CN107347632B - Breeding method of cabbage type rape radish cytoplasmic sterility restoring line and application of cabbage type rape radish cytoplasmic sterility restoring line in cabbage type rape breeding - Google Patents
Breeding method of cabbage type rape radish cytoplasmic sterility restoring line and application of cabbage type rape radish cytoplasmic sterility restoring line in cabbage type rape breeding Download PDFInfo
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Abstract
The invention relates to a breeding method of a cabbage type rape radish cytoplasm sterility restoring line, which is characterized in that specific molecular markers of a restoring gene in CLR650 and a linked exogenous radish fragment thereof are developed by comparing whole genome re-sequencing data of a cabbage type rape radish cytoplasm sterility restoring material CLR650 with radish genome draft information; then, the target individual plant of the cabbage type rape radish cytoplasmic sterility restoring material CLR650 backcross population is bred by the assistance of the specific molecular marker to obtain BC4Isolating the population; mixing BC4Carrying out multi-generation mixed selection on fertile plants in the population by adopting a mixing method until a mixed separation population with the fertile plant accounting for more than 75 percent is obtained; finally, carrying out homozygous stabilization through multi-generation selfing to obtain a new cytoplasmic sterile restoring line of the brassica napus radish. The invention overcomes the obstacles of the prior method that the restored gene heritability is seriously low and the characters are difficult to be homozygous and stable.
Description
Technical Field
The invention belongs to the technical field of rape breeding, and particularly relates to a method for breeding a cabbage type rape radish cytoplasm sterility restoring line based on a conventional breeding technology of hybridization, sister hybridization and selfing and a molecular marker-assisted selection technology.
Background
The rape has obvious heterosis, and is the most potential oil crop for coping with the shortage of edible vegetable oil and developing renewable biological energy sources at home and abroad. The radish cytoplasmic male sterility ogu CMS is a cytoplasmic male sterility system established by a sterility source found in radish in 1968 by the Japan warehouse, has very important value in heterosis utilization of cruciferous crops due to very stable and complete sterility of the cytoplasmic male sterility system, and is an ideal way for heterosis utilization of brassica napus at present. However, since its restorer gene is only present in radish, there is no source of restoration available throughout brassica crops, and the rape ogu CMS restorer gene must be transferred from radish.
Heyn in 1976 successfully introduced radish restorer genes into Brassica napus by intergeneric hybridization (Heyn.1976). Subsequently, Pelletier et al, France, also introduced the restorer gene in Raphanus sativus into Brassica napus by protoplast fusion technique. However, when the restoring gene is transferred from radish to brassica napus, redundant radish segments also permeate into rape genomes, so that the restoring material has the problems of unsatisfactory restoring capability, partial female sterility, linkage of higher glucosinolates and the restoring gene and the like. Since the exogenous radish segments introgressed into the genome of rape are difficult to identify from appearance, the restorer line transformation and improvement must be carried out by means of molecular marking technology. However, the foreign ogu CMS restoration gene related marker technology is mostly protected by patent, and the application range is limited. With the continuous and deep research of rape molecular biology, the genome sequencing technology is perfected, the sequencing cost is further reduced, the development of molecular markers and the application of auxiliary selection technology are increasingly mature and practical, and the technology support is provided for the independent research and development of cabbage type rape radish cytoplasm sterility restoring materials in China. Since the introduction of the ogara-CMS in the last 80 th century, a great deal of research is carried out by rape breeding units in China. For example, the plum Asahi peak reports that the restoring gene is introduced into rape from radish variety Lanhua, but the restoring gene of the obtained material is in episome and can not be stably inherited; in the year 2012, Liu Gung Xia utilizes the existing cabbage type rape radish cytoplasm sterility restoring line as a female parent to be hybridized with an examined winter rape variety, and a novel restoring line with improved agronomic characters is screened out, but the research is only conventional hybridization and homozygous breeding, and the source of the restoring source cannot be determined. Because the transformation and restoration system is very difficult, China is always lack of available restoration sources.
Disclosure of Invention
The invention aims to solve the technical problems that the defects and defects mentioned in the background technology are overcome, and a method for cultivating the cabbage type rape radish cytoplasmic sterility restoring line and subsequent application of the restoring line are provided, wherein the specific marker of a target gene developed by a genome re-sequencing technology and a comparative genomics technology is combined with conventional hybridization, backcross, mixed selection and selfing breeding technologies to solve the problems that the genetic power of distant hybridization exogenous target genes is seriously low, the characters are difficult to be homozygous and stable and the like in the breeding process of the cabbage type rape radish cytoplasmic sterility restoring line in China at present, and a quick and effective breeding method is provided for the breeding of the cabbage type rape radish cytoplasmic sterility restoring line.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for selectively breeding the cytoplasmic sterile restoring line of cabbage-type rape radish includes such steps as developing the specific molecular marker of exogenous target segment, hybridizing, backcrossing, sister cross-breeding, selfing, conventional molecular marker-aided selection, and culturing the cytoplasmic sterile restoring line of cabbage-type rape radish, including:
1) hybridizing the cabbage type rape radish cytoplasmic male sterile recovery material CLR650 serving as a female parent with the conventional cabbage type rape strain A to obtain F1A population; selection of multiple plants F1Backcrossing the generation plants and the conventional cabbage type rape line A as recurrent parents to construct BC1Isolating the population;
2) at seedling stage, at least 1 exogenous radish segment is used for specifically marking BC1The segregating population is subjected to molecular marker assisted selection,removing the individual plants with negative marker analysis results; selection of multiple plants BC in flowering time1Separating a single plant of the conventional cabbage type rape line A with a phenotype close to (i.e. similar in character) in the population as a female parent, and continuing backcrossing the conventional cabbage type rape line A; repeating the above steps until obtaining BC of cabbage type rape CLR6504A fertility segregating population;
3) pulling out BC4Performing mixed pollination and harvesting on the remained fertile single plants in the isolation region, and investigating fertility performance of the progeny population in the full-bloom stage until a population with the fertile plant accounting for more than 75% is obtained;
4) and (3) carrying out isolation selfing on all the fertile plants in the segregation population with the fertile plant proportion of more than 75%, and continuously selecting the fertile plants from the selfed progeny for isolation selfing until obtaining the cabbage type rape radish cytoplasm sterile restorer line CLR095 with inseparable fertility and consistent phenotype.
Preferably, according to the breeding method of the cabbage type rape radish cytoplasmic sterility restorer line, the conventional cabbage type rape line A in the step 1) is 20B. The conventional strain genome is relatively homozygous, so that the filial generation does not have a violent segregation phenomenon, and the influence of the progeny character segregation on the homozygous stability of the restorer line is reduced to the maximum extent. In addition, the conventional strain does not contain other types of restoring genes carried by hybrids, and does not have other adverse cytoplasmic effects, so that the molecular marker screening of the progeny population and the acquisition of the target strain are facilitated.
Preferably, according to the breeding method of the cabbage type rape radish cytoplasmic sterility restorer line, a plurality of plants F are selected in the step 1)1Backcrossing the generation plants and the conventional cabbage type rape line A as recurrent parents means that 2-4F plants are selected1The generation plants and the conventional cabbage type rape line 20B are used as recurrent parents for backcross.
Preferably, according to the breeding method of the cabbage type rape radish cytoplasmic sterility restorer line, BC is constructed in the step 1)1The separation population refers to the BC with the size of 200-250 single plants1Isolating the population.
It is preferable thatAccording to the breeding method of the cabbage type rape radish cytoplasmic sterility restoring line, a plurality of plants BC are selected in the step 2)1The method for separating a single plant with a phenotype close to that of the conventional cabbage type rape line A in a population as a female parent refers to selecting BC18-10 individuals with phenotypes close to 20B in the segregating population were used as female parents.
Preferably, according to the breeding method of the cabbage type rape radish cytoplasmic sterility restorer line, in the step 4), the number of generations of isolation selfing is 2-3 generations.
In the method for breeding the cytoplasmic sterility restorer line of brassica napus, the method for preparing the specific marker of the exogenous radish fragment comprises the following steps:
a) hybridizing the cabbage type rape radish cytoplasmic male sterile recovery material CLR650 serving as a female parent with the conventional cabbage type rape strain A to obtain F1Population, selection of multiple plants F1Bagging, isolating and selfing the plant group to obtain F2Separating individual plants of the group with fertility, and extracting the total DNA of the genome of each individual plant; survey in full bloom F2Respectively establishing fertile plant and sterile plant gene mixing pools when the group single plant is fertile; performing whole genome re-sequencing on the brassica napus radish cytoplasmic sterility restoring material CLR650, comparing a sequencing result with a radish genome sketch and radish cytoplasmic sterility restoring gene sequences to obtain a restoring gene of the CLR650 and flanking exogenous radish sequence information thereof, and designing a specific primer according to a target region;
b) screening the obtained specific primers by using the gene mixing pool, reserving the primers which are positive in PCR amplification of the fertile plant gene mixing pool and negative in PCR amplification of the sterile plant gene mixing pool, and screening the screened primers in F2And carrying out coseparation verification on the single plant of the fertility segregation population, and taking a primer for coseparation of PCR amplification positive expression and fertility expression as a specific marker primer of the exogenous radish fragment.
Preferably, according to the breeding method of the cytoplasmic sterility restorer line of Brassica napus radish, the step a) of selecting a plurality of F1 population plants for bagging isolation selfing is to select 6-8F 1 plants1And bagging, isolating and selfing the plant groups.
A great deal of molecular biological and cytogenetic analysis is carried out on novel brassica napus Ogu CMS restoration material CLR650 autonomously cultivated by the research institute of crops in Hunan province, and the result shows that the restoration material has stable chromosome set and the fertility restoration gene is the Rfo gene. Based on the problems of serious fertility segregation and difficult stability of characters of CLR650 filial generations, a specific molecular marker of an exogenous radish fragment is autonomously developed through genome re-sequencing and reference genome sequence comparison. The specific marker of the exogenous radish fragment comprises one or more of CLR9-1-CLR9-20, and the nucleic acid sequences of CLR9-1-CLR9-20 are respectively shown in SEQ ID No: 1 to SEQ ID No: 20.
as a general technical concept, the invention also provides the application of the cabbage type rape radish cytoplasmic male sterile restorer line CLR095 obtained by the breeding method in cabbage type rape breeding, and hybrid production is carried out by adopting the cabbage type rape sterile line, the maintainer line thereof and the restorer line CLR095 through a three-line matching method.
Compared with the prior art, the invention has the beneficial effects that:
1. the novel cabbage type rape radish cytoplasm sterility restoring line obtained by the invention has stable heredity, eliminates distant hybridization unfavorable characters carried by CLR650 to a certain extent, and can provide a cabbage type rape radish cytoplasm sterility restoring source with pioneering significance for China;
2. the molecular markers are exogenous radish specific markers which are independently developed according to CLR650 genome re-sequencing results, and can provide a new molecular marker technical support for the cytoplasmic sterility restoration source transformation of brassica napus radishes in China;
3. the breeding is carried out by adopting the conventional breeding means and the molecular marker assisted selection method, the initial selection can be carried out in the seedling stage, on one hand, non-target single plants can be pulled out in advance, and the workload of the test land and later-stage 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 eliminated in time;
4. the method adopts the first backcross directional transformation, then the mixed selection, increases the homozygous probability of the target gene to the maximum extent, and finally overcomes the obstacles of serious low heritability of the restored gene, difficult stability of characters and the like caused by distant hybridization through a breeding strategy of homozygous stability by selfing, and can avoid the selfing decline phenomenon caused by continuous multi-generation selfing.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic diagram of the transformation process of Brassica napus Ogu CMS restorer line CLR095 in the present invention.
FIG. 2 shows F of radish specific marker in Brassica napus radish sterility restoring material CLR6502PCR amplification between fertility segregating population gene mixing pools
FIG. 3 shows F of radish specific marker in Brassica napus radish sterility restoring material CLR6502The PCR amplification result of the isolated population single plants, wherein F represents fertile plants and S represents sterile plants.
FIG. 4 is a comparison between the cytoplasmic sterility restorer line CLR095 and CLR650 of Brassica napus radish obtained in the present invention, and in FIG. 4: a: comparing the leaves in the seedling stage; b: comparing the flower organs; c comparing the fruits of the horns. FIG. 4 shows that the CLR650 is used as a comparison object, and the lengths of leaves, flowers and horns of the CLR095 are obviously changed in FIG. 4.
FIG. 5 is a comparison of the cytoplasmic sterility restorer line CLR095 of Brassica napus radish and CLR650 obtained in the present invention. FIG. 5 shows that the CLR650 is used as a comparison object, and the lengths of leaves, flowers and horns of the CLR095 are obviously changed in FIG. 5.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art 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 limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1:
the breeding method of the cabbage type rape radish cytoplasmic sterility restorer line CLR095 provided by the embodiment of the invention is shown in figure 1, and specifically comprises the following steps.
1. Radish cytoplasmic male sterility restoring gene specific molecular marker and linked marker development
1.1 materials of embodiment
The cabbage type rape radish sterile restoring material CLR650 (radish male sterile cytoplasm, zigzag leaf margin, sharper leaf tip, dark green leaf color, thick and short silique, see the literature: Chenweijiang et al, creation of cabbage type rape radish cytoplasmic male sterile restoring material, Chinese agricultural science 2012, 45 (8): 1465-1474) is obtained by hybridizing and breeding radish-blue (AACCRR, 2n ═ 56) and cabbage type rape by a grafting technology, and the number of chromosomes is between 38 and 40. The cytoplasm is male sterile cytoplasm of the radish, and the nucleus contains a radish cytoplasm restoring gene Rfo of CLR650, so that the radish cytoplasm is normally fertile; the conventional cabbage type rape line 20B (conventional cabbage type rape cytoplasm, cut at leaf margin, medium leaf tip, light green leaf color, slender silique, see the literature: plum berry and the like, breeding of new cabbage type hybrid rape Fengyou 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 restorer gene F2Construction of segregating populations
In 2011 spring, when CLR650 enters the initial flowering stage, the unopened bud on CLR650 plant is removed stamen and hybridized with 20B to obtain F1Seeds; f to be obtained in 5-month middle of 20111Sowing seeds in a nutrition pot in a climate chamber to obtain 20F seeds1Selecting 8 strong individual plants in the early flowering period, bagging with parchment paper bags for isolated selfing to obtain F2Separating the population of seeds; 2011 in 10 months selects 1 part of the F2Sowing seeds in rape test field after long sand paddy-upland rotation to obtain F2Separating the plant population with fertility, tagging the individual plants at 3-5 leaf stage, and selecting 2-3cm from each individual plant2Total DNA extraction was carried out on young leaves of large size by the method reported in Lijia et al (Lijia et al, a method for efficiently extracting total DNA from rape leaves, proceedings of Huazhong university of agriculture, 1994, 13 (5): 521-. Investigating and recording F when the rape in the next year enters the full bloom stage2Fertility expression of individual plants in the population. The F2 segregation population obtained by the invention has 210 individuals, wherein fertile individuals are 74 individuals, and sterile individuals are 136 individuals. From F according to fertility survey results2In the segregating population, respectively selecting 10 fertile single plants and 10 sterile single plants to carry out equal-quantity mixing on total DNA, respectively obtaining 1 fertile plant mixing pool and 1 sterile plant mixing pool, and developing target fragment molecular markers for later use.
1.3 radish cytoplasmic sterility restoring gene specific primer design based on CLR650 genome resequencing
Sowing CLR650 seeds in nutrition pot, and taking 3-4cm when 3-4 true leaves grow2The young leaves are sent to Shanghai Europe-Yi biomedicine science and technology Limited company to carry out re-sequencing analysis by utilizing an Illumina MiSeq platform, so that sequencing data of the unidirectional 67.169G base number and the 447,797,399 fragment number are obtained. Wherein the clean reads ratio reaches 96.09%, the ratio of high-quality bases reaches 99.21%,>71% of the genome reached a sequencing depth of 50X or more. These sequences were then separately compared with the sequences published on NCBIThe radish genome is compared with a 127Kb region of the radish cytoplasmic sterility restoring gene Rfo to obtain the sequence information of the radish genome Scaffold 131 of the radish cytoplasmic sterility restoring gene, and the exogenous radish cytoplasmic sterility restoring gene in CLR650 is deduced to be positioned in the radish genome R9On a chromosome.
1.4 screening and verification of specific molecular marker of radish cytoplasmic male sterility restoring gene
For radish R9The published sequences of Scaffold 131 and Rfo in 127Kb were combined to design 80 primers specific to radish using Primer3 Primer design tool. Firstly, carrying out PCR amplification on the 80 pairs of primers in the fertile plant mixing pool and the sterile plant mixing pool in the step 1.2, screening the primers with positive performance in the fertile plant mixing pool and negative performance in the sterile plant mixing pool, and then putting the obtained primers in the F2The fertility segregation population is subjected to coseparation verification (see the attached figures 2 and 3), and 20 specific markers of exogenous radish fragments are determined through verification: CLR9-1, CLR9-2, CLR9-3, CLR9-4, CLR9-5, CLR9-6, CLR9-7, CLR9-8, CLR9-9, CLR 9-10, CLR9-11, CLR9-12, CLR9-13, CLR9-14, CLR9-15, CLR9-16, CLR9-17, CLR9-18, CLR9-19 and CLR9-20 (the primer sequences and products of each specific marker are shown in tables 1 and 2 below).
Table 1: the invention relates to a primer sequence of a radish specific marker and the size of a product fragment
TABLE 2 PCR product sequence of radish specific marker of the present invention
2. Breeding of radish cytoplasmic male sterility restoring line
2.1 molecular marker assisted selection backcross Breeding
2011 spring, F obtained in step 1.2 above1Backcrossing the plant with 20B to obtain BC1Seeds; BC to be obtained in 10 months of 20111After the seeds are sown in the long-sand rape test field, BC of CLR650/20B is obtained1And (4) separating the group plants. Because the radish germplasm sterile restoring gene has a serious segregation phenomenon in CLR650 offspring, the invention combines the specific markers CLR9-3, CLR9-12 and CLR9-18 of the exogenous radish segment obtained in the step 1.4 to carry out BC treatment1The 3-5 leaf stage in the segregating population begins to select, and the individuals with negative results of marker analysis are pulled out. In spring rape full-bloom stage of 2012, fertility investigation is carried out on the reserved fertile single plants, the fertile plant main inflorescence with the phenotypic character close to 20B is bagged, isolated and selfed by a parchment bag, unopened buds on branches are backcrossed with 20B after stamens are removed, and BC is obtained2Isolating seeds of the population. And (3) investigating the shape, the length and the grain number of each pod of each backcross individual plant under the condition of natural pollination in the maturation period, harvesting the naturally pollinated seeds by individual plants, analyzing three quality characters of oil content, erucic acid content and glucosinolate content by using a near infrared analyzer, keeping the individual plant with the phenotype and the quality character closest to 20B, and carrying out backcross continuously by using 20B as a recurrent parent until a backcross population with the phenotype consistent with 20B and improved quality character is obtained.
The invention proceeds from BC1In separate populations 16Of the 3 individuals, 31 fertile individuals with positive molecular marker analysis results were obtained, and 10 phenotypic characters were relatively close to 20B. The 10 BC obtained were subjected to the near-infrared analyzer (Matrix-1, Bruker, Germany, OPUS/QUANT5.5 software)1Performing three quality character analyses of oil content, erucic acid content and glucosinolate content on the selfing seed of the fertile single plant (the result is shown in table 3), and reserving BC of backcrossed single plant with lowest glucosinolate content, oil content of more than 35 percent and erucic acid content of less than 5 percent2Isolating the population of seeds. In 5 Yuehu of 2012, the BC is added2Separating group seeds, sowing in Qinghai Xining rape test field to obtain BC2Segregating the population of plants and proceeding according to BC1Further backcrossing until a phenotypically identical BC is obtained4And (4) separating the group plants. BC with relatively consistent phenotype is obtained in Qinghai in 2014 in the invention4Segregating a population of plants, the segregating population comprising 191 individuals, wherein 52 individuals exhibit normal fertility and 139 individuals exhibit cytoplasmic male sterility in radish.
TABLE 3 Brassica napus radish sterility restoring material BC obtained by the present invention1-BC4Quality analysis result of selected fertile individual plants in group
2.2 homozygous and stable cytoplasmic sterility restoring line of Brassica napus and radish
In the full-bloom stage, according to the result of the fertility survey of the initial bloom stage, the BC obtained in the step 2.14And (3) removing sterile single plants in the segregating population, carrying out isolation mixed pollination on all the reserved fertile plants, carrying out mixed harvest after the fertile plants are mature, and continuously reserving the fertile plants in the segregating population for the next generation to carry out isolation mixed pollination until the segregating population with the fertile plant proportion exceeding 75% is obtained. Then isolating and selfing all fertile individual plants in the obtained segregation population by a parchment bag until selfing is obtainedA line with inseparable fertility, namely a new cabbage type rape radish cytoplasm sterility restoring line.
In the invention, all the reserved 54 fertile plants are subjected to isolated mixed pollination in 2014 spring, and are subjected to mixed harvest after being matured. Sowing seeds obtained by mixed harvest in 10 months in 2014 in a long sand rape test field to obtain fertility segregating groups of 155 single plants, wherein in the full-bloom stage of 2015, the fertility survey of the single plants of the groups shows that the proportion of fertile plants is about 30 percent, and the proportion of fertile plants of 1 segregating group (field number 14Q017) is about 50 percent; 5 fertile strains in the segregating population 14Q14 are selected to continue to carry out pairwise crossing to obtain 10 brother and sister crossing segregating populations. Sowing 10 segregating populations in 10 months in 2014 in the Hunan Changsha rape test field, and investigating fertility performances of the populations at the full bloom stage of the next year, wherein the fertile plant proportion of 8 segregating populations (field numbers 14C041, 14C043, 14C044, 14C045, 14C046, 14C047, 14C048 and 14C050) is about 30 percent, and the fertile plant proportion of 1 segregating population (field number 14Q042) is about 50 percent; the ratio of fertile plants of 1 segregating population (field number 14C049) is about 70 percent; all fertile individuals in segregating population 14Q049 were segregatively selfed with parchment bags to obtain seeds of 32 selfed lines. And (3) sowing the seeds of the 32 inbred lines in the Qinghai Xining rape test field in 5 months in 2015, and performing fertility investigation on each inbred line in the full-bloom stage to obtain an inbred line (field number 15Q028) with the fertile plant proportion of about 90%. Fertile individuals of line 15Q028 were segregatively selfed with parchment bags to obtain seeds of 28 selfed lines. In 2015, 10 parts of seeds of the 28 selfing lines are sown in a test field of the Hunan Changsha rape. And (3) performing fertility investigation on each selfing line at the next full-bloom stage to obtain a fully-fertile selfing line (field number 15C048), and performing three quality character analyses of oil content, erucic acid content and glucosinolate content on the single selfing fructification seed of the line 15Q028 by using a near infrared analyzer (Matrix-1, Bruker, Germany, OPUS/QUANT5.5 software), wherein the oil content is 38.90%, the glucosinolate content is 68.65 mu mol/g, and the erucic acid content is 0.7%.
10 parts of 2016, sowing the selfed seeds of the line 15C048 in a test field of the Hunan Changsha rape, and performing fertility investigation in the full bloom stage of the next year to show that the fertility of each plant of the line is all normal (the field number is 16C095), wherein the line is named as the radish sterility restoring line CLR095 of the cabbage rape.
2.3 survey of characters of cytoplasmic sterility restorer line of Brassica napus and radish
The restorer line CLR095 obtained in step 2.2 was sown in a single row area 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 compared with CLR650 and CLR 20B, respectively. Leaf color and leaf margin shape are inspected in the seedling stage, free pollination is performed in the flowering stage, the shape and the growing state of petals are inspected, the plant height, the number of branches at one time, the number of siliques at one time, the length of the siliques and the number of grains per silique are inspected in the mature stage, three quality characters of oil content, erucic acid content and glucosinolate content are analyzed, and specific results are shown in a table 4. The restorer line CLR095 obtained by the invention removes the adverse effect of exogenous redundant radish fragments in the genome to a greater extent, and is obviously different from the original cabbage type rape radish sterile restorer material CLR650 in phenotypic agronomic characters such as quality, leaf type, leaf color, pod shape and the like (see attached figure 4, figure 5 and table 4), thereby showing that the breeding method of the invention is effective in breeding the cabbage type rape radish restorer line.
TABLE 4 Property Performance of Brassica napus radish sterility restorer line CLR095
Interpretation of related terms:
cytoplasmic male sterility: the term "professional" is a natural phenomenon widely present in higher plants and is manifested by maternal inheritance, pollen abortion and normal pistil.
Strain: the term "material" used earlier in the breeding process, which has a certain number of individuals of the same type, is used as a generic term before the variety comparison test.
And (3) PCR: the term "generic term," a molecular biological technique for amplifying a specific DNA fragment.
Low-sulfur glycoside: in professional terms, thioglucoside content < 30. mu. mol/g is used as a criterion for a material with low glucosinolates.
The term "Shuanghuang" is the term used in the present invention, i.e., the abbreviation of the term "canola" or "thioglycoside".
Carrying out sister hybridization: the term of art refers to the cross-breeding of sister lines or individuals of similar shape, which are bred from the same original parent.
Sequence listing
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<120> a breeding method of cabbage type rape radish cytoplasm sterility restoring line and application in cabbage type rape breeding
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<170>SIPOSequenceListing 1.0
<210>1
<211>323
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
ggctcaacgc ggaaagcaga tgctctgcaa ggacactcac ctgcggtctt tcctattgag 60
tctctctacc tttaattgaa tagacttcca tttctttgga cagattgtag cctgtcctgt 120
gtgtggttga ctcctcgttt tctttgatta ttattgttat caatcacacg cagagtgaca 180
atcggcacag agaaatattt atttcgtttg tcggcatgtg accgtcccca aagaaagaga 240
gtgatcgaga ttcaattcga catggagtgt ctgctgtgac tgtgccctgg acatcggagt 300
gcctcaattt caacgaatac aca 323
<210>2
<211>354
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
aagttcggac gaaagagagg gcaagttacc tcttgaaggt ttgacctcca tcctttcacc 60
gctatcaagg aagaaaatga ccatatattt gtttgggcac ctgattttac accaagacat 120
tttcactaaa aaaaaaaaaa aaagtgtccc ttttcggggt tattgggggt ttttattttt 180
ccccgtaagg ttttccgggg atccccttta aaaaataaaa aaaagtgatt gcgtccggtt 240
tgaaatacac ctggccaccc agacgtaacc tacgacgggt ttcaatcttt ttcaatgttt 300
taaagcaatc atattaatac cgtaactagg tgagaccggt gggtaggagg gatt 354
<210>3
<211>439
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
gccgcgagta gaggaagaag agcttatcta aaaactaact tgccttgtct aagctcctga 60
ttaaccggta taattgtttc tggagaattt aaaaatactg atctaaatgc ttaaatattt 120
ggaatactct ctttaaaaat ttccaaaata ttttttaaat tattattgaa attttttaac 180
gtataatatc ccgcgcttct taagctcgga tcaaaatctt ttaattttat ccattaacac 240
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
cattttttcctggaacataa 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 (7)
1. A breeding method of a cabbage type rape radish cytoplasm sterility restoring line comprises the following steps:
1) hybridizing the cabbage type rape radish cytoplasmic male sterile recovery material CLR650 serving as a female parent with a conventional cabbage type rape strain 20B to obtain F1A population; selection of multiple plants F1Backcrossing the generation plants and the conventional cabbage type rape line 20B as recurrent parents to construct BC1Isolating the population;
2) at seedling stage, at least 1 exogenous radish segment is used for specifically marking BC1Carrying out molecular marker-assisted selection on the segregating population, and removing the individual plants with negative marker analysis results; selection of multiple plants BC in flowering time1Separating a single plant with a phenotype close to the conventional cabbage type rape line 20B in the population as a female parent, and continuing backcrossing the conventional cabbage type rape line 20B; repeating the above steps until obtaining BC of cabbage type rape CLR6504A fertility segregating population;
3) pulling out BC4Sterile single plants in the fertility segregating population are subjected to mixed pollination and harvesting in the isolation area, and investigation is carried out in the full-bloom stagePerforming fertility expression on the progeny population until a population with the ratio of fertile strains of more than 75% is obtained;
4) separating and selfing all fertile plants in segregation populations with fertile plant proportion over 75 percent, continuously selecting the fertile plants from selfing offspring for separating and selfing all the fertile plants until obtaining the cabbage type rape radish cytoplasm sterile restoring line CLR095 with inseparable fertility and consistent phenotype;
the preparation method of the exogenous radish fragment specific marker comprises the following steps:
a) hybridizing the cabbage type rape radish cytoplasmic male sterile recovery material CLR650 serving as a female parent with a conventional cabbage type rape strain 20B to obtain F1Population, selection of multiple plants F1Bagging, isolating and selfing the plant group to obtain F2Separating individual plants of the group with fertility, and extracting the total DNA of the genome of each individual plant; survey in full bloom F2Respectively establishing fertile plant and sterile plant gene mixing pools when the group single plant is fertile; performing whole genome re-sequencing on the brassica napus radish cytoplasmic sterility restoring material CLR650, comparing a sequencing result with a radish genome sketch and radish cytoplasmic sterility restoring gene sequences to obtain a restoring gene of the CLR650 and flanking exogenous radish sequence information thereof, and designing a specific primer according to a target region;
b) screening the obtained specific primers by using the gene mixing pool, reserving the primers which are positive in PCR amplification of the fertile plant gene mixing pool and negative in PCR amplification of the sterile plant gene mixing pool, and screening the screened primers in F2Carrying out coseparation verification on individual plants of the fertility segregation population, and taking a primer for coseparation of PCR amplification positive performance and fertility performance as a specific marker primer of the exogenous radish segment;
the specific marker of the exogenous radish fragment comprises one or more of CLR9-1-CLR9-20, and the nucleic acid sequences of CLR9-1-CLR9-20 are respectively shown in SEQ ID No: 1-SEQ ID No: 20.
2. the method for breeding the cytoplasmic sterility restorer line of Brassica napus according to claim 1, wherein multiple plants F are selected in step 1)1The generation plants and the conventional cabbage type rape line 20B are used as recurrent parentsThe backcrossing is carried out by selecting 2-4F strains1The generation plants and the conventional cabbage type rape line 20B are used as recurrent parents for backcross.
3. The method for breeding the cytoplasmic sterility restorer line of Brassica napus radish according to claim 1, wherein the BC is constructed in the step 1)1The separation population refers to the BC with the size of 200-250 single plants1Isolating the population.
4. The method for breeding the cytoplasmic sterility restorer line of Brassica napus according to claim 1, wherein the step 2) comprises selecting multiple BC plants1The single plant with the phenotype close to the conventional cabbage type rape line 20B in the segregating population is used as a female parent to select BC18-10 individuals with phenotypes close to 20B in the segregating population were used as female parents.
5. The method for breeding the cytoplasmic sterility restorer line of Brassica napus according to claim 1, wherein in step 4), the number of generations of segregating selfing is 2-3 generations.
6. The method for breeding the cytoplasmic sterility restorer line of Brassica napus according to claim 1, wherein said step a) comprises selecting multiple F plants1The bag-covering isolation selfing of the population plants refers to the selection of 6-8F plants1And bagging, isolating and selfing the plant groups.
7. The application of the cabbage type rape radish cytoplasmic sterility restorer line CLR095 obtained through the selective breeding method of any one of claims 1 to 6 in cabbage type rape breeding is characterized in that hybrid production is carried out by adopting a cabbage type rape sterile line and a maintainer line thereof as well as the restorer line CLR095 through a three-line matching method.
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| CN109197575A (en) * | 2018-11-16 | 2019-01-15 | 贵州省油料研究所 | The method of Brassica napus recessive gms line and radish selection cross white flower rape |
| CN109402295B (en) * | 2018-12-26 | 2021-06-11 | 江西省农业科学院作物研究所 | Method for accelerating backcross breeding process of brassica napus by using orange flower color molecular markers |
| CN109566129B (en) * | 2019-01-21 | 2021-01-29 | 什邡市孝楠农业科技有限责任公司 | Breeding method of red flower rape variety |
| CN111575399B (en) * | 2020-06-09 | 2022-03-04 | 上海市农业科学院 | Cabbage type rape wax powder gene positioning method based on whole genome re-sequencing |
| 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 |
| CN113016606B (en) * | 2021-03-24 | 2022-06-07 | 华中农业大学 | Breeding method of cabbage type rape radish cytoplasm restoring line |
| CN114568294A (en) * | 2022-01-25 | 2022-06-03 | 华中农业大学 | Method for breeding clubroot-resistant variety based on brassica napus Ogu-CMS restorer line |
| CN115443907B (en) * | 2022-07-26 | 2023-04-21 | 开封市农林科学研究院 | High-yield large-fruit peanut hybrid combination selection method based on whole genome selection |
| CN116267586A (en) * | 2023-04-12 | 2023-06-23 | 广州市农业科学研究院 | Breeding method of cabbage heart restorer line material |
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