CN116083617A - InDel marker and primer of bright green gene BrWAX3 of Chinese cabbage and application of InDel marker and primer - Google Patents
InDel marker and primer of bright green gene BrWAX3 of Chinese cabbage and application of InDel marker and primer Download PDFInfo
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Abstract
The invention relates to an InDel marked detection primer and application thereof in breeding of Chinese cabbages without wax powder, belonging to the field of molecular genetic breeding. The invention locates a new bright green wax-free powder gene BrWAX3 by BSA-seq (bulked segregant analysis sequencing) method. The first time discloses InDel markers A09-24305871 of the bright green wax-free powder gene BrWAX3 and a detection primer BrWAX3-KASP1 thereof, wherein the detection primer comprises three primers. Through PCR amplification of DNA of genetic population and detection by using KASP gene analysis system, it is found that BrWAX3-KASP1 can obviously distinguish waxy powder and bright green waxy powder-free material, and can be used for breeding new variety of bright green waxy powder-free white vegetable.
Description
Technical Field
The invention belongs to the technical field of crop molecular marker assisted breeding, and particularly relates to an InDel marker, a primer and application of a Chinese cabbage bright green gene BrWAX3.
Background
The wax on the surface of the plant is also called wax powder, and is a hydrophobic layer formed by lipophilic compounds and covered outside the epidermal cells of the plant. The wax powder has the main functions of reducing the water loss of the non-stomata, enhancing the resistance of plants to biotic stress and abiotic stress, and the like. In addition, the waxes also affect the growth and development of plants, and mutations in genes associated with wax loss can cause organ fusion and reduced fertility.
Cabbage crops (Brassica rapa l.) belong to Brassica (Brassica) of the Cruciferae family (cricifera), including chinese cabbage (b.rapa. Pekinensis), chinese cabbage (b.rapa. Chinensis), turnip (b.rapa. Rapa), cabbage (b.rapa. L. Chinesis var. Utilis Tsen et Lee) and the like, have a common genome (a genome), are robust to cross each other without reproductive isolation, and excellent genes identified in chinese cabbage can be transferred to the cabbage and chinese cabbage by hybridization and backcrossing. The fresh and tender stems and leaves of cabbage crops are used as edible organs, and the waxy characters of the leaves and the stem skins are important commodity characters. The leaves and the stem epidermis of the waxy-deficient vegetables are covered with little wax, have bright green color, luster, good commodity, good edible quality, fresh and tender sense and are popular with consumers. However, the waxy character of the cabbage vegetables can be shown only in the bolting and flowering period, and the time and the labor are wasted in the process of breeding the bright green wax-free powder material, so that the breeding process is greatly delayed. Therefore, in order to accelerate the breeding speed of bright green wax-free powder varieties, molecular marker assisted breeding is necessary by utilizing the modern molecular biology technology.
Competitive allele-specific PCR (Kompetitive allele specific PCR, KASP) genotypes SNP or InDel based on specific matching of primer terminal bases, and comprises 2 forward primers and 1 reverse primer, wherein the 5' -ends of the two forward primers respectively carry fluorescent linker sequences FAM or HEX with different sequences, so that the specificity of SNP or InDel can be detected according to different fluorescent signals. Has the advantages of high accuracy, low cost, high flux and the like, and is one of the mainstream methods for genotyping SNP and InDel at present internationally. Currently, the KASP-SNP typing technology is widely applied to the aspects of genetic map construction, gene localization, germplasm resource analysis, molecular marker assisted breeding, seed purity identification and the like of plants (Allen et al, 2011; wu et al, 2018; shi et al, 2015).
Disclosure of Invention
The invention digs a new bright green wax-free powder gene in the Chinese cabbage, develops a closely linked KASP mark aiming at the gene, and lays a foundation for carrying out molecular auxiliary breeding of bright green wax-free powder varieties by using the mark and accelerating the breeding process.
The invention provides an InDel marker of a Chinese cabbage bright green wax-free powder gene BrWAX3, which is shown as SEQ ID NO: 1.
The invention provides a KaSP detection primer of InDel mark of a Chinese cabbage bright green wax-free powder gene BrWAX3, which comprises the following components: specific primers homozygous for the wax powder trait such as seq id NO:2 is shown in the figure; homozygous bright green wax-free pink specific primers such as seq id NO:3 is shown in the figure; reverse primer such as seq id NO: 4.
The application of the primer in cabbage vegetable breeding.
The breeding refers to the breeding of the homozygous bright green non-wax powder shape
The application comprises the use of the detection primer for preparing a detection kit.
The bred homozygous bright green wax-powder-free character is used as a parent for breeding.
A breeding method of a Chinese cabbage bright green wax-free powder variety comprises the following steps:
(1) Extracting genome of the Chinese cabbage to be detected to obtain genome solution;
(2) Performing KASP-PCR amplification on the genome solution obtained in the previous step;
(3) KASP-PCR amplified products were read for endpoint fluorescence signal using a Roche fluorescent quantitative PCR Instrument, lightCycler 480Instrument II (LC 480 II). SNP typing results were analyzed using LC480 software v 1.5.1: the signal point of the homozygous wax powder material is blue, the 5' end is connected with the primer competitive amplification of FAM fluorescent label sequence, and the primer is polymerized near the X axis; the signal point of the homozygous bright green wax-free powder material is green, and the primer with the HEX fluorescent tag sequence connected with the 5' end is amplified in a competitive manner and polymerized near the Y axis; the signal points of the hybrid wax powder material are red, and are polymerized near the diagonal.
The invention discloses a novel bright green wax-free powder gene BrWAX3 of cabbage vegetables, invents InDel markers A09-24305871 of the bright green wax-free powder gene BrWAX3, and discloses a detection primer BrWAX3-KASP1 for detecting the markers. The InDel marker and the detection primer thereof can accurately and efficiently detect the genotypes of the bright green and the waxy powder. The screening method is not influenced by environmental factors, can greatly reduce the field selection workload, and is helpful for helping to accelerate the breeding of Chinese cabbages.
Drawings
FIG. 1 shows the BSA-Seq localization results of the light green wax-free gene BrWAX 3; and (3) injection: the drawing window is 1Mb, the step length is 50kb, the yellow line is 95% confidence interval, and the blue line is 99% confidence interval. The localization interval is a candidate region (19.65-26.15 Mb) on the A09 chromosome;
FIG. 2 cloning and sequence differences of BrWAX 3;
FIG. 3 KASP genotyping the BC1P1 population using the marker BrWAX3-KASP 1;
FIG. 4 KASP genotyping of (SD 369 XSD 2135) -F2 population using the marker BrWAX3-KASP1.
Detailed Description
1. Investigation of test materials and disease resistance
Selecting green and bright Chinese cabbage wax-free powder DH series material SD369 (P) 1 )(Yang et al，Map-Based Cloning and Characterization of Br-dyp1,a Gene Conferring Dark Yellow Petal Color Trait inChinese cup (Brassica rapa L. Ssp. Pekinensis), frontiers in Plant Science, 13:84328) and waxy powder material R16-11 (P) 2 ) (Yang Shuangjuan et al 2020, kasp marker development of the cabbage bolting-related gene BrFLC1, nuclear agronomy report, 34 (2): 0265-0272) as a parent, and hybridization of two materials to obtain F 1 Seed generation, F 1 Selfing the material to obtain F 2 Seed generation, F 1 Respectively with SD369 (P) 1 ) And R16-11 (P) 2 ) Hybridization to obtain BC 1 P 1 And BC (binary code) 1 P 2 Seed. All materials are planted in an original male experiment field of a gardening institute of Henan agricultural academy of sciences, field unified management is carried out, and whether wax powder exists on each plant flower stem and each plant leaf is investigated after all the materials are bolting.
Chinese cabbage wax powder sex-state genetic law: by P pair 1 、P 2 、F 1 、F 2 、BC 1 P 1 And BC (binary code) 1 P 2 Six generations of wax powder shape investigation show that 10 strains F 1 The leaves, stems and buds of the plants show wax powder, F 2 102 plants of wax powder and 40 plants of wax powder-free plants in the population, and the separation ratio of the wax powder to the BC is 3:1 through a square test 1 P 1 The colony has 540 plants of wax powder and 494 plants of wax powder-free plants, and the separation ratio of the wax powder to the water based on the square test of the card is 1:1, and BC 1 P 2 All plants in the population had wax powder. In summary, genetic analysis shows that the Chinese cabbage has wax powder and no wax powder, the property is controlled by 1 pair of nuclear genes, and the wax powder is dominant to the no wax powder.
TABLE 1 Chinese cabbage parent and its progeny wax powder and wax powder-free plant segregation
2. Genomic DNA extraction
Extraction of two by improved CTAB methodParent, F 1 And F 2 Genomic DNA of each individual strain. The specific steps are as follows.
Fresh leaves were taken in 2.0mL Eppendorf centrifuge tubes, 1 shot of steel balls (5 MM diameter) was placed into each tube, after which 1000. Mu.L of 2% CTAB extraction buffer was added and the cells were broken on a tissue breaker (model: retsch MM400, germany) at a frequency of 30 times/sec for 1min. After cooling, adding 500 mu L of 24:1 chloroform/isoamyl alcohol extract, shaking up and down for 30 times, standing for delamination, centrifuging (12000 r/min) for 10min, sucking 400 mu L of supernatant into a new 1.5mL centrifuge tube, adding 400 mu L of isopropanol for precipitating DNA, centrifuging (12000 r/min) for 5min after up and down mixing, discarding supernatant, adding 70% ethanol for 750 mu L for cleaning DNA precipitation, centrifuging (12000 r/min) for 2min, discarding supernatant, airing DNA at room temperature, adding 100 mu L of ddH2O for dissolving DNA, and placing in a refrigerator at-20 ℃ for standby.
3. Localization of new gene BrWAX3 of Chinese cabbage bright green wax-free powder
With F as above 2 The separated groups are test materials, wax powder and bright green wax-free powder F are selected 2 And constructing wax powder and brilliant green two gene mixed pools of 50 plants each, and carrying out whole genome re-sequencing on the two mixed pools by using a BSA-Seq technology. The genome sequence (V1.5) of Chinese cabbage Chiifu-401-42 is used as a reference sequence, the parents and clear reads of two mixed pools are respectively compared to the reference genome by BWA (v0.7.17) software, and SNP cloning is carried out by using Samtools (V1.9) and Bcftools (V1.9) to obtain SNP loci of each sample. And respectively calculating SNP-index of the wax powder mixing pool and the bright green mixing pool, subtracting the SNP-index of the wax powder pool from the SNP-index of the bright green pool to obtain delta SNP-index between the two mixing pools, and carrying out sliding window analysis by taking 1Mb as a window and 50kb as a step length to determine a candidate interval of the bright green wax powder-free gene. By significance analysis, the bright green waxy powder-free gene was located within the interval of 19.65-26.15Mb of the A09 chromosome (FIG. 1), with an interval length of 6.5Mb. The localization interval of this gene was reported by the former to be BrWax1 (A01 chromosome, zhang et al 2013.Fine mapping of BrWax1,a gene controlling cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. Ssp. Pekinensis)) and BrWAX2 (A09 chromosome 37.35-38.88Mb,Yang et al, 2022.BrWAX2 plays an essential role in cuticular wax biosynthesis i)n Chinese cabbage (Brassica rapa L.ssp. Pekinensis)) is not identical in location interval, and is a bright green wax-free gene, and is temporarily designated as BrWAX3.
4. KASP molecular marker of new gene BrWAX3 of Chinese cabbage bright green wax-free powder
The candidate gene of BrWAX3 was determined by the gene localization method, and the candidate gene was sequenced and sequence aligned, and it was found that there was a 5567-bp insertion at position 24305871 of the A09 chromosome, as shown in FIG. 2, at position 590bp, a 5567-bp large fragment insertion in the light green waxy powder material SD369, and not in the waxy powder material R16-11, and this variation was referred to as InDel marker A09-24305871. Artificial sequence (Artificial Sequence)
The KASP detection primer BrWAX3-KASP1 is designed for the InDel marker A09-24305871 and comprises three primers BrWAX3-KASP1Fa 5'GAAGGTGACCAAGTTCATGCTAAACCTAAAGACATCGACATACTC-3'；
BrWAX3-KASP1Fb:5'-GAAGGTCGGAGTCAACGGATTTACCCGTAAATGTTACCGTCGC-3'；
BrWAX3-KASP1R:5'-ACTTGTTGATTATCATGGCCGAGAG-3'。
BrWAX3-KASP1Fa and BrWAX3-KASP1Fb are two allele-specific forward primers, brWAX3-KASP1Fa is a specific primer of homozygous waxy powder property, brWAX3-KASP1Fb is a specific primer of homozygous bright green waxy powder property, and FAM and HEX fluorescent sequence tag sequences (underlined parts) are added to the 5' end respectively. BrWAX3-KASP1R is a common reverse primer.
Parent SD369, R16-11 and BC thereof using the KASP primer BrWAX3-KASP1 1 P 1 The offspring were tested in single plants. The KASP-PCR reaction was performed on a 96-well PCR apparatus with 8. Mu.L of the reaction system: 1.5. Mu.L of DNA (80 ng. Mu.L-1), 4. Mu. L KASP Master mix (2X), 0.14. Mu.L of primer mix (from a concentration of 100. Mu. Mol. L) -1 BrWAX3-KASP1Fa, brWAX3-KASP1Fb, brWAX3-KASP1R and ddH of (B) 2 O is mixed according to the volume ratio of 12:12:30:46, and the rest is ddH 2 And (3) supplementing O.
The KASP-PCR amplification procedure was: denaturation at 94℃for 15min in the first stage; the second stage of denaturation at 94℃for 20s and annealing at 61℃for 60s, for a total of 10 cycles (each cycle reduced by 0.6℃from the second cycle); the third stage is denaturation at 94 ℃ for 20s and annealing at 55 ℃ for 60s, 26 cycles are carried out; the fourth stage is carried out at 37 ℃ for 1min.
KASP-PCR amplified products were read for endpoint fluorescence signal using a Roche fluorescent quantitative PCR Instrument, lightCycler 480Instrument II (LC 480 II). SNP typing results were analyzed using LC480 software v 1.5.1: the signal point of the homozygous wax powder material is blue, the 5' end is connected with the primer competitive amplification of FAM fluorescent label sequence, and the primer is polymerized near the X axis; the signal point of the homozygous bright green wax-free powder material is green, and the primer with the HEX fluorescent tag sequence connected with the 5' end is amplified in a competitive manner and polymerized near the Y axis; the signal points of the hybrid wax powder material are red and aggregate near the diagonal (fig. 3). The BrWAX3-KASP1 marker can be used for distinguishing two homozygous genotypes obviously, and can be used for distinguishing heterozygous genotypes, and has the characteristic of co-dominant marker, and the marker is successfully developed.
5. Application of KASP molecular marker BrWAX3-KASP1
The extraction of leaf DNA was performed by CTAB method. Material selection F constructed from SD369 (light green wax-free powder) and SD2135 (wax powder) 2 A population. Labelling of BrWAX3-KASP1 with KASP in F containing 96 individuals 2 Verification in the population (SD 369×SD 2135-F2) showed that the genotype of BrWAX3-KASP1 and the phenotype of wax powder were completely matched (FIG. 4), the phenotype of bright green and wax-free materials were all polymerized near the Y axis, the genotype was matched with SD369, and the phenotype of wax powder individuals were polymerized either near the X axis or the diagonal. The marker BrWAX3-KASP1 has good universality and accuracy, and can be used for molecular marker assisted selection of bright green wax-free powder of Chinese cabbage vegetables.
The invention discloses a novel bright green wax-free powder gene BrWAX3 of cabbage vegetables, invents InDel markers A09-24305871 of the bright green wax-free powder gene BrWAX3, and discloses a detection primer BrWAX3-KASP1 for detecting the markers. The InDel marker and the detection primer thereof can accurately and efficiently detect the genotypes of the bright green and the waxy powder. The screening method is not influenced by environmental factors, can greatly reduce the field selection workload, and is helpful for helping to accelerate the breeding of Chinese cabbages.
Claims (7)
1. An InDel marker of a Chinese cabbage bright green wax-free powder gene BrWAX3, which is characterized in that: the InDel marker is shown in SEQ ID NO: 1.
2. A KASP detection primer marked by InDel of a Chinese cabbage bright green wax-free powder gene BrWAX3 is characterized in that the detection primer comprises: the specific primer with the homozygous wax powder property is shown as SEQ ID NO:2 is shown in the figure; the homozygous bright green wax-free powder specific primer is shown as SEQ ID NO:3 is shown in the figure; the reverse primer is shown in SEQ ID NO: 4.
3. The use of the primer as claimed in claim 2 in breeding of cabbage vegetables.
4. A use according to claim 3, wherein: the breeding refers to breeding of homozygous bright green without wax powder.
5. A use according to claim 3, characterized in that: the application comprises the use of the detection primer for preparing a detection kit.
6. The use according to claim 4, wherein the bred homozygous bright green wax-powder-free character is used as a parent for breeding.
7. A breeding method of a Chinese cabbage bright green wax-free powder variety is characterized by comprising the following steps:
(1) Extracting genome of the Chinese cabbage to be detected to obtain genome solution;
(2) Performing KASP-PCR amplification on the genome solution obtained in the previous step;
(3) KASP-PCR amplified products were read for endpoint fluorescence signal using a Roche fluorescent quantitative PCR Instrument, lightCycler 480Instrument II (LC 480 II). SNP typing results were analyzed using LC480 software v 1.5.1: the signal point of the homozygous wax powder material is blue, the 5' end is connected with the primer competitive amplification of FAM fluorescent label sequence, and the primer is polymerized near the X axis; the signal point of the homozygous bright green wax-free powder material is green, and the primer with the HEX fluorescent tag sequence connected with the 5' end is amplified in a competitive manner and polymerized near the Y axis; the signal points of the hybrid wax powder material are red, and are polymerized near the diagonal.
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