WO2024050761A1 - Marqueur de pcr pour détecter un fragment de radis exogène dans une lignée d'introgression de brassica oleracea-radis, amorce et utilisation de celui-ci - Google Patents

Marqueur de pcr pour détecter un fragment de radis exogène dans une lignée d'introgression de brassica oleracea-radis, amorce et utilisation de celui-ci Download PDF

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WO2024050761A1
WO2024050761A1 PCT/CN2022/117836 CN2022117836W WO2024050761A1 WO 2024050761 A1 WO2024050761 A1 WO 2024050761A1 CN 2022117836 W CN2022117836 W CN 2022117836W WO 2024050761 A1 WO2024050761 A1 WO 2024050761A1
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radish
cabbage
pcr
exogenous
fragments
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PCT/CN2022/117836
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Chinese (zh)
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张扬勇
任文静
司劲超
方智远
杨丽梅
庄木
吕红豪
王勇
季家磊
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中国农业科学院蔬菜花卉研究所
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Priority to PCT/CN2022/117836 priority Critical patent/WO2024050761A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

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  • the invention belongs to the field of molecular breeding technology, and specifically relates to PCR markers and primers for detecting exogenous radish fragments in cabbage-radish introgression systems and their applications.
  • Cabbage (Brassica oleracea L.var.capitata), referred to as cabbage, is an important vegetable crop of the genus Brassica in the Brassicaceae family.
  • the hybrid vigor is obvious. Most of the cabbage varieties produced are hybrids, and the utilization of hybrid vigor mainly relies on two methods: self-incompatible lines and male sterile lines. In the middle and late part of the last century, self-incompatible lines were mainly used. After entering this century, male sterile lines have been widely used. It is estimated that more than 80% of cabbage varieties are male sterile in recent years.
  • the widely used types of male sterility in cabbage mainly include dominant male sterility (DGMS) and Ogura cytoplasmic male sterility (Ogura CMS) (Fang Zhiyuan, Liu Yumei, Yang Limei, Wang Xiaowu, Zhuang Mu, Zhang Yangyong, Sun Peitian. Cabbage Dominant Male Sterility) Breeding and seed production of genetic male sterile and cytoplasmic male sterile lines. Chinese Agricultural Sciences, 2004, 37(5):717-723.). Ogura CMS has the characteristics of easy transfer, stable sterility, and complete abortion. At present, most domestic and foreign breeders use Ogura CMS (Yu Hailong. 2018.
  • Rfo fertility restorer genes were introduced from radish to Brassica napus through protoplast fusion, distant hybridization and other means (Heyn F W. 1976. Transfer of restore genes from Raphanus to cytoplasmic male sterile Brassica napus. Cruciferae Newsletter, 1:15-16), and then continuously improved (Delourme R, Foisset N, Horvais R, Barret P, Champagne G, Cheung W Y, Landry B S, Renard M. 1998. Characterization of the radish introgression carrying the Rfo restorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed(Brassica napus L.).
  • Ogura CMS fertility restoration material has currently obtained BC 6 generations of high-generation backcross introgression line populations through continuous backcrossing (Yu H L, Fang Z Y, Liu Y M, Yang L M, Zhuang M, Lv H H, Li Z S, Han F Q ,Liu Z Y, Yang L M, Liu Y M, Zhuang M, Zhang L G, Lv H H, Li Z S, Han F Q, Liu X P, Fang Z Y, Zhang Y Y. 2017. Morphological and molecular characterization of the second backcross progenies of Ogu-CMS Chinese kale and rapeseed.
  • MAAL_3B Has excellent fiber strength and fineness (Meng S, Xu Z, Xu P, Chen A, Guo Q, Zhao L, Chen X, Wen T, Zhang X, Zhang G, Ni W, Shen X.2020.A complete set of monosomic alien addition lines developed from Gossypium anomalum in a Gossypium hirsutum background:genotypic and phenotypic characterization. Breeding Science, 70(4):494-501).
  • the cabbage-radish introgression line population with 14Mb heterologous radish fragment will be an excellent genetic material population for broadening the cabbage gene pool, and will play an important role in mining and utilizing germplasm resources between radish genera and revealing the interaction of chromosome fragments between genera.
  • the technical problem to be solved by the present invention is how to quickly and accurately identify the presence, absence and/or sequence of exogenous radish fragments in the cabbage-radish introgression system.
  • the present invention provides PCR markers for detecting exogenous radish fragments in the cabbage-radish introgression system, which are characterized in that they include one or more of the PCR markers BoRa1 to BoRa26; the BoRa1 to BoRa26
  • the nucleotide sequence is shown in SEQ ID NO: 1 to 26.
  • the present invention also provides a primer set for amplifying the above-mentioned PCR marker, which is characterized in that it includes one or more pairs of primer pairs BoRa1p to BoRa26p;
  • BoRa1p ⁇ BoRa26p are respectively used to amplify the exogenous radish fragments whose nucleotide sequences are shown in SEQ ID NO: 1 ⁇ 26;
  • BoRa1p ⁇ BoRa26p The sequence information of BoRa1p ⁇ BoRa26p is as follows:
  • the present invention also provides a kit for detecting exogenous radish fragments in the cabbage-radish introgression system, characterized in that the kit includes the above primer set.
  • the primers in the kit can be in liquid or powder form.
  • the kit further contains universal reagents for performing PCR and/or electrophoresis.
  • Universal reagents for performing PCR can be DNA polymerase, PCR buffer and dNTP solution, or PCR master mix; the PCR master mix contains DNA polymerase, PCR buffer and dNTP.
  • Common reagents used for electrophoresis can be agarose, electrophoresis loading buffer, DNA ladder and nucleic acid dyes.
  • the invention also provides a method for identifying exogenous radish fragments in the cabbage-radish introgression system, which is characterized by comprising:
  • the genomic DNA of the sample can be used as a template, and one or more pairs of BoRa1p to BoRa26p are used to perform PCR respectively; the PCR product is detected to determine the presence of exogenous radish fragments in the sample. Deletions and/or sequences.
  • the PCR product can be detected by sequencing or electrophoresis.
  • the electrophoresis may be agarose gel electrophoresis. For example, 1-1.5% agarose gel electrophoresis is used. After electrophoresis separation at 130V constant power for 20 minutes, the bands are observed and photographed using a UV gel imager. If a characteristic band of the corresponding fragment size amplified by the primer pair appears in the PCR product, then the cabbage-radish introgression system sample to be tested contains the corresponding exogenous radish fragment.
  • the PCR reaction system may include: 0.5-1 ⁇ mol/L upstream primer, 0.5-1 ⁇ mol/L downstream primer and 4-10 ng/ ⁇ L genomic DNA.
  • the PCR reaction system is: 0.5 ⁇ L of 10 ⁇ mol/L upstream primer, 0.5 ⁇ L of 10 ⁇ mol/L downstream primer, PCR SuperMix 5 ⁇ L, template DNA 2 ⁇ L, ddH 2 O 2 ⁇ L.
  • the PCR reaction program can be: pre-denaturation at 94°C for 3-5 minutes; denaturation at 94°C for 30 seconds, annealing at 55-62°C for 30 seconds, extension at 72°C for 45-60 seconds, 30-40 cycles; extension at 72°C for 5-10 minutes.
  • the PCR reaction program is: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles; and extension at 72°C for 5 minutes.
  • the identification of exogenous radish fragments can be carried out at the seedling stage of the cabbage-radish introgression line to be tested.
  • the present invention successfully developed PCR markers BoRa1 to BoRa26 that can accurately identify the 14 Mb exogenous radish fragments that have penetrated into the cabbage genome, and obtained PCR markers for amplifying these 26
  • the PCR labeled primer pair BoRa1p ⁇ BoRa26p has the advantages of strong specificity and good stability. Experiments have shown that the primer pair BoRa1p ⁇ BoRa26p can amplify the target fragment of corresponding size in Brassica napus "Y403" and radish variety "RFRs" which are known to contain 14Mb radish heterologous fragments.
  • the present invention uses primer pairs BoRa1p to BoRa26p to assist in the identification of exogenous radish fragments in the cabbage-radish introgression system.
  • the operation is simple and easy, and the exogenous radish fragments contained in the target strain can be quickly identified at the seedling stage, thereby being targeted.
  • the method of the present invention does not need to wait for 2-3 months for the plant growth stage, is not affected by environmental conditions and human factors, greatly shortens the breeding cycle of the cabbage-radish introgression line, and improves improve breeding efficiency.
  • Figures 1 to 3 show the amplification of the primer pair BoRa1p to BoRa26p on cabbage varieties that are known to contain no exogenous radish fragments and Brassica napus varieties and radish varieties that are known to contain 14 Mb of exogenous radish fragments.
  • lane M is a 2000bp DNA ladder
  • lanes 1-4 are the cabbage variety "J34" (negative control), the inbred cabbage variety “Jinzaosheng” (negative control), and the Brassica napus variety " Y403” (positive control), radish variety “RFRs” (positive control).
  • the primer pair BoRa1p ⁇ BoRa26p in the positive control amplified the sizes of 514bp, 514bp, 539bp, 502bp, 574bp, 344bp, 585bp, 317bp, 377bp, 384bp, 520bp, 379bp, 362bp, 348bp, 558bp, 332bp, 378bp, 371bp respectively.
  • Figures 4 to 6 show the amplification of the cabbage-radish introgression line single plant 6GH5-14 by the primer pair BoRa1p ⁇ BoRa26p at different annealing temperatures.
  • lane M is a 2000bp DNA ladder
  • lanes 1-4 are the amplification products of the primer pairs at 55°C, 58°C, 60°C and 62°C annealing temperatures.
  • Figures 7 to 9 show the results of using the primer pair BoRa1p to BoRa26p to detect exogenous radish fragments in the cabbage-radish introgression line population.
  • Lane M is a 2000bp DNA ladder
  • lane 1 is the cabbage variety "J34" (negative control)
  • lane 2 is the radish variety “RFRs” (positive control)
  • lanes 3-8 are 6 BC 6 high-generation backcross introgression lines. Group single plant.
  • the primer pair BoRa1p ⁇ BoRa26p in the positive control amplified the sizes of 514bp, 514bp, 539bp, 502bp, 574bp, 344bp, 585bp, 317bp, 377bp, 384bp, 520bp, 379bp, 362bp, 348bp, 558bp, 332bp, 378bp, 371bp respectively.
  • Cabbage "Jinzaosheng” is a known cabbage variety and is commercially available.
  • Cabbage "J34" is a known cabbage material that has been disclosed in non-patent literature (Ren Wenjing, Creation and utilization of high-generation backcross Ogura CMS fertility restoration material of cabbage, 2021 master's thesis of the Chinese Academy of Agricultural Sciences).
  • Brassica napus "Y403" is a known Brassica napus material and has been reported in non-patent literature (Ren Wenjing, Creation and utilization of high-generation backcross Ogura CMS fertility restoration material of cabbage, 2021 Master's thesis of the Chinese Academy of Agricultural Sciences) in public.
  • Radish "RFRs" is a known radish variety. It is the Ogura cytoplasmic fertility restored radish used in this laboratory. It has been reported in non-patent literature (Ren Wenjing, Creation and development of high-generation backcross Ogura CMS fertility restoration materials in cabbage). Utilized, published in 2021 Master's Thesis of the Chinese Academy of Agricultural Sciences).
  • 96 cabbage inbred line materials They are high-generation inbred lines of cabbage, created by our laboratory, and have been reported in non-patent literature (Li Zhiyuan, KASP markers are used to construct cabbage fingerprints and classify hybrid groups, 2018 Master's thesis of the Chinese Academy of Agricultural Sciences ).
  • BC 3 to BC 6 generation backcross introgression lines The Brassica napus variety "Y403” is used as the male parent and the kale variety "Y101" is used as the female parent.
  • the hybrid F 1 generation plants obtained are mixed with the kale material "Y101""After two generations of backcrossing, the BC 2nd generation single plant 16Q2-11 was obtained, and then continued backcrossing with the cabbage material "J34" to obtain BC 3 to BC 6 generations of cabbage-radish introgression population materials, which has been reported in non-patent literature (Ren Wenjing) , Creation and utilization of fertility restoration materials for high-generation backcrossing of cabbage Ogura CMS, disclosed in the 2021 Master's Thesis of the Chinese Academy of Agricultural Sciences).
  • the key single plant of the BC 6 -generation high-generation backcross introgression line 6GH5-14 It is the key single plant in the above-mentioned BC 6- generation high-generation backcross introgression line. Its genetic background and morphological characteristics are close to the backcross cabbage parent “J34” and Fertility recovery is stable and natural fruiting is better.
  • the above biological materials can be obtained from the Cabbage Research Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.
  • PCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd., catalog number: AS111-11.
  • the reagents used in the following examples are all conventional reagents in the field and can be purchased commercially or prepared according to conventional methods in the field.
  • the specifications are laboratory pure grade.
  • the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in this field. Please refer to relevant experimental manuals, publicly known literature or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • the cabbage variety "J34”, the cabbage inbred line variety “Jinzaosheng”, the cabbage type rape variety “Y403”, and the radish variety “RFRs” stored in our laboratory with known sizes of exogenous radish fragments were used to screen and designed for expansion.
  • Add PCR labeled primers (referred to as PCR labeled primers).
  • "J34” and “Jin Zaosheng” are known to contain no heterologous radish fragments
  • "Y403" and “RFRs” are known to contain all 14Mb of exogenous radish fragments.
  • the screening method is as follows:
  • the CTAB (Hexadecyl trimethyl ammonium Bromide) method was used to extract the cabbage variety "J34", the cabbage inbred line variety “Jinzaosheng”, the cabbage rape variety "Y403", and the radish variety “RFRs” ” leaf genomic DNA.
  • genomic DNA extraction methods see Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321-4325.
  • PCR reaction system (10 ⁇ L): 0.5 ⁇ L of upstream primer (10 ⁇ mol/L), 0.5 ⁇ L of downstream primer (10 ⁇ mol/L), PCR SuperMix 5 ⁇ L, template DNA (20ng/ ⁇ L) 2 ⁇ L, ddH 2 O 2 ⁇ L.
  • PCR reaction program pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles; extension at 72°C for 5 minutes.
  • the PCR products were detected by electrophoresis using 1% agarose gel at a constant voltage of 130V for 20 min, and photographed using a UV gel imager.
  • Lanes 1 to 3 are the cabbage variety "J34", the inbred cabbage variety “Jinzaosheng”, the Brassica napus variety “Y403”, and the radish variety “RFRs”.
  • the results showed that the primer pair BoRa1p ⁇ BoRa26p amplified target bands of corresponding sizes in the Brassica napus variety "Y403” and the radish variety “RFRs” containing exogenous radish fragments, indicating that the Brassica napus variety "Y403” and radish The variety "RFRs” contains 14Mb of exogenous radish fragment.
  • the primer pair BoRa1p ⁇ BoRa26p had no amplification products in the cabbage variety "J34" that does not contain exogenous radish fragments and the inbred cabbage variety "Jinzaosheng".
  • the primer pair BoRa1p ⁇ BoRa26p to detect 96 cabbage inbred line materials stored by our research group using the same method, and no target band with a characteristic size was amplified. Therefore, the primer pair BoRa1p to BoRa26p developed in the present invention has strong specificity for exogenous radish fragments in the cabbage-radish introgression line, and there is no non-specific amplification in the genome of the cabbage inbred line material.
  • the CTAB method was used to extract the genomic DNA of the key single plant 6GH5-14 of the BC 6- generation high-generation backcross introgression line.
  • genomic DNA of a single strain 6GH5-14 as a template, PCR reactions were performed on BoRa1p to BoRa26p using the primers shown in Table 2 of Example 1.
  • 4 temperature gradients in the temperature range of 55-62°C performed PCR according to the following reaction system and reaction procedures, and compared the amplification of each primer pair in the cabbage-radish introgression system material. Increased stability over time.
  • the PCR reaction system is: total volume 10 ⁇ L, including 0.5 ⁇ L upstream primer (10 ⁇ mol/L) and 0.5 ⁇ L downstream primer (10 ⁇ mol/L).
  • the PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C, 58°C, 60°C or 62°C for 30 s, extension at 72°C for 45 s, 35 cycles; extension at 72°C for 5 min.
  • Genomic DNA of seedlings was extracted using CTAB method. Using the primer pair BoRa1p to BoRa26p shown in Table 2, a total of 21,600 cabbage-radish introgressions were conducted with the cabbage variety "J34" (negative control), the radish variety “RFRs” (positive control), and BC 3 to BC 6 generations. Using the genomic DNA of a single strain as a template, perform the PCR reaction according to the following system and procedures:
  • PCR reaction system total volume 10 ⁇ L, including 0.5 ⁇ L upstream primer (10 ⁇ mol/L) and 0.5 ⁇ L downstream primer (10 ⁇ mol/L).
  • PCR SuperMix 5 ⁇ L, template DNA (20ng/ ⁇ L) 2 ⁇ L, ddH 2 O 2 ⁇ L.
  • PCR reaction program pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles; extension at 72°C for 5 minutes.
  • the PCR reaction system is: 0.5 ⁇ L of upstream primer (10 ⁇ mol/L), 0.5 ⁇ L of downstream primer (10 ⁇ mol/L), PCR SuperMix 5 ⁇ L, genomic DNA (20ng/ ⁇ L) 2 ⁇ L, ddH 2 O 2 ⁇ L;
  • the PCR reaction program was: pre-denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, and extension at 72°C for 5 min.

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Abstract

La présente invention concerne un marqueur de PCR pour détecter un fragment de radis exogène dans une lignée d'introgression de Brassica oleracea-radis, comprenant un ou plusieurs marqueurs de PCR BoRa1-BoRa26. Les séquences nucléotidiques du BoRa1-BoRa26 sont telles que présentées dans les SEQ ID NO : 1-26, respectivement. L'invention concerne également un ensemble d'amorces pour amplifier le marqueur de PCR, comprenant une ou plusieurs paires de paires d'amorces BoRa1p-BoRa26p.
PCT/CN2022/117836 2022-09-08 2022-09-08 Marqueur de pcr pour détecter un fragment de radis exogène dans une lignée d'introgression de brassica oleracea-radis, amorce et utilisation de celui-ci WO2024050761A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008123714A1 (fr) * 2007-04-06 2008-10-16 Dongbu Hitek Co., Ltd. Procédé de production d'une semence hybride utilisant une plante de la nouvelle lignée raphanus sativus à stérilité mâle génique cytoplasmique et marqueurs d'adn utilisés pour sélectionner la plante de ladite lignée raphanus sativus
CN104805212A (zh) * 2015-05-08 2015-07-29 中国农业科学院蔬菜花卉研究所 用于甘蓝Ogura胞质不育系育性恢复基因Rfo筛选的PCR标记
CN107541517A (zh) * 2017-08-24 2018-01-05 湖南省作物研究所 一种外源萝卜片段特异标记及其制备方法和应用

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2008123714A1 (fr) * 2007-04-06 2008-10-16 Dongbu Hitek Co., Ltd. Procédé de production d'une semence hybride utilisant une plante de la nouvelle lignée raphanus sativus à stérilité mâle génique cytoplasmique et marqueurs d'adn utilisés pour sélectionner la plante de ladite lignée raphanus sativus
CN104805212A (zh) * 2015-05-08 2015-07-29 中国农业科学院蔬菜花卉研究所 用于甘蓝Ogura胞质不育系育性恢复基因Rfo筛选的PCR标记
CN107541517A (zh) * 2017-08-24 2018-01-05 湖南省作物研究所 一种外源萝卜片段特异标记及其制备方法和应用

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Title
QI-DONG YANG, XI KUN; DONG JUN-GANG; ZHANG BO; XU TING: "Molecular markers of Ogura CMS restorers R2572 in Brassica napus", JOURNAL OF NORTHWEST A & F UNIVERSITY(NATURAL SCIENCE EDITION), vol. 44, no. 4, 14 March 2016 (2016-03-14), pages 57 - 63, XP093146896 *
WANG, TONGHUA ET AL.: "Genetic characterization of a new radish introgression line carrying the restorer gene for Ogura CMS in Brassica napus", PLOS ONE, vol. 15, no. 7, 28 July 2020 (2020-07-28), XP055919347, DOI: 10.1371/journal.pone.0236273 *

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