcrossing the primary foreign additional line population and the hot pickled mustard tuber to obtain candidate foreign additional line population BC 3 ；
(3) Screening a monomer or a disomy additional system by using a specific molecular marker;
(4) TuMV inoculation is carried out on the tuber mustard-purple cabbage monomer or the disomic additional line, and tuber mustard-purple cabbage monomer or the disomic additional line resisting TuMV is screened.
2. The method according to claim 1, characterized in that: in the step (1), the hot pickled mustard tuber is a homozygous restorer line material.
3. The method according to claim 1, characterized in that: in the step (1), the heterohexaploid genome obtained by distant hybridization of the hot pickled mustard tuber and the red cabbage is AABBCC, the AABB represents the hot pickled mustard tuber genome, the CC represents the red cabbage genome, and the chromosome number is 2n=54.
4. The method according to claim 1, characterized in that: in the step (1), the preserved szechuan pickle and the purple cabbage are obtained through distant hybridization and have the characteristics of large plants, dark green leaves, high TuMV resistance and high total glucosinolate content.
5. The method according to claim 1, characterized in that: in the step (2), the heterologous Chinese gall has the characteristics of large plants, dark green leaves and purple stems.
6. The method according to claim 1, characterized in that: in the step (3), a specific molecular marker adopts an EST-SSR identification method, and the nucleotide sequence of a molecular marker primer pair aiming at the purple cabbage chromosome is as follows:
CC01 forward primer is shown as SEQ ID NO. 1;
the reverse primer is shown as SEQ ID NO. 2;
the CC02 forward primer is shown as SEQ ID NO. 3;
the reverse primer is shown as SEQ ID NO. 4;
CC03 forward primer is shown as SEQ ID NO. 5;
the reverse primer is shown as SEQ ID NO. 6;
CC04 forward primer is shown as SEQ ID NO. 7;
the reverse primer is shown as SEQ ID NO. 8;
the CC05 forward primer is shown as SEQ ID NO. 9; the method comprises the steps of carrying out a first treatment on the surface of the
The reverse primer is shown as SEQ ID NO. 10;
CC06 forward primer is shown as SEQ ID NO. 11;
the reverse primer is shown as SEQ ID NO. 12;
the CC07 forward primer is shown as SEQ ID NO. 13;
the reverse primer is shown as SEQ ID NO. 14;
the CC08 forward primer is shown as SEQ ID NO. 15;
the reverse primer is shown as SEQ ID NO. 16;
the CC09 forward primer is shown as SEQ ID NO. 17;
the reverse primer is shown as SEQ ID NO. 18.
7. The method according to claim 1, characterized in that: in the step (4), tuMV disease resistance identification is carried out on the candidate foreign additional line group by adopting a friction inoculation method.
8. The method according to claim 7, wherein: the virus strain identified by TuMV disease resistance is TuMV-UK.
9. Use of a novel method of obtaining TuMV-resistant hot pickled mustard tuber-purple cabbage differential addition line by distant hybridization according to any one of claims 1-8 in hot pickled mustard tuber breeding.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3322200A (en) * | 2000-01-28 | 2001-08-07 | Dhara Vegetable Oil And Foods Company Limited | Novel 'oxy' cms brassica napus plant corrected for chlorosis using hexaploid bridging material generated through protoplast fusion |
| CN103651111A (en) * | 2013-12-16 | 2014-03-26 | 浙江大学 | Pickle and purple cabbage trigenomic species allohexaploid vegetable germplasm and acquisition method |
| CN107568059A (en) * | 2017-10-30 | 2018-01-12 | 四川农业大学 | The method that low temperature resistant germination corn variety is cultivated using corn allopolyploid |
-
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- 2022-12-20 CN CN202211643395.8A patent/CN116158344A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3322200A (en) * | 2000-01-28 | 2001-08-07 | Dhara Vegetable Oil And Foods Company Limited | Novel 'oxy' cms brassica napus plant corrected for chlorosis using hexaploid bridging material generated through protoplast fusion |
| CN103651111A (en) * | 2013-12-16 | 2014-03-26 | 浙江大学 | Pickle and purple cabbage trigenomic species allohexaploid vegetable germplasm and acquisition method |
| CN107568059A (en) * | 2017-10-30 | 2018-01-12 | 四川农业大学 | The method that low temperature resistant germination corn variety is cultivated using corn allopolyploid |
Non-Patent Citations (4)
| Title |
|---|
| LI JX 等: "Production of Brassica tri-genomic vegetable germplasm by hybridization between tuber mustard(Brassica juncea)and red cabbage(B.oleracea)", 《EUPHYTICA》, pages 1 - 11 * |
| MEI, JQ 等: "Transfer of sclerotinia resistance from wild relative of Brassica oleracea into Brassica napus using a hexaploidy step", 《THEOR APPL GENET》, pages 1 - 6 * |
| 万华方;魏帅;冯宇霞;钱伟;: "以六倍体(A~nA~nC~nC~nC~oC~o)为桥梁创制抗旱新型甘蓝型油菜(A~nA~rC~nC~o)", 中国农业科学, no. 16 * |
| 王爱凡;康雷;李鹏飞;李再云;: "我国甘蓝型油菜远缘杂交和种质创新研究进展", 中国油料作物学报, no. 05, pages 691 - 698 * |
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