WO2022160427A1 - 一种抗根肿病橙色大白菜新种质的选育方法 - Google Patents

一种抗根肿病橙色大白菜新种质的选育方法 Download PDF

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WO2022160427A1
WO2022160427A1 PCT/CN2021/081285 CN2021081285W WO2022160427A1 WO 2022160427 A1 WO2022160427 A1 WO 2022160427A1 CN 2021081285 W CN2021081285 W CN 2021081285W WO 2022160427 A1 WO2022160427 A1 WO 2022160427A1
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orange
clubroot
chinese cabbage
marker
resistant
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PCT/CN2021/081285
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French (fr)
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张鲁刚
卢倩倩
孟艳
张妮南
王亚秀
和禹廷
张学芬
刘小愿
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西北农林科技大学
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Publication of WO2022160427A1 publication Critical patent/WO2022160427A1/zh
Priority to US18/047,286 priority Critical patent/US20230189733A1/en

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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
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/1255—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for fungal resistance
    • 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
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • 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
    • A01H6/204—Brassica rapa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

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  • the invention belongs to the field of vegetable variety selection and germplasm resource innovation, and in particular relates to a new germplasm selection method of orange Chinese cabbage resistant to clubroot.
  • Chinese cabbage (Brassica campestris syn.rapa L.ssp.pekinensis) is one of the important vegetable crops of the Brassica family. With the improvement of people's living standards, the attention to the quality of Chinese cabbage has gradually increased. High-quality Chinese cabbage must also be resistant to diseases. However, high-quality Chinese cabbage is susceptible to diseases. The use of pesticides will not only pollute the environment, but also make vegetables resistant to pesticides if they are used for a long time. Therefore, high-quality disease-resistant Chinese cabbage germplasm innovation. become a research hotspot in recent years.
  • Clubroot is a devastating soil-borne disease caused by the infection of Plasmodiophora brassicae Woron. In recent years, clubroot has occurred in many parts of my country and has become the main disease of Chinese cabbage production. . It is not possible to fundamentally prevent clubroot through field management and biochemical control. Cultivating Chinese cabbage varieties resistant to clubroot is an important measure to solve the problem of clubroot. Yang Zheng et al. used the Chinese cabbage anti clubroot gene CRa and CRb molecular marker primer sets to identify 78 Chinese cabbage materials with anti clubroot molecular markers, and screened out 17 homozygous disease resistance loci materials. Zhu Mingzhao et al.
  • Molecular marker-assisted selective breeding is a plant selective breeding method that determines whether the target gene exists by analyzing the genotype of the molecular marker closely linked to the target gene at the molecular level, and selects the target gene.
  • the object of the present invention is to: for the report that utilizes molecular marker selection to polymerize resistance to clubroot and orange Chinese cabbage that has not yet appeared in the above-mentioned prior art, provide a kind of breeding method for new germplasm of orange Chinese cabbage with resistance to clubroot , through the combination of molecular marker-assisted selection and field trait selection, genotype screening of individual plants of isolated populations of Chinese cabbage, and then continuous selfing to obtain Chinese cabbage inbred lines containing both the Chinese cabbage orange gene and the clubroot disease resistance gene , to create high-quality germplasm resources for Chinese cabbage breeding.
  • a method for breeding new germplasm of orange Chinese cabbage resistant to clubroot comprising the following steps:
  • the dominant orange gene marker Br530 is:
  • PCR-specific amplification primers for the dominant orange gene marker Br530 are:
  • PCR-specific amplification primers of anti-tumor root marker SC2930-T are:
  • PCR-specific amplification primers of anti-tumor root marker SC2930Q are:
  • PCR amplification system is:
  • the total volume was 10 ⁇ L, of which 5 ⁇ L of 2 ⁇ Taq Master Mix for PAGE (Dining), 1 ⁇ L of DNA, 0.5 ⁇ L of upstream primer, 0.5 ⁇ L of downstream primer, and the rest were filled with ddH 2 O.
  • golden brocade of Chinese cabbage with resistance to clubroot is used as the male parent.
  • the orange phenotype of Chinese cabbage is controlled by a single recessive gene, and the ratio of orange leaf bulbs to white leaf bulbs in the F2 generation population of the present invention is about 1:3, which fully shows that the orange trait is controlled by a recessive single gene independent inheritance, in line with Mendelian inheritance.
  • Several different markers have been developed based on sequence differences between the Br-or genes of white and orange Chinese cabbage varieties. However, these markers cannot be universally used in all varieties, as these sequences are not exclusively present in orange Chinese cabbage, the deletions have also been detected in other white cabbage.
  • the molecular marker used in the present invention is developed based on the insertion of a large fragment of 501 bp in the Brcrtiso gene of orange Chinese cabbage compared with the 3' end of the Brcrtiso gene of the white leaf bulb, and can directly detect the functional variation of the gene. There are 146 individual strains of the globular gene, and 57 individual strains without the orange marker. The separation of molecular markers is also in line with Mendelian inheritance.
  • the specific molecular marker of clubroot disease resistance gene can quickly detect the disease resistance gene contained in Chinese cabbage material.
  • the present invention uses CRa specific molecular marker to detect 203 plants.
  • the homozygous susceptible marker plants were 61, 102 and 36, respectively, close to 1:2:1.
  • the homozygous germplasm resources for clubroot disease resistance can be obtained, which are further applied in the practice of clubroot disease resistance breeding.
  • the genes that control orange leaf balls are a pair of recessive genes located in the A9 linkage group
  • the gene CRa that controls clubroot disease is a pair of dominant genes located in the A3 linkage group
  • non-homologous chromosomes are located in the offspring gene recombination. Free combination of non-alleles on F2, theoretically, the ratio of the four phenotypes (white resistance: orange resistance: white susceptible: orange susceptible) is 9:3:3:1.
  • the number of white disease-resistant plants was 104, the orange disease-resistant plants were 34, the white susceptible plants were 45, and the orange susceptible plants were 16.
  • the present invention combines molecular marker-assisted selection and field trait selection to perform marker screening on individual plants of Chinese cabbage isolated populations, so as to select Chinese cabbage inbred lines containing both the Chinese cabbage orange gene and the clubroot disease resistance gene. It provides a new method for Chinese cabbage breeding to create high-quality germplasm resources.
  • the present invention adopts molecular marker-assisted selection polymerization breeding to realize direct selection of genotypes, which is fast and accurate, is not easily affected by the environment, can be carried out in any period of field growth, and effectively shortens the breeding years.
  • Fig. 1 is the breeding flow chart of the present invention
  • Fig. 2 is the breeding flow chart of embodiment
  • Figure 3 shows the amplification results of primer Br530 in 22 samples; M molecular weight standard DL2000; the serial number is the number of the individual plant;
  • Figure 4 shows the amplification results of primer SC2930T in 22 samples; M molecular weight standard DL2000; the serial number is the number of the individual plant;
  • Figure 5 shows the amplification results of primer SC2930Q in 22 samples; M molecular weight standard DL2000; the serial number is the number of the individual plant;
  • Figure 6 is a picture of leaf balls of orange Chinese cabbage resistant to clubroot
  • Figure 7 is a picture of the leaf bulb section of the clubroot resistant orange Chinese cabbage.
  • the orange Chinese cabbage inbred line 13S93 in the following examples is the orange Chinese cabbage inbred line selected and bred by this research group, and its plant characteristics are: yellow-green outer leaves, semi-erect plants, leaf balls huddled together, middle stake, cannonball type , bulbous leaves orange.
  • the clubroot resistant Chinese cabbage variety "Jinjin” in the following examples is a commercial variety.
  • parent and female parent are not limited to the orange Chinese cabbage inbred line 13S93 and the clubroot disease resistant Chinese cabbage variety "Jinjin” as an example but not limited to this variety.
  • a preferred embodiment of the present invention provides a method for breeding new germplasm of orange Chinese cabbage resistant to clubroot, the specific steps are as follows:
  • Plant F 1 (15ZF4) in the spring of the following year to obtain F 2 by selfing a single plant, and plant the F 2 segregated population 15ZF4-2, with a total of 205 plants. Plant seeds in plug trays and transplant to open field after 30 days. The row spacing is 45cm ⁇ 50cm, and conventional field management is adopted;
  • CTAB 2% Tris-HC1 100mmol/L, EDTA 20mmol/L, NaCl 1.4mol/L, and add 8 ⁇ L of ⁇ -mercaptoethanol and shake well; take 0.2g of fresh tender leaves from each individual plant with the main vein removed, add liquid nitrogen to grind to powder, and then put it in a 1.5ml centrifuge tube
  • the extract was added to the mortar, mixed and transferred to a centrifuge tube, and the centrifuge tube was shaken constantly; then the centrifuge tube was placed in a 65°C water bath, shaken every few minutes in the middle, and the water bath was 30min; take out the centrifuge tube , add an equal volume of a mixture of phenol:chloroform:isoamyl alcohol, the composition is:
  • PCR amplification was carried out with primers of dominant orange gene marker Br530 and anti-tumor root marker SC2930-T/SC2930Q to identify individual genotypes;
  • the PCR-specific amplification primers for the dominant orange gene marker Br530 are:
  • the PCR-specific amplification primers for anti-tumor marker SC2930-T are:
  • the PCR-specific amplification primers for anti-tumor root marker SC2930Q are:
  • the PCR amplification system is:
  • the total volume is 10 ⁇ L, of which 5 ⁇ L of 2 ⁇ Taq Master Mix for PAGE (Dining), 1 ⁇ L of DNA, 0.5 ⁇ L of upstream primer, 0.5 ⁇ L of downstream primer, and the rest are filled with ddH 2 O;
  • the PCR amplification program of the dominant orange gene marker Br530 is:
  • 51 individual plants contained homozygous orange leaf bulb genes; 102 individual plants contained clubroot resistance genes, and 36 individual plants contained homozygous clubroot resistance genes, among which, There are 34 individual plants of orange Chinese cabbage with resistance to clubroot, the individual plant numbers are 4, 5, 8, 17, 21, 39, 45, 50, 51, 54, 59, 62, 75, 76, 83, 105, 107, 119, 124, 125, 128, 134, 143, 147, 154, 155, 156, 161, 165, 168, 169, 197, 200, 202, 7 of which were homozygous for clubroot resistance orange Chinese cabbage, the individual plant numbers are 8, 17, 50, 107, 134, 155, and 168, and all the 7 individual plants are reserved.
  • the new germplasm of orange Chinese cabbage with resistance to clubroot disease 19CR26 and other horticultural characters obtained by the selection and breeding of the present invention has neat horticultural characters, good bulbing, completes the aggregation of the two characters of orange character and resistance to clubroot disease, and enriches the germplasm resources of Chinese cabbage , laying the foundation for the cultivation of new varieties of orange Chinese cabbage resistant to clubroot.

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Abstract

本发明公开了一种抗根肿病橙色大白菜新种质的选育方法，包括以下步骤：（1）以橙色大白菜为母本，以抗根肿病大白菜为父本，杂交获得F1，F1单株自交获得F2，种植F2群体，提取单株DNA；（2）观察F2园艺形状、切球进行球色表型性状观察统计；（3）使用显性橙色基因标记Br530和抗根肿标记SC2930-T/SC2930Q进行PCR扩增，鉴定单株基因型；（4）依据标记检测结果和园艺形状观察进行综合评价，选择双位点纯合的抗根肿病橙色大白菜植株连续单株自交2代；（5）自交3代后，即得。本发明通过分子标记辅助选择和田间性状选择，对大白菜分离群体单株进行筛选，筛选出同时含有大白菜橙色基因和抗根肿病基因的大白菜，为大白菜育种创制优质的种质资源。

Description

一种抗根肿病橙色大白菜新种质的选育方法 技术领域
本发明属于蔬菜品种选育与种质资源创新领域，尤其涉及一种抗根肿病橙色大白菜新种质的选育方法。
背景技术
大白菜(Brassica campestris syn.rapa L.ssp.pekinensis)是十字花科芸薹属重要的蔬菜作物之一，随着人们生活水平的提高，对大白菜品质的关注度也逐渐增加，大白菜要求高品质同时还要兼具抗病，然而品质高的大白菜又容易感病，使用农药不仅会污染环境，而且长时间使用农药会使蔬菜产生抗药性，因此高品质抗病大白菜种质创新成为近年来研究的热点。
近年来育种家培育出的橙色大白菜不仅色泽鲜艳而且含有较高的类胡萝卜素，质地柔嫩风味鲜美，营养品质丰富。为了加快橙色大白菜育种步伐，国内外学者在橙色大白菜分子标记方面开展了许多工作。王绮获得了与大白菜橙色叶球基因遗传距离为3.7cM的分子标记SC82-591。Feng等获得了遗传距离1.3cM的分子标记syau15，Zhang等将橙色大白菜Br-or基因定位在A09连锁群16.7kb的区间，获得了连锁距离为0.1和0.2cM的分子标记InDel2和Br-InDel1，并进一步根据橙色大白菜和普通大白菜BrCRTISO启动子序列的差异设计了橙色叶球标记，证明该标记为与橙色性状的共显性标记。李佩荣等在橘红心材料Br-or基因的cDNA水平上编码区发现53个SNPs和6个碱基缺失，并根据6bp的缺失开发了分子标记GAA2用于辅助选择育种。
根肿病是由芸薹根肿菌(Plasmodiophora brassicae Woron)侵染引起的一种专性寄生的毁灭性土传病害，近年来根肿病在我国多地发生，已经成为大白菜生产的主要病害。通过田间管理和生物化学防治不能从根本上防治根肿病，培育抗根肿病大白菜品种是解决根肿病问题的重要措施。杨征等利用大白菜抗根肿基因CRa和CRb分子标记引物组对78份大白菜材料进行了抗根肿病分子标记鉴定，筛选出纯合抗病位点材料17份。朱明钊等对24份大白菜材料进行根肿病分子标记鉴定，获得了5份对多种根肿菌生理小种有优良抗性的材料。陈丽潇等以10个抗根肿病大白菜品种为试材，利用根肿抗性基因CRa的特异分子标记CRaEXON4-3筛选DH群体获得27个携带抗性基因植株。分子标记辅助选择育种是通过在分子水平上分析与目标基因紧密连锁的分子标记的基因型来判别目标基因是否存在，并对目标基因进行选择的一种植物选择育种方法。
目前尚未见到应用分子标记辅助选择橙色叶球及抗根肿病的相关报道。
发明内容
本发明的目的在于：针对上述现有技术中尚未出现的利用分子标记选择将抗根肿病与橙 色大白菜聚合方面的报道，提供一种抗根肿病橙色大白菜新种质的选育方法，通过分子标记辅助选择和田间性状选择相结合，对大白菜分离群体单株进行基因型筛选、再连续自交，以获得同时含有大白菜橙色基因和抗根肿病基因的大白菜自交系，为大白菜育种创制优质的种质资源。
本发明采用的技术方案如下：
一种抗根肿病橙色大白菜新种质的选育方法，包括以下步骤：
(1)以橙色大白菜为母本，以抗根肿病大白菜为父本，杂交获得F 1，F 1单株自交获得F 2，种植F 2群体，苗期单株编号、提取DNA；
(2)田间定植F 2群体，在F 2结球晚期，观察园艺形状、切球进行球色表型性状观察统计；
(3)使用显性橙色基因标记Br530和抗根肿标记SC2930-T/SC2930Q进行PCR扩增，鉴定单株基因型；
(4)依据标记检测结果和园艺形状观察进行综合评价，选择双位点纯合的抗根肿病橙色大白菜植株连续单株自交2代；
(5)种植双位点纯合的抗根肿病橙色大白菜植株自交3代后，即获得园艺性状稳定的纯合的抗根肿病橙色大白菜植株自交系。
进一步地，显性橙色基因标记Br530为：
Figure PCTCN2021081285-appb-000001
进一步地，显性橙色基因标记Br530的PCR特异性扩增引物为：
上游引物(F)：5’－CAGAAACATCAGGGTTGAAATC－3’；
下游引物(R)：5’－TTACTGCCGAAAGCGAAA－3’。
进一步地，抗根肿标记SC2930-T的PCR特异性扩增引物为：
上游引物(F)：5’－TAGACCTTTTTTTTGTCTTTTTTTTTACCT－3’；
下游引物(R)：5’－AAGGCCATAGAAATCAGGTC－3’。
进一步地，抗根肿标记SC2930Q的PCR特异性扩增引物为：
上游引物(F)：5’－CAGACTAGACTTTTTGTCATTTAGACT－3’；
下游引物(R)：5’－AAGGCCATAGAAATCAGGTC－3’。
进一步地，PCR扩增体系为：
总体积为10μL，其中2×Taq Master Mix for PAGE(Dining)5μL，DNA 1μL，上游引物0.5μL，下游引物0.5μL，其余用ddH 2O补齐。
进一步地，显性橙色基因标记Br530的PCR扩增程序为：
95℃ 3min，
95℃ 30s，
58℃ 30s，
72℃ 1min，
38个循环，
72℃ 10min，
6℃，
∞。
进一步地，抗根肿标记SC2930-T/SC2930Q的PCR扩增程序为：
94℃ 3min，
94℃ 1min，
55℃ 1.5min，
72℃ 2min，
30个循环，
72℃ 7min，
6℃，
∞。
进一步地，以橙色大白菜13S93为母本。
进一步地，以抗根肿病大白菜金锦为父本。
综上所述，由于采用了上述技术方案，本发明的有益效果是：
大白菜橙色表型是由单个隐性基因控制的，本发明的F2代群体中橙色叶球单株与白色叶球单株的比例约为1﹕3，充分说明橙色性状为隐性单基因控制的独立遗传，符合孟德尔遗传规律。根据白色和橙色大白菜品种Br-or基因之间的序列差异，已经开发了几种不同标记。但 是这些标记不能普遍用于所有品种，因为这些序列并非仅存在于橙色大白菜中，在其他白色白菜中也检测到了这种缺失。本发明所使用的分子标记是基于橙色大白菜Brcrtiso基因较白色叶球的Brcrtiso基因3’末端有501bp的大片段插入开发的，能够直接检测该基因的功能变异，结果分子标记检测出携带橙色叶球基因的单株为146株，不含橙色标记单株57株，分子标记分离也符合孟德尔遗传。
利用抗根肿病基因特异分子标记可以快速检测大白菜材料含有的抗病基因,本发明利用CRa特异分子标记检测的203个植株,结果纯合抗病标记植株、抗感共显性标记植株与纯合感病标记植株分别是61、102和36接近1﹕2﹕1。通过本发明的方法能够获取抗根肿病纯合种质资源,进而应用于抗根肿病育种实践当中。
本发明中控制橙色叶球的基因为位于A9连锁群的一对隐性基因，控制抗根肿病的基因CRa为位于A3连锁群的一对显性基因，在后代基因重组中非同源染色体上的非等位基因自由组合，理论上F2的4种表现型(白色抗病：橙色抗病：白色感病：橙色感病)比例为9﹕3﹕3﹕1。在标记鉴定结合表型统计中白色抗病单株为104，橙色抗病单株为34，白色感病单株为45，橙色感病单株为16。卡方检验符合9﹕3﹕3﹕1的自由组合定律(2＝3.461<χ2(0.05，1)＝7.815)，在34个橙色抗病单株中筛选了7个单株为纯合抗根肿病橙色大白菜植株，连续两代自交观测获得了3个稳定自交系，说明本发明的分子标记辅助选择方法简单高效。
综上，本发明通过分子标记辅助选择和田间性状选择相结合，对大白菜分离群体单株进行标记筛选，达到了选出同时含有大白菜橙色基因和抗根肿病基因的大白菜自交系的目标，为大白菜育种创制优质的种质资源提供了新方法。此外，本发明采用分子标记辅助选择聚合育种实现对基因型直接选择，快速准确，不易受环境影响，可以在田间生长的任何时期进行，有效缩短了育种年限。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明选育流程图；
图2为实施例的选育流程图；
图3为引物Br530在22个样品中的扩增结果；M分子量标准DL2000；编号为单株编号；
图4为引物SC2930T在22个样品中的扩增结果；M分子量标准DL2000；编号为单株编号；
图5为引物SC2930Q在22个样品中的扩增结果；M分子量标准DL2000；编号为单株编号；
图6为抗根肿病橙色大白菜的叶球图片；
图7为抗根肿病橙色大白菜的叶球剖面图片。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，术语“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
下述实施例中的橙色大白菜自交系13S93是本课题组选育的橙色大白菜自交系，其植株特征为：外叶黄绿色、植株半直立，叶球合抱、中桩、炮弹型，球叶橙色。
下述实施例中的抗根肿病大白菜品种“金锦”是商业品种。
需要说明的是，本发明以父母和母本不限于橙色大白菜自交系13S93和抗根肿病大白菜品种“金锦”为例但不限于该品种。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例
本发明较佳的实施例提供一种抗根肿病橙色大白菜新种质的选育方法，具体步骤如下：
1、以橙色大白菜自交系13S93为母本，以抗根肿病大白菜“金锦”为父本，杂交获得F 1；
2、次年春季种植F 1(15ZF4)单株自交获得F 2，种植F 2分离群体15ZF4-2，共计205株；大白菜栽种地平坦，肥力均匀，土壤为碱性土。穴盘播种，30d后移栽到露地。株行距45cm×50cm，采取常规田间管理；
大白菜幼苗期，田间单株插牌编号，单株取样，提取DNA；在1.5ml的离心管中加入750uL的1×CTAB提取液，其组成为：CTAB 2％，Tris-HC1 100mmol/L，EDTA 20mmol/L，NaCl 1.4mol/L，并加入8μL的β-巯基乙醇摇匀；每单株取0.2g去掉主脉的新鲜嫩叶片，加入液氮研磨至粉末，然后将1.5ml离心管中提取液加入到研钵中，混匀后转入到离心管中，并不断地摇动离心管；随后将离心管放入65℃水浴中，中间每间隔几分钟摇一次，水浴30min；取出离心管，加入等体积的酚：氯仿：异戊醇的混合物，其组成为：酚：氯仿：异戊醇＝25：24：1，摇匀10min后，常温下12000r/min离心10min；将上层液相转移到另一离心管中，加入等体积的氯仿：异戊醇的混合物，其比例为24：1，摇匀10min，常温下12000r/min离心10min；取上清加入2倍体积的预冷的无水乙醇，混匀后，使DNA抱团，-20℃条件下沉淀30min，4℃条件下12000r/min离心10min；弃上清，加入1ml 75％乙醇洗涤沉淀2-3次，沉淀物室温晾干；加入400-500μL TE缓冲液溶解DNA，加入1.5uL浓度为10μg/uL的RNaseA，使终浓度达10μg/mL，混匀后37℃保温30min；待DNA完全溶解后，加入3mol/L NaAC溶液40-50μL和等体积的异丙醇，摇匀使DNA充分沉淀，冰浴15min；在4℃，12000r/m条件下离心10min，弃上清，加入75％的乙醇清洗沉淀1-2次，后加入1200μL DNA贮存液，DNA贮存液由75％乙醇，0.3mol/L NaAC溶液构成；提取的DNA，经纯度检测，证实纯化较好，一般用灭菌的400-500μL ddH 2O稀释，存于4℃中备用；
使用显性橙色基因标记Br530和抗根肿标记SC2930-T/SC2930Q的引物进行PCR扩增，鉴定单株基因型；
显性橙色基因标记Br530的PCR特异性扩增引物为：
上游引物(F)：5’－CAGAAACATCAGGGTTGAAATC－3’；
下游引物(R)：5’－TTACTGCCGAAAGCGAAA－3’；
抗根肿标记SC2930-T的PCR特异性扩增引物为：
上游引物(F)：5’－TAGACCTTTTTTTTGTCTTTTTTTTTACCT－3’；
下游引物(R)：5’－AAGGCCATAGAAATCAGGTC－3’；
抗根肿标记SC2930Q的PCR特异性扩增引物为：
上游引物(F)：5’－CAGACTAGACTTTTTGTCATTTAGACT－3’
下游引物(R)：5’－AAGGCCATAGAAATCAGGTC－3’；
PCR扩增体系为：
总体积均为10μL，其中2×Taq Master Mix for PAGE(Dining)5μL，DNA 1μL，上游引物0.5μL，下游引物0.5μL，其余用ddH 2O补齐；
显性橙色基因标记Br530的PCR扩增程序为：
95℃ 3min，
95℃ 30s，
58℃ 30s，
72℃ 1min，
38个循环，
72℃ 10min，
6℃，
∞；
抗根肿标记SC2930-T/SC2930Q的PCR扩增程序为：
94℃ 3min，
94℃ 1min，
55℃ 1.5min，
72℃ 2min，
30个循环，
72℃ 7min，
6℃，
∞。
使用单显性橙色叶球标记Br530对群体进行单株鉴定结果显示(图3)，有146个单株含有橙色标记，57个单株不含橙色标记。经卡方值检验，橙色基因符合3﹕1的孟德尔遗传规律(χ 2＝1.026<χ 2(0.05，1)＝3.84)。在大白菜结球后期剖开叶球统计单株球色，在表型统计中，橙色叶球有51个，白色叶球有152个，符合1﹕3的分离比例。说明橙色对白色为隐性遗传。分子标记结合球色表型筛选，在203个有效单株中有51个单株为等位基因纯合的橙色单株。
由图4、5可知，有4株两种引物均没有扩增条带，用CRa抗根肿病基因引物对群体的抗性基因鉴定结果显示，163个单株含有SC2930-Q感病标记，其中61个单株只含有SC2930Q标记，有102个为杂合抗病单株；有138个单株含有SC2930-T抗病标记，其中有36个单株 只含有SC2930-T标记，表明这些单株中含有等位基因纯合的抗根肿病基因CRa。经卡方值检验，抗根肿病基因符合3﹕1的孟德尔遗传规律(χ 2＝3.392<χ 2(0.05，1)＝3.84)。
综上，在F 2分离群体中，51个单株含有纯合的橙色叶球基因；102个单株含有抗根肿病基因，36个单株含有纯合的抗根肿病基因，其中，有34个单株为抗根肿的橙色大白菜，单株编号为4、5、8、17、21、39、45、50、51、54、59、62、75、76、83、105、107、119、124、125、128、134、143、147、154、155、156、161、165、168、169、197、200、202，其中有7个单株为纯合抗根肿病橙色大白菜，单株编号为8、17、50、107、134、155、168，这7个单株全部留种。
3、下一年春季，栽培上年选留的7个单株，单株自交，得到F 2S 1群体的种子7份，分别编号17CR1到17CR7。秋季田间种植F 2S 1群体的种子(17CR1到17CR7各30株，总共210株)，观察园艺性状一致性，在17CR1到17CR7中选择结球性好、园艺性状一致的单株各10株，自然通过春化。
4、下一年春季，栽培从17CR1到17CR7中入选的单株，单株自交(70株)，得到F 2S 2群体的种子70份，分别编号18CR1到18CR70。秋季田间种植F 2S 2群体的种子(18CR1到18CR70各30株，总共2100株)，观察园艺性状一致性，从中选择稳定株系10个，各选择5株单株，自然通过春化。
5、下一年春季，栽培从18CR1到18CR70中入选的10个稳定株系，各5株单株，单株自交(50株)，得到F 2S 3群体的种子50份，分别编号19CR1到19CR50。秋季田间种植F 2S 3群体的种子(19CR1到19CR50各30株，总共1500株)，观察园艺性状一致，发现3个株系(19CR5、26、38)园艺性状整齐一致，分子标记鉴定携带纯合的橙色基因和抗根肿病，其中19CR26抗根肿病橙色大白菜新种质的表型如图6和图7所示。
本发明选育得到的抗根肿病橙色大白菜新种质19CR26等园艺性状整齐、结球性好、完成了橙色性状与抗根肿病性状两个性状的聚合，丰富了大白菜种质资源，为培养抗根肿病橙色大白菜新品种奠定了基础。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种抗根肿病橙色大白菜新种质的选育方法，其特征在于，包括以下步骤：
    (1)以橙色大白菜为母本，以抗根肿病大白菜为父本，杂交获得F 1，F 1单株自交获得F 2，种植F 2群体，苗期单株编号、提取DNA；
    (2)田间定植F 2群体，在F 2结球晚期，观察园艺形状、切球进行球色表型性状观察统计；
    (3)使用显性橙色基因标记Br530和抗根肿标记SC2930-T/SC2930Q进行PCR扩增，鉴定单株基因型；
    (4)依据标记检测结果和园艺形状观察进行综合评价，选择双位点纯合的抗根肿病橙色大白菜植株连续单株自交2代；
    (5)种植双位点纯合的抗根肿病橙色大白菜植株自交3代后，即获得园艺性状稳定的纯合的抗根肿病橙色大白菜植株自交系。
  2. 根据权利要求1所述的抗根肿病橙色大白菜新种质的选育方法，其特征在于，所述显性橙色基因标记Br530为：
    CAGAAACATCAGGGTTGAAATCTAAACCCAGAAAATAAACCCAATATGGTATAGGTTTACCCGTGGGTACCCAAAGTATTATCTTATTTATTCTGAAGATCATGTAAAACTCATTTATGGTTTTAACGAGAAAACTTGTAAAGTTGTTTTTGTGGTTTTAGCGGAAATTTTTCTTTTTGCGGTTTTTGGTCGGTAATTTTATTTTGTGGCTTGGTTGGAAAACTCATTTTTGCGGTTTGCGGGAAAAATAATCTTTCTGGTTTTGACGAAAAAATTCGGTTTTACGGTTTTTGCGAGAAAATTCGGTTTAGCAGTTTTGGCAGGAAACCTCGCTTTTGCGGTTTTGGCGGAAAAACTCGTTTTTGATTTTGACGGAAAAACTTGTTTTTACGGTTTTGGGGAAACTCGGTTTTCGGCTTTGACGGGAAAACTCGATTTTTCGATTTTGGCGGGAAAACTCGATTTTGCGGTTTTGGCGGGAAAACTCGGTTTTTCTGTTTTGGCGGAAAAACCATGTTTTTCGCTTTCGGCAGTAA。
  3. 根据权利要求1所述的抗根肿病橙色大白菜新种质的选育方法，其特征在于，所述显性橙色基因标记Br530的PCR特异性扩增引物为：
    上游引物(F)：5’－CAGAAACATCAGGGTTGAAATC－3’；
    下游引物(R)：5’－TTACTGCCGAAAGCGAAA－3’。
  4. 根据权利要求1所述的抗根肿病橙色大白菜新种质的选育方法，其特征在于，所述抗根肿标记SC2930-T的PCR特异性扩增引物为：
    上游引物(F)：5’－TAGACCTTTTTTTTGTCTTTTTTTTTACCT－3’；
    下游引物(R)：5’－AAGGCCATAGAAATCAGGTC－3’。
  5. 根据权利要求1所述的抗根肿病橙色大白菜新种质的选育方法，其特征在于，所述抗 根肿标记SC2930Q的PCR特异性扩增引物为：
    上游引物(F)：5’－CAGACTAGACTTTTTGTCATTTAGACT－3’；
    下游引物(R)：5’－AAGGCCATAGAAATCAGGTC－3’。
  6. 根据权利要求1所述的抗根肿病橙色大白菜新种质的选育方法，其特征在于，所述PCR扩增体系为：
    总体积为10μL，其中2×Taq Master Mix for PAGE(Dining)5μL，DNA 1μL，上游引物0.5μL，下游引物0.5μL，其余用ddH 2O补齐。
  7. 根据权利要求1所述的抗根肿病橙色大白菜新种质的选育方法，其特征在于，所述显性橙色基因标记Br530的PCR扩增程序为：
    95℃ 3min，
    95℃ 30s，
    58℃ 30s，
    72℃ 1min，
    38个循环，
    72℃ 10min，
    6℃，
    ∞。
  8. 根据权利要求1所述的抗根肿病橙色大白菜新种质的选育方法，其特征在于，所述抗根肿标记SC2930-T/SC2930Q的PCR扩增程序为：
    94℃ 3min，
    94℃ 1min，
    55℃ 1.5min，
    72℃ 2min，
    30个循环，
    72℃ 7min，
    6℃，
    ∞。
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