WO2017219634A1 - 油菜双单倍体诱导系选育十字花科蔬菜材料及品种的方法 - Google Patents

油菜双单倍体诱导系选育十字花科蔬菜材料及品种的方法 Download PDF

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WO2017219634A1
WO2017219634A1 PCT/CN2016/111356 CN2016111356W WO2017219634A1 WO 2017219634 A1 WO2017219634 A1 WO 2017219634A1 CN 2016111356 W CN2016111356 W CN 2016111356W WO 2017219634 A1 WO2017219634 A1 WO 2017219634A1
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line
sterile
plants
stable
rapeseed
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PCT/CN2016/111356
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English (en)
French (fr)
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杨进
付绍红
匡成兵
陈玲
李云
唐祖君
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 KR1020187031446A priority Critical patent/KR102198083B1/ko
Priority to EP16906171.0A priority patent/EP3485725A4/en
Priority to JP2018556894A priority patent/JP6670008B2/ja
Priority to US16/310,627 priority patent/US20190327923A1/en
Priority to AU2016410430A priority patent/AU2016410430B2/en
Publication of WO2017219634A1 publication Critical patent/WO2017219634A1/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
    • 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
    • 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
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • 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
    • A01H4/008—Methods for regeneration to complete plants
    • 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
    • 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
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
    • A01N33/02—Amines; Quaternary ammonium compounds
    • A01N33/08—Amines; Quaternary ammonium compounds containing oxygen or sulfur
    • A01N33/10—Amines; Quaternary ammonium compounds containing oxygen or sulfur having at least one oxygen or sulfur atom directly attached to an aromatic ring system

Definitions

  • the invention relates to agriculture, in particular to the breeding of new hybrid varieties of cruciferous vegetables and the rapid breeding method of the sterile lines and maintainers.
  • Cruciferous vegetables are a large class of winter vegetables in China, and also the main source of winter vegetables in China; mainly including cabbage, cauliflower (cauliflower), cabbage (cabbage, cabbage), radish, mustard cruciferous vegetables (green vegetables, Mustard, kohlrabi, kale, etc.).
  • the main heterosis pathways are mainly cytoplasmic infertility types.
  • Cabbage, cauliflower (cauliflower) and radish are mainly radish cytoplasmic infertility types, other cross flowers.
  • the main vegetables are mainly cytoplasmic infertility to breed hybrids.
  • cultivars Because of the main purpose of cruciferous vegetables to obtain vegetative bodies, the breeding of cultivars is mainly for the purpose of breeding sterile lines and maintaining lines, and no restoration system is needed. New hybrid lines and sterile lines can be selected for breeding combinations or new varieties.
  • breeding new inbred lines or genetically stable homozygous lines - homozygous lines breeding new inbred lines or genetically stable homozygous lines - homozygous lines (inbred lines).
  • the homozygous strain is tested with the cytoplasmic sterile line to determine the relationship between the restoration and the maintenance. If the hybrid line is crossed with the sterile line, the hybrid line is crossed with multiple generations of backcross. The characteristic sterile line, the new sterile line and multiple maintenance lines are tested and matched, and a good combination or variety can be selected. The homozygous strains and the sterile lines are tested and tested, and the progeny of the test are not restored, and are generally eliminated, or enter the next round of maintenance.
  • the homozygous inbred lines currently selected can be used for breeding in production, but for resource conservation, breeders tend to maintain and maintain the line. .
  • Stable inbred lines are then tested with the sterile line, and multiple generations of backcrossing to breed new sterile lines require backcrossing for 5-6 generations. Therefore, it is necessary to use conventional means to realize the second-line breeding new varieties of cruciferous vegetables, which takes about 10-12 years, and the efficiency of breeding new hybrids or new varieties is very low.
  • induction 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% (calculated as the number of haploids obtained in harvested seeds).
  • the object of the present invention is to provide a method for rapidly and effectively breeding cruciferous vegetable breeding materials and varieties; the method only requires three generations (two or three years) to obtain a genetically stable strain of cruciferous vegetables. Improve the efficiency and pertinence of breeding materials and hybrid varieties of cruciferous vegetables.
  • the method for breeding a cruciferous vegetable material and variety by the double haploid induction line of the rapeseed of the present invention comprises the following steps:
  • Flower family vegetable breeding resources collect and identify the characteristics of resource materials, including disease resistance, stress resistance, agronomic traits, yield characteristics, quality characteristics, etc., and classify resource materials with different genetic differences between different sources and agronomic traits.
  • the flowering time was investigated, and according to the flowering time, the double haploid induction line of rape was planted.
  • the sowing time of rapeseed double haploid is between October 20 and November 5 of the previous year, which can guarantee the next year to meet the flowering period of cruciferous vegetables such as cabbage, cabbage, radish and cauliflower;
  • step 4 planting the test cross seed in the above step 4), identifying the fertility of the test progeny, and measuring the progeny such as total infertility, corresponding to the parent in the step 4) being the maintainer of the sterile type, and simultaneously
  • the maintainer line is identified by the self-cultivation rate;
  • the test parent progeny such as the full fertility corresponding step 4
  • the restorer of the sterile type corresponding to the recovery
  • the complex should be eliminated; if the offspring are incompletely infertile (half-recovery and semi-insurance), the father in the corresponding step 4) is not restored, and should be eliminated or hybridized to transfer a new maintainer;
  • step 6) The above-mentioned step 5) the selected maintainer line is matched with the same type of sterile line, and the new hybrid combination or variety having the yield, quality and disease resistance characteristics is selected;
  • test line selected in the above step 5) is tested and tested with the same type of sterile line, according to the agronomic traits of the tested progeny, the maintainer is used to make back-crossing or multi-generation backcross or cross-generation or Test 1 generation, use the rapeseed double haploid induction line to test the cross, backcross or multi-generation backcross infertility single plant pollination, and isolate the bag;
  • step 8) is subjected to secondary induction and sterile individual offspring to determine the stability and consistency of the strain through agronomic traits, molecular markers (SSR or SRAP).
  • SSR or SRAP molecular markers
  • the sterile line formed after the second induction is pollinated with the rapeseed double haploid induction line to maintain its infertility state, and the genetic characteristics of the sterile line (or stable sterile line) It has nothing to do with the double haploid induction of Brassica napus.
  • the sterile line has the property characteristics of the maintainer (temporary maintainer) in the above step 7), has a certain genetic difference with the maintainer (temporary maintainer), or contains 50% to 99% of the maintainer (temporary retention)
  • the nuclear gene which contains the number of nuclear genes in the maintainer (temporary maintainer), depends on the backcrossing algebra of the maintainer (temporary maintainer) and the sterile individual, except for the nucleus containing different degrees of maintainer (temporary maintainer) In addition to the gene, the nuclear gene of the stable cytoplasmic male sterile line in step 4) is also included;
  • Cytoplasmic sterile lines (stable sterile lines) with different genetic differences (or different genetic backgrounds) formed in the above step 9), according to the induction ability of the rapeseed double haploid induction line to the cytoplasmic male sterile line (greater than 98) %) can be maintained by the same maintainer line (canola double haploid induction line), and the double haploid induction line of Brassica napus is a universal maintainer of the cytoplasmic male sterile line. At the same time, a maintainer is used to maintain a plurality of genetically stable cytoplasmic male sterile lines with different genetic backgrounds;
  • the breeding potential has the potential to produce disease resistance. Resistant to the new cruciferous vegetable varieties, to achieve a new hybrid variety of cruciferous vegetables.
  • the stable genetic progeny of cruciferous vegetables obtained by the invention can induce parthenogenesis in the F1 generation by the double haploid induction system of the rapeseed, and form a stable double haploid individual in the F 2 generation, and stabilize the F 3 generation. Sexuality, consistency identification, and stable genetic offspring.
  • the method for breeding the above-mentioned rapeseed double haploid induction line comprises the following steps:
  • 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;
  • plants with dominant polyploids are mainly ploidy genetically stable, good in fruiting, have parthenogenetic genetic characteristics, and have dominant traits (such as dominant dwarfs, Hexaploid or octoploid rapeseed plants of purple leaves, mosaics, yellow leaves, high erucic acid, etc.;
  • 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 above-mentioned rapeseed double haploid induction line is selected by mixing two parental materials with F 1 generation seeds or early generation stable lines with parthenogenetic genetic characteristics and hybrid F 1 generation seeds obtained by hybridizing dominant traits in culture medium. Artificial chromosome doubling is performed with a chromosome doubling inducer as follows:
  • the surface of the seed is disinfected with 75% alcohol for 25-40 seconds, disinfected with 0.1% mercuric chloride for 12-17 minutes, then rinsed with mercury on the surface of the seed with sterile water, and the surface of the seed is treated with sterile paper. Moisture drying, and then seeding the seed on the first medium;
  • the first medium described above consists of the following components:
  • the pH of the first medium is 5.8-6.0
  • the second medium described above consists of the following components:
  • the pH of the second medium is 5.8-6.0
  • the third medium described above consists of the following components:
  • the pH of the third medium is 5.8-6.0
  • the above soaking buffer consists of the following components:
  • the double haploid induction system of Brassica napus can directly induce double haploid progeny in rapeseed and cruciferous vegetables, without artificial chromosome doubling to obtain homozygous lines; and the induction efficiency is high, up to 100%, and the general induction efficiency is More than 50%.
  • the main principle of double haploid induction inducing the production of double haploid in maternal plants is that the induced line can induce maternal plants, the megaspore germ cells (egg cells) produce parthenogenetic effects, and the egg cells can perform chromosome doubling, ie egg cell parthenogenesis
  • the offspring of reproduction are double haploids, and the mechanism by which this phenomenon occurs is still unclear.
  • the above chromosome doubling inducer is at least one of colchicine, trifluralin, and amsulfame.
  • rapeseed double haploid induction line hexaploid or octoploid plant
  • the induced line has a parthenogenetic induction gene, and when the induced line is used as a male parent, the induced line chromosome (or gene) is not associated with the mother plant. Chromosomal fusion, but induce maternal plants (ie, egg cells, diploid) to produce parthenogenetic effects, and the mother plant egg cell chromosome itself doubled to form a double haploid.
  • the method of the invention can be rapidly used for the breeding of the crucifer vegetable breeding material inbred line (DH line), the maintainer line and the new cytoplasmic male sterile line.
  • DH line crucifer vegetable breeding material inbred line
  • maintainer line the maintainer line
  • new cytoplasmic male sterile line The above materials can be obtained in 2 or 3 generations, greatly saving the breeding time of cruciferous vegetables and improving breeding efficiency.
  • the method of the invention can rapidly select the cruciferous vegetable inbred line (maintaining line), obtain the stable inherited inbred line (maintaining line) in 3 years, and quickly select the new sterile line, the fastest. 4 years to obtain a stable sterile line, in the 5-7 years to achieve the second line of cruciferous vegetables, breeding new varieties, saving more than half of the breeding cycle of cruciferous vegetables, raising the cruciferous vegetables The efficiency of breeding new varieties, saving manpower and material resources;
  • the method of the invention can be applied to the entire cruciferous vegetable, and the application field is wide;
  • the method of the invention is suitable for breeding of cruciferous vegetable hybrid varieties, especially for the selection of cytoplasmic sterility system materials of cruciferous vegetables, such as radish cytoplasmic sterility, cytoplasmic interaction cytoplasmic male sterile line. And maintain the breeding system.
  • Figure 1 is a flow chart of a method for rapidly breeding new varieties of cruciferous vegetables 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.
  • Fig. 6 is a flow chart showing the breeding of the rapeseed early generation stable line P3-2.
  • Figure 7 is a selection diagram of the cabbage-maintaining system Ronggan B012.
  • Figure 8 is a diagram showing the selection of the cytoplasmic male sterile line Ronggan A105.
  • Figure 9 is a breeding diagram of the radish sterile line Luorong A007.
  • Figure 10 is a ploidy map of the root tip of P3-2 tetraploid rapeseed.
  • Figure 11 is a diagram showing the ploidy identification of P3-2 tetraploid rapeseed.
  • Figure 12 is a Y3380 flow cytoplasmic identification map.
  • Figure 13 is a Y3560 flow cytoplasmic identification map.
  • Fig. 1, Fig. 2, Fig. 5 and Fig. 7 the planting of the cabbage resources collected for many years is carried out in the field, and the traits are observed.
  • Gan 336 is found, which has good appearance, high yield and disease resistance, but within the group. Unstable, with genetic separation.
  • the early flowering period of Gan 336 was artificially emasculated, and bagging was isolated.
  • the offspring were inoculated in the field, and it was found that 25 of the progeny were 100% identical to cabbage, and the ploidy was identified by flow cytometry, which was all diploid.
  • the offspring of the offspring were forced to self-crossing, and they were bagged and isolated, and 18 plants were collected from each plant.
  • the induced offspring lines were identified, and all 18 lines were found to be consistent, and there were some differences among the lines. 18 strains were tested with the cabbage sterile line (radish cytoplasmic infertility), and the 18 stable strains were highly infertile, and 18 stable strains were maintained in the sterile line.
  • the rapeseed double haploid induction line was obtained by the following method:
  • the tetraploid early generation stable line P3-2 of Brassica napus obtained by the applicant is reciprocally intersected with 20 homozygous cabbage type tetraploid rapeseed.
  • Three positive and negative F 1 generations were isolated, and the three combined F 2 generations showed stable strains, indicating that P3-2 had parthenogenetic genetic characteristics.
  • Cross-P3-2 with high erucic acid, Dwarf canola 4247 (dwarf, high erucic acid for dominant traits) then hybrid F 1 generation seeds for chromosome doubling, double progeny identified by flow cytometry or root It was identified by sharp-microscopic observation to show a dwarf octaploid plant named Y3560.
  • 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 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:
  • the pH of the first medium is 5.8-6.0
  • 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 pH of the second medium is 5.8-6.0
  • the third medium described above consists of the following components:
  • the pH of the third medium is 5.8-6.0
  • 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%.
  • Y3560 was used as the male parent, and the cytoplasmic sterile line (0464A) of Brassica napus L. was tested and tested, and 80 progeny were tested, all of which were high poles, and 76 were tetraploid rapeseed, 2 The strain was diploid and the two were octoploid; 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%.
  • 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 a large leaf, no split leaves, and a compact leaf, and an 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 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:
  • the pH of the first medium is 5.8-6.0
  • 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 pH of the second medium was 5.8-6.0.
  • the third medium described above consists of the following components:
  • the pH of the third medium is 5.8-6.0
  • the above soaking buffer consists of the following components:
  • the cabbage self-incompatibility line is selected.
  • the high-complexity line) Gan 121 is hybridized with the storage and transportation line Gan 051, the artificial F1 generation of hybrid F 1 generation is artificially emasculated, pollinated with the rapeseed double haploid induction line Y3560, and 15 plants are obtained by F 2 generation (induced 1 generation).
  • the rapeseed double haploid induction line Y3380 was used to continue pollinating the corresponding test offspring of 105 lines, and isolated individual seeds were isolated. Planting offspring of 105 test crosses were planted in the field, and 45 plants were obtained. 42 plants were found, which were completely characterized by cabbage and diploid. The three plants looked like plants mixed with rapeseed, and the ploidy was 3 times. 42 plants were highly infertile, and Y3560 was used to pollinate single plants with storage resistance and flat head characteristics, and bagging was isolated. Seeds were harvested from each plant, and 30 plant seeds were obtained, and 30 strains were obtained.
  • SRAP Appearance morphology, agronomic traits and molecular markers
  • New sterile line cabbage radish cytoplasm
  • the sterile line Ronggan A105 which can be used for pollination and reproduction of the sterile line by Y3560.
  • the cells were identified by using the cells to identify the ploidy of the progeny. Ten individual plants were found, one of which was haploid and 9 diploid, and the shape was radish, and 9 single flowering sets. The bag was self-crossed, and the self-crossing progeny was identified for consistency and stability in the lines. Nine lines and strains were highly consistent and stable, and 9 lines of pollen were used to test the radish sterile line Rongluo A001.
  • Consistency and ploidy identification were carried out on the progeny of the test crosses, and the 9 progenies were all infertile. Among them, the strain 7 had the highest disease resistance and yield characteristics, and continued to use the rapeseed double haploid induction line Y3560 pollen to No. 7 The lines of the tested offspring were pollinated, and bagged and isolated. The morphological separation was found in the offspring. Four sterile plants with the characteristics of the 7th line were selected and pollinated by Y3380 to form 4 lines and 4 lines were carried out. Identification of the consistency and stability of the strains, and found that each strain is highly consistent and stable, and each strain is slightly The differences in agronomic traits were mainly in leaf color and plant height.
  • the two high-induction sterility lines were found to be the same sterile line, and the sterile line could be Y3560 and Y3380 maintains infertility, the new sterile line is radish cytoplasmic infertility, and its genotype comes from the above-mentioned stable strain 7 (genotype from round white radish Y23 Korean radish H22) and Rongluo A001, named Luo Rong A007.
  • the breeding method of the rapeseed double induction line in the third embodiment is the same as in the first embodiment.
  • the method of the invention can obtain cruciferous vegetable materials with application value in breeding or basic research quickly (3 generations), high efficiency and large scale; and the patented technology has wide application range and is suitable for the entire cruciferous crop, including cabbage, Cauliflower (cauliflower), cabbage (cabbage, pakchoi), radish, mustard cruciferous vegetables (green vegetables, mustard, kohlrabi, kale, etc.), which have a wide application area and promote high yield and quality of cruciferous vegetable crops. Quality breeding has a positive effect.

Abstract

本申请提供了一种利用油菜双单倍体诱导系选育十字花科蔬菜品种的方法，包括：1）收集十字花科蔬菜资源，鉴定归类，调节诱导系的播种时间，保证花期相遇；2）用诱导系对十字花科蔬菜授粉；3）套袋自交或蕾期剥蕾强制自交；4）进行株系稳定性鉴定；5）稳定株系与不育系测交；6）测交后代不育度调查，选育优良保持系；7）用诱导系给测交后代不育株授粉；8）调查诱导后代育性，继续用诱导系给不育单株授粉选育新的不育系；9）不育系与保持系测配选育杂交新组合。

Description

油菜双单倍体诱导系选育十字花科蔬菜材料及品种的方法 技术领域：
本发明与农业有关，特别与十字花科蔬菜杂交新品种选育及不育系、保持系的快速选育方法有关。
背景技术：
十字花科蔬菜是我国冬季蔬菜的一大类，也是我国冬季蔬菜的主要来源；主要包括甘蓝、花菜(花椰菜)、白菜(大白菜、小白菜)、萝卜、芥菜类十字花科蔬菜(青菜、榨菜、大头菜、芥蓝等)。目前，十字花科蔬菜基本实现了杂交品种选育，主要利用的杂种优势途径还是以细胞质不育类型为主，甘蓝、花菜(花椰菜)、萝卜主要以萝卜细胞质不育类型为主，其他十字花科蔬菜主要还是以细胞质不育为主来选育杂交品种。由于十字花科蔬菜以获得营养体为主要目的，品种选育主要以选育不育系和保持系为首要目的，不需要恢复系。新保持系和不育系测配就可以选育好的杂交组合或新品种。
十字花科蔬菜新品种选育，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)。其次，纯合株系与细胞质不育系测交，判断恢保关系，如果是保持系与不育系杂交测配新的杂交品种，或利用该保持系多代回交选育具有该保持系特性的不育系，新的不育系与多个保持系测配，可选育出一批好的组合或品种。纯合株系与不育系测交，测交后代不恢不保，一般进行淘汰，或进入下一轮保持系选育。为了实现十字花科蔬菜的杂交化推广，目前选育出的纯合自交系具有恢复效率在生产上仍然可以用于品种选育，但为了资源保护，育种者更趋向与保持系的选育。
常规十字花科蔬菜自交系的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F5—F6代才能获得稳定的十字花科蔬菜新品系，以1年1代计算，所花时间大概在6—7年的时间。稳定的自交系再与不育系测配，多代回交选育新的不育系需要回交5—6代。因此，用常规手段来实现十字花科蔬菜的二系选育新品种，需要10—12年左右的时间，选育杂交新组合或新品种的效率很低。
目前，在在十字花科蔬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：
本发明的目的是为了提供一种能快速、有效，选育十字花科蔬菜育种材料及品种的方法；该方法只需3代(2年或3年)获得十字花科蔬菜遗传稳定株系，提高十字花科蔬菜选育育种材料、杂交品种的效率及针对性。
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十字花科蔬菜材料及品种的方法，包括如下步骤：
1)花科蔬菜育种资源收集并鉴定资源材料的特性，包括抗病、抗逆、农艺性状、产量特性、品质特性等，对不同来源、农艺性状遗传差异大的资源材料进行归类编号。调查开花时间，并根据开花时间，推后播种油菜双单倍体诱导系。一般油菜双单倍体的播种时间在上一年10月20—11月5号之间，能保证下一年与甘蓝、白菜、萝卜、花菜等十字花科蔬菜花期相遇；
2)对上述步骤1)中鉴定优良且遗传不稳定的资源材料在初花期进行人工去雄(不育资源可直接授粉)，并套袋隔离，2－4天后，用油菜双单倍体诱导系授粉，并套袋单株收获诱导后代；
3)对上述步骤2)中收获的诱导后代进行种植，并在苗期用流氏细胞仪鉴定诱导后代的倍性，淘汰多倍体、单倍体、以及具有油菜特性的植株，选择育性正常、倍性正常的单株套袋自交或蕾期剥蕾强制自交；
4)对上述步骤3)中诱导后代正常自交后代进行株系种植，鉴定株系内的稳定性和一致性，并用分子标记(SSR或SRAP)鉴定株系内的一致性，用稳定株系作父本与稳定细胞质不育系(萝卜胞质不育等胞质不育类型)作母本测交，并收获测交后代种子；
5)对上述步骤4)中测交种子进行种植，鉴定测交后代的育性，测交后代如全不育，对应步骤4)中的父本为该不育类型的保持系，同时对该保持系进行自交结实率鉴定；测交后代如全可育对应步骤4)中的父本为该不育类型的恢复系，对应的恢 复系应淘汰；测交后代如不育不彻底(半恢半保)，对应步骤4)中的父本为不恢不保，应进行淘汰或进行杂交转育新的保持系；
6)上述步骤5)选育出的保持系与同类型的不育系侧配，选育具有产量、品质、抗病特性的新杂交组合或品种；
7)上述步骤5)中选育出的保持系与同类型的不育系测交后，根据测交后代的农艺性状，用该保持系与测交后代进行回交或多代回交或在测交1代，用油菜双单倍体诱导系给测交、回交或多代回交不育单株授粉，并隔离套袋；
8)将上述步骤7)中收获单株种子进行种植，在花期鉴定育性，对不育单株继续用油菜双单倍体诱导系进行授粉诱导，并单株套袋隔离，并单株收种；
9)将上述步骤8)中经过二次诱导和不育单株后代通过农艺性状、分子标记(SSR或SRAP)进行株系内稳定性、一致性鉴定。对农艺性状一致，株系内稳定的不育株系，形成新的十字花科蔬菜细胞质不育系，用油菜双单倍体诱导系第三次对该不育株系进行诱导授粉鉴定诱导效率；
10)对上述步骤9)中第三次诱导后代鉴定诱导系对该不育株系的诱导能力，如果第三次诱导后代株系内农艺性状、不育度高度一致，且诱导能力超过98％以上(株系内农艺性状、高度不育度植株占总诱导后代的比例)，最终，用油菜双单倍体诱导系保持新形成的不育株系(或稳定的不育系)遗传特性和不育状态；
11)上述步骤8)中，二次诱导后形成的不育株系，用油菜双单倍体诱导系授粉保持其不育状态，该不育株系(或稳定的不育系)的遗传特性与油菜双单倍体诱导系无关。该不育株系具有上述步骤7)中保持系(临时保持系)的性状特性，与该保持系(临时保持系)具有一定的遗传差异，或含有50％—99％的保持系(临时保持系)核基因，含有保持系(临时保持系)核基因的多少，取决于保持系(临时保持系)与不育单株的回交代数，除含有不同程度保持系(临时保持系)的核基因外，还含有步骤4)中稳定细胞质不育系的核基因；
12)上述步骤9)中形成的不同遗传差异(或不同遗传背景)的细胞质不育株系(稳定不育系)，根据油菜双单倍体诱导系对细胞质不育系的诱导能力(大于98％)，可以用同一个保持系(油菜双单倍体诱导系)进行保持，油菜双单倍体诱导系成为细胞质不育系的万能保持系。同时用一个保持系，保持多个遗传稳定、含有不同遗传背景的细胞质不育系；
13)根据步骤1)中不同来源、农艺性状遗传差异的材料特性以及对应来源诱导稳定的保持系与步骤12)形成的稳定的新的不育系测配，选育具有产量潜力、抗病、抗逆的新的十字花科蔬菜品种，实现十字花科蔬菜二系配套选育杂交新品种。
采用本发明获得十字花科蔬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。
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，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(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。
上述油菜双单倍体诱导系选育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 PCTCN2016111356-appb-000001
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 PCTCN2016111356-appb-000002
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 PCTCN2016111356-appb-000003
Figure PCTCN2016111356-appb-000004
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。
油菜双单倍体诱导系能直接诱导油菜和十字花科蔬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主要原理是：诱导系能诱导母体植株，大孢子生殖细胞(卵细胞)产生孤雌生殖效应，且卵细胞能进行染色体加倍，即卵细胞孤雌生殖产生的后代就双单倍体，产生该现象的机理目前尚不明确。
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。
上述油菜双单倍体诱导系(六倍体或八倍体植株)的基本原理是：诱导系具有孤雌生殖诱导基因，诱导系作父本时，诱导系染色体(或基因)没有与母体植株染色体融合，而是诱导母体植株(即卵细胞，二倍体)产生孤雌生殖效应，且母体植株卵细胞染色体自身加倍形成双单倍体。
本发明的方法可以快速用于十字花科蔬菜育种材料自交系(DH系)、保持系、新细胞质不育系的选育。可以在2年或3代的时间内获得上述材料，大大节约十字花科蔬菜的育种时间，提高育种效率。
本发明方法具有以下优点：
1、本发明方法可以快速选育十字花科蔬菜自交系(保持系)，最快可3年获得稳定遗传的自交系(保持系)，也可快速选育新不育系，最快4年获得稳定的不育系，在5—7年的时间内实现十字花科蔬菜的二系配套、选育新品种，节约十字花科蔬菜的选育周期一半以上，提高了十字花科蔬菜新品种选育的效率，节约人力物力；
2、本发明方法可以在整个十字花科蔬菜中应用，应用领域广；
3、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；
4、本发明方法适用于十字花科蔬菜杂交品种选育，特别是用于十字花科蔬菜细胞质不育系统材料的选育，如萝卜胞质不育、核质互作类性细胞质不育系、保持系的选育。
附图说明：
图1为油菜双单倍体诱导系快速选育十字花科蔬菜新品种的方法流程图。
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为甘蓝保持系蓉甘B012的选育图。
图8为甘蓝萝卜胞质不育系蓉甘A105的选育图。
图9为萝卜不育系萝蓉A007的选育图。
图10为P3—2四倍体油菜根尖染色体倍性鉴定图。
图11为P3—2四倍体油菜法式细胞倍性鉴定图。
图12为Y3380流式细胞倍性鉴定图。
图13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，对多年收集的甘蓝资源进行田间种植于进行，并对性状观察，其中发现甘336，外观形态、丰产性、抗病性较好，但群体内不稳定，有遗传分离。上年秋季8月中旬播种，9月中旬定植，12月下旬去营养生长重新定植，第一年3月低初花期对甘336人工去雄，并套袋隔离，去雄3天后，用油菜双单倍体诱导系Y3380诱导授粉，并套袋隔离。当年8月在田间播种诱导后代，发现后代25株外形100％与甘蓝一致，并用流氏细胞仪鉴定倍性，全为二倍体。第二年花期诱导后代单株剥蕾强制自交，并套袋隔离，单株收种18株。第三年对诱导后代株系进行鉴定，发现18株株系全部一致，各株系之间存在部分差异。18个株系与甘蓝不育系(萝卜胞质不育)蓉萝A019测交，18个稳定株系测交后代都高度不育，18个稳定株系对不育系都为保持系，通过形态观察及产量调查，发现012株系产量优势、抗病性、抗逆性更好，最终形成蓉甘B012的保持系，可直接与甘蓝萝卜胞质不育系进行组合配制，选育新品种。
本实施例中，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7、图13，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10、图11、图12，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250C，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250C，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 PCTCN2016111356-appb-000005
第一培养基的pH＝5.8—6.0，
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 PCTCN2016111356-appb-000006
Figure PCTCN2016111356-appb-000007
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 PCTCN2016111356-appb-000008
第三培养基的pH＝5.8—6.0，
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10、图11，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250C，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250C，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 PCTCN2016111356-appb-000009
第一培养基的pH＝5.8—6.0，
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 PCTCN2016111356-appb-000010
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 PCTCN2016111356-appb-000011
Figure PCTCN2016111356-appb-000012
第三培养基的pH＝5.8—6.0，
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 PCTCN2016111356-appb-000013
Figure PCTCN2016111356-appb-000014
实施例2：
参见图1、图2、图4、图5、图8，为改造原有甘蓝不育系，提升不育系的配合力合抗病及耐储运特性，选用甘蓝自交不亲和系(高配合力品系)甘121与耐储运品系甘051杂交，杂交F1代花期人工去雄，用油菜双单倍体诱导系Y3560授粉诱导，F2代(诱导1代)获得15个单株，经调查发现15个单株全为甘蓝外形，且都为二倍体，15个单株套袋(剥蕾)自交，并取花粉与甘蓝不育系(萝卜胞质不育)蓉萝A019测交，自交后代进行株系内稳定性一致性鉴定，测交后代进行一致性，不育度鉴定，发现自交后代株系内都整齐一致，都为二倍体，测交后代也高度一致，都为不育，但只有一个株系105具有耐储运、平头特性，且测交后代产量高、抗病性好。用油菜双单倍体诱导系Y3380继续给105株系对应的测交后代单株授粉，并隔离收获单株种子。对105测交株诱导后代进行田间种植，获得45株植株，发现42株植株，完全具有甘蓝的特性，且为二倍体，3株植株外观像与油菜杂交的植株特性，倍性为3倍体，42株单株高度不育，继续用Y3560给具有耐储运、平头特性 不育单株授粉，并套袋隔离，单株收获种子，获得30个株系植株种子，对30个株系用外观形态、农艺性状、分子标记(SRAP)进行株系内一致性稳定性鉴定，其中10个株系，株系内一致性稳定性高度一致，其中2个株系具有耐储运、抗病、丰产特性，2个株系每个株系选10株用Y3560和Y3380分别授粉5株，并套袋隔离，5个株系混合授粉，鉴定两个诱导系对稳定的不育系的诱导效率，发现Y3560对其中一个不育系的诱导效率为100％，另外一个不育系诱导效率97％，Y3380对两个不育系的诱导效率分别为97％和96％，因此，最终只形成一个新的不育系，甘蓝萝卜胞质不育系蓉甘A105，该不育系可用Y3560进行授粉繁殖该不育系。
实施例3：
图1、图2、图4、图5、图9，用圆形白萝卜Y23与韩国萝卜H22杂交，F1代花期人工去雄，用油菜双单倍体诱导系Y3380授粉，F2代诱导后代苗期用流氏细胞以鉴定诱导后代倍性，发现10个单株，有1个单株为单倍体，9个位二倍体，且外形都为萝卜特性，9个单株花期套袋自交，对自交后代进行株系内一致性及稳定性鉴定，9个株系，株系内都高度一致和稳定，并用9个株系花粉与萝卜不育系蓉萝A001测交，对测交后代进行一致性、倍性鉴定，9个测交后代全为不育，其中，7号株系抗病性，产量特性最高，继续用油菜双单倍体诱导系Y3560花粉给7号株系测交后代授粉诱导，并套袋隔离，诱导后代发现形态分离，选具有7号株系特性的4个不育单株继续用Y3380授粉诱导，形成4个株系，4个株系进行株系内一致性、稳定性鉴定，发现每个株系内高度一致和稳定，每个株系间略有农艺性状差异，主要表现在叶色和株高上，4个稳定株系用Y3560和Y3380各选5个单株授粉，并套袋隔离，5个株系混和收种，鉴定诱导系对稳定不育系的诱导效果，发现Y3560的诱导效率在94－99.8％之间，其中1个株系的诱导效率在99.8％，该株系可以用Y3560进行不育性保持，Y3380的诱导率93－99.7％之间，其中1个株系的诱导效率在99.7％，可以用Y3380进行不育性保持，最后发现这两个高诱导效率的不育系为同一不育系，该不育系可用Y3560和Y3380保持不育，新不育系为萝卜胞质不育，其基因型来之上述选育的7号稳定株系(基因型来自圆形白萝卜Y23韩国萝卜H22)和蓉萝A001，定名为萝蓉A007。
本实施例2，实施例3中油菜双倍诱导系的选育方法同实施例1。
本发明方法可以快速(3代)、高效、规模化获得育种上或基础研究中具有应用价值的十字花科蔬菜材料；且该专利技术应用范围广，适应于整个十字花科作物，包括甘蓝、花菜(花椰菜)、白菜(大白菜、小白菜)、萝卜、芥菜类十字花科蔬菜(青菜、榨菜、大头菜、芥蓝等)，其应用面积广，对推动十字花科蔬菜作物的高产、品 质育种具有积极的促进作用。
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十字花科蔬菜材料及品种的方法，包括以下步骤：
    1)十字花科蔬菜育种资源收集并鉴定资源材料的特性，对不同来源、农艺性状遗传差异大的资源材料进行归类编号，调查开花时间，并根据开花时间，推迟播种油菜双单倍体诱导系，一般油菜双单倍体的播种时间在上一年10月20—11月5号之间，能保证下一年与十字花科蔬菜花期相遇；
    2)对上述步骤1)中收集的十字花科蔬菜资源材料在初花期进行人工去雄，不育资源直接授粉，并套袋隔离，2－4天后，用油菜双单倍体诱导系授粉，并套袋单株收获诱导后代；
    3)对上述步骤2)中收获的诱导后代进行种植，并在苗期用流氏细胞仪鉴定诱导后代的倍性，淘汰多倍体、单倍体、以及具有油菜特性的植株，选择育性正常、倍性正常的单株套袋自交或蕾期剥蕾强制自交；
    4)对上述步骤3)中诱导后代正常自交后代进行株系种植，鉴定株系内的稳定性和一致性，并用分子标记鉴定株系内的一致性，用稳定株系作父本与稳定细胞质不育系作母本测交，并收获测交后代种子；
    5)对上述步骤4)中测交后代种子进行种植，鉴定测交后代植株的育性，测交后代为全不育，对应步骤4)中的父本为该不育类型的保持系，同时对该保持系进行自交结实率鉴定；测交后代为全可育对应步骤4)中的父本为该不育类型的恢复系，对应的恢复系应淘汰；测交后代为不育不彻底，对应步骤4)中的父本为不恢不保，应进行淘汰或进行杂交转育新的保持系；
    6)上述步骤5)选育出的保持系与同类型的不育系测配，选育具有产量、品质、抗病特性的新杂交组合或品种；
    7)上述步骤5)中选育出的保持系与同类型的不育系测交后，根据测交后代的农艺性状，用该保持系与测交后代进行回交或多代回交或在测交1代，用油菜双单倍体诱导系给测交、回交或多代回交不育株单株授粉，并隔离套袋；
    8)将上述步骤7)中收获单株种子进行种植，在花期鉴定育性，对不育单株继续用油菜双单倍体诱导系进行授粉诱导，并单株套袋隔离，并单株收种；
    9)将上述步骤8)中经过二次诱导的不育单株后代通过农艺性状、分子标记进行株系内稳定性、一致性鉴定，对农艺性状一致，株系内稳定的不育株系，形成新的十字花科蔬菜细胞质不育系，用油菜双单倍体诱导系第三次对该不育株系进行诱导授 粉鉴定诱导效率；
    10)对上述步骤9)中第三次诱导后代鉴定诱导系对该不育株系的诱导能力，第三次诱导后代株系内农艺性状、不育度高度一致，且诱导能力即株系内农艺性状、高度不育度植株占总诱导后代的比例超过98％以上，最终用油菜双单倍体诱导系保持新形成的不育株系或稳定的不育系遗传特性和不育状态；
    11)上述步骤8)中，二次诱导后形成的不育株系，用油菜双单倍体诱导系授粉保持其不育状态，该不育株系或稳定的不育系的遗传特性与油菜双单倍体诱导系无关，该不育株系具有上述步骤7)中保持系即临时保持系的性状特性，与该保持系即临时保持系具有一定的遗传差异，或含有50％—99％的保持系即临时保持系核基因，含有保持系即临时保持系核基因的多少，取决于保持系即临时保持系与不育单株的回交代数，除含有不同程度保持系即临时保持系的核基因外，还含有步骤4)中稳定细胞质不育系的核基因；
    12)上述步骤9)中形成的不同遗传差异的稳定细胞质不育系，根据油菜双单倍体诱导系对细胞质不育系的诱导能力大于98％，则用同一个保持系即油菜双单倍体诱导系进行保持，油菜双单倍体诱导系成为细胞质不育系的万能保持系，同时用一个保持系，保持多个遗传稳定、含有不同遗传背景的稳定的细胞质不育系；
    13)根据步骤1)中不同来源、农艺性状遗传差异的材料特性以及对应来源诱导稳定的保持系与步骤12)形成的稳定的新的多个细胞质不育系测配，选育具有产量潜力、抗病、抗逆的新的十字花科蔬菜品种，实现十字花科蔬菜二系配套选育杂交新品种；
    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；显性性状的多倍体的植株是倍性遗传稳定、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十字花科蔬菜材料及品种的方法，其特征在于油菜双单倍体诱导系选育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 PCTCN2016111356-appb-100001
    Figure PCTCN2016111356-appb-100002
    上述的第二培养基由以下配比的组分组成：
    Figure PCTCN2016111356-appb-100003
    上述的第三培养基由以下配比的组分组成：
    Figure PCTCN2016111356-appb-100004
    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十字花科蔬菜材料及品种的方法，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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