NL2026261B1 - Molecular Marker Closely Linked to Chinese Cabbage Turnip Mosaic Virus Resistance Gene retrcs03 and Application Thereof - Google Patents
Molecular Marker Closely Linked to Chinese Cabbage Turnip Mosaic Virus Resistance Gene retrcs03 and Application Thereof Download PDFInfo
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Abstract
The present invention provides a molecular marker closely linked to the Chinese cabbage turnip mosaic virus resistance gene retrcs03 and application thereof and belongs to the technical field of biology. The molecular marker provided by the present invention further maps the Chinese cabbage TuMV resistance gene retrcs03, which is helpful for better use in Chinese cabbage TuMV-resistant molecular marker-assisted selection. Meanwhile, the gene can be mapped, which lays a foundation for the cloning and in-depth utilization and research of the gene. At the same time, the molecular marker is applied in breeding work, which will greatly reduce the economic loss caused by TuMV prevalence to production of the Chinese cabbage, is conducive to reducing production cost and has great application potential and high economic value.
Description
Molecular Marker Closely Linked to Chinese Cabbage Turnip Mosaic Virus Resistance Gene retrcs03 and Application Thereof Technical Field The present invention belongs to the technical field of biology, and particularly relates to a molecular marker closely linked to the Chinese cabbage turnip mosaic virus resistance gene retrcs03 and application thereof.
Background The disclosure of the background information only aims to increase the understanding of the overall background of the present invention and is not necessarily regarded as an acknowledgment or any form of implication that the information constitutes the prior art well known by those of ordinary skill in the art.
The turnip mosaic virus (TuMV for short) belongs to Potyviruse Y of Potyviridae and is mainly propagated by aphis or sap contact. TUMV is the most harmful virus with the widest host range in Potyviridae and is widely distributed around the world. TuMV is distributed in all the continents except Antarctica, and the host range is very wide. TuMV can infect 318 dicotyledons (including cruciferae, compositae, chenopodiaceae, leguminosae, caryophyllaceae and the like) in 156 genera in 43 families and part of monocotyledons (Walsh and Jenner 2002}. In the production of Chinese cabbages in China, an average annual yield loss is 5%. In some years, the yield is reduced by more than 10%, and plots with serious diseases almost have no yield. The disease is difficult to control, and the chemical control effect is not ideal. The most effective and sustainable control measure is to cultivate disease-resistant varieties (Hughes et al.2002). The use of molecular marker-assisted selection or genetic engineering means to improve or innovate germplasm can greatly accelerate the breeding process and is the development trend of modern breeding.
Researches show that the Chinese cabbage turnip mosaic virus has a very complicated genetic law (Tan Qimeng 1980; Providenti 1980; Leung and Williams 1983; Niu Xinke 1984; Suh 1995; Yan Jingi 2000; Yoon et al. 1993; Han Heping 2003; Rusholme et al. 2007; Pan Chunging 2007; Zhang Xiaowei et al. 2009; Qu Shuping et al. 2009; Li et al. 2011; Li Qiaoyun et al. 2012; Qian Wei et al. 2012), and has multiple variations at multiple sites.
TuMV resistance genes TURBOIb (Rusholme et al. 2000), retr01 and ConTROI (Rusholme et al. 2007), and TURBCHO1 (Wang et al. 2011), retr02 (Qian et al. 2013), TURBO? (Jin et al. 2014) and TuURBCS01 (Li et al. 2015) mapped on the Chinese cabbage are respectively located on chromosomes A06, A04, A08, AOS, A04, AOS and A04. In addition, Zhang Xiaowei et al. (2009) detects three QTLs related to the resistance of the Chinese cabbage TuMV-C4 strain, which are respectively located on Chinese cabbage chromosomes A03, A04 and A06; Tian Xihui et al. (2014) detects one major QTL related to the resistance of the Chinese cabbage TuMV-C4 strain,
which is located on chromosome A09; and Li Guoliang et al. (2019) detects two QTLs related to the resistance of the Chinese cabbage TuMV-C4 strain, which are respectively located on Chinese cabbage chromosomes A07 and AGS. In the previous researches, a disease-resistant material '73' and a disease-susceptible material '06-247' are used as parents to construct a segregating population so as to identify a recessive TUMV resistance gene, named retrcs03. The previous researches only initially screen out three molecular markers BrID90143 (4.2 cM), BrSSR4068 (4.2 cM) and BriD10645 (10.1 cM) linked to the gene (Zeng Qiang et al. 2014), but the above molecular markers are not highly linked. Therefore, it is necessary to further screen molecular markers more closely linked to the gene retrcs03.
Summary Aiming at the above prior art, the present invention provides a molecular marker closely linked to the Chinese cabbage turnip mosaic virus resistance gene retrcs03 and application thereof. The molecular marker provided by the present invention further maps the Chinese cabbage TuMV resistance gene retrcs03, which is helpful for better use in Chinese cabbage TuMV-resistant molecular marker-assisted selection. Meanwhile, the gene can be mapped, which lays a foundation for the cloning and in-depth utilization and research of the gene and is conducive to the breeding of the Chinese cabbage resistant to the turnip mosaic virus.
The present invention has the following technical solution: The first aspect of the present invention is to provide the application of a substance for detecting the molecular marker closely linked to the Chinese cabbage turnip mosaic virus resistance gene retrcs03 in the genome of a Chinese cabbage to be detected.
The application of a substance for detecting the molecular marker closely linked to the Chinese cabbage turnip mosaic virus resistance gene retrcs03 in the genome of a Chinese cabbage to be detected provided by the present invention in any of the following 1)-9): 1). identifying or assisting in identifying the turnip mosaic virus resistance of the Chinese cabbage to be detected; 2). preparing a product for identifying or assisting in identifying the turnip mosaic virus resistance of the Chinese cabbage to be detected; 3). identifying or assisting in identifying that the Chinese cabbage to be detected is resistant to the turnip mosaic virus or susceptible and resistant to the turnip mosaic virus; 4). preparing a product for identifying or assisting in identifying that the Chinese cabbage is resistant to the turnip mosaic virus or susceptible and resistant to the turnip mosaic virus; 5). breeding the Chinese cabbage; 8). breeding the variety of Chinese cabbage resistant to the turnip mosaic virus; 7). preparing a product for breeding the variety of Chinese cabbage resistant to the turnip mosaic virus;
8). identifying or assisting in identifying the turnip mosaic virus resistance properties of the Chinese cabbage to be detected; 9). preparing a product for identifying or assisting in identifying the turnip mosaic virus resistance properties of the Chinese cabbage to be detected.
In the above application, the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, and the fragment size of the marker is 191 bp.
In the above application, the substance for detecting the molecular marker closely linked to the Chinese cabbage turnip mosaic virus resistance gene retrcs03 in the genome of a Chinese cabbage to be detected is the following 1) or 2): 1) the substance as shown comprises a set of primers, and the set of primers is composed of a primer 1 and a primer 2; 2) the substance as shown comprises a PCR reagent or kit containing the set of primers; the primer 1 is the following a1) or a2): a1) a single-stranded DNA molecule shown in SEQ ID No.2; a2) a single-stranded DNA molecule which is obtained by deleting, inserting and/or changing one or a plurality of bases in the single-stranded DNA molecule defined in a1) and has the same functions as the single-stranded DNA molecule defined in a1); the primer 2 is the following b1) or b2): b1) a single-stranded DNA molecule shown in SEQ ID No.3; b2) a single-stranded DNA molecule which is obtained by deleting, inserting and/or changing one or a plurality of bases in the single-stranded DNA molecule defined in b1) and has the same functions as the single-stranded DNA molecule defined in b1); In the above application, the final concentration of each primer in the set of primers in the PCR reagent is 1.0 HM.
The second aspect of the present invention is to provide a product.
The product provided by the present invention is the substance for detecting the molecular marker closely linked to the Chinese cabbage turnip mosaic virus resistance gene retrcs03 in the genome of a Chinese cabbage to be detected.
In the above product, the product is any of the following 1)-4): 1) a product for identifying or assisting in identifying the turnip mosaic virus resistance of the Chinese cabbage to be detected; 2) a product for identifying or assisting in identifying that the Chinese cabbage is resistant to the turnip mosaic virus or susceptible and resistant to the turnip mosaic virus; 3) a product for breeding the variety of Chinese cabbage resistant to the turnip mosaic virus; 4) a product for identifying or assisting in identifying the turnip mosaic virus resistance properties of the Chinese cabbage to be detected.
The third aspect of the present invention is to provide a method for identifying or assisting in identifying that the Chinese cabbage to be detected is resistant to the turnip mosaic virus or susceptible to the turnip mosaic virus.
The method provided by the present invention comprises the following steps: conducting PCR amplification on the genomic DNA of the Chinese cabbage to be detected by using the product to obtain an amplification product; and detecting the amplification product.
If the amplification product contains a fragment of 191 bp, the Chinese cabbage to be detected is selected as the Chinese cabbage resistant to the turnip mosaic virus; If the amplification product contains a fragment of 199 bp, the Chinese cabbage to be detected is selected as the Chinese cabbage susceptible to the turnip mosaic virus; Further, if the nucleotide sequence of the amplification product is shown as SEQ ID No.1, the Chinese cabbage to be detected is selected as the Chinese cabbage resistant to the turnip mosaic virus; Further, if the nucleotide sequence of the amplification product is shown as SEQ ID No.4, the Chinese cabbage to be detected is selected as the Chinese cabbage susceptible to the turnip mosaic virus.
The fourth aspect of the present invention is to provide a method for breeding the variety of Chinese cabbage resistant to the turnip mosaic virus.
The method provided by the present invention comprises the following steps: conducting PCR amplification on the genomic DNA of the Chinese cabbage to be detected by using the product to obtain an amplification product; detecting the amplification product; and selecting the Chinese cabbage with the amplification product containing the fragment of 191 bp selected as parents for breeding.
Further, the nucleotide sequence of the amplification product is shown as SEQ ID No.1.
Further, the Chinese cabbage to be detected is an individual or a population.
The present invention has the following advantageous effects that: 1) The molecular marker of the present invention further maps the Chinese cabbage TuMV resistance gene retrcs03. The marker has strong specificity, high stability, simple and rapid screening method and low requirements for the quality of detection equipment and primer template and has the advantages of small amount of test reagents, high speed, low cost and suitability for large batch, high throughput and automation. The molecular marker is very suitable for the molecular breeding trend in modern agriculture.
2) The present invention provides very important molecular genetic information for the map- based cloning of the Chinese cabbage TuMV resistance gene retrcs03.
3) The molecular marker primer of the Chinese cabbage TuMV resistance gene retrcs03 of the present invention is applied in breeding work, which will greatly reduce the economic loss caused by TuMV prevalence to production of the Chinese cabbage, is conducive to reducing production cost and has great application potential and high economic value.
4) The present invention screens out the SSR marker linked to the gene retrcs03, which can be effectively used for marker-assisted selection of the gene retrcs03. The close linkage distance between the molecular marker and the Chinese cabbage TuMV resistance gene retrcs03 is 1.1 cM. The gene retrcs03 can be finely mapped by using the physical position information of the 5 marker in the genome of the Chinese cabbage and can be approached by the chromosome walking method to improve the selection accuracy, shorten the breeding time and lay a foundation for the cloning of the gene retrcs03, so the marker has good practical application value.
Description of Drawings Drawings of the Description forming a part of the present application are used for providing further understanding of the present invention. Exemplary embodiments of the present invention and the description are used for explaining the present invention, but do not constitute a limitation to the present invention.
Fig. 1 is a diagram showing the amplification result of the primer SAAS_mBr4174 involved in embodiment 1 of the present invention in two parents and resistant and susceptible pools, wherein P1 is the parent '73', P2 is the parent '06-247', A is the susceptible pool, and B is the resistant pool.
Fig. 2 is a marker linkage diagram of the gene retrcs03 in embodiment 1 of the present invention, wherein the left numbers represent linkage distances in cM.
Fig. 3 is a diagram showing the amplification results of the primer combination SAAS_mBr4174 in part of individual plants of the BC1 segregating population in embodiment 1 of the present invention, wherein P1 is the parent '73'; P2 is the parent '06-247'; 1-15 are 15 individual backcross plants; for the individual plants numbered 1, 3, 4, 6, 9, 11, 12 and 14, disease-resistant bands are amplified; and for the individual plants numbered 2, 5, 7, 8, 10, 13 and 15, disease-susceptible hybrid bands are amplified.
Fig. 4 is a diagram showing the sequence alignment result of the disease-resistant marker and the disease-susceptible marker amplified with the primer combination SAAS_mBr4174 in embodiment 1 of the present invention; and the figure shows that the two sequences have differences in six positions, among which the difference in five positions is a base substitution, and the difference in one position is a base deletion, that is, the disease-resistant marker lacks the base sequence CTATCTAT at 121-128.
Detailed Description It should be pointed out that the following detailed descriptions are all illustrative and are intended to provide further description of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the technical field to which the present invention belongs.
It should be noted that the terms used herein are intended to merely describe specific embodiments, not to limit the exemplary embodiments according to the present invention. As used herein, unless the context otherwise clearly indicates, the singular form is also intended to include the plural form. In addition, it should also be understood that when being used in the Description, the terms "comprise" and/or "include" indicate the existence of features, steps, operations, devices, components and/or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the following specific implementation solutions. It should also be understood that the terms used in the embodiments of the present invention are intended to describe specific implementation solutions, not to limit the protection scope of the present invention. Experimental methods in which specific conditions are not specified in the following specific embodiments are carried out according to the conventional methods and conditions of the molecular biology in the art, and such technologies and conditions are fully explained in the literature. Refer to, for example, the technologies and conditions in Sambrook et al., "Molecular Cloning: Laboratory Manual", or follow the conditions recommended by the manufacturer In a specific embodiment of the present invention, a molecular marker closely linked to the Chinese cabbage TuMV resistance gene retrcs03 is provided, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, and the fragment size of the marker is 191 bp. In another specific embodiment of the present invention, a primer pair for amplifying a molecular marker closely linked to the Chinese cabbage TuMV resistance gene retrcs03 is provided, wherein the sequences of the primer pair are respectively shown as SEQ ID NO.2 and SEQ ID NO.3. In another specific embodiment of the present invention, a detection method for a molecular marker closely linked to the Chinese cabbage TuMV resistance gene retrcs03 is provided, which uses the genomic DNA of the Chinese cabbage material resistant and susceptible to TUMV as the template to obtain specific bands that can identify the genotypes of the male parent, the female parent and the hybrid thereof simultaneously, through PCR amplification. The specific bands include a plant-specific band carrying a resistance gene and a plant-specific band carrying no resistance gene, and the plant band carrying a resistance gene is a molecular marker with the nucleotide sequence as shown as SEQ ID No.1. The nucleotide sequence of the plant band carrying no resistance gene is shown as SEQ ID No.4. In the above method, the PCR reaction system is that: 10 pL of reaction system comprises
1.0 HM of forward primer, 1.0 uM of reverse primer, 50-70 ng of DNA template and 5.0 uL of 2xEs Taq MasterMix (containing Tag DNA polymerase, 3 mM MgCl 2 and 400uM dNTPs). In the above method, the PCR amplification procedure is: pre-denaturing at 95°C for 5 min, denaturing at 94°C for 1 min, annealing at 50°C for 45 s and extending at 72°C for 45 s which are cycled for 30 times; and extending at 72°C for 10 min, and storing at 4°C.
The following embodiment is intended to further describe the present invention, not to limit the present invention. It should be understood that the embodiments are only used for illustrating the present invention, not used for limiting the scope of the present invention. In addition, the molecular biology methods not described in detail in the embodiments are all conventional methods in the art. For specific operations, refer to the molecular biology guide or product specification.
Embodiment 1 Screening, Sequencing and Gene Mapping of Molecular Marker Closely Linked to Gene retrcs03 Plant material: in the present invention, the disease-resistant parent material is a high- generation inbred line material '73' of Chinese cabbage with high resistance to TuMV, the disease-susceptible material is 06-247, and the segregating population is a '06- 247'x*73’x'73’backcross population. After being sterilized with a PINDSTRUP seedling substrate imported from Denmark, the above materials are sown in a 6.5 cm plastic seedling bowl and placed in an artificial climate room for cultivation. Culture conditions: the temperature is 25°C, the humidity is 60%, and the light intensity is 8000-2000 lux.
Toxic source material: the C4 strain of TuMV, introduced from Vegetable and Flower Research Institute of Chinese Academy of Agricultural Sciences, and cultured on the disease- susceptible material for toxin reproduction one month before use.
TuMV inoculation identification: when the material for test grows to three leaves and one heart, the two parents and the BC1 population are respectively inoculated with TuMV-C4. The inoculation method is a friction inoculation method. See Li Qiaoyun et al. (2009) for details. Disease-resistance identification is performed after 2-3 weeks. The TuMV resistance identification of individual plants is performed by means of biological observation. For the identification standard, refer to GB/T 19557.5-2004. Multiple identification results are comprehensively analyzed to determine the resistance of each plant so as to calculate the disease index of the population or the separation ratio of an individual resistant and susceptible plant and analyze the resistance classification and resistance inheritance. The result shows that among 180 individual plants, the number of individual disease-resistant plants is 89, and the number of individual disease-susceptible plants is 91, x 2 c =0.02<x 2 0.05 = 3.84, which is in line with the separation ratio of 1:1.
Primer source: 150 pairs of SSR primers are designed according to the screening result of the molecular marker of the Chinese cabbage TuMV resistance gene retrcs03 in Zeng Qiang et al. (2014), by reference to the marker information on the Chinese cabbage genome website http://brassicadb.org/brad/ and based on the sequence information near the gene marker.
The specific method for designing primers is: selecting the above genome sequence of about 600 bp, logging in to the website http://www.gramene.org/gremene/searches/ssrtool, and screening SSR on line by using the software SSRIT (Simple Sequence Repeat Identification Tool). The screening standard is: not less than 10 times for mononucleotide repeat, not less than
6 times for dinucleotide repeat, not less than 4 times for trinucleotide repeat, and not less than 3 times for tetranucleotide, pentanucleotide, hexanucleotide and other polynucleotide repeats. Primers are designed by using primer premier 5.0 software according to the sequences at both ends of the SSR. The primers are synthesized by Shanghai Bioengineering Co., Ltd. The sequences of the above 150 pairs of primers are used for screening molecular markers closely linked to the gene retrcs03.
Extraction and detection of genomic DNA: the genomic DNAs of the materials used in the present invention are extracted by using the rapid plant genomic DNA extraction kit of TIANGEN Company, and the specific method is as follows:
1. Processing materials: taking 0.2 g of fresh plant tissue and adding liquid nitrogen to fully grind the fresh plant tissue. Adding 400 pl of buffer solution FP1 and 8 pl of RNase A (10 mg/ml), conducting vortex oscillation for 1 min, and standing at room temperature for 10 min.
2. Adding 130 HI of buffer solution FP2, mixing well, and conducting vortex oscillation for 1 min.
3. Centrifuging at 12,000 rpm (~13,400xg) for 5 min and transferring the supernatant to a new centrifuge tube.
4. Centrifuging the supernatant at 12,000 rpm (~13,400xg) for 5 min again and transferring the supernatant to a new centrifuge tube.
5. Adding 0.7 times volume of isopropyl alcohol to the supernatant and mixing well. At this time, flocculent genomic DNA appears. Centrifuging at 12,000 rpm (-13,400xg) for 2 min, discarding the supernatant, and retaining the precipitate.
6. Adding 600 pl of 70% ethanol, conducting vortex oscillation for 5 sec, centrifuging at 12,000 rpm (~13,400xg) for 2 min, and discarding the supernatant.
7. Repeating step 6.
8. Opening the lid, placing upside down, and drying the remaining ethanol thoroughly at room temperature for 5-10 min.
9. Adding 100 pl of double distilled water and dissolving the DNA in a 65°C water bath for 10-60 min during which the centrifuge tube is turned upside down for mixing well several times to aid dissolution to finally obtain the DNA solution.
The concentration and purity of the extracted DNA are measured with a spectrophotometer, and 1% agarose gel is used for electrophoresis detection. Finally, the extracted DNA is diluted with deionized water to the concentration of 50 ng/L before use.
Construction of resistant and susceptible pools: the genomic DNAs of 10 individual extremely disease-resistant plants and 10 individual extremely disease-susceptible plants in the BC1 segregating population are selected, and resistant and susceptible pools are constructed after respective mixing for polymorphism screening of primers and linkage analysis of the marker.
PCR amplification and amplification product detection: the PCR reaction system is that: 10 ML of reaction system comprises 1.0 uM of forward primer, 1.0 uM of reverse primer, 50-70 ng of
DNA template and 5.0 pL of 2xEs Taq MasterMix (containing Taq DNA polymerase, 3 mM MgCl 2 and 400uM dNTPs). The PCR amplification procedure is: pre-denaturing at 95°C for 5 min, denaturing at 94°C for 1 min, annealing at 50°C for 45 s and extending at 72°C for 45 s which are cycled for 30 times; and extending at 72°C for 10 min, and storing at 4°C. The result is detected by silver staining after electrophoresis. The result shows that with the genomic DNA of the two parents as the template, amplification is performed with 150 pairs of primers in the target region of the Chinese cabbage genome, wherein 138 pairs are successfully amplified, 42 pairs have polymorphism between the two parents, and 21 pairs have consistent polymorphism between the two parents and the resistant and susceptible pools. Finally, verification is performed among the 180 individual plants in the BC1 population in combination with the disease resistance identification result, and the markers amplified with 11 pairs of primers are linked to the gene retrcs03, wherein the linkage between the marker amplified with the primer SAAS_mBr4174 and the gene retrcs03 is the closest, and the linkage distance is 1.1 cM. Among the 180 individual plants, the disease resistance identification results of 177 individual plants are consistent with the amplification result of the primer SAAS_mBr4174, and only three individual plants No. 149, No.
163 and No. 173 are exchanged.
Data statistics and analysis: the primers that have polymorphism between the two parents and the resistant and susceptible pools are screened out and verified among the 180 individual plants in the BC1 population, and the banding patterns of all individual plants are counted. A disease-susceptible band is recorded as "A", a disease-resistant band is recorded as "H", and a band that is unclear or not amplified is recorded as "-". Genetic linkage analysis is performed by using JoinMap4.0 software to calculate the linkage distance and determine the relative position of each marker and the gene. The result is shown in Fig. 2, wherein the linkage distance between the amplification site of the primer SAAS_mBr4174 and the gene retrcs03 is only 1.1 cM, which is close linkage.
Cloning and sequencing of PCR product: after the amplification of the target band is confirmed by electrophoresis, the target band is amplified with high-fidelity enzyme; the amplification product is recovered with the gel recovery kit of Thermo Fisher Scientific, and then 4 pL of gel recovery product, 1 HL of pMD19-T cloning Vector of TaKaRa Company and 4 ul of Solution | are added into a microcentrifuge tube, mixed by flick, ligated at constant temperature of 16°C for 1 h, and ligated at 4°C overnight. E. coli competent cells DH5a are transformed, and the transformed bacteria are cultured upside down on an LB solid plate containing 50 pg/ml kanamycin at 37°C for about 16 h. After colonies are detected by PCR, positive colonies are selected for cloning and sequencing. The sequencing result of the band amplified with the primer SAAS _mBr4174 in the disease-resistant parent 73 is shown as SEQ ID No. 1, and the size is 191 bp. The band amplified in the disease-susceptible parent 06-247 is shown as SEQ ID No. 4, and the size is 199 bp.
Sequence alignment and analysis: the above marker sequence is aligned with the published Chinese cabbage complete genome sequence (http://brassicadb.org/brad/) to determine the specific position of each marker on the chromosome, and then the target gene is mapped according to the relationship between each marker and the Chinese cabbage TuMV resistance gene.
As shown in Fig. 2, the gene retrcs03 is located between the two markers BrlD101487 and SAAS_mBr4174_191. The sequence of the disease-resistant marker SAAS_mBr4174_191 is aligned with the Chinese cabbage genome sequence (v1.5), and the result shows that the marker is located between 6371496 and 6371694 on Chinese cabbage chromosome 4. The sequence of the disease-resistant marker BrID101487 (see http://brassicadb.org/brad/ for details) is aligned with the Chinese cabbage genome sequence (v1.5), and the result shows that the marker is located between 3229172 and 3229304 on chromosome 4. Therefore, the gene retrcs03 is located in the region of about 3.14 Mb between 3229304 and 6371496 on chromosome 4 of the Chinese cabbage genome.
Table 1 shows the disease resistance identification results of part of individual plants in the above BC1 population and the amplification results of using the primer combination SAAS_mBr4174, wherein R represents disease-resistant, S represents disease-susceptible, the amplification banding pattern 1 represents a band consistent with the disease-resistant parent '73', 2 represents a band consistent with the disease-susceptible parent '06-247', 3 represents a hybrid band, and 0 represents a band not amplified.
The amplification results of most of individual plants are consistent with the disease resistance identification results, only the individual plant No. 1 is inconsistent, which appears to be disease-susceptible, but the disease-resistant band is amplified.
Table 1 Number oft 2 3 4 5 7 10 11 112 13 14 15 saar [IEEE PEPE Rees S RRS RES RS RRS RE SAAS mBr4174 1 B 1 N B 1 BB 3 1 3 1 1 3 3 Amplification Banding Pattern
The above mapping results of the gene retrcs03 can be used for further fine mapping and map-based cloning of the gene; and the closely linked molecular markers on both sides of the gene can be used for molecular marker-assisted selection of the gene during the breeding process, which is helpful for the breeding of TuMV resistant Chinese cabbage varieties.
With the closely linked markers on both sides of the above gene retrcs03 as foreground markers, part of markers on each chromosome in the Chinese cabbage genome are selected as background markers to construct near-isogenic lines of precious breeding materials (such as 06-247), which can improve or innovate germplasm.
It should be noted that the above embodiment is only used for describing the technical solution of the present invention rather than limitation.
Although the present invention is described in detail with reference to the given embodiment, those ordinary skilled in the art can amend or equivalently replace the technical solution of the present invention without departing from the spirit and the scope of the technical solution of the present invention.
<110> Vegetable and Flower Research Institute of Shandong Academy of Agricultural Sciences <120> Molecular Marker Closely Linked to Chinese Cabbage Turnip Mosaic
Virus Resistance Gene retrcs@3 and Application Thereof
<130> Retrcse3 NL <150> CN 201910764572.X <151> 2019-08-19 <160> 4 <170> PatentIn version 3.5 <2105 1 <211> 191 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized <400> 1 catccaaatt ctacccaatc tatggtgtga atagatacag ttctaacctg gaaacgtagc 60 atcaaatagc ttctgaaagt ttccttcatg gactcgtgtc tcaatcaaac gaccaatcta 120 ttacaataaa atagacttgc ctctctcctc atgaagccac atcagcatgg agaggtgagc 180 attttaggac a 191 <2105 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized <400> 2 tgtcctaaaa tgctca 16 <2105 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Articiciall synthesized <400> 3 catccaaatt ctaccc 16 <210> 4
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<212> DNA
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<220>
<223> Artificially synthesized
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| CN103060338A (en) * | 2012-12-27 | 2013-04-24 | 中国农业科学院蔬菜花卉研究所 | TuMV resistance gene retr02 of Chinese cabbage and allele retr02 Retr02, and encoded protein and application thereof |
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