CN114350655B - Exogenous radish fragment specific marker and preparation method and application thereof - Google Patents
Exogenous radish fragment specific marker and preparation method and application thereof Download PDFInfo
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Abstract
The application relates to a preparation method and application of a specific marker of exogenous radish fragments of cabbage type rape and a specific marker of exogenous radish fragments, and specific molecular markers CLR9-1 to CLR9-20 of exogenous radish fragments are developed through comparing complete genome re-sequencing data of a cytoplasmic sterile restoring material CLR650 of the cabbage type rape with genome sketch information of the radish. Obtaining BC through a target single plant of a cytoplasmic sterile restoring material CLR650 backcross population 4 Isolating the population; BC will be added 4 The fertile plants in the population are mixed and selected for multiple generations by adopting a mixing method until a mixed separation population with the proportion of the fertile plants exceeding 75% is obtained; and finally, carrying out homozygous stabilization through multi-generation selfing to obtain the cytoplasmic sterile restorer line of the brassica napus radish. The application overcomes the defects of serious lower genetic transmission and difficult homozygosity and stability of the restoring gene in the prior method.
Description
(the application relates to a division application of an exogenous radish fragment specific marker, a preparation method and application thereof, the application date of the original application is 2017, 8 and 24, the application number is 201710735768.7, and the application creates the name of the exogenous radish fragment specific marker, the preparation method and application thereof.)
Technical Field
The application belongs to the technical field of rape breeding and the field of molecular biology, and particularly relates to an exogenous radish fragment specific marker and a preparation method of the exogenous radish fragment specific marker, and further relates to application of the exogenous radish fragment specific marker in cabbage type rape breeding.
Background
Rape has obvious heterosis, and is an oil crop with the most potential for coping with edible vegetable oil shortage and developing renewable biological energy sources at home and abroad. The radish cytoplasmic male sterility (Ogu CMS) is an ideal type of sterility for utilizing hybrid vigor of brassica napus, and is a cytoplasmic male sterility system established by the sterile source found in radishes in 1968 in the japanese ministrator. The sterility of the sterility system is very stable and thorough, so that the sterility system has very important value in the utilization of heterosis of cruciferae crops. With the continuous deep research of rape molecular biology, the genome sequencing technology is perfected, the sequencing cost is further reduced, and the development of molecular markers and the application of auxiliary selection technology are increasingly mature and practical, so that the technology support is provided for researching the cytoplasmic sterile recovery material of the brassica napus.
The recovery gene of radish was successfully introduced into brassica napus by intergeneric crossing in 1976 by Heyn (Heyn, 1976). Subsequently, pelletier et al, france, also introduced restorer genes in European radishes into Brassica napus by protoplast fusion techniques. However, when the restoring gene is transferred from the radish to the brassica napus, redundant radish fragments also permeate into the genome of the brassica napus, so that the restoring material has the problems of unsatisfactory restoring capability, partial female sterility, linkage of higher glucosides and the restoring gene and the like.
The cabbage type rape ogura-CMS system was introduced in the 80 s of the last century in China, and a great deal of research was subsequently carried out by rape breeding institutions in China. As Li Xufeng, it has been reported that the restorer gene was introduced into rape from radish variety Lan Huazi, but the restorer genes of the obtained material are all on episomes, but are not stably inherited; in 2015, hua Jinshui et al hybridized with the sterile line using the brassica napus restorer R2000 from INRA, france, then F 1 The R2000 is genetically improved by recovering individual plants with changed generation radiation mutation screening markers, but the recovery source and the molecular markers used by the strain are all from the research in foreign countries. There is no molecular marker developed autonomously in China that can be used for the breeding of the ogura-CMS restorer.
Disclosure of Invention
The application aims to solve the technical problems and overcome the defects and shortcomings in the background technology, and provides a target gene specific marker developed based on a genome re-sequencing technology and a comparative genomics technology, a preparation method thereof and application thereof in cabbage type rape breeding. The specific molecular marker is derived from exogenous radish fragments, the preparation method is simple, feasible, accurate and effective, and can be combined with conventional hybridization, backcross, mixed selection and selfing breeding technologies, so that the problems of serious low genetic ability, difficult homozygous stability of the character and the like of distant hybridization exogenous target genes in the breeding process of the cabbage rape radish cytoplasmic sterile restorer in China can be effectively solved.
A great deal of molecular biology and cytogenetic analysis are carried out on a novel cabbage type rape Ogu CMS restoring material CLR650 which is autonomously cultivated by the crop institute of Hunan province, and the result shows that the restoring material chromosome group is stable, and the fertility restoring gene is an Rfo gene. Based on the problems of serious fertility bias separation and difficult stability of characters of the CLR650 hybrid offspring, specific molecular markers of exogenous radish fragments are independently developed through genome resequencing and reference genome sequence comparison. The specific markers of the radish segments comprise CLR9-1, CLR9-2, CLR9-3, CLR9-4, CLR9-5, CLR9-6, CLR9-7, CLR9-8, CLR9-9, CLR9-10, CLR9-11, CLR9-12, CLR9-13, CLR9-14, CLR9-15, CLR9-16, CLR9-17, CLR9-18, CLR9-19 and CLR9-20. The exogenous radish fragment specific marker CLR9-1 has the sequence shown in SEQ ID No:1, and a nucleotide sequence shown in the specification.
The exogenous radish fragment specific marker CLR9-2 has the sequence shown in SEQ ID No:2, and a nucleotide sequence shown in the following formula.
The exogenous radish fragment specific marker CLR9-3 has the sequence shown in SEQ ID No:3, and a nucleotide sequence shown in 3.
The exogenous radish fragment specific marker CLR9-4 has the sequence shown in SEQ ID No:4, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-5 has the sequence shown in SEQ ID No:5, and a nucleotide sequence shown in SEQ ID NO.
The exogenous radish fragment specific marker CLR9-6 has the sequence shown in SEQ ID No:6, and a nucleotide sequence shown in FIG. 6.
The exogenous radish fragment specific marker CLR9-7 has the sequence shown in SEQ ID No: 7.
The exogenous radish fragment specific marker CLR9-8 has the sequence shown in SEQ ID No:8, and a nucleotide sequence shown in SEQ ID NO.
The exogenous radish fragment specific marker CLR9-9 has the sequence shown in SEQ ID No: 9.
The exogenous radish fragment specific marker CLR9-10 has the sequence shown in SEQ ID No:10, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-11 has the sequence shown in SEQ ID No:11, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-12 has the sequence shown in SEQ ID No:12, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-13 has the sequence shown in SEQ ID No: 13.
The exogenous radish fragment specific marker CLR9-14 has the sequence shown in SEQ ID No:14, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-15 has the sequence shown in SEQ ID No:15, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-16 has the sequence shown in SEQ ID No:16, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-17 has the sequence shown in SEQ ID No:17, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-18 has the sequence shown in SEQ ID No:18, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-19 has the sequence shown in SEQ ID No:19, and a nucleotide sequence shown in seq id no.
The exogenous radish fragment specific marker CLR9-20 has the sequence shown in SEQ ID No:20, and a nucleotide sequence shown in seq id no.
The application also provides a preparation method of the exogenous radish fragment specific marker, which comprises the following steps of:
(1) Hybridization is carried out between the cabbage type rape radish cytoplasmic sterile restoring material CLR650 serving as a female parent and the conventional cabbage type rape strain A to obtain F 1 Population, select multiple strains F 1 The group plants are subjected to bagging, isolation and selfing to obtain F 2 Fertility separation of colony single plants, and extracting genome total DNA of each single plant; investigation of the full flowering phase F 2 The fertility of single plant of the colony, set up the gene mixing pool of the fertility plant and sterile plant separately;
(2) Carrying out whole genome re-sequencing on a cabbage type rape radish cytoplasmic sterile restoring material CLR650, comparing a sequencing result with a radish genome sketch and a radish cytoplasmic sterile restoring gene sequence to obtain a restoring gene of the CLR650 and exogenous radish sequence information beside the restoring gene, and designing a specific primer according to a target region;
(3) Specific primers to be obtainedScreening by using a gene mixing pool, reserving primers positive for PCR amplification of the fertility gene mixing pool and negative for PCR amplification of the fertility gene mixing pool, and placing the screened primers in F 2 Co-separation verification is carried out on individual plants of fertility isolated population, and primers for co-separation of PCR amplification positive expression and fertility expression are used as exogenous radish fragment specific markers.
Preferably, the conventional line A of Brassica napus in the step (1) is 20B. The genome of the conventional strain is in a relatively homozygous state, the filial generation does not have severe segregation phenomenon, and adverse effects of segregation of the offspring character on homozygosity and stability of the restorer are reduced to the greatest extent. In addition, the conventional strain does not contain other types of restorer genes, does not have other bad cytoplasmic effects, and is more beneficial to molecular marker screening of offspring population and obtaining of target strains.
Preferably, in the step (1), a plurality of strains F are selected 1 The group plants are subjected to bagging isolation selfing, namely 6-8 plants F are selected 1 And (5) bagging, isolating and selfing the group plants.
The molecular marker is obtained by comparing a genome resequencing result with a radish genome, is derived from the information of the radish genome, is not only limited to the region where a restoring gene is located, but also relates to a sequence adjacent to the restoring gene, and can more effectively detect the change of exogenous radish fragments permeated into the rape genome in the process of transformation.
As a general technical concept, the application also provides an application of any one of the exogenous radish fragment specific markers CLR 9-1-20 in cabbage type rape restorer line breeding, which comprises the following steps:
1) Hybridization is carried out between the cabbage type rape radish cytoplasmic sterile restoring material CLR650 serving as a female parent and the conventional cabbage type rape strain A to obtain F 1 A population; selection of multiple strains F 1 The generation plant and the conventional line A of the cabbage type rape are used as recurrent parent to carry out backcross, and BC is constructed 1 Isolating the population;
2) In seedling stage, BC is specifically marked by using at least 1 exogenous radish fragment 1 The molecular marker assisted selection is carried out on the separation group,removing the single plant with negative analysis result; selection of multiple plants BC during flowering phase 1 The single plant with the surface form close to (i.e. similar in character to) the conventional line A of the cabbage type rape in the segregating population is used as a female parent, and the conventional line A of the cabbage type rape is continuously used for backcross; repeating the above steps until BC of cabbage type rape CLR650 is obtained 4 Fertility segregating populations;
3) Pulling out BC 4 Sterile single plants in fertility isolated population, carrying out mixed pollination and harvesting on all the reserved fertility single plants in an isolation area, and investigating fertility expression of offspring population in a full-bloom period until a population with the proportion of the fertility single plants exceeding 75% is obtained;
4) And (3) carrying out all isolation selfing on the fertile plants in the segregating population with the proportion of the fertile plants exceeding 75%, and continuously selecting all isolation selfing on the fertile plants in the selfing offspring until the cabbage type rape radish cytoplasmic sterile restorer line CLR095 with the insemination and consistent phenotype is obtained.
Compared with the prior art, the application has the beneficial effects that:
1. the molecular markers related by the application are exogenous radish fragment specific markers which are independently developed according to the CLR650 genome resequencing result, have pioneering significance, and can provide a novel molecular marker technical support for the transformation of cabbage type rape radish cytoplasmic sterility restoration sources in China;
2. the breeding is performed by adopting a conventional breeding means and a molecular marker assisted selection method, so that preliminary selection can be performed in a seedling stage, on one hand, non-target single plants can be pulled out in advance, and the workload of test land and later phenotype identification is reduced; on the other hand, the selection efficiency and accuracy can be greatly improved, and the influence of mechanical mixing and biological mixing in the breeding process can be timely eliminated;
3. the novel cabbage type rape radish cytoplasmic sterile restorer obtained by the application has stable heredity, eliminates the distant hybridization unfavorable character carried by CLR650 to a certain extent, and can provide a cabbage type rape radish cytoplasmic sterile restorer with pioneering significance for China;
4. the application adopts the directional transfer of backcross, then through mixed selection, the probability of target gene homozygosity is increased to the greatest extent, finally through the self-copulation and stable breeding strategy, the barriers of serious low gene genetic inheritance, hard stability of the character and the like caused by distant hybridization can be overcome, the self-copulation recession phenomenon caused by continuous multi-generation self-copulation can be avoided, and the strategy is proved to be easier to obtain the homozygosity stable radish cytoplasmic sterile restorer line by practice.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the transformation procedure of the Brassica napus Ogu CMS restorer CLR095 according to an embodiment of the application.
FIG. 2 is the F of the present application in the cabbage type rape radish sterility restoring material CLR650 2 PCR amplification between mixed pools of fertility segregating population genes.
FIG. 3 is the F of the present application in the cabbage type rape radish sterility restoring material CLR650 2 And (3) separating a population single-strain PCR amplification result, wherein F represents a fertile strain in the figure, and S represents a sterile strain.
FIG. 4 is a comparison of the cytoplasmic sterile restorer line CLR095 of Brassica napus radish obtained in the application with CLR650, FIG. 4: a: leaf comparison in seedling stage; b: flower organ comparison; c: and comparing the fruits. Fig. 4 is a graph of CLR650 as a comparison, and it can be seen from fig. 4 that CLR095 has significantly changed leaf, flower and pod lengths.
FIG. 5 is a comparison of the cytoplasmic sterile restorer line CLR095 of Brassica napus radish obtained in the application with CLR 650. Fig. 5 is a graph of CLR650 as a comparison, and it can be seen from fig. 5 that CLR095 has significantly changed leaf, flower and pod lengths.
Detailed Description
The present application will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the application, but the scope of the application is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present application.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.
Example 1:
the breeding method of the cabbage type rape radish cytoplasmic sterile restorer line CLR095 is shown in the figure 1, and specifically comprises the following steps.
1. Development of specific molecular marker and linkage marker of radish cytoplasmic male sterility restoring gene
1.1 implementation materials
The cabbage type rape radish cytoplasmic sterile restoring material CLR650 (which is radish male sterile cytoplasm, zigzag leaf margin, sharp leaf tip, dark green leaf color and thick and short horn, see document Chen Weijiang and the like, is prepared by the steps of preparing the cabbage type rape radish cytoplasmic male sterile restoring material, chinese agricultural science 2012, 45 (8): 1465-1474), and is obtained by hybridizing and breeding of scindapsus aureus-blue (AACRR, 2n=56) and cabbage type rape by a grafting technology, wherein the chromosome number is 38-40. The cytoplasm of the strain is the radish male sterile cytoplasm, and the nucleus contains the radish cytoplasm recovery gene Rfo of CLR650, so that the strain is normally fertile; the conventional line 20B of the cabbage type rape (the cytoplasm of the conventional cabbage type rape, the leaf margin being cut, the leaf tip being medium, the leaf color being light green, the pod being slender, see document Li Mei, etc., the breeding of the new variety of the cabbage type hybrid rape being rich in oil 730, hunan agricultural science, 2008, (6): 19-20) is a double low conventional line of the semi-winter cabbage type rape.
1.2 radish cytoplasmic Male sterility restoring gene F 2 Construction of segregating populations
Spring 2011, when the CLR650 enters the early flowering phase, the CLR650 plant is still onRemoving stamens of unopened bud, and hybridizing with 20B to obtain F 1 Seed; f to be obtained in 5 th 2011 1 Seeds are sown in a nutrition pot in a climate chamber to obtain 20F 1 Selecting 8 strong single plants in the early flowering stage, and performing bagging isolation selfing by using parchment bags to obtain F 2 Separating the colony seeds; 1 part of F is selected in 10 months 2011 2 Sowing seeds in a rape test field after the paddy-upland rotation of the long sand to obtain F 2 Separating plant of colony for fertility, separating individual plants in 3-5 leaf stage, hanging, and selecting 2-3cm from each individual plant 2 The total DNA was extracted from young leaves of a size as described in Li Jia et al (Li Jia et al, a method for efficiently extracting total DNA from rape leaves, university of Huazhou agricultural journal, 1994, 13 (5): 521-523), and the DNA concentration was measured by agarose gel electrophoresis of 0.8% to measure the DNA quality and an ultraviolet spectrophotometer (Pharmacia Biotech, geneQuant II). Investigation and recording F when the next year rape enters the full bloom stage 2 Fertility performance of individual plants in the population. The F2 isolated population obtained by the application has 210 single plants, wherein the number of the fertile single plants is 74, and the number of the sterile single plants is 136. From F according to fertility investigation result 2 In the isolated population, 10 fertile single plants and 10 sterile single plants are respectively selected for equal amount mixing of total DNA, 1 fertile plant mixing pool and 1 sterile plant mixing pool are respectively obtained, and the target fragment molecular marker is developed for standby.
1.3 design of specific primers for cytoplasmic sterile restorer genes of radish based on CLR650 genome re-sequencing
Sowing CLR650 seeds in a nutrition pot, but taking 3-4cm when growing into 3-4 true leaves 2 The young leaves were sent to a re-sequencing analysis by Shanghai European biomedical technologies Inc. using the Illumina Miseq platform to obtain sequencing data of one-way 67.169G base number and 447,797,399 fragment number. Wherein the clear ready ratio reaches 96.09%, the high quality base ratio reaches 99.21%,>71% of the genome reaches a sequencing depth of 50X or more. Then the sequences are respectively aligned with the published radish genome on NCBI and the 127Kb region of the radish cytoplasmic sterility restoring gene Rfo,obtaining sequence information of radish genome Scaffold 131 where radish cytoplasmic sterility restoring gene is located, and deducing that exogenous radish cytoplasmic sterility restoring gene in CLR650 is located in radish genome R 9 On the chromosome.
1.4 selection and verification of specific molecular markers of radish cytoplasmic male sterility restorer genes
R for radish 9 The published sequence of Scaffold 131 and the region sequence of 127Kb where Rfo is located were together designed with 80 pairs of radish specific primers using Primer3 Primer design tools. Firstly, carrying out PCR amplification on the 80 pairs of primers in the fertile plant mixing tank and the sterile plant mixing tank in the step 1.2, screening primers with positive performance of the fertile plant mixing tank and negative performance of the sterile plant mixing tank, and then, putting the obtained primers in F 2 Co-segregation verification was performed on fertility segregating populations (see FIGS. 2, 3), and 20 specific markers labeled as exogenous radish fragments were determined by verification: CLR9-1, CLR9-2, CLR9-3, CLR9-4, CLR9-5, CLR9-6, CLR9-7, CLR9-8, CLR9-9, CLR9-10, CLR9-11, CLR9-12, CLR9-13, CLR9-14, CLR9-15, CLR9-16, CLR9-17, CLR9-18, CLR9-19, CLR9-20 (primer sequences and product sequences for each specific tag are shown in table 1, table 2, respectively).
Table 1: primer sequence and product fragment size of radish specific marker of the application
TABLE 2 PCR product sequence of radish specific markers of the application
2. Breeding of radish cytoplasmic male sterile restoring line
2.1 molecular marker assisted selection backcross breeding
Spring 2011, F obtained in the step 1.2 is obtained 1 Backcrossing the plant with 20B to obtain BC 1 Seed; BC to be obtained 10 months 2011 1 After seeds are sown in a long sand rape test field, BC of CLR650/20B is obtained 1 And (5) separating the plant of the colony. Because of the serious segregation phenomenon of the radish cytoplasmic sterility restoring gene in the CLR650 offspring, the application utilizes the combination of the exogenous radish fragment specific markers CLR9-3, CLR9-12 and CLR9-18 obtained in the step 1.4 to perform BC 1 And 3-5 leaf stages in the isolated population are selected, and the single plants with negative marker analysis results are pulled out. The fertility investigation is carried out on the reserved fertile single plants in the full bloom stage of the spring rape in 2012, the main inflorescence of the fertile plants with the phenotype character close to 20B is subjected to sleeve isolation selfing by using a parchment bag, and the non-opened flower buds on branches are subjected to backcross with 20B after stamens is removed, thus obtaining BC 2 Seed of the isolated population. The shape, the length and the number of grains of the horns of each backcrossed individual plant under the natural pollination condition are investigated in the maturity period, the natural pollination seeds of the backcrossed individual plants are harvested by the individual plants, three quality character analyses of oil content, erucic acid content and sulfuric glucoside content are carried out by utilizing a near infrared analyzer, the individual plants with the phenotype and the quality character closest to 20B are reserved, and the backcrossed population with the phenotype consistent with 20B and the improved quality character is obtained by continuously using 20B as a recurrent parent.
The application is from BC 1 Out of 163 individuals in the isolated population, 31 fertile individuals with positive molecular marker analysis results are obtained, wherein 10 phenotypic traits are relatively close to 20B. The 10 BC obtained were subjected to a near infrared analyzer (Matrix-1, bruker, germany, OPUS/QUANT5.5 software) 1 The selfing seed of the fertile single plant is subjected to three quality character analyses (the results are shown in table 3) of oil content, erucic acid content and glucosinolate content, and BC of the backcross single plant with the lowest glucosinolate content, oil content of more than 35% and erucic acid content of less than 5% is reserved 2 And (5) separating the group seeds. The BC is prepared in the last 5 th year of 2012 2 Separating group seeds, sowing the seeds in Qinghai Xining rape test field to obtain BC 2 Segregating population plants and continuing according to BC 1 Further backcrossing until a phenotypically consistent BC is obtained 4 And (5) separating the plant of the colony. BC with relatively consistent phenotype was obtained in Qinghai in 2014 4 And (3) separating plant population, wherein the total of 191 single plants in the separating population is 191, 52 single plants show normal fertility and 139 single plants show cytoplasmic male sterility of the radish.
TABLE 3 cabbage type rape radish quality sterility recovering material BC obtained by the application 1 -BC 4 Quality analysis results of selected fertile single plants in population
2.2 homozygous and stable cytoplasmic sterile restorer line of Brassica napus radish
In the full bloom stage, according to the fertility investigation result of the initial bloom stage, the BC obtained in the step 2.1 is obtained 4 Pulling out sterile single plants in the isolated population, carrying out isolated mixed pollination on the whole reserved fertile plants, carrying out mixed harvesting after maturation, and continuing to reserve the fertile plants in the isolated population for isolated mixed pollination for the next generation until the isolated population with the proportion of the fertile plants exceeding 75% is obtained. Then, isolating and selfing all the fertile single plants in the obtained isolated population by using a parchment paper bag until a plant line with insemination not isolated is obtained, namely new cabbage type rape radish cytoplasmSterile restorer line.
The application fully carries out isolation mixed pollination on the 54 reserved fertile plants in 2014 spring, and carries out mixed harvest after maturation. Sowing the mixed harvested seeds in a long-sand rape test field in 10 months in 2014 to obtain 155 individual fertility isolated groups, wherein in a full-bloom period in 2015, the individual fertility survey of the groups shows that the proportion of the fertile plants is about 30%, and the proportion of the fertile plants of 1 isolated group (field number 14Q 017) is about 50%; and selecting 5 fertile strains in the segregating population 14Q14 to continue paired sister crossing, so as to obtain 10 sister crossing segregating populations. 10 segregations were sown in the Hunan Changsha rape test field in 10 months 2014, fertility performance of each group was investigated in the next year full bloom period, the proportion of fertile plants of 8 segregations (field numbers 14C041, 14C043, 14C044, 14C045, 14C046, 14C047, 14C048, 14C 050) was about 30%, and the proportion of fertile plants of 1 segregations (field number 14Q 042) was about 50%; the proportion of the fertile plants of 1 segregating group (field number 14C 049) is about 70%; all fertile individuals in the segregating population 14Q049 were isolated for selfing with parchment bags to obtain seeds of 32 inbred lines. Seeds of the 32 inbred lines are sown in Qinghai-Xining rape test fields in 5 months of 2015, fertility investigation is carried out on each inbred line in full bloom period, and an inbred line with a fertility rate of about 90 percent is obtained (field number 15Q 028). The fertile single plants of strain 15Q028 were isolated for selfing with parchment bags to obtain seeds of 28 selfing strains. 10 parts in 2015, seeds of the 28 inbred lines were sown in a Hunan Changsha rape test field. In the next full bloom stage, fertility investigation is carried out on each inbred line to obtain a fully-fertile inbred line (field number 15C 048), and three quality trait analyses of oil content, erucic acid content and sulfuric acid content of the inbred seed of the line 15Q028 single plant are carried out by utilizing a near infrared analyzer (Matrix-1, bruker, germany, OPUS/QUANT5.5 software), wherein the oil content is 38.90%, the sulfuric acid content is 68.65 mu mol/g and the erucic acid content is 0.7%.
10 parts of 10-year selfing seeds of a strain 15C048 are sown in a Hunan Changsha rape test field, and fertility investigation in the next year full bloom stage shows that the fertility of the single plant of the strain is all normal (field number 16C 095), and the strain is named as a cabbage type rape radish sterile recovery line CLR095.
2.3 investigation of the Properties of the cytoplasmic sterile restorer line of Brassica napus radish
The restorer line CLR095 obtained in step 2.2 was sown in a single row under natural conditions, repeated 2 times with a row spacing of 0.27m and a plant spacing of 0.15m (10 plants per row), and CLR650 and 20B were used as controls, respectively. Leaf color, leaf margin shape and free pollination in flowering phase are inspected in seedling stage, petal shape and setting state are inspected, plant height, primary branch number, primary branch horn number, horn length and fruit grain number per horn are inspected in mature stage, and three quality characters of oil content, erucic acid content and glucosinolate content are analyzed, and specific results are shown in table 4. The recovery line CLR095 obtained by the application removes the adverse effect of exogenous redundant radish segments in genome to a large extent, and the phenotypic agronomic characters such as quality, leaf shape, leaf color and pod shape are obviously different from the original cabbage type rape radish quality sterile recovery material CLR650 (see fig. 4, 5 and table 4), which shows that the breeding method of the application is effective in breeding the cabbage type rape radish quality recovery line.
TABLE 4 trait expression of Brassica napus radish sterility restorer CLR095
Interpretation of related terms:
cytoplasmic male sterility: the term of art is a natural phenomenon widely present in higher plants, manifested by maternal inheritance, pollen abortion and pistil normal.
Strain: the technical term is that the early-stage materials in the breeding process have a certain number of similar individuals and are collectively called before the variety comparison test.
And (2) PCR: in technical terms, a molecular biological technique for amplifying specific DNA fragments.
Low sulfur glycoside: the technical term is that the thioglucoside content is less than 30 mu mol/g as the standard of low-sulfur glucoside materials.
Double low-grade technical terms, namely the technical terms of the application, namely short for low erucic acid and low glucosinolate.
Sister cross: in terms of the technical terms, sister lines or single plants which are similar in shape and are cultivated by the same original parent are hybridized with each other.
Sequence listing
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ggaatactct ctttaaaaat ttccaaaata ttttttaaat tattattgaa attttttaac 180
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ataaccctgg ccaccccgac aaaagctacg actggtttcc gttttttact atgtattaat 300
gcaatcttat tattattatt aactaggtaa gaccgttgcg atatggaatt ccaatccccc 360
tagagaataa aactcctcca atccaaaccc aaggtggggg tcaaccgaat tcggtaaacc 420
ccggccggta aacaaagaa 439
<210> 4
<211> 364
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
caggggggac aacaaacaaa tatgagtaat taccaagatc tttttttttt tgccccccat 60
tgcccctgaa atttgttcaa tttcccccct gtctaactat ttgggaaccc ccccttatgc 120
ccgaacccca aaaaaccgaa aggccccgcg ggggggctcc cccaaggggg gacccccccc 180
ccgcgcctcc ccgttttttg gggatccccc cctacatggg gccccccccc aacttaaccc 240
acggccggat tttaaccctt ttattttctt ccggggccca aaaaaaaaaa aatttgggaa 300
aaaaaaaaat ttttttaaat tttttggggg gggaaacttt tgttttcccc aaagtggaaa 360
aaaa 364
<210> 5
<211> 367
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tcggaaaggg caagaagcaa gttgctgttt aattccggat cctttttgct tcgtcatttt 60
atattgattt acaaaatttc tttaaatgtc tactttaatt gggctccctg aggttttccc 120
agttagggcc cttactggca aacacatcca gagcccaatt ccagtttacc cttaatttaa 180
ctttttcaaa tcaatttaaa aagaagatta tcaaaggggt ccggcgtgaa atcagccccg 240
gaaaaaaaaa aacaaaagaa aaaaaaatct ggaacagcgc tgccccacga ggattatcgg 300
ggtgccatga cttcacataa aaacatgaac taggtaatac cccggccaaa atggaaaaaa 360
aaatgat 367
<210> 6
<211> 349
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tcgcgaggtg caagagggag gcaaggacgc gtaaaaagta ctggtatggt attaattcgc 60
gtagaataag tgacaaaata tttagtttta atatggcaca caagattaga caagaagacg 120
ttgacgaata aaaaaaaaaa aaaaaaaact ttaacggggt cgttgggggt ttaaattttg 180
gtttttaccc tttccccgga aaaccctttt taaaaaaaaa aaaaagatcc atcatgtagg 240
actttgacct ggccacccag acaatgcaac gactggtttc cttttttact atgtattaat 300
gcaatcatat tattaccatt aactaagtaa gaccctgcca aaatggaat 349
<210> 7
<211> 364
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ccgaagggaa caaaagaagg aaaacgaaaa ataaaaaaaa gaagttaaga catctttttc 60
ttttgttaat ataaataaac aaccagacca acattgatct gccttttcct ttgtggatta 120
aaatcaattg ggccaccaaa atcaaataat ccccaatcat gggtattaat cccttctgga 180
ggccccactc aattttgccc aacaaaaaaa tcgctttact ttttttgctt tgtttatttt 240
tttatattac caaaaaaaaa aaattttttc cccattttaa ttaaaaggga aaagcgggaa 300
aataaaaatt tctttttttt ttattatggg gtctaaccct tttcctttgg tttaaattaa 360
aaaa 364
<210> 8
<211> 374
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ccgatcggaa aaagctgaag gacgttcaat ccttattccc tttttgattt gtaaactatg 60
gcatctattt tttttattaa tattaacaaa caaacaaaaa aaaattgatc ttgcttttcc 120
attgatgatc aaaatcgatt ggacgaccaa gaccagatta atacccgaca tctgcgatta 180
tccatgaagg aggcacaact caaattatca caacaagaaa aatgctttac tttttgtgct 240
ctgcttattt atttatataa ccaaaaaaaa aaaatttttt tccctattta attaaaatgg 300
aaaaaaagga aaaacaaaaa ttccttgtat tttattattg tgtcttacct tttttccttg 360
ttttccaata aaaa 374
<210> 9
<211> 392
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gcgccgcgcg aagagagaaa gcgcagtgta tgttagtgat gatataaact tctatttgag 60
gctgatgcct gaaaaaaatt ggatattttc ttatactttc gatattttgg ataaaacata 120
ttaggaaaat tttggatgat caaatatttg attctacttt aatttttcaa atatgttata 180
tcttttaaat acattttttt tttttatatc tattttttgt tatgccacta cctcatatgt 240
atgtttatat cttctctttc tacattccca ttgaggggat gtgcgaggcg gggattgtgt 300
aagacgtaca actctcctgt ttcgctcttt gattttgggc gggggggtca acccgttttt 360
ctggggatca aaaaaaaaaa aagggggggg gg 392
<210> 10
<211> 348
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
taggagacgg gagaaccaaa ctgtgagtaa tactgcgtga gagaaatggt cttcctgctc 60
agatctctcc ctttcactaa agataaataa accctaaaac tttcccgcag tttctttctc 120
cttccgagtt tccctaaaag tacgaactac tcctcttcaa agacccatct gcttgaatta 180
ccaaaatcat cagcaaaaat acccaggtaa aaattactgc ttttacttct tcatctttat 240
aagggccttt cacttttttt tttttttttt cccgtttttg gttttttacc cggggggggg 300
gggttttccc tttttaactc tttcccttaa aagggggggt cgaaaaaa 348
<210> 11
<211> 369
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tccgagccga gagaaagaag agtgcaacga atttcatcga actggtcgtc ctcctttggg 60
cggcatgact cctcctcttt cttcatccat cttgtcttac gtcgcttcga ctctcgcggg 120
gggttcttct tcctgaacct ctcacccatt catctctcct tccttttttc ccttactctc 180
tctctctctc tctctatctc ttcctccccc atagacgtgc ttcacgttgt taggttcttt 240
cattttttcc tggaacataa agaatacacg tgtcttcttc ctcacccctc ccgttattta 300
taaaaaaaga tttctttgta ttttataggc gggggggccc cctttccttt tcgagtgcaa 360
aaaaaaaaa 369
<210> 12
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
tcggtgctgg taggcgggaa ctttacctcg atcctaaggg tatatttcaa gattgctgta 60
agttcggtga catatatatg ataatatatt gttccaattt tttaatatat atatatatat 120
atatatatat agagagtgta tttaaattat ttttttactg tatacccatc tgaaaaaaat 180
gtgtacttga ttttcgtaca aaaattactc tccctcccgt ttctccgggg gggtatataa 240
aaaaatctat cctgggatgg cccccgccct ctaaatatag ggtttaaagg aaatttcaaa 300
accgttacgt tttttacgcc ccccgccccc cccggtttcc ggggagtaaa ataaaaaaag 360
<210> 13
<211> 385
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tacaatataa atgaaatttt atttgtgatc ttgccgctcc caactcgagc gaaaccaggt 60
acgcaaaacg ttacatatat gttaggttgt tgccctttgg cccaattttt tctaatttgc 120
ccctcccttt aaaaaaaaac caaattaaaa ccgcctttaa aaaaaatttt cccccaagac 180
gggcaaaaaa aaaaaggggt ttttaatttt ttttcaaaca aaaaaaattt gccaaaattt 240
ttctttgttt ctttccaaaa aaaaaccata ataattttga attttttttg acccggtttc 300
cccccgaggg ggggccccac aacccgcccc cccagggggg ggaaaattcc cccccccctt 360
tttgaaccga cataagggaa aattt 385
<210> 14
<211> 367
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
taactataaa aaattttttc cgcgttacca ctcttatact accagttgct tatggtaaac 60
tactattgga tagtaatcta gtggatttat gctattctgc ctactggtgg ttttccttgg 120
ctgttgaaaa aaaaccccga ggggacgaag aaaaaacatg tatctatata tatatatata 180
tatatatata tatacatata tctatatacc catatatcca aaacccgggt tgtctttttt 240
ctttctcaca atacattatt acaacaggga cactttttcc tctcgctccc aatcaaacgg 300
gggagaaggc accaacaaaa aaaaaaaaaa aggagcctgg tctcaagccg attaaaaagg 360
ggccaat 367
<210> 15
<211> 566
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gaaggttaaa gataagacga gcttgctttg caggagaatc tatggaattc cttaagcttg 60
gccgacgaac ccgaagcctg aggacacgcc gctttctcct ccggcgactt cgcctccgcc 120
gtcaaccact tcaccgaagc gatcaacctc gccccgacca accacgtcct ctactccaac 180
cgctccgccg cccacgcctc cctcctccgc tacgaggaag ctctctccga cgccaagaag 240
accgtcgagc tcaaacccga ctgggccaag ggctacagcc gcctcggcgc cgctcacctc 300
ggcctcaacc aatccgacga agccgtcgag gcctgctcca agggtctcga gatcgatcca 360
agcaacgacg cgctttaatc gggttctgca gacggcttcg agggtccgcg ccgcccctcc 420
ccccccgaat ccgtttgggg acgcgttcaa ggggccagag catgtgggcg aaggtgaggg 480
cggatccgtc gacgaggggg ttcttgtgtg agcctgactt cgtcaacatg atgcaggaga 540
tgcagaggaa tcctataacg ggaaaa 566
<210> 16
<211> 311
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
taaggcatag gcagaagagc aactgctcaa gatttctact taaactgaaa cattttctgt 60
gacacactca ttgtagccgg aagaaaagag attgtcgtat ctattcactc ctcacagcag 120
ctctctcgat catgaacatg accacagtga gagagagaga gagagagaga tatccaaatt 180
tactttcact atctctcctt caagtttttc agatgcttca tttgcttcct cttgacttta 240
aaaacaactt cggcgctata tcaacctcct accttctttg gtggctgcac aaacatgatc 300
ctctctacga a 311
<210> 17
<211> 346
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
taaatatttt tcttttctcc ggatagatac tccagggagt cgtgcaagtg gccgactctg 60
tgcgcccact cgggcagaga aggcgggcct caattggtgg ttttcgtggc tgtgcttgta 120
tgattgttga ctttcggttg tctccatgtc tctttgtgat atgttcttcc tccaggaatt 180
tcccccagct attgaccaag atatccccgg cctggaggtt cttttttctt tttcacgata 240
cggtactaca acaggcagat aggattccag tgtctccctc acaacgaaga agcttattcc 300
gttgctgagt ttgctcagga gcctggtcac aagcgggatg agaagg 346
<210> 18
<211> 346
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
tacggaaaca agggacgaag agcaatggag tcaacagatg acttatctgt tgcttttttt 60
tgaaatcctc acaaaaatcc gaagaacaac cgaactgaaa acatatgagt tttgtagttt 120
ccataattga tttcatcaat cgaacctaaa atataatttt ttcttggtct gctgatgaaa 180
tccaatatta gaatcacaaa atctctatat ctctcatttc gtttttcttt ttatttatga 240
taaattatta tttttaaaaa ttattgataa ctttttcatg aataacttgt tttttttttg 300
gaacgtcttt gagaaaaata atgtgccata tgttgtaccg aaaaaa 346
<210> 19
<211> 343
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
tccgggaagg aaagcgaaca tgaaccgttg taaacgatat cttatctgtt gctttttttt 60
gaaatcttca caataatccg aacaacaacc gaacagaaaa catatgagta tcgtaatatc 120
cataattgat ttcatcaacc gaacctaaaa tataatttta tcttggtctg ctgatgaaat 180
ccaatattgg aataacaaaa tatatatatc ttttatttcg ttgttctttt tatttatgat 240
aaattattat ttttaaaaat tattgataac tttttcatga ataacttgtt ttttttttgg 300
aacgtctttg agaaaaataa tgtgccatag gttgtaccga gga 343
<210> 20
<211> 535
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
tacacaagaa aagcgagaca gagagttctt ttagtatttt gacgggtact tagactttca 60
tacttctatt acctaacgtt ttggaagaat aacaatctct actccatgtg aggagctagt 120
attaagtgta aggaaaaatt tcttccgaaa gcgctctgtc tctcactcta tatagataaa 180
tatatagata tttaaatatt tatatataaa tatccagatt tgtaaaaaat taaaaacctt 240
cccattttca atttcggaaa atcaaaaaaa tttttttttt tttatatata tatatatata 300
tataaaaaaa taaaaaaata tatatatata tatatataga gagagacgtg ttccactctc 360
tcaaaaaaat atctatatat atatagagag acaaaagaga gagagagagt gtgtgtcact 420
cccacacaca ctcgaaaaag agagagagtg tgtgtgtgtc tctgtgacaa aaaaaaattt 480
tgtgagacac tgtgtgtatt tttatcaaca ccccctctct gtgtgtatag agaaa 535
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gaacttatgg cactccgatc tc 22
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ggtcattttc ttccttgata gc 22
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
atatcgcacg ggttccttac 20
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tgttaaaacc gagggaaaag ag 22
<210> 25
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
cgtagggcag ctttgatttt ag 22
<210> 26
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
aaagaagtct cgcctgaaca ag 22
<210> 27
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
aagagaaaac cagagcgaca ag 22
<210> 28
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gcagcgatag gaaattggat aa 22
<210> 29
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
cgaacagaat tgaaaccgaa c 21
<210> 30
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cagaagcaag tcgagagaga ca 22
<210> 31
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
tataaaacct ggggattgtt gc 22
<210> 32
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
aaatgccttc cttgataact gg 22
<210> 33
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
tggtggtgtc tcaaaatggt a 21
<210> 34
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
tgctttgtat tcatctctcc cc 22
<210> 35
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
tgctagggtt cctctggatc t 21
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
ctgagaggat catgttttgt gc 22
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ctttatctgc ttctgctgtt gc 22
<210> 38
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
aatagcttcc tcacctgtca cc 22
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
tgcatacaaa ccgagaatca 20
<210> 40
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
ggacaaacaa ggatggagtt tc 22
<210> 41
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
cgaagcaagt aagaaacaca cg 22
<210> 42
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
atataactag gtgttttgcc cg 22
<210> 43
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
catccatcag ttcaatcggt ta 22
<210> 44
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
tctgcacttg ggtcactaca at 22
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gcttgtacgc ttcttccaga tt 22
<210> 46
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
atgagaatgg ctagtccggt ta 22
<210> 47
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
gcagcgatag gaaattggat aa 22
<210> 48
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
aagagaaaac cagagcgaca ag 22
<210> 49
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
gttgtacgtc ttccactttc cc 22
<210> 50
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
aggaaccgac attcagagag ag 22
<210> 51
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
aattaacctt gtcgggtgaa ga 22
<210> 52
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
cgaagatttc attgctgata cg 22
<210> 53
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
catggtactc ctgagcttat ttg 23
<210> 54
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
cgactcttca atgtgcatct ct 22
<210> 55
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
tctccttcaa agcaatctct cc 22
<210> 56
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
gcagagactt cttcaccgtc tt 22
<210> 57
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
tttctccctg atgacctttt gt 22
<210> 58
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
ggtttagacg gcacctagtc ag 22
<210> 59
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
cggtctaaca tattgcacat tc 22
<210> 60
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
ccaaatctga atgcgagaga at 22
Claims (2)
1. An exogenous radish fragment specific marker, which is characterized in that the specific marker is CLR9-20, and the nucleotide sequence of the specific marker is SEQ ID No: shown at 20.
2. Use of the exogenous radish fragment-specific marker of claim 1 in the selection of brassica napus restorer line.
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| CN202110220860.6A CN114350655B (en) | 2017-08-24 | 2017-08-24 | Exogenous radish fragment specific marker and preparation method and application thereof |
| CN201710735768.7A CN107541517B (en) | 2017-08-24 | 2017-08-24 | Exogenous radish fragment specific marker and preparation method and application thereof |
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| CN112259164B (en) * | 2020-10-28 | 2022-07-05 | 湖南省作物研究所 | Development method of competitive INDEL molecular marker for co-separation of radish cytoplasmic sterility restorer line |
| CN113355453B (en) * | 2021-08-09 | 2021-10-29 | 华智生物技术有限公司 | Cabbage type rape radish cytoplasm sterility restoring geneRfoSNP molecular marker and application thereof |
| WO2024050761A1 (en) * | 2022-09-08 | 2024-03-14 | 中国农业科学院蔬菜花卉研究所 | Pcr marker for detecting exogenous radish fragment in brassica oleracea-radish introgression line, primer, and use thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2283493A1 (en) * | 1997-06-10 | 1998-12-17 | Pioneer Hi-Bred International, Inc. | Use of molecular markers for genotype determination of the ogura rf gene in brassica napus |
| CN1849879A (en) * | 2006-06-05 | 2006-10-25 | 华中农业大学 | Selective breeding method preserved sichuan pickle cytoplasm male sterile line |
| WO2011020698A1 (en) * | 2009-08-03 | 2011-02-24 | Nickerson International Research | Brassica plant for restoring fertility in an ogura cytoplasmic male-sterility system, method for producing same, and use of said plant |
| CN102220316A (en) * | 2011-04-20 | 2011-10-19 | 中国农业科学院油料作物研究所 | Preparation method and application of molecular marker of rape male sterile restoring gene |
| CN102640700A (en) * | 2012-05-04 | 2012-08-22 | 湖南省作物研究所 | Cabbage type rape Ogura cytoplasmic male sterility (OGU CMS) restorer and transformation method and application thereof |
| CN105002290A (en) * | 2015-08-06 | 2015-10-28 | 华中农业大学 | Molecular detection kit and application for brassica plant cytoplasmic type |
| CN105248273A (en) * | 2015-11-04 | 2016-01-20 | 浙江省农业科学院 | Improved cabbage type oilseed rape radish cytoplasmic male sterility restoring line and improvement method thereof |
| CN105830906A (en) * | 2015-12-15 | 2016-08-10 | 山东省农作物种质资源中心 | Ogura-type radish male-sterile cytoplasm and seed selection method thereof |
| CN106613920A (en) * | 2017-02-20 | 2017-05-10 | 陕西省杂交油菜研究中心 | Method for breeding cabbage type rape radish cytoplasmic male sterility three-line |
| CN106718822A (en) * | 2016-11-15 | 2017-05-31 | 江西省农业科学院作物研究所 | A kind of cabbage type rape cytoplasmic sterility restorer population improvement method |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2193938A1 (en) * | 1996-12-24 | 1998-06-24 | David G. Charne | Oilseed brassica containing an improved fertility restorer gene for ogura cytoplasmic male sterility |
| EP2247734A2 (en) * | 2008-02-06 | 2010-11-10 | Pioneer Hi-Bred International Inc. | New brassica ogura restorer lines with shortened raphanus fragment (srf) |
| CN101422133B (en) * | 2008-12-23 | 2011-12-21 | 华中农业大学 | Cabbage type rape self-incompatibility two-line hybrid seed-selection method |
| CA2765353A1 (en) * | 2012-01-23 | 2013-07-23 | Viterra Inc. | Hybrid canola quality brassica juncea |
| CN102586244B (en) * | 2012-03-09 | 2013-08-14 | 西北农林科技大学 | Radish cytoplasmic male sterility molecular markers and method for assisting selective breeding using same |
| CN102696474B (en) * | 2012-05-15 | 2013-11-06 | 西北农林科技大学 | Breeding and application of cytoplasmic male sterility restoring line of brassica napus rapeseed and radish |
| CN106967803B (en) * | 2017-03-21 | 2020-09-29 | 北京市农林科学院 | High-throughput molecular marker for detecting fertility restorer gene of radish Ogura-CMS (fertility restorer gene) and application of high-throughput molecular marker |
-
2017
- 2017-08-24 CN CN202110220860.6A patent/CN114350655B/en active Active
- 2017-08-24 CN CN201710735768.7A patent/CN107541517B/en active Active
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Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2283493A1 (en) * | 1997-06-10 | 1998-12-17 | Pioneer Hi-Bred International, Inc. | Use of molecular markers for genotype determination of the ogura rf gene in brassica napus |
| CN1849879A (en) * | 2006-06-05 | 2006-10-25 | 华中农业大学 | Selective breeding method preserved sichuan pickle cytoplasm male sterile line |
| WO2011020698A1 (en) * | 2009-08-03 | 2011-02-24 | Nickerson International Research | Brassica plant for restoring fertility in an ogura cytoplasmic male-sterility system, method for producing same, and use of said plant |
| CN102220316A (en) * | 2011-04-20 | 2011-10-19 | 中国农业科学院油料作物研究所 | Preparation method and application of molecular marker of rape male sterile restoring gene |
| CN102640700A (en) * | 2012-05-04 | 2012-08-22 | 湖南省作物研究所 | Cabbage type rape Ogura cytoplasmic male sterility (OGU CMS) restorer and transformation method and application thereof |
| CN105002290A (en) * | 2015-08-06 | 2015-10-28 | 华中农业大学 | Molecular detection kit and application for brassica plant cytoplasmic type |
| CN105248273A (en) * | 2015-11-04 | 2016-01-20 | 浙江省农业科学院 | Improved cabbage type oilseed rape radish cytoplasmic male sterility restoring line and improvement method thereof |
| CN105830906A (en) * | 2015-12-15 | 2016-08-10 | 山东省农作物种质资源中心 | Ogura-type radish male-sterile cytoplasm and seed selection method thereof |
| CN106718822A (en) * | 2016-11-15 | 2017-05-31 | 江西省农业科学院作物研究所 | A kind of cabbage type rape cytoplasmic sterility restorer population improvement method |
| CN106613920A (en) * | 2017-02-20 | 2017-05-10 | 陕西省杂交油菜研究中心 | Method for breeding cabbage type rape radish cytoplasmic male sterility three-line |
Non-Patent Citations (3)
| Title |
|---|
| Genetic characterization of a new radish introgression line carrying the restorer gene for Ogura CMS in Brassica napus;Wang Tonghua等;PLOS ONE;第15卷(第7期);第1-14页 * |
| 甘蓝型油菜Ogu CMS恢复材料1575R的分子初步鉴定;许婷;董振生;张搏;高永祥;段海峰;黄伟男;董军刚;;华北农学报(06);第40-44页 * |
| 甘蓝型油菜萝卜细胞质雄性不育杂种优势利用研究进展;王同华等;分子植物育种;第15卷(第7期);第2777-2783 页 * |
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| CN114350655A (en) | 2022-04-15 |
| CN114350832A (en) | 2022-04-15 |
| CN107541517A (en) | 2018-01-05 |
| CN114350832B (en) | 2023-09-26 |
| CN107541517B (en) | 2021-04-06 |
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