CN106591355B - Breeding method of high-content 4-methyl sulfur oxygen butyl thioglycoside cabbage crop - Google Patents

Breeding method of high-content 4-methyl sulfur oxygen butyl thioglycoside cabbage crop Download PDF

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CN106591355B
CN106591355B CN201611224588.4A CN201611224588A CN106591355B CN 106591355 B CN106591355 B CN 106591355B CN 201611224588 A CN201611224588 A CN 201611224588A CN 106591355 B CN106591355 B CN 106591355B
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王晓武
梁建丽
武剑
程峰
刘志远
张冀芳
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Abstract

The invention relates to a breeding method of a cabbage crop with high content of 4-methyl sulfur oxygen butyl thioglycoside. The breeding method comprises the following steps: step A, transferring a gene losing functions in a donor material into a receptor material to be bred to obtain a polymerized single plant; b, performing systematic breeding on the polymerized single plant to obtain a cabbage crop with high content of 4-methyl sulfur oxygen butyl thioglycoside; wherein the loss-of-function gene comprises at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3. The obtained high-content 4-methyl sulfur butyl sulfur glucoside cabbage contains loss-of-function protein, and the loss-of-function protein comprises at least two of BrAOP2.1, BrAOP2.2 and BrAOP2.3. The breeding method has the advantages that the molecular marker-assisted selection technology is adopted, the breeding cost is saved, the breeding time is shortened, and the breeding efficiency is improved.

Description

Breeding method of high-content 4-methyl sulfur oxygen butyl thioglycoside cabbage crop
Technical Field
The invention belongs to the technical field of breeding, and particularly relates to a breeding method of a cabbage crop with high content of 4-methyl thiooxybutylsulroside.
Background
Brassica rapa (l.syn. Brassica campestis) belong to the Brassica genus of the brassicaceae family and contain many important vegetable, forage and oil crops. China is the origin and evolution center of Chinese cabbage crops. Chinese cabbage crops have been cultivated for more than two thousand years in China, and a plurality of types including Chinese cabbages, pakchoi, turnips, cabbage hearts, flowering cabbages and the like are gradually evolved, so that the Chinese cabbage crops become one of the vegetable crops with the widest distribution and the largest planting area in China, and have a very important position in agricultural production.
Glucosinolates (called glucosinolates for short) are a unique secondary metabolite in cruciferous plants, and after plant cells are damaged, the metabolite is combined with extracellular myrosinase to form a specific degradation product immediately, and the degradation products not only help the plants to protect against plant diseases and insect pests, but also are important reasons for generating special taste and flavor of cruciferous vegetables. In recent years, studies on degradation products of glucosinolates have found that some of the degradation products of glucosinolates, particularly the degradation product of 4-methylthiobutylglucosinolate (GRA), sulforaphane, can inhibit tumor growth by blocking the cell cycle and promoting apoptosis. More and more researches show that the sulforaphane can effectively inhibit the growth of various tumor cells of intestinal cancer, gastric cancer, lung cancer, prostatic cancer, liver cancer, colon cancer and the like. In cruciferous vegetables, GRAs are detected in high content in cabbage vegetables such as broccoli and cauliflower, however, in most white vegetables, only trace amount of GRAs can be detected, even the content of GRAs cannot be detected.
The enzyme encoded by AOP2 gene is a key factor that catalyzes the GRA conversion of beneficial glucosinolates to NAP (3-butenyl glucosinolates). Although the cabbage and cabbage crops have a genome-wide replication event and 3 AOP2 genes, 3 AOP2 genes in the cabbage crops have activity, and 2 AOP2 genes in the cabbage have base deletion, so that early termination is caused and the cabbage loses functions. This may be the reason for the cabbage crop to have a higher GRA. In addition, due to the existence of the redundancy phenomenon of AOP2 gene, the loss of copy function may be caused and the accumulation of glucosinolate GRA in vivo cannot be influenced significantly. Therefore, the creation of Chinese cabbage crops carrying two or three BrAOP2 genes with lost functions has important significance for breeding new varieties of various Chinese cabbage crops with high content of beneficial glucosinolates GRA.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art and provides a method for breeding cabbage crops with high content of 4-methyl thiooxybutylsulroside, which breeds cabbage crops simultaneously carrying two or three BrAOP2 gene loss functions by utilizing the technologies of polymerization hybridization and molecular marker-assisted selection, thereby rapidly breeding cabbage materials with high content of beneficial glucosinolate GRA.
Therefore, the invention provides a breeding method of a cabbage crop with high content of 4-methyl thiooxybutyl glucosinolate, which comprises the following steps:
step A, transferring a gene losing functions in a donor material into a receptor material to be bred to obtain a polymerized single plant;
b, performing systematic breeding on the polymerized single plant to obtain a cabbage crop with high content of 4-methyl sulfur oxygen butyl thioglycoside;
wherein the loss-of-function gene comprises at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3.
In some embodiments of the invention, the polymerizing individual contains a loss-of-function gene that includes at least two of BrAOP2.1, BrAOP2.2, and BrAOP2.3.
In other embodiments of the invention, the high 4-methylsulfanylbutylsulfatide cabbage plant comprises a loss-of-function protein comprising at least two of BrAOP2.1, BrAOP2.2, and BrAOP2.3.
In some embodiments of the invention, the donor material is at least one strain, and when the donor material is two or more strains, the nonfunctional genes differ between the donor materials.
In other embodiments of the invention, the loss-of-function gene in the donor material is obtained by natural mutation, EMS mutagenesis, RNAi interference, or by using CRISPR/Cas9 knock-out method.
In some embodiments of the invention, a nonfunctional gene in the donor material is introduced into the recipient material to be bred by convergent hybridization, and the aggregate individual is screened by molecular marker assisted selection.
In other embodiments of the invention, the aggregate individual is selected systematically by molecular marker assisted selection.
In some embodiments of the invention, the generation number of the breeding of the system is 2-4 generations.
According to the invention, both the donor material and the recipient material to be bred are cabbage crops.
According to the invention, the content of the 4-methyl thiooxybutylsulfan in the high-content 4-methyl thiooxybutylsulfan cabbage crops is 5-10 times of the content of the 4-methyl thiooxybutylsulfan in common cabbage crops.
The invention has the beneficial effects that: the breeding method of the invention breeds the cabbage crop carrying two or three BrAOP2 gene loss functions simultaneously by polymerization hybridization and molecular marker assisted selection technology, thereby rapidly breeding the cabbage material with high content of beneficial glucosinolate GRA, and the bred cabbage material with high content of beneficial glucosinolate GRA can be directly used for preparing the cabbage composition containing high content of beneficial glucosinolate GRA. Meanwhile, the breeding method avoids measuring the GRA content of each material by an instrument through a molecular marker assisted selection technology, greatly saves the breeding cost, shortens the breeding time and improves the breeding efficiency.
Drawings
The invention is described below with reference to the accompanying drawings.
FIG. 1 is a flow chart of a method for breeding high content GRA Chinese cabbage plants in example 1; wherein the numbers in parentheses indicate the number of plants planted.
FIG. 2 is a schematic diagram showing the identification of the enzyme activities of BrAOP2.1, BrAOP2.2 and BrAOP2.3 in the donor material R-O-18.
FIG. 3 is a schematic diagram showing the typing results of BrAOP2.2 tags; wherein, FAM represents a plant with FAM fluorescence; hex represents plants with Hex fluorescence; box represents a heterozygous plant with Both FAM and Hex fluorescence; NTC represents a negative control.
FIG. 4 is a schematic diagram showing the molecular detection results of BrAOP2.3 labeling.
FIG. 5 shows a braop2.2braop2.3 individual, BrAOP2.2BrAOP2.3 individual, braop2.2BrAOP2.3 individual, BrAOP2.2braop2.3, L58, R-O-18, F1Determination of medium GRA glucosinolate contentThe results are shown schematically.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below.
As mentioned above, the creation of cabbage material carrying two or three BrAOP2 genes with lost functions has important significance for breeding new varieties of various cabbage crops with high content of beneficial glucosinolates GRA, and no rapid breeding method for the new varieties of the cabbage crops exists at present.
The former carries out RNAi interference on the AOP2 gene of the rape, reduces the activity of AOP2, thereby obviously improving the GRA content in the rape, namely the activity of AOP2 is in negative correlation with the GRA content; in addition, three BrAOP2 genes in the cabbage crops are distributed on three chromosomes (A02, A03 and A09), and are suitable for carrying out polymerization hybridization to introduce the mutant BrAOP2 genes dispersed in different varieties into the same cabbage variety. The present invention has been made based on the above.
Therefore, the invention relates to a breeding method of a cabbage crop with high content of 4-methyl thiooxybutyl glucosinolate, which comprises the following steps:
(1) firstly, obtaining donor materials possibly carrying a gene with loss function by natural mutation, EMS mutagenesis, RNAi interference or using a CRISPR/Cas9 knock-out method; screening out donor materials carrying the gene with lost functions through gene sequence analysis, prokaryotic expression and in-vitro enzyme activity experiments; the loss-of-function gene comprises at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3;
(2) introducing the gene losing functions in the donor material into a receptor material to be bred in a polymerization hybridization mode, and screening successfully introduced positive plants by using a molecular marker-assisted selection method to obtain a polymerization single plant;
the obtained polymeric single strain contains the loss-of-function genes, the loss-of-function genes comprise at least two of BrAOP2.1, BrAOP2.2 and BrAOP2.3, and the loss-of-function genes are in a heterozygote form, namely, the encoded protein still has functions;
at least one strain of donor material; when the donor materials are two or more plants, the genes losing functions are different among the donor materials; the loss-of-function gene comprises at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3;
the specific mode for screening the polymerized single plant by using the molecular marker-assisted selection method is as follows:
firstly, designing molecular markers (such as KASP markers and InDel markers) according to sequence differences between the loss-of-function genes and the normal-function genes, wherein the markers are obviously different in plants with the loss-of-function genes and plants with the normal-function genes (such as different amplified fragments in size and different fluorescent signals);
then extracting DNA of the plant to be screened, carrying out PCR amplification by using the designed molecular marker, and detecting a PCR amplification product; if the marker in the PCR amplification product is in accordance with the marker of the loss-of-function gene, it indicates that the plant to be screened contains the target gene (the loss-of-function gene), and the plant containing the corresponding target gene is selected to obtain the polymerized single plant.
(3) Performing systematic breeding on the polymerized single plant to obtain a cabbage crop with high content of 4-methyl sulfur oxygen butyl thioglycoside;
the high-content 4-methyl sulfur butyl sulfur glucoside cabbage crops contain protein losing functions, and the protein losing functions comprises at least two of BrAOP2.1, BrAOP2.2 and BrAOP2.3; the loss-function gene in the high-content 4-methyl sulfur oxygen butyl sulfur glucoside cabbage crop obtained after the systematic breeding is in a homozygote shape;
in the system breeding process, a molecular marker auxiliary selection method is combined to perform optimal selection, so that stable Chinese cabbage crops with high GRA content are obtained; the generation number of the system breeding is 2-4 generations; the generation number of general system breeding can be shortened to 2 generations;
the specific mode of carrying out systematic breeding by utilizing a molecular marker assisted selection method is as follows:
firstly, designing molecular markers (for example, KASP markers and InDel markers) according to sequence differences between loss-of-function genes and normal-function genes;
extracting DNA of a plant to be selected, performing PCR amplification by using the designed molecular marker, and detecting a PCR amplification product; if the marker in the PCR amplification product is in accordance with the marker of the gene function-loss material, it is indicated that the plant to be bred contains the target gene (the gene with lost function), and the plant containing the corresponding target gene is selected.
According to the invention, both the donor material and the recipient material to be bred are cabbage crops.
The inventor finds that the content of 4-methyl thiooxybutyl glucosinolate in the bred and bred high-content 4-methyl thiooxybutyl glucosinolate cabbage crops is 0.999 +/-0.150 umol/g of DW, while the content of 4-methyl thiooxybutyl glucosinolate in the common cabbage crops is 0.109 +/-0.040 umol/g of DW through detection; the content of the 4-methyl thiooxybutyl glucosinolate in the high-content 4-methyl thiooxybutyl glucosinolate cabbage crops is 5-10 times of the content of the 4-methyl thiooxybutyl glucosinolate in common cabbage crops.
The term "nonfunctional" as used herein means "non-functional or reduced functional".
The term "braop 2.2" as used herein means a nonfunctional BrAOP2.2 gene; "braop 2.3" means a loss of function BrAOP2.3 gene; "the individual strain of braop2.2braop 2.3" means that the genotype is the individual strain of braop2.2braop2.3braop 2.3; "BrAOP2.2BrAOP2.3 individual plant" means that the genotype is BrAOP2.2BrAOP2.2BrAOP2.3BrAOP2.3 individual plant; "the braop2.2BrAOP2.3 individual" means that the genotype is braop2.2braop2.2BrAOP2.3BrAOP2.3 individual; "BrAOP2.2braop2.3 individual" means that the genotype is BrAOP2.2BrAOP2.2braop2.3braop2.3 individual.
Examples
In order that the invention may be more readily understood, the invention will now be described in further detail with reference to the accompanying drawings and examples, which are given by way of illustration only and are not limiting to the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
Example 1: breeding method of high-content 4-methyl sulfur oxygen butyl sulfur glucoside cabbage crop
The flow chart of the breeding method is shown in figure 1, and the specific operation steps are as follows:
(1) selection of donor material and recipient material to be bred
By gene sequence analysis, R-O-18 material (one of Yellow Sarson types) carrying possible loss of BrAOP2.2 and BrAOP2.3 functions was selected; the enzyme activities of BrAOP2.1, BrAOP2.2 and BrAOP2.3 in the R-O-18 material are detected through prokaryotic expression and in-vitro enzyme activity experiments, and the result is shown in figure 2; as can be seen from FIG. 2, BrAOP2.1 in the R-O-18 material has catalytic activity, while BrAOP2.2 and BrAOP2.3 lose catalytic activity, so the R-O-18 material is selected as the donor material;
selecting an L58 material (four nine cabbage heart) with activity of BrAOP2.1, BrAOP2.2 and BrAOP2.3 as a breeding receptor material;
(2) obtaining of Polymer Individual
Designing KASP marker and InDel marker according to the difference of BrAOP2.2 and BrAOP2.3 gene sequences in parent L58 material and R-O-18 material;
f was obtained by hybridizing the R-O-18 material with the L58 material1(first filial generation); planting F1Backcrossing with parent L58 material to obtain BC1(backcross generation) seeds;
planting BC1Obtaining 424 strains of BC1At BC1Extracting DNA of each strain by using an improved CTAB method in the seedling stage, and using the two markers to pair BC1Screening single plants; wherein, the gene BrAOP2.2 is specially marked with KASP for detection, and PCR amplification is carried out by using KASP marking primers which are respectively:
hex fluorescent primer: 5'-GAAGGTCGGAGTCAACGGATTGTGGTGCTGAATACTTCATCAGTCA-3' (SEQ ID NO: 1);
FAM fluorescent primer: 5'-GAAGGTGACCAAGTTCATGCTGGTGCTGAATACTTCATCAGTCG-3' (SEQ ID NO: 2);
the general primer is as follows: 5'-AGAACCTCAAGTCAATGAATTACCGTCTA-3' (SEQ ID NO: 3);
the reaction product was detected by qPCR (the type of the used instrument is ABI 7900HT-fast, ABI Quantstrudio 12K or Roche Lightcycler II 480, etc.), and the detection result is shown in FIG. 3, and BC with FAM and Hex dual fluorescent labels was selected1And (4) single plants.
The gene BrAOP2.3 is specially marked with InDel for marking detection, and PCR amplification is carried out by using InDel marking primers which are respectively as follows:
an upstream primer F: 5'-GTGCTGGTGATGGTGCTAATGATG-3' (SEQ ID NO: 4);
InDel marker R: 5'-CAACACCAGCACTACTGAGAGTAC-3' (SEQ ID NO: 5);
the reaction product was detected by agarose gel, and the detection result is shown in FIG. 4, and BC having 268bp and 476bp was selected1And (4) single plants.
Analysis of the results of the detection of the two markers in combination, at BC1Selecting 112 polymeric individuals with the marker genotype of BrAOP2.2braop2.2BrAOP2.3braop2.3 from the large population (424), and carrying out background marker selection on the polymeric individuals to obtain the polymeric individual BC with the background closest to the L58 material1018 (one individual in a backcross generation); the methods used for background marker selection are described in: menglin, QTLs analysis of the nitrogen utilization efficiency of cabbage crops, Master's academic thesis of Yunnan university of agriculture, pages 2012 and 38-39.
(3) Systematic breeding method for obtaining high-content 4-methyl sulfur oxygen butyl sulfur glucoside cabbage crops
Planting BC1018, backcrossing with parent L58 material to obtain BC2(backcross second generation) seeds;
planting BC2Obtaining 224 strains BC2In seedling stage, the KASP marker and the InDel marker are used for marking on BC2Screening 64 individuals with the marker genotype of BrAOP2.2braop2.2BrAOP2.3braop2.3 from the large population, and carrying out background marker selection on the individuals to obtain BC with the background closest to the L58 material2Material BC2227 (one single plant in the second backcross generation).
BC2-227 selfing to obtain BC2S1(backcross second generation selfing first generation) seed, planting BC2S1Obtaining 400 BC2S1In seedling stage, the KASP marker and the InDel marker are used for marking on BC2S1Screening pure single plants with marker gene type of braop2.2braop2.2braop2.3braop2.3 from the large population, namely the cabbage crops with high content of 4-methyl sulfur oxygen butyl sulfur glycoside.
For the obtained braop2.2braop2.3 single strain, L58, R-O-18 and F1GRA glucosinolates were measured on BrAOP2.2BrAOP2.3 individuals, braop2.2BrAOP2.3 individuals, and BrAOP2.2braop2.3 individuals, and the measurement results are shown in FIG. 5. As can be seen from FIG. 5, the average GRA content of individual plants with genotype braop2.2braop2.3 was 1.02. mu. mol/g DW, which is significantly higher than the GRA content of parent L58 (0.13. mu. mol/g DW).
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
SEQUENCE LISTING
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> a breeding method of high-content 4-methyl sulfur-oxygen butyl sulfuric glucoside cabbage crop
<130>2016
<160>5
<170>PatentIn version 3.3
<210>1
<211>46
<212>DNA
<213> KASP-labeled Hex fluorescent primer
<400>1
gaaggtcgga gtcaacggat tgtggtgctg aatacttcat cagtca 46
<210>2
<211>44
<212>DNA
<213> KASP-labeled FAM fluorescent primer
<400>2
gaaggtgacc aagttcatgc tggtgctgaa tacttcatca gtcg 44
<210>3
<211>29
<212>DNA
<213> KASP-tagged Universal primer
<400>3
agaacctcaa gtcaatgaat taccgtcta 29
<210>4
<211>24
<212>DNA
<213> InDel-labeled upstream primer
<400>4
gtgctggtga tggtgctaat gatg 24
<210>5
<211>24
<212>DNA
<213> InDel-labeled downstream primer
<400>5
caacaccagc actactgaga gtac 24

Claims (4)

1. A method for breeding a cabbage crop with high 4-methyl sulfur oxygen butyl thioglycoside comprises the following steps:
step A, transferring a gene with lost function in donor material R-O-18 into acceptor material L58 to be bred to obtain a polymerized single plant;
b, performing systematic breeding on the polymerized single plant to obtain a cabbage crop with high content of 4-methyl sulfur oxygen butyl thioglycoside;
wherein the loss-of-function genes are BrAOP2.2 and BrAOP2.3;
wherein, a gene with lost function in a donor material R-O-18 is introduced into a receptor material L58 to be bred through polymerization hybridization, and a polymerization single plant is screened through a molecular marker-assisted selection method;
the specific mode for screening the polymerized single plant by using the molecular marker-assisted selection method is as follows:
firstly, designing KASP and InDel molecular markers respectively according to sequence differences between genes of BrAOP2.2 and BrAOP2.3 which lose functions and normal genes, wherein the markers are obviously different between plants of the genes which lose functions and plants of the normal genes;
then extracting DNA of the plant to be screened, carrying out PCR amplification by using the designed molecular marker, and detecting a PCR amplification product; if the marker in the PCR amplification product is in accordance with the marker of the gene with lost function, the fact that the plant to be screened contains the gene with lost function is indicated, and the plant containing the corresponding gene with lost function is selected to obtain a polymerized single plant;
wherein the KASP labeled primers are respectively as follows:
hex fluorescent primer: 5'-GAAGGTCGGAGTCAACGGATTGTGGTGCTGAATACTTCATCAGTCA-3', respectively;
FAM fluorescent primer: 5'-GAAGGTGACCAAGTTCATGCTGGTGCTGAATACTTCATCAGTCG-3', respectively;
the general primer is as follows: 5'-AGAACCTCAAGTCAATGAATTACCGTCTA-3');
the primers for the InDel labeling were:
an upstream primer F: 5'-GTGCTGGTGATGGTGCTAATGATG-3', respectively;
a downstream primer R: 5'-CAACACCAGCACTACTGAGAGTAC-3' are provided.
2. The selective breeding method of claim 1, wherein the high-content 4-methylsulfanylbutylthionin cabbage plant contains a loss-of-function protein, and the loss-of-function protein is BrAOP2.2 and BrAOP2.3.
3. The breeding method according to claim 1 or 2, wherein the generation number of the systematic breeding is 2-4.
4. The breeding method according to claim 1 or 2, wherein the content of 4-methylsulfuroxybutylthioside in the high-content 4-methylsulfuroxybutylthioside cabbage crop is 5-10 times higher than the content of 4-methylsulfuroxybutylthioside in a normal cabbage crop.
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