WO2017219633A1 - 油菜双单倍体诱导系选育甘蓝型油菜品种及材料的方法 - Google Patents

油菜双单倍体诱导系选育甘蓝型油菜品种及材料的方法 Download PDF

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WO2017219633A1
WO2017219633A1 PCT/CN2016/111327 CN2016111327W WO2017219633A1 WO 2017219633 A1 WO2017219633 A1 WO 2017219633A1 CN 2016111327 W CN2016111327 W CN 2016111327W WO 2017219633 A1 WO2017219633 A1 WO 2017219633A1
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plants
line
rapeseed
hybrid
stable
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PCT/CN2016/111327
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English (en)
French (fr)
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付绍红
李云
杨进
王继胜
邹琼
陶兰蓉
康泽明
唐蓉
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成都市农林科学院
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Priority to EP16906170.2A priority Critical patent/EP3485724A4/en
Priority to AU2016410429A priority patent/AU2016410429B2/en
Priority to CA3028501A priority patent/CA3028501A1/en
Priority to US16/310,497 priority patent/US20190254247A1/en
Publication of WO2017219633A1 publication Critical patent/WO2017219633A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A01H6/202—Brassica napus [canola]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A01H1/08—Methods for producing changes in chromosome number
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor

Definitions

  • the invention relates to agriculture, and in particular to a method for selecting a variety and material of Brassica napus L. in a double haploid induction line of Brassica napus.
  • breeding new inbred lines or genetically stable homozygous Strains-homozygous lines such as these homozygous lines meet the production needs in terms of resistance, yield, quality, etc., and finally identified or approved as a new new rapeseed variety (conventional variety) through regional trials.
  • the homozygous strains are tested with the sterile line to determine the relationship between the restoration and the restoration. If the restorer and the sterile line are crossed to match the new hybrid, if the maintainer and the sterile line are tested, the breeding has the retention. A new sterile line with the characteristics of a line.
  • the stable line is not restored (the test offspring cannot fully restore fertility or cannot maintain high degree of infertility completely), nor can it be used as a conventional variety used in production. Strains are either eliminated or linked to other maintainers or Compound hybridization enters the next round of breeding material selection. Under normal circumstances, it takes 6-7 generations to breed a conventional rapeseed variety through conventional artificial hybridization. If breeding hybrids are selected, stable sterile lines and maintainer lines need to be cultivated.
  • inducing line means that the plant is used as a male parent to pollinate similar plants with its pollen, which can induce the corresponding effects of the same plant (mother), such as haploid, double haploid (DH), etc. .
  • Maize is most commonly used in plants for the selection of introductory lines, but the induction line in maize is only a haploid induction line.
  • the earliest maize haploid induction line was stock6, which induced only haploid production in maize, and then the haploid plants were doubled to form homozygous diploid (double haploid), and the induction efficiency. Lower, generally the induction efficiency is below 10% (in The haploid number was calculated in the harvested seeds).
  • the object of the present invention is to provide a method for rapidly selecting and breeding a genetically stable Brassica napus restorer line, a maintainer line or a conventional variety of Brassica diploid induction line for breeding Brassica napus varieties and materials.
  • the method for selecting a Brassica napus variety and material by the rapeseed double haploid induction line of the present invention comprises the following steps:
  • step 2) For the hybridization, polymeric hybrid progeny or backcross progeny material obtained in step 1), the flower bud is artificially emascated at the flowering stage, and bagged and isolated;
  • step 2) Using pollen double haploid to induce pollen, artificially pollinating the plants in step 2) for 2-4 days after emasculation, and bagging and isolating, and harvesting the pollinated seeds after harvesting;
  • the induced seeds obtained in the above step 3) are planted at a single plant, and the ploidy is identified by flow cytometry at the seedling stage, and the polyploid, haploid or the plants having the dominant characteristics of the rapeseed double haploid induction line are eliminated. Select normal fertility tetraploid plants, and self-crossing the individual plants;
  • the sterile line is tested, or tested with the Brassica napus nucleus sterility (GMS), and the fertility identification of the offspring is tested to determine the relationship between the test and the father;
  • step 6 the offspring of the cross-test are completely infertile, and the corresponding parent is the maintainer; the test offspring is fully fertile, and the corresponding test is the restorer;
  • the maintenance line identified in the above step 7) continues to backcross with the sterile line for several generations, and the stable sterile line that is consistent with the maintainer line nuclear gene is selected; the restorer line of the test cross identification is directly related to the corresponding system.
  • the sterile line is tested and matched, the hybrid combination is selected, and the hybrid combination is entered into the variety comparison test.
  • the yield, resistance, high yield and quality traits are better than those of large-scale application in production, and can be formed by satisfying the identification (or validation) standard.
  • Hybrid rapeseed varieties which are identified (or approved) by provincial or national seed management departments, can be used for production promotion;
  • the method for breeding the above-mentioned rapeseed double haploid induction line comprises the following steps:
  • F 1 plants were self-crossed or forced self-crossing to obtain F 2 generation, F 2 generation field planting observation, and identification of fertility of each individual plant, selection of fertile offspring self-crossing to obtain F 3 generation,
  • the F 3 generation was identified for homozygosity.
  • the morphological, cytological and molecular marker identification was used to polymerize the progeny DNA by polymerase chain reaction. The DNA banding and number of bands of each individual amplified by specific primers were observed by electrophoresis. Each hybrid was shown to be a hybrid progeny of two parents, and the molecular marker maps of each individual were identical, indicating that these individuals were homozygous---the early generation stable system;
  • the obtained early generation stable system was reciprocally cross-linked with at least 10 conventional homozygous stable lines of rapeseed, and the F 1 and F 2 generations were used to identify the genetic characteristics of the early generation stable lines, that is, whether there were parthenogenetic characteristics; If there is F 1 separation, F 2 generations appear partially stable strains, and the corresponding early generation stable lines are early generation stable lines with parthenogenetic genetic characteristics;
  • the early generation stable line with parthenogenetic hereditary characteristics is crossed with the dominant trait rapeseed (such as dominant dwarf, purple leaf, mosaic, yellow leaf, high erucic acid and other traits), and the hybrid F 1 seed is obtained.
  • the hybrid F 1 seed is subjected to artificial chromosome double doubling on the medium with a chromosome doubling inducer to obtain a doubled F 1 plant with a dominant trait;
  • Dominant traits in polyploid plants that are genetically stable, have parthenogenetic genetic characteristics, and have dominant traits can remove hybrids produced in the test progeny, if dominant traits are present in the test progeny, or Aneuploid plant, indicating that the plant is produced by crossing a polyploid plant and a female parent, and removing the plant;
  • the dominant polyploid plant is a double haploid induction line of Brassica napus.
  • the double haploid induction line of Brassica napus L. can directly induce the double haploid progeny of rapeseed, without artificial chromosome doubling to obtain homozygous lines; and the induction efficiency is high, up to 100%, and the general induction efficiency is above 50%.
  • double haploid induction induces the production of double haploids in maternal plants is that the induced lines can induce maternal plants, and the megaspore germ cells (eggs) undergo chromosome doubled and produce parthenogenetic effects, ie, parthenogenetic reproduction after egg cell doubling
  • eggs megaspore germ cells
  • the stable progeny of Brassica napus obtained by the present invention can induce parthenogenesis in the F1 generation by the double haploid induction line of the Brassica napus, and form a stable double haploid individual in the F 2 generation, and the F 3 generation is stable and consistent.
  • the method can be rapid and effective, and only three generations (two or three years) can obtain stable homozygous rapeseed lines, and the efficiency and pertinence of the rapeseed breeding breeding materials and conventional rapeseed varieties are improved.
  • Brassica napus L. is the most widely used rapeseed cultivar at present. More than 90% of the promoted Brassica napus L. is a hybrid variety.
  • the breeding of hybrid varieties is mainly based on the selection of sterile lines (corresponding to maintainer lines) and restorer lines.
  • the hybridization between sterile line and restorer line realizes the utilization of heterosis, and forms a hybrid variety with excellent yield improvement potential, disease resistance and lodging resistance.
  • the key to breeding Brassica napus hybrids is to select and culture a variety of excellent traits.
  • the genetic stability is restored to the restorer line and the maintainer line, while the breeding and restoring system, the maintenance system has a long time period and consumes a lot of manpower and material resources. Therefore, it is difficult to breed excellent hybrid varieties of Brassica napus L.
  • F hybrid seed having a dominant trait in hybrid rapeseed medium obtained Artificial chromosome doubling with chromosome doubling inducer, the specific method is as follows:
  • the first medium described above consists of the following components:
  • the second medium described above consists of the following components:
  • the third medium described above consists of the following components:
  • the above soaking buffer consists of the following components:
  • the above chromosome doubling inducer is at least one of colchicine, trifluralin, and amsulfame.
  • the method described above can be rapidly applied to the breeding of Brassica napus hybrids, especially the rapid selection of restorer lines and maintainer materials, and can also be used for rapid selection of conventional varieties.
  • the above materials or varieties can be obtained in 2 or 3 generations, which greatly saves the breeding time of rapeseed and improves breeding efficiency.
  • the method of the invention can rapidly select new materials or varieties of hybrid rape breeding of Brassica napus L., and has great application potential in the breeding of restorer lines and maintainer lines of Brassica napus L., the fastest 3 generations (2 years), and obtain genetic stability.
  • Cytoplasmic sterile CMS Bomus cytoplasmic sterile polima CMS, radish cytoplasmic sterile ogura CMS, mustard-type oil cytoplasmic sterility Hau CMS, JA cytoplasmic sterility JACMS) restorer line and maintainer line, 4 Generation (2-4 years) formed a new hybrid rapeseed combination (new variety), and also obtained the GMS restorer line of Brassica napus L. var.
  • the invention can also rapidly select the conventional varieties of Brassica napus L., and the third generation of breeding conventional rapeseed varieties with production potential.
  • This method can rapidly select the parent material of Brassica napus L. hybrids (recovery line and maintainer line), and the 4th generation (2-4 years) breed a new hybrid rapeseed combination with potential for promotion.
  • the earth has improved the breeding speed and efficiency of Brassica napus hybrids;
  • the method can rapidly (2 or 3 generations), large-scale selection of conventional varieties of Brassica napus L., greatly improving the breeding speed and efficiency of Brassica napus varieties;
  • the method can be applied to Brassica napus L., especially the utilization of different heterosis in breeding of hybrid varieties.
  • Cytoplasmic sterility system of Brassica napus L. (Bolioma cytoplasmic sterility polima CMS, radish cytoplasmic sterility ogura CMS, mustard type oil cytoplasmic sterility Hau CMS, JA cytoplasmic sterility JA CMS), Brassica napus sterility system (GMS) can be applied;
  • the double haploid induction of Brassica napus directly induces the production of double haploids in the maternal plants, eliminating the need for artificial chromosome doubling, and forming stable progeny in one step.
  • Figure 1 is a flow chart showing the selection of restorer lines, maintainer lines and conventional varieties of Brassica napus L. in the double haploid induction line of Brassica napus L.
  • Figure 2 is a flow chart showing the breeding of the double haploid induction line of Brassica napus L.
  • Figure 3 is a flow chart of a method for obtaining a stable line of rapeseed in the early generation.
  • Figure 4 is a flow chart showing the breeding of rapeseed double haploid induction line Y3560.
  • Figure 5 is a flow chart showing the breeding of rapeseed double haploid induction line Y3380.
  • Figure 6 is a flow chart showing the breeding of P3-2 in the early generation of rapeseed.
  • Figure 7 is a selection diagram of the cytoplasmic polimaCMS restorer line C2859 of Brassica napus L.
  • Figure 8 is a selection diagram of the GMS restorer line C2994 of Brassica napus L.
  • Figure 9 is a selection diagram of the cytoplasmic polioCMS maintainer line B4653 of Brassica napus L.
  • Figure 10 is a selection diagram of the cytoplasmic ogura CMS restorer line C4707 of Brassica napus L.
  • Figure 11 is a diagram showing the selection of the cytoplasmic ogura CMS retention line of the Brassica napus L.
  • Figure 12 is a graph showing the ploidy cell ploidy of P3-2 tetraploid rapeseed.
  • Figure 13 is a graph showing the ploidy cell ploidy of P3-2 tetraploid rapeseed.
  • Figure 14 is a Y3380 flow cytoplasmic identification map.
  • Figure 15 is a graph showing the flow ploidy of Y3560 flow cytometry.
  • F 11 generation (induced offspring) were planted and tested by flow cytometry, normal breeding, normal ploidy (tetraploid), non-inducible dominant traits (dwarf) plants, single bagging, self-crossing, and selection
  • the excellent single line was tested with the polima cytoplasmic male sterile line "Rong A0068", and the tested progeny were fully fertile and the traits of the individual plants within the line were consistent, indicating that the line was a restorer line. Therefore, after the induction of 2 generations, a stable cytoplasmic male sterility restorer line "Chun C2859" of Brassica napus L. was developed. And with the "Rong A0068" configuration hybrid combination miscellaneous 1256, and in 2014, 2015 through the Sichuan province rapeseed regional test and national zone test, entered the 2016 annual production test, pending validation of the variety.
  • the rapeseed double haploid induction line was obtained by the following method:
  • the tetraploid early generation stable line P3-2 of Brassica napus L. obtained by the applicant, and 20 homozygous cabbage type tetraploid rapeseed are positive Backcrossing, the three positive and negative F 1 generations were separated, and the three combined F 2 generations showed stable strains, indicating that P3-2 had parthenogenetic genetic characteristics.
  • P3-2 and tetraploid Brassica napus D3-5 reciprocal dwarf (dwarf dominant trait) then the F 1 hybrid seeds chromosome doubling, doubling the offspring identified by flow cytometry or by apical Microscopic observation identified it as a dwarf octaploid plant named Y3380.
  • Example P3-2 with the present embodiment will dwarf rapeseed D3-5 hybrid F 1, P3-2 and dwarf, high erucic acid rapeseed 4247 F 1 hybrid seed specific artificial chromosome doubling is carried out with colchicine in the culture medium as follows:
  • the surface of the seed is sterilized with 75% alcohol for 25 seconds, disinfected with 0.1% liter of mercury for 12 minutes, then rinsed with mercury in the surface of the seed with sterile water, and the surface of the seed is blotted with sterile paper.
  • the seed is then seeded on the first medium (chromosome double induction medium);
  • the culture conditions are: temperature 25 ° C, daylight illumination 16 hours, light intensity 2000 lux, dark culture at night for 8 hours, when growing to 1-2 true leaves, the plants are Hypocotyl cutting continues to grow on the second medium;
  • the cut plants are continuously inserted into the second medium to continue the culture, and after the lateral buds are differentiated, the lateral buds and the plants are transferred to the third medium (rooting medium) for rooting culture;
  • the first medium described above consists of the following components:
  • MS medium was invented by Murashige and Skoog, abbreviated as MS, and its formulation is shown in Schedule 1.
  • the second medium described above consists of the following components:
  • the third medium described above consists of the following components:
  • the above soaking buffer consists of the following components:
  • Y3380 was used as the male parent, and the cytoplasmic sterile line (0464A) of Brassica napus L. was tested and tested, and 50 progeny were tested, all of which were high poles, and all were tetraploid rapeseed, among which 49 strains were all sterile, 1 strain was semi-sterile, and the morphological characteristics were identical to those of 0464A.
  • Y3380 was used as the male parent and the rapeseed 3954 thawing polymerization hybridization (3954 is F 1 , which is derived from the hybridization of Zhongshuang 11 and CAX), the polymeric hybrid progeny F 1 is separated, each F 1 is selfed, and F 1 is selfed. 45 strains. 45 F 2 generation strains were planted, and 45 stable strains appeared. The stable strains showed a ratio of 100% and the induction rate was 100%.
  • Y3380 was used as the male parent and the rapeseed 3968 was deagglomerated and polymerized (3968 was F 1 , which was hybridized from Zhongshuang 11 and 1365). The polymerized hybrid progeny F 1 was isolated, each F 1 was selfed, and F 1 was selfed. 52 strains. 52 F 2 generation strains were planted, 28 stable strains appeared, the proportion of stable strains was 53.85%, and the induction rate was 53.85%.
  • Y3380 made with paternal and 11 Brassica bis (conventional varieties, homozygous lines) emasculation hybridization, hybridization obtained 70 plants F 1, F 1 70 11 identical shape and double, and from each plant After the crossing, the F 2 generation did not separate, and it was a stable strain, which was identical to the form of Zhongshuang 11 , indicating that the F 1 generation was pure. That is, the process of hybridization between Y3380 and Zhongshuang 11 induces parthenogenesis in Zhongshuang 11 and the F 1 produced is parthenogenetic selfing, which is a homozygous line, so F 1 is stable, F 2 is also stable, and The morphology of 11 is identical, and the induction rate is 100%.
  • cytoplasmic sterile line (0464A) of Brassica napus L. was tested and tested, and 80 progeny were tested, all of which were high rods, and 76 strains were tetraploid rapeseed. 2 strains were diploid and 2 strains were octaploid; 76 of the tetraploid plants were completely sterile, 4 were semi-sterile, and the morphological characteristics were identical to those of 0464A.
  • testcross progeny 153, 102 appear dwarf, high pole 51, The fertility separation was large, with 65 full-fertility, 35 semi-sterile, and 53 sterile. It indicated that the gene in Y3560 did not enter the test cross, and the progeny of the test was 4646A parthenogenetic, and the induction rate was 95%.
  • emasculation hybridization to obtain F 1 hybrid plants 124, 123 and GW Form F 1 is identical, and each plant selfing
  • the morphology of the post-F 2 generation is tetraploid and the shape is consistent with YH, indicating that the hybridization process between Y3560 and GW induces parthenogenesis in GW, and the resulting F 1 is parthenogenetic self-crossing, and is identical to GW morphology.
  • the induction rate was 99.2%.
  • the dominant dwarf octoploid plant Y3560 was identified as the rapeseed double haploid induction line.
  • the method for obtaining the early generation stability system P3-2 is as follows:
  • F 1 generation hybrid seeds were artificially chromosome doubled with colchicine on the medium. Of the F 1 plants were selfed doubled (selfing or forced) to obtain the F 2, planted in the field for the observation of the F 2, i.e.
  • pollen fertility is determined, three kinds of occurrence Situation (1, haploid plants, pollen is very rare, and fertility is extremely low; 2, polyploid plants are completely sterile, flower organ development is blocked, can not normally flower, no pollen; 3, normal fertile plants, The amount of pollen is more than 95% of pollen fertility.
  • F 3 generations of normal fertile plants were selfed to obtain F 3 generation. The homozygous degree of F 3 generation was identified, and the F 3 generation single plant line was planted. 32% of the fertile lines were uniform and the flowering was normal. The cytological identification of the uniform lines was consistent, the number of chromosomes was consistent (38), and the chromosome morphology did not appear abnormal.
  • SSR molecular markers by DNA polymerase chain reaction, electrophoresis observation of each specific primer amplification of single DNA band type, showing that each individual plant is a hybrid progeny of F009 and YH, and each individual DNA amplification band The number and band type are consistent, and it can be judged that these lines are homozygous, that is, early generation stable lines.
  • One of the early-generation stable lines of Brassica napus (Chromosome 38) with large leaves, no lobes, and compact leaves, and oil content of 55% was named P3-2.
  • the specific method for artificial chromosome doubling of the F1 hybrid seed on the medium with colchicine is as follows:
  • the surface of the seed is sterilized with 75% alcohol for 25 seconds, disinfected with 0.1% liter of mercury for 12 minutes, then rinsed with mercury in the surface of the seed with sterile water, and the surface of the seed is blotted with sterile paper.
  • the seed is then seeded on the first medium (chromosome double induction medium);
  • the culture conditions are: temperature 25 ° C, daylight illumination 16 hours, light intensity 2000 lux, dark culture at night for 8 hours, when growing to 1-2 true leaves, the plants are Hypocotyl cutting continues to grow on the second medium;
  • the cut plants are continuously inserted into the second medium to continue the culture, and after the lateral buds are differentiated, the lateral buds and the plants are transferred to the third medium (rooting medium) for rooting culture;
  • the first medium described above consists of the following components:
  • MS medium was invented by Murashige and Skoog, abbreviated as MS, and its formulation is shown in Schedule 1.
  • the second medium described above consists of the following components:
  • the third medium described above consists of the following components:
  • the above soaking buffer consists of the following components:
  • the early generation stable line P3-2 of Brassica napus L. is crossed with Zhongshuang 11 and the hybrid progeny F1 is artificially emasculated.
  • the rapeseed double haploid induction line Y3380 obtained by the applicant is used. Pollination, inducing progeny bagging self-crossing, F 2 generation (inducing offspring) for planting and flow cytometry, normal breeding, ploidy (tetraploid) normal, no induced line dominant trait (dwarf) plants The self-crossing of the individual plants was carried out, and the purity was identified in the F 3 generation.
  • the stable strain 4653 was selected and tested with the polimaCMS cytoplasmic male sterile line "Rong A0068", and the progeny were tested as complete sterile, indicating that the induced stable strain 4653 was a maintainer.
  • the polimaCMS cytoplasmic retention system has high oil content (more than 49%), lodging resistance, good disease resistance, early maturity, and complete infertility.
  • the infertile offspring is less affected by temperature, and is currently undergoing multiple generations of backcross replacement.
  • the nuclear gene of the breeding line is formed to correspond to the sterile line A4653 of the line of the B4653.
  • the test showed that the male parent 4707 was the restorer line, and the cytoplasmic ogura CMS restorer line C4707 was formed.
  • the recovery system restores the radish cytoplasmic sterile line, complete double low quality, anti-falling, antibacterial disease, and the oil content is more than 45%.
  • the radish cytoplasmic male sterile line of common rapeseed is its maintainer line, hybridized with Chuanyou 36 and P3-2, to obtain polymeric hybrid F1, F1 to use the applicant
  • the obtained rapeseed double haploid induction line Y3560 was pollinated.
  • F 2 generation (inducing offspring) was planted and tested by flow cytometry, normal breeding, ploidy (tetraploid) normal, no induced line dominant traits (dwarf) plants, single bagging, F 3 Purity identification was carried out, and a stable strain 4700 was obtained.
  • the cabbage type double low material "925100” is hybridized with "Huza No. 3 selection line (F5)", and the excellent individual plants are selfed.
  • F 5 F 6 generation of artificial emasculation, with rapeseed obtained by the present applicant double haploid inducer Y3560 pollination
  • F 7 generation Progenies planting and flow cytometry, selected from The normal fertility, ploidy (tetraploid) normal, non-inducible dominant traits (dwarf) plants were self-crossed, and the selected single plant was tested with the nuclear (GMS) sterile line "Rong A4979".
  • the progeny of the tested progeny were fully fertile and the traits of the individual plants within the line were consistent. This indicates that the line is a nuclear (GMS) restorer line. Therefore, after the induction of 2 generations, a stable restorer line of the canola nuclear sterility was established. Rong C2994". The restorer line was combined with "Rong A4979" to produce Brassica napus hybrid combination 15149, which is a combination of early maturity and is currently undergoing the first year regional trial.
  • the breeding method of the rapeseed double haploid inducing line in each of the above examples is the same as in the first embodiment.

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Abstract

本申请提供了一种利用油菜双单倍体诱导系选育甘蓝型油菜的方法，包括：1）确定甘蓝型油菜恢复系、保持系、常规品种选育的目标性状；2）将两个或多个具有目标性状的甘蓝型油菜杂交或聚合杂交；3）用油菜双单倍体诱导系对杂交或回交后代授粉；4）诱导后代稳定性鉴定；5）稳定后代测交鉴定或产量、抗性鉴定；6）形成稳定的恢复系、保持系进行杂交育种配组或形成常规品种。

Description

油菜双单倍体诱导系选育甘蓝型油菜品种及材料的方法 技术领域：
本发明与农业有关，特别与油菜双单倍体诱导系选育甘蓝型油菜品种和材料的方法有关。
背景技术：
油菜是我国主要的油料作物，甘蓝型油菜(Brassica napus，芸苔(aa，n＝10)与甘蓝(cc，n＝9)通过自然种间杂交后双二倍化进化而来的一种复合种，根据染色体来源判断为四倍体(2n＝38)，是目前油菜生产中主要的栽培油菜类型。甘蓝型油菜新品种选育，首先是选育新的自交系或遗传稳定的纯合株系-纯合系(自交系)，如这些纯合株系在抗性、产量、品质等要求上满足生产需要，通过区域试验最后认定或审定为新的油菜新品种(常规品种)。其次，纯合株系与不育系测交，判断恢保关系，如果是恢复系与不育系杂交测配新的杂交品种，如果是保持系与不育系测配，选育具有该保持系特性的新的不育系，如果稳定株系不恢不保(测配后代不能完全恢复育性或不能完全保持高度不育)，也不能行成生产上使用的常规品种，这样的纯合株系要么淘汰，要么与其他保持系或恢复系杂交进入下一轮育种材料选育。正常情况下通过常规的人工杂交选育一个常规油菜品种需要6-7代的时间，如果选育杂交品种，需要培育稳定的不育系、保持系、恢复系，油菜新品种选育时间更长，10-15年。常规油菜新品系选育是通过两个或多个遗传背景不同的品系杂交、聚合杂交或回交形成杂交F1代(或回交BC1代，根据目标性状的选择要求可进行多代回交形成BC2、BC3……等等)，回交后代或F1代自交形成F2代，F2代再选择优良单株自交形成F3代，F3再选单株自交，一直到F6-F7代才能获得稳定的油菜新品系，以1年1代计算，所花时间大概在7-8年的时间，通过异地加代也需要4年左右的时间。
目前，在油菜中还未有诱导系或双单倍体诱导系的报道。所谓“诱导系”是指，用该植物作为父本用其花粉对同类植株授粉，能诱导同类植株(母本)产生相应的效应，如产生单倍体、双单倍体(DH系)等。在植物中运用诱导系进行新品种选育最多的是玉米，但玉米中的诱导系也只是单倍体诱导系。最早出现的玉米单倍体诱导系为stock6，该诱导系只能诱导玉米产生单倍体，然后单倍体植株再进行人工染色体加倍形成纯合二倍体(双单倍体)，且诱导效率较低，一般诱导效率在10％以下(以 收获种子中获得单倍体数计算)。
发明内容：
本发明的目的为了提供一种可快速选育遗传稳定的甘蓝型油菜恢复系、保持系或常规品种的油菜双倍体诱导系选育甘蓝型油菜品种及材料的方法。
本发明的目的是这样来实现的：
本发明油菜双单倍体诱导系选育甘蓝型油菜品种及材料的方法，包括以下步骤：
1)确定甘蓝型油菜恢复系、保持系、常规品种选育的目标性状，将至少两个具有目标性状的甘蓝型油菜杂交或聚合杂交，根据目标性状要求进行回交或多代回交，形成杂交后代、聚合杂交后代或回交后代；
2)对步骤1)中获得的杂交、聚合杂交后代或回交后代材料，在花期，对花蕾进行人工去雄，并套袋隔离；
3)用油菜双单倍体诱导系花粉对步骤2)中去雄后2-4天植株进行人工授粉，并套袋隔离，收获其授粉后诱导结实种子；
4)对上述步骤3)获得的诱导种子进行单株种植，苗期利用流式细胞仪鉴定倍性，淘汰多倍体、单倍体或具有油菜双单倍体诱导系显性性状特征植株，选择正常育性四倍体植株，单株套袋自交；
5)上述步骤4)中获得的正常育性四倍体单株自交种子进行株系种植，调查株系形态一致性，并通过分子标记(SSR或SRAP)鉴定株系一致性及稳定性；
6)对上述步骤5)中鉴定的稳定四倍体株系与甘蓝型油菜细胞质(玻里马细胞质不育polima CMS、萝卜细胞质不育ogura CMS、芥菜型油胞质不育Hau CMS、JA胞质不育JA CMS)不育系进行测交，或与甘蓝型油菜核不育(GMS)系测交，并对测交后代进行育性鉴定，判断测交父本的恢保关系；
7)上述步骤6)中测交后代全不育，对应测交父本为保持系；测交后代全可育，对应测交父本为恢复系；
8)上述步骤7)中测交鉴定的保持系继续与不育系多代回交，选育稳定的与该保持系核基因一致的不育系；测交鉴定的恢复系，直接与对应系统的不育系测配，选育杂交组合，杂交组合进入品种比较试验，产量、抗性、丰产性、品质性状好于生产上大面积应用的品种，满足品种认定(或审定)标准即可形成杂交油菜品种，并通过省级区域或国家种子管理部门认定(或审定)登记，可生产上推广应用；
9)对上述步骤6)中获得的稳定株系进行品比及生产试验，产量、抗性、丰产性、品质性状优于对照，满足品种认定(或审定)标准即可形成常规品种，并通过省级区域或国家种子管理部门认定(或审定)登记，可生产上推广应用；
上述油菜双单倍体诱导系的选育方法，包括如下步骤：
(1)选育具有孤雌生殖遗传特性的早代稳定系：
①将两个油菜亲本材料杂交F1代种子在培养基上用染色体加倍诱导剂进行人工染色体加倍获得加倍后的F1代植株；
②加倍后的F1代植株进行自交或强制自交获得F2代，对F2代进行田间种植观察，并鉴定每个单株的育性，选择可育后代自交获得F3代，对F3代进行纯合度鉴定，通过形态、细胞学以及分子标记鉴定，对后代DNA进行聚合酶链反应扩增，电泳观察每个特异引物扩增下单株的DNA带型及条带数目，显示每个单株都是两个亲本的杂交后代，每个单株之间分子标记图谱一致，说明这些单株是纯合系---早代稳定系；
③获得的早代稳定系与至少10个油菜常规纯合稳定系进行正反交，F1代、F2代鉴定早代稳定系的遗传特性，即是否有孤雌生殖特性；上述正反交，如有F1分离，F2代出现部分稳定株系，对应的早代稳定系是具有孤雌生殖遗传特性的早代稳定系；
(2)选育携带显性遗传性状、具有孤雌遗传特性且倍性遗传稳定的多倍体油菜
①具有孤雌生殖遗传特性的早代稳定系与具有显性性状油菜杂交(如显性矮杆、紫叶、花叶、黄叶、高芥酸等性状)，得到杂交F1代种子。上述杂交F1种子在培养基上用染色体加倍诱导剂进行人工染色体加倍，得到加倍后的带显性性状的F1植株；
②对加倍的带显性性状的F1植株，通过显微观察或流式细胞仪进行染色体倍性鉴定，选择带显性性状的多倍体的植株，淘汰非正常加倍株、非整倍体植株、以及不带显性性状加倍植株，带显性性状的多倍体植株主要是倍性遗传稳定、结实性好、具有孤雌生殖遗传特性、带显性性状(如显性矮杆、紫叶、花叶、黄叶、高芥酸等性状)的六倍体或八倍体油菜植株；
(3)油菜双单倍体诱导系鉴定及诱导能力测定：
①倍性遗传稳定、具有孤雌生殖遗传特性、带显性性状的多倍体植株中的显性性状能去除测交后代中产生的杂交株，如果测交后代中出现显性性状植株、或非整倍体植株，说明该植株是多倍体植株和母本杂交产生的，去除该植株；
②上述单株测交后代如果出现全不育、为正常倍性(二倍体或四倍体)油菜、且不带显性性状，说明该测交后代对应的父本基因未进入测交后代中，显性多倍体植株为油菜双单倍体诱导系。油菜双单倍体诱导系能直接诱导油菜产生双单倍体后代，无需进行人工染色体加倍来获得纯合系；且诱导效率高，最高可达100％，一般的诱导效率都在50％以上。双单倍体诱导系诱导母体植株产生双单倍体的可能原理是：诱导系能诱导母体植株，大孢子生殖细胞(卵细胞)进行染色体加倍并产生孤雌生殖效应，即卵细胞加倍后孤雌生殖产生双单倍体，产生该现象的确切机理目前尚不明确。
采用本发明获得油菜稳定遗传后代借助了油菜双单倍体诱导系能诱导母体植株在F1代发生孤雌生殖，在F2代形成稳定的双单倍体个体，F3代进行稳定性、一致性鉴定，获得稳定遗传后代。该方法能快速、有效，只需3代(2年或3年)获得稳定纯合系油菜株系，提高了甘蓝型油菜选育育种材料、常规油菜品种的效率及针对性。甘蓝型油菜是目前生产上应用最为广泛的油菜栽培种，推广的甘蓝型油菜90％以上为杂交品种，杂交品种选育主要以不育系(对应保持系)、恢复系选育为主，通过不育系与恢复系的杂交实现杂种优势利用，形成生产上具有产量提升潜力、抗病、抗倒性优良的杂交品种；选育甘蓝型油菜杂交品种的关键是选育聚合多种优良性状且遗传稳定得恢复系、保持系，而选育恢复系、保持系时间周期长、耗费人力物力大，因此选育优良甘蓝型油菜杂交品种难度较大。
上述获得油菜双单倍体诱导系是将两个亲本材料杂交F1代种子或具有孤雌生殖遗传特性的早代稳定系与具有显性性状油菜杂交得到的杂交F1代种子在培养基上用染色体加倍诱导剂进行人工染色体加倍，具体方法如下：
1)用纯度为75％酒精进行种子表面消毒25-40秒，用0.1％升汞消毒12-17分钟，然后用无菌水将种子表面的升汞冲洗干净，用无菌纸将种子表面的水分吸干，然后将种子接种在第一培养基上；
2)让种子在第一培养基上生根发芽，培养条件：温度23-25℃，白天光照12-16小时，光照强度2000-3000勒克斯，夜晚暗培养8-12小时，待植株长到1—2片真叶时，从下胚轴处剪下植株继续在第二培养基上生长；
3)将剪下的植株继续插入第二培养基上继续培养，待有侧芽分化后，将侧芽及植株转入第三培养基中进行生根培养；
4)生根培养二周后，植株长出粗壮的根后，将植株在室温炼苗3-7天，取出植株将植株上的培养基用自来水冲洗干净，并在浸泡缓冲液中浸泡15—30分钟后移栽到温室中，温室温度16℃—25℃，相对湿度60-80％，能保证移栽成活率在95％以上；
上述的第一培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000001
上述的第二培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000002
上述的第三培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000003
上述的浸泡缓冲液由以及下配比的组分组成：
水                     1L
易保或克露              0.6-1.2g
α-萘乙酸             0.5—1mg。
上述的染色体加倍诱导剂采用秋水仙素、氟乐灵、氨磺乐灵中的至少一种。
以上描述的方法可以快速用于甘蓝型油菜杂交品种选育，特别是恢复系、保持系材料的快速选育，也可用于常规品种的快速选育。可以在2年或3代的时间内获得上述材料或品种，大大节约油菜的育种时间，提高育种效率。
本发明方法能快速选育甘蓝型油菜杂交育种新材料或品种,特别是对甘蓝型油菜恢复系、保持系的选育中具有巨大的应用潜力，最快3代(2年)，获得遗传稳定的甘蓝型油菜细胞质不育CMS(玻里马细胞质不育polima CMS、萝卜细胞质不育ogura CMS、芥菜型油胞质不育Hau CMS、JA胞质不育JACMS)恢复系和保持系，可4代(2－4年)形成杂交油菜新组合(新品种)，也可最快3代获得甘蓝型油菜核不育GMS恢复系。本发明也能快速选育甘蓝型油菜常规品种，3代选育具有生产潜力的常规油菜品种。
本发明具有以下优点：
1、该方法可快速(2年或3代)选育甘蓝型油菜杂交种亲本材料(恢复系、保持系)，4代(2-4年)选育具有推广潜力的杂交油菜新组合，极大地提高了甘蓝型油菜杂交品种选育速度和效率；
2、该方法可快速(2年或3代)、规模化选育甘蓝型油菜常规品种，极大地提高了甘蓝型油菜品种选育速度和效率；
3、该方法可运用于甘蓝型油菜，特别是杂交品种选育的不同杂种优势利用途径。甘蓝型油菜细胞质不育系统(玻里马细胞质不育polima CMS、萝卜细胞质不育ogura CMS、芥菜型油胞质不育Hau CMS、JA胞质不育JA CMS)，甘蓝型油菜核不育系统(GMS)均可应用；
4、油菜双单倍体诱导系直接诱导母体植株产生双单倍体，无需进行人工染色体加倍，可一步形成稳定后代。
附图说明：
图1为油菜双单倍体诱导系选育甘蓝型油菜恢复系、保持系、常规品种流程图。
图2为油菜双单倍体诱导系选育流程图。
图3为获得油菜早代稳定系的方法流程图。
图4为油菜双单倍体诱导系Y3560选育流程图。
图5为油菜双单倍体诱导系Y3380选育流程图。
图6为油菜早代稳定系P3-2选育流程图。
图7为甘蓝型油菜细胞质polimaCMS恢复系蓉C2859的选育图。
图8为甘蓝型油菜细胞核GMS恢复系蓉C2994的选育图。
图9为甘蓝型油菜细胞质polimaCMS保持系蓉B4653的选育图。
图10为甘蓝型油菜萝卜胞质ogura CMS恢复系蓉C4707的选育图。
图11为甘蓝型油菜萝卜胞质ogura CMS保持系蓉B萝4700的选育图。
图12为P3-2四倍体油菜倍流式细胞倍性鉴定图。
图13为P3—2四倍体油菜倍流式细胞倍性鉴定图。
图14为Y3380流式细胞倍性鉴定图。
图15为为Y3560流式细胞倍性鉴定图。
具体实施方式：
实施例1
参见图1、图2、图5、图7，甘蓝型双低材料“925100”与恢复材料“2150×里今特”杂交，并选优良单株自交，到F9代时仍有分离，F10代时人工去雄，用由本申请人获得的油菜双单倍体诱导系Y3380授粉，获得大量诱导后代种子。F11代(诱导后代)进行种植并进行流式细胞检测、选育性正常、倍性(四倍体)正常、无诱导系显性性状(矮秆)植株单株套袋自交，并选优良单株系与polima细胞质不育系“蓉A0068”测交，测交后代全可育且株系内部单株之间性状一致说明该株系为恢 复系。因此在诱导2代后育成了稳定的甘蓝型双低油菜细胞质雄性不育恢复系“蓉C2859”。且与“蓉A0068”配置杂交组合杂1256，并于2014、2015年度通过四川省油菜区试和国家区试试验，进入2016年度生产试验，待审定品种。
以上实施例中，油菜双单倍体诱导系是通过以下方法获得的：
参见图2、图4、图6、图12、、图13、图15，由本申请人获得的甘蓝型油菜四倍体早代稳定系P3—2，与20个纯合甘蓝型四倍体油菜正反交，3个正反交F1代出现分离，且这3个组合F2代出现稳定株系，说明P3—2具有孤雌生殖遗传特性。用P3—2与高芥酸、矮杆油菜4247正反交(矮杆、高芥酸为显性性状)，然后将杂交F1代种子进行染色体加倍，加倍后代用流式细胞仪鉴定或根尖显微镜观察鉴定为显示矮杆八倍体植株，该植株定名为Y3560。
参见图2、图5、图6、图12、图13、图14，由本申请人获得的甘蓝型油菜四倍体早代稳定系P3—2，与20个纯合甘蓝型四倍体油菜正反交，3个正反交F1代出现分离，且这3个组合F2代出现稳定株系，说明P3—2具有孤雌生殖遗传特性。用P3—2与四倍体甘蓝型矮杆油菜D3—5正反交(矮杆为显性性状)，然后将杂交F1代种子进行染色体加倍，加倍后代用流式细胞仪鉴定或根尖显微镜观察鉴定为显示矮杆八倍体植株，该植株定名为Y3380。
本实施例中将P3-2与矮杆油菜D3—5杂交F1、P3-2与矮杆、高芥酸油菜4247杂交F1种子在培养基上用秋水仙素进行人工染色体加倍的具体方法如下：
1)用纯度为75％酒精进行种子表面消毒25秒，用0.1％升汞消毒12分钟，然后用无菌水将种子表面的升汞冲洗干净，用无菌纸将种子表面的水分吸干，然后将种子接种在第一培养基(染色体加倍诱导培养基)上；
2)让种子在第一培养基上生根发芽，培养条件：温度25℃，白天光照16小时，光照强度2000勒克斯，晚上暗培养8小时,待长到1—2片真叶时，将植株从下胚轴剪下继续在第二培养基上生长；
3)将剪下的植株继续插入第二培养基上继续培养，待有侧芽分化后，将侧芽及植株转入第三培养基(生根培养基)中进行生根培养；
4)生根培养二周后，植株长出粗壮的根后，将植株在室温炼苗3天后，取出植株将植株上的培养基冲洗干净，并在浸泡缓冲液中浸泡15分钟后移栽到温室中，温室温度25℃，相对湿度60％，能保证移栽成活率在95％以上；
上述的第一培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000004
Figure PCTCN2016111327-appb-000005
MS培养基由Murashige和Skoog发明，简写为MS，其配方参见附表1，
上述的第二培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000006
上述的第三培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000007
上述的浸泡缓冲液由以下配比的组分组成：
水                     1L
易保或克露             0.6g
α—萘乙酸            0.5mg。
参见图2、图3、图5，用Y3380作父本，与甘蓝型油菜细胞质不育系(0464A)测交，测交后代50株，全为高杆,且全为四倍体油菜，其中49株为全不育，1株半不育，且形态特征与0464A完全相同。同时用P3-2与矮杆油菜D3-5杂交F1(非加倍株)做父本与0464A测交作为对照验证，测交后代102株，出现矮杆62株、高杆40株、且育性分离较大，出现全可育73株、半不育20株、全不育9株。说明Y3380中的基因并未进入测交株，测交后代为0464A孤雌生殖而来，诱导率98％。用Y3380做父本与甘蓝型油菜3954去雄聚合杂交(3954为F1，由中双11与CAX杂交而来)，聚合杂交后代F1分离，每个F1自交，收获F1自交株45个。种植F2代株系45个，出现稳定株系45个，稳定株系出现比列100％，诱导率100％。
用Y3380做父本与甘蓝型油菜3968去雄聚合杂交(3968为F1，由中双11与1365杂交而来)，聚合杂交后代F1分离，每个F1自交，收获F1自交株52个。种植F2代株系52个，出现稳定株系28个，稳定株系出现比例53.85％，诱导率53.85％。
用Y3380做父本与甘蓝型油菜中双11(常规品种，纯合系)去雄杂交，获得杂交F1植株70株，70株F1形态与中双11完全相同，且每个单株自交后F2代未发生分离，为稳定株系，与中双11形态也完全相同，说明F1代就为纯系。即Y3380与中双11杂交过程，诱导中双11发生了孤雌生殖，所产生的F1为孤雌生殖自交，是纯合系，因此F1稳定、F2也稳定，且与中双11形态完全相同，该诱导率100％。
同样用Y3380做父本与白菜型油菜雅安黄油菜YH(二倍体油菜，2n＝20)去雄杂交，获得杂交F1植株98株，97株F1形态与YH完全相同，且每个单株自交后F2代形态都为二倍体、外形与YH一致，说明Y3380与YH杂交过程，诱导YH发生了孤雌生殖，所产生的F1为孤雌生殖自交，且与YH形态完全相同，该诱导率98.9％。最终，显性矮杆八倍体植株Y3380确定为油菜双单倍体诱导系。
参见图2、图3、图4，用Y3560作父本，与甘蓝型油菜细胞质不育系(0464A)测交，测交后代80株，全为高杆,且76株为四倍体油菜、2株为二倍体、2株为八倍体；其中76株四倍体植株为全不育，4株半不育，且形态特征与0464A完全相同。同时用P3-2与矮杆、高芥酸油菜4247杂交F1(非加倍株)做父本与0464A测交作为对照验证，测交后代153株，出现矮杆102株、高杆51株、且育性分离较大，出现全可育65株、半不育35株、全不育53株。说明Y3560中的基因并未进入测交株，测交后代为0464A孤雌生殖而来，诱导率95％。
用Y3560做父本与白菜型油菜雅安黄油菜YH(二倍体油菜，2n＝20)去雄杂交，获得杂交F1植株145株，143株F1形态与YH完全相同，且每个单株自交后F2代形态都为二倍体、外形与YH一致，说明Y3560与YH杂交过程，诱导YH发生了孤雌生殖，所产生的F1为孤雌生殖自交，且与YH形态完全相同，该诱导率98.6％。
同样用Y3560做父本与芥菜型油菜GW(四倍体油菜，2n＝36)去雄杂交，获得杂交F1植株124株，123株F1形态与GW完全相同，且每个单株自交后F2代形态都为四倍体、外形与YH一致，说明Y3560与GW杂交过程，诱导GW发生了孤雌生殖，所产生的F1为孤雌生殖自交，且与GW形态完全相同，该诱导率99.2％。最终，显性矮杆八倍体植株Y3560确定为油菜双单倍体诱导系。
参见图3、图6、图12、图13，获得早代稳定系P3-2方法如下：
甘蓝型油菜F009(四倍体，染色体2n＝38)与白菜型油菜YH(二倍体，雅安黄油菜，染色体2n＝20)剥蕾进行人工去雄杂交获得F1代杂交种子。F1代杂交种子在培 养基上用秋水仙素进行人工染色体加倍。对加倍后的F1代植株进行自交(或强制自交)获得F2代，对F2代进行田间种植观察、育性鉴定即通过醋酸洋红对花粉染色，判断花粉育性，出现三种情况(1、单倍体植株，花粉极少，且育性极低；2、多倍体植株完全不育，花器官发育受阻，不能正常的开花，无花粉；3、正常可育的植株，花粉量多，花粉育性95％以上)。对F2代正常可育单株进行自交获得F3代。对F3代进行纯合度鉴定，种植F3代单株株系，32％的可育株系单株植株整齐一致，开花结实正常。对整齐一致株系进行细胞学鉴定，染色体条数一致(38条)，染色体形态未出现异常。SSR分子标记，通过DNA聚合酶链反应，电泳观察每个特异引物扩增下单株DNA带型，显示每个单株都是F009与YH的杂交后代，且每个单株DNA扩增条带数目及带型一致，可以判断这些株系为纯合系，即早代稳定系。将其中1个叶片较大、无裂叶、叶片着生紧凑、含油率55％的甘蓝型(染色体38条)油菜早代稳定系定名为P3-2。
本实施例中将F1代杂交种子在培养基上用秋水仙素进行人工染色体加倍的具体方法如下：
1)用纯度为75％酒精进行种子表面消毒25秒，用0.1％升汞消毒12分钟，然后用无菌水将种子表面的升汞冲洗干净，用无菌纸将种子表面的水分吸干，然后将种子接种在第一培养基(染色体加倍诱导培养基)上；
2)让种子在第一培养基上生根发芽，培养条件：温度25℃，白天光照16小时，光照强度2000勒克斯，晚上暗培养8小时,待长到1—2片真叶时，将植株从下胚轴剪下继续在第二培养基上生长；
3)将剪下的植株继续插入第二培养基上继续培养，待有侧芽分化后，将侧芽及植株转入第三培养基(生根培养基)中进行生根培养；
4)生根培养二周后，植株长出粗壮的根后，将植株在室温炼苗3天后，取出植株将植株上的培养基冲洗干净，并在浸泡缓冲液中浸泡15分钟后移栽到温室中，温室温度25℃，相对湿度60％，能保证移栽成活率在95％以上；
上述的第一培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000008
MS培养基由Murashige和Skoog发明，简写为MS，其配方参见附表1。
上述的第二培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000009
上述的第三培养基由以下配比的组分组成：
Figure PCTCN2016111327-appb-000010
上述的浸泡缓冲液由以下配比的组分组成：
水                    1L
易保或克露             0.6g
α—萘乙酸            0.5mg。
附表1 MS培养基成分配方
Figure PCTCN2016111327-appb-000011
Figure PCTCN2016111327-appb-000012
实施例2：
参见图1、图2、图5、图9，甘蓝型油菜早代稳定系P3-2与中双11杂交，杂交后代F1人工去雄，用由本申请人获得的油菜双单倍体诱导系Y3380授粉，诱导后代套袋自交，F2代(诱导后代)进行种植并进行流式细胞检测、选育性正常、倍性(四倍体)正常、无诱导系显性性状(矮秆)植株单株套袋自交，F3代进行纯度鉴定，其中选择稳定株系4653与polimaCMS细胞质不育系“蓉A0068”测交，测交后代为全不育，说明诱导稳定株系4653为保持系，该polimaCMS细胞质保持系含油率高(49％以上)、抗倒性、抗病性好、熟期早，保持不育彻底，不育后代受温度影响小，目前正在进行多代回交置换不育系细胞核基因，形成与蓉B4653保持系对应不育系蓉A4653。
实施例3：
参见图1、图2、图4、图10，萝卜胞质ogura CMS类型，其恢复基因不易获得甘蓝型油菜川油36为四川省长江上中下游审定的萝卜胞质三系杂交油菜，其中含有萝卜胞质恢复基因。用川油36与P3-2杂交，获得聚合杂交F1，F1去雄用本申请人获得的油菜双单倍体诱导系Y3560授粉。F2代(诱导后代)进行种植并进行流 式细胞检测、选育性正常、倍性(四倍体)正常、无诱导系显性性状(矮秆)植株单株套袋自交，F3代进行纯度鉴定，获得稳定株系4707，同时用4707花粉与稳定萝卜胞质不育系萝A100测交，测交后代全可育，说明测交父本4707为其恢复系，形成甘蓝型油菜萝卜胞质ogura CMS恢复系蓉C4707。该恢复系，恢复萝卜胞质不育系彻底、双低品质、抗倒、抗菌病，含油率45％以上。
实施例4：
参见图1、图2、图4、图11，萝卜胞质不育系普通的油菜都为其保持系，用川油36与P3-2杂交，获得聚合杂交F1，F1去雄用本申请人获得的油菜双单倍体诱导系Y3560授粉。F2代(诱导后代)进行种植并进行流式细胞检测、选育性正常、倍性(四倍体)正常、无诱导系显性性状(矮秆)植株单株套袋自交，F3代进行纯度鉴定，获得稳定株系4700，同时用4700花粉与稳定萝卜胞质不育系萝A100测交，测交后代全不育，说明测交父本4700为其保持系，该保持系自交稳定，不分离不育株，含油率高(47％)，双低品质、抗倒、抗菌病，目前正在与萝A100不育系回交，置换该不育系核基因，形成与蓉B萝4700核基因一致的不育系蓉A萝4700。
实施例5：
参见图1、图2、图4、图8，甘蓝型双低材料“925100”与“华杂3号选系(F5)”杂交，并选优良单株自交。到F5代时仍有分离，F6代时人工去雄，用由本申请人获得的油菜双单倍体诱导系Y3560授粉，F7代(诱导后代)进行种植并进行流式细胞检测、选育性正常、倍性(四倍体)正常、无诱导系显性性状(矮秆)植株单株套袋自交，选优良单株与细胞核(GMS)不育系“蓉A4979”测交，测交后代全可育且株系内部单株之间性状一致说明该株系为细胞核(GMS)恢复系，因此在诱导2代后育成了稳定的甘蓝型双低油菜细胞核雄性不育恢复系“蓉C2994”。该恢复系与“蓉A4979”组配产生甘蓝型油菜杂交组合杂15149，该组合为早熟组合，目前正在进行第一年区域试验。
上述各实施例中油菜双单倍体诱导系的选育方法同实施例1。
上述实施例是对本发明的上述内容作进一步说明，但不应将此理解为本发明上述主题的范围仅限于上述实施例。凡基于上述内容所实现的技术均属于本发明的额范围。

Claims (3)

  1. 油菜双单倍体诱导系选育甘蓝型油菜品种及材料的方法，包括以下步骤：
    1)确定甘蓝型油菜恢复系、保持系、常规品种选育的目标性状，将至少两个具有目标性状的甘蓝型油菜杂交或聚合杂交，根据目标性状要求进行回交或多代回交，形成杂交后代、聚合杂交后代或回交后代；
    2)对步骤1)中获得的杂交、聚合杂交后代或回交后代材料，在花期，对花蕾进行人工去雄，并套袋隔离；
    3)用油菜双单倍体诱导系花粉对步骤2)中去雄后2-4天植株进行人工授粉，并套袋隔离，收获其授粉后诱导结实种子；
    4)对上述步骤3)获得的诱导结实种子进行种植，苗期利用流式细胞仪鉴定倍性，淘汰多倍体、单倍体或具有油菜双单倍体诱导系显性性状特征植株，选择正常育性四倍体植株，单株套袋自交；
    5)上述步骤4)中单株自交种子进行株系种植，调查株系形态一致性，并通过分子标记鉴定株系一致性及稳定性；
    6)对上述步骤5)中鉴定的稳定四倍体株系与甘蓝型油菜细胞质不育系进行测交，或与甘蓝型油菜核不育系测交，并对测交后代进行育性鉴定,判断测交父本的恢保关系；
    7)上述步骤6)中如测交后代全不育，对应测交父本为保持系；测交后代全可育，对应测交父本为恢复系；
    8)上述步骤7)中鉴定的保持系继续与不育系多代回交，选育稳定的与该保持系核基因一致的不育系；上述步骤7)中鉴定的恢复系，直接与对应系统的不育系测配，选育杂交组合，杂交组合进入品种比较试验，产量、抗性、丰产性、品质性状好于生产上大面积应用的品种，满足品种认定或审定标准即可形成杂交油菜品种，并通过省级区域或国家种子管理部门认定或审定登记，在生产上推广应用；
    9)对上述步骤6)中获得的稳定四倍体株系进行品比及生产试验，产量、抗性、丰产性、品质性状优于生产上大面积应用的品种，满足品种认定或审定标准即形成常规品种，并通过省级区域或国家种子管理部门认定或审定登记，在生产上推广应用；
    上述油菜双单倍体诱导系的选育方法，包括如下步骤：
    (1)选育具有孤雌生殖遗传特性的早代稳定系：
    ①将两个油菜亲本材料杂交F1代种子在培养基上用染色体加倍诱导剂进行人工 染色体加倍获得加倍后的F1代植株；
    ②加倍后的F1代植株进行自交或强制自交获得F2代、对F2代进行田间种植观察、并鉴定每个单株的育性，选择可育后代自交获得F3代，对F3代进行纯合度鉴定、通过形态、细胞学以及分子标记鉴定，对后代DNA进行聚合酶链反应扩增，电泳观察每个特异引物扩增下单株的DNA带型及条带数目，显示每个单株都是两个亲本的杂交后代，每个单株之间分子标记图谱一致，说明这些单株是纯合系---早代稳定系；
    ③获得的早代稳定系与至少10个油菜常规纯合稳定系进行正反交，F1代、F2代鉴定早代稳定系的遗传特性，即是否有孤雌生殖特性；上述正反交，如有F1分离，F2代出现部分稳定株系，对应的早代稳定系是具有孤雌生殖遗传特性的早代稳定系；
    (2)选育携带显性遗传性状、具有孤雌遗传特性且倍性遗传稳定的多倍体油菜：
    ①具有孤雌生殖遗传特性的早代稳定系与具有显性性状油菜杂交得到杂交F1代种子，杂交F1种子在培养基上用染色体加倍诱导剂进行人工染色体加倍，得到加倍后的带显性性状的F1植株；
    ②对加倍的带显性性状的F1植株，通过显微观察或流式细胞仪进行染色体倍性鉴定，选择带显性性状的多倍体的植株，淘汰非正常加倍株、非整倍体植株、以及不带显性性状加倍植株，带显性性的多倍体的植株主要是倍性遗传稳定、结实性好、具有孤雌生殖遗传特性、带显性性状的六倍体或八倍体油菜植株。
    (3)油菜双单倍体诱导系鉴定及诱导能力测定：
    ①倍性遗传稳定、具有孤雌生殖遗传特性、带显性性状的多倍体植株中的显性性状能去除测交后代中产生的杂交株，如果测交后代中出现显性性状植株、或非整倍体植株，说明该植株是多倍体植株和母本杂交产生的，去除该植株；
    ②上述单株测交后代如果出现全不育、为正常倍性即二倍体或四倍体油菜、且不带显性性状，说明该测交后代对应的父本基因未进入测交后代中，显性多倍体植株为油菜双单倍体诱导系。
  2. 如权利要求1所述的油菜双单倍体诱导系选育甘蓝型油菜品种及材料的方法，其特征在于是油菜双单倍体诱导系选育两个亲本材料杂交F1代种子或具有孤雌生殖遗传特性的早代稳定系与具有显性性状油菜杂交得到的杂交F1代种子在培养基上用染色体加倍诱导剂进行人工染色体加倍，具体方法如下：
    1)用纯度为75％酒精进行种子表面消毒25-40秒，用0.1％升汞消毒12-17分钟，然后用无菌水将种子表面的升汞冲洗干净，用无菌纸将种子表面的水分吸干，然后将种子接种在第一培养基上；
    2)让种子在第一培养基上生根发芽，培养条件：温度23-25℃，白天光照12-16 小时，光照强度2000-3000勒克斯，夜晚暗培养8-12小时，待植株长到1—2片真叶时，从下胚轴处剪下植株继续在第二培养基上生长；
    3)将剪下的植株继续插入第二培养基上继续培养，待有侧芽分化后，将侧芽及植株转入第三培养基中进行生根培养；
    4)生根培养二周后，植株长出粗壮的根后，将植株在室温炼苗3-7天，取出植株将植株上的培养基用自来水冲洗干净，并在浸泡缓冲液中浸泡15—30分钟后移栽到温室中，温室温度16℃—25℃，相对湿度60-80％，能保证移栽成活率在95％以上；
    上述的第一培养基由以下配比的组分组成：
    Figure PCTCN2016111327-appb-100001
    第一培养基的pH＝5.8-6.0，
    上述的第二培养基由以下配比的组分组成：
    Figure PCTCN2016111327-appb-100002
    第二培养基的pH＝5.8-6.0，
    上述的第三培养基由以下配比的组分组成：
    Figure PCTCN2016111327-appb-100003
    第三培养基的pH＝5.8-6.0，
    上述的浸泡缓冲液由以及下配比的组分组成：
    水          1L
    易保或克露  0.6-1.2g
    α-萘乙酸   0.5—1mg。
  3. 如权利要求1或2所述的油菜双单倍体诱导系选育甘蓝型油菜品种及材料的方法，其特征在于染色体加倍诱导剂采用秋水仙素、氟乐灵、氨磺乐灵中的至少一种。
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