CN116179742A - KASP (KASP-labeled primer combination for detecting dwarf trait of watermelons and application of KASP-labeled primer combination - Google Patents

KASP (KASP-labeled primer combination for detecting dwarf trait of watermelons and application of KASP-labeled primer combination Download PDF

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CN116179742A
CN116179742A CN202211357421.0A CN202211357421A CN116179742A CN 116179742 A CN116179742 A CN 116179742A CN 202211357421 A CN202211357421 A CN 202211357421A CN 116179742 A CN116179742 A CN 116179742A
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金炳奎
刘金宝
王海艳
王昌盛
由守昌
杨猛
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Younaier Biotechnology Co ltd
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Abstract

The invention belongs to the field of molecular biology, and provides a KASP (KASP-labeled primer combination for detecting dwarf traits of watermelons and application thereof. The KASP-labeled primer combination for detecting the dwarf trait of the watermelon comprises the following three primers: an upstream primer 1, an upstream primer 2 and a downstream primer. The invention also provides application of the KASP mark primer combination for detecting the dwarf trait of the watermelon, and the primer combination provided by the invention is used for detecting the dwarf genotype of the watermelon, so that the operation flow is simple, the human error is reduced, and the analysis flux is high; the method is applied to the transformation of the watermelon dwarf gene, can greatly save time and labor cost, improve the breeding efficiency of molecular marker assisted selection and accelerate the breeding of the watermelon dwarf trait.

Description

KASP (KASP-labeled primer combination for detecting dwarf trait of watermelons and application of KASP-labeled primer combination
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a KASP (Kompetitive Allele-Specific PCR, i.e. competitive allele-Specific PCR) marking primer combination for detecting the dwarf trait of watermelons and application thereof.
Background
Watermelon (citrullus lanatus (thunder.) Matsum. Et Nakai) is an annual vining herb of the genus Citrullus of the family Cucurbitaceae. Dwarf traits are a very important property in watermelon planting, as in many crops, a high decrease is associated with lodging resistance, increased economic pool size and stable yield increase. Many genes controlling plant height have been identified and located in cucurbitaceae plants. The molecular marker has very important effect in crop breeding, can identify the plant characteristics in the seedling period, and can finely select parents with the required characteristics, thereby achieving the purpose of accurate breeding. Currently, the commonly used molecular marking methods include CAPS, dCAPS, SSR, RFLP, and the technologies all need enzyme digestion or electrophoresis verification, which has the disadvantages of long time, high cost and complex process, so that the application of the markers in molecular breeding is greatly limited.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a KASP labeled primer combination for detecting the dwarf trait of watermelons and application thereof, and achieves the following purposes: the dwarf trait of the watermelon is detected through the KASP marked primer combination, so that the detection time is greatly shortened, and the aim of accurate breeding is fulfilled.
In order to achieve the above object, the following technical scheme is adopted:
the KASP marking primer combination is used for detecting the dwarfing property of the watermelon, the dwarfing gene of the watermelon is Dsurf gene, and the KASP marking core primer combination comprises the following three primers:
core sequence of the upstream primer 1:
5'-tatgcttatactctcactggactta-3'；
core sequence of the upstream primer 2:
5'-tatgcttatactctcactggacttc-3'；
sequence of the downstream primer:
5'-gcaattatgccagcattggcataggattc-3'。
the KASP mark primer combination for detecting the Dwarf of the watermelon is Dwarf gene, and comprises the following three primers:
sequence of the upstream primer 1: 5'-gaaggtgaccaagttcatgcttatgcttatactctcactggactta-3';
sequence of the upstream primer 2: 5'-gaaggtcggagtcaacggatttatgcttatactctcactggacttc-3';
sequence of the downstream primer: 5'-gcaattatgccagcattggcataggattc-3'.
Use of a KASP marker primer combination for detecting a dwarf trait of a watermelon, the use: PCR detection of the dwarf trait of watermelons is carried out by adopting a KASP marked primer combination, and the method comprises the following steps:
(1) Extracting genome DNA of a watermelon variety to be detected;
(2) PCR amplification of watermelon genomic DNA using KASP-labeled primer combinations;
(3) According to the difference of PCR fluorescent signals, the genotype of each watermelon to be detected is identified by utilizing software, namely the watermelon variety belonging to homozygous AA, CC and heterozygous AC genotypes is identified.
The PCR amplification: the PCR reaction system adopted is as follows: 20-100 ng/. Mu.L of watermelon genomic DNA 2.5. Mu.L, KASP Master Mix 2.5. Mu.L, primer Mix 0.075. Mu.L, and 5.075. Mu.L in total.
The Primer mix: the primers upstream primer 1, upstream primer 2 and downstream primer were each subjected to ddH 2 After diluting O to 50. Mu.M, the upstream primer 1, the upstream primer 2 and the downstream primer were mixed in a molar concentration ratio of 1:1:3.
The PCR amplification: the reaction procedure adopted is: pre-denaturation at 95℃for 10min; 15s at 95 ℃ and 45s at 61 ℃ for 10 cycles, each cycle being reduced by 0.6 ℃; 15s at 95℃and 1min at 55℃for 34 cycles.
Use of a KASP marker primer combination for detecting a dwarf trait of a watermelon, the use: cultivating a new plant with the dwarf trait of the watermelon; the method comprises the following steps:
step 1, preparing F1 generation hybrid combination by using a receptor parent P1 and a donor parent P2; then backcrossing is carried out by taking the receptor parent P1 as a recurrent parent to obtain a BC1F1 generation backcrossing segregation population;
step 2, carrying out genotype detection on the BC1F1 generation backcross segregating colony by adopting a method for carrying out PCR detection on the dwarf trait of the watermelon by adopting a KASP (KASP-labeled primer) combination, and selecting a single plant with the genotype of AC to continuously backcross to obtain a BC2F1 generation backcross segregating colony;
step 3, carrying out genotype detection on the BC2F1 generation backcross segregating colony by adopting a method for carrying out PCR detection on the dwarf trait of the watermelon by adopting a KASP (sequence-specific amplified fragment) marked primer combination, selecting a single plant with the genotype of AC, and continuously backcrossing for n generations to obtain a BCnF1 generation backcross segregating colony; and n is an integer of 3-8, preferably 4.
And 4, carrying out genotype detection on the BCnF1 generation backcross segregating population by adopting a method for PCR detection of the dwarf trait of the watermelon by adopting KASP (KASP-related sequence) labeled primer combination, and selecting a single plant with the genotype of AC for selfing to obtain a homozygous dwarf strain of the parent P1 variety.
The beneficial effects of the invention are as follows:
1. the KASP mark primer combination for detecting the dwarf of the watermelon is used for detecting the dwarf character of the watermelon, has simple operation flow, short time, reduced human error and high analysis flux, and is very suitable for simultaneous detection of a large number of samples. The method can detect the difference of 1bp, is very accurate and simple, can finish a large amount of detection in a short time, and has higher advantages. Five and six hours are required to perform screening by trait using the conventional molecular detection method, and one to two months are required by the method of the present invention.
2. The KASP mark primer combination for detecting the dwarf trait of the watermelon is applied to transformation of a new dwarf variety of the watermelon, so that the time and labor cost can be greatly saved, the breeding efficiency of molecular mark assisted selection is improved, and the breeding process of the watermelon is accelerated.
Drawings
FIG. 1 is an allelic discrimination diagram in example 2; in the figure, blue dots represent that the locus is homozygous genotype AA; red dots represent that the locus is homozygous genotype CC; the green dots represent that the locus is heterozygous genotype AC;
the detection result in experiment 1 of fig. 2 is the DNA sequencing result of the watermelon sample represented by blue dots;
the detection result in experiment 1 of fig. 3 is the DNA sequencing result of the watermelon sample represented by red dots;
the detection result in experiment 1 of FIG. 4 is the result of DNA sequencing of the watermelon sample represented by the green dots.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
EXAMPLE 1 KASP-labeled primer combinations for detection of dwarf Properties of watermelon
Designing a molecular marker DWARF related to the DWARF trait of the watermelon, and specifically adopting the following method to obtain the watermelon:
1) Comparing genome sequences in Dwarf watermelon plants and non-Dwarf watermelon plants, identifying that Dwarf genes of the Dwarf watermelon plants and the non-Dwarf watermelon plants have 1bp mutation at genome level, and finding that the 1bp mutation occurs in an exon region through sequence analysis, wherein the 1bp mutation can cause amino acid change, thereby causing character difference.
2) Based on the variation of 1bp of Dsurf gene in non-Dwarf watermelons, designing primer combinations (3) in the area nearby the variation; the specific sequences of the primer combinations are as follows:
FAM-labeled upstream primer 1:
5'-gaaggtgaccaagttcatgcttatgcttatactctcactggactta-3'；
VIC-tagged upstream primer 2:
5'-gaaggtcggagtcaacggatttatgcttatactctcactggacttc-3'；
common sequence downstream primer:
5'-gcaattatgccagcattggcataggattc-3'；
the primers were synthesized by the company Highway, inc. (Shanghai) of biological engineering.
Namely, the KASP labeled primer combination for detecting the dwarf trait of the watermelon is a combination of an upstream primer 1, an upstream primer 2 and a downstream primer.
3) The CTAB method is adopted to extract genomic DNA of parent seedlings and filial generation seedlings, and the specific steps are as follows:
(1) 0.5g of tender leaves are taken in a 2mL centrifuge tube, 600 mu L of CTAB extraction buffer solution is added into each tube, 2 steel balls are placed into each tube, the tubes are crushed by a tissue lyser-192 crusher, and the frequency is set to be 60.00HZ and the time is set to be 240s.
(2) After crushing, placing in a water bath at 65 ℃ for 60min, and reversing and uniformly mixing every 15 min.
(3) After water bath, 200. Mu.L of 3M ammonium acetate is added, the mixture is placed on ice for cooling for 10min, 600mL of chloroform and isoamyl alcohol mixed solution (the volume ratio of the chloroform to the isoamyl alcohol is 24:1) is added, and the mixture is fully and uniformly mixed, and the mixture is centrifuged at 12000rpm for 10min.
(4) 600. Mu.L of the supernatant was added to 600. Mu.L of pre-chilled isopropanol (in a 96-well deep well plate), gently mixed, and left at-20℃for 30min.
(5) Centrifuging at 12000rpm for 10min, discarding supernatant, washing the precipitate with 70% ethanol for 1 time, and air drying at room temperature until no ethanol smell is present.
(6) 200 mu LddH was added 2 O65 ℃ water bath for 20min. The ddH 2 O: contains 10mg/mL of RNase 1. Mu.L.
(7) 1 μl of the sample was taken in
Figure SMS_1
The concentration of the sample was measured on N50 and adjusted to be uniform.
Extracting genome DNA of non-dwarf plants and dwarf plants respectively by adopting the method; PCR amplified Dwarf gene sequences of non-Dwarf plants and Dwarf plants, and the PCR amplified products were submitted to sequencing by Shanghai, inc.
The PCR amplification method comprises the following steps: and (3) taking the extracted genome DNA as a template, and carrying out PCR amplification by adopting a primer F and a primer R to obtain PCR amplification products respectively.
The primer F sequence is as follows: 5'-tcaagacccattagatgggaaa-3';
the sequence of the primer R is as follows: 5'-gaaaatagtaatcagaacaattcacaa-3';
the primers were synthesized by the company Shanghai, inc.
The PCR reaction system adopted is as follows: 100 ng/. Mu.L -1 2. Mu.L of watermelon genomic DNA, 10. Mu.M of primer F, 10. Mu.M of primer R, 2. Mu.L of 2 XTaqMastermix, 25. Mu.L of ddH2O 19. Mu.L, and a total volume of 50. Mu.L.
The PCR amplification reaction comprises the following steps: 94 ℃ for 5min;94 ℃ for 30s,56 ℃ for 30s and 72 ℃ for 30s, and 35 cycles are total; and at 72℃for 5min.
4) The sequencing shows that the 1bp variation of the Dsurf gene is completely matched with the dwarfing property of the watermelon, so that the designed KASP mark primer combination can be used for identifying the dwarfing property of the watermelon.
EXAMPLE 2 method for PCR detection of dwarf Properties of watermelon Using KASP-labeled primer combination
1. Extraction of genomic DNA
Genomic DNA of watermelon material was extracted according to the CTAB procedure of example 1.
2. PCR amplification
PCR amplification was performed using the genomic DNA extracted in the step one as a template and the KASP-labeled primer combination (upstream primer 1, upstream primer 2, downstream primer) for detecting the dwarf trait of watermelon of example 1 to obtain a PCR amplification product.
The PCR reaction system is as follows: 50 ng/. Mu.L of watermelon genomic DNA 2.5. Mu.L, KASP Master Mix 2.5. Mu.L, primer Mix 0.075. Mu.L, and 5.075. Mu.L in total;
the KASP Master mix is manufactured by Guangzhou solid Biotechnology Co., ltd, catalog number GBS-1016-002.
The Primer mix: the primers upstream primer 1, upstream primer 2 and downstream primer were each subjected to ddH 2 After diluting O to 50. Mu.M, the upstream primer 1, the upstream primer 2 and the downstream primer were mixed in a molar concentration ratio of 1:1:3.
The PCR amplification reaction comprises the following steps: pre-denaturation at 95℃for 10min; 15s at 95 ℃ and 45s at 61 ℃ for 10 cycles, each cycle being reduced by 0.6 ℃; 15s at 95℃and 1min at 55℃for 34 cycles.
Meanwhile, sterile water without template DNA is used as a blank control in the reaction system, and 4 blank controls are arranged on each PCR plate.
3. Fluorescent scanning of PCR amplified products
PCR reactions were performed on Quantum studio 6Flex instrument using Qantstudio TM The Real-Time PCR Software software checks the parting condition, so that parting results are directly obtained, and the software automatically divides the detection sample into homozygous AA, CC genotypes and heterozygous AC genotypes according to different genotypes, so as to obtain an allele discrimination diagram.
When the software analysis occurs with points distributed in the upper left and lower right corners, the corresponding material is homozygous, wherein the blue dots represent that the site is homozygous genotype "AA", and the red dots represent that the site is homozygous genotype "CC"; when present in the centered (green) dot, this locus is indicated to be heterozygous genotype "AC"; black x marks represent NTC, i.e. water control. When no heterozygotes appear, dots do not appear in the middle position, the blue dots at the upper left corner become green dots, but the display materials are homozygotes. Since the 2 upstream primers (upstream primer 1 and upstream primer 2) are respectively provided with different fluorescent linkers, FAM and VIC respectively. If the detected material is homozygous genotype, a corresponding primer is amplified during amplification, and whether the detected material is AA or CC is distinguished according to the difference of fluorescence; if the detected material is heterozygous, 2 primers can be amplified during amplification, and the generated fluorescence is different from that of the material with homozygous genotype, so that the aim of distinguishing heterozygous genotypes is fulfilled.
Test 1, detecting 154 watermelon samples of different plants according to the method of example 2, and obtaining an allele discrimination chart through detection, wherein the allele discrimination chart is shown in figure 1; statistics of all samples the KASP test results are shown in table 1:
TABLE 1 statistical table of results of DWARF detection of 154 watermelon samples
Figure SMS_2
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Figure SMS_3
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Figure SMS_4
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Figure SMS_5
In Table 1, the watermelon plants with the results of AA and AC are non-dwarf varieties in morphology, and the watermelon plants with the results of CC are dwarf varieties in morphology, so that the molecular marker provided by the invention can be proved to have high accuracy in selecting the dwarf varieties of watermelons.
Test 2 sequencing by selecting materials from different spots
Selecting samples with red, blue and green detection results in the experiment 1, amplifying DNA fragments, and then submitting the samples to sequencing by a biological engineering (Shanghai) stock company (Qingdao), wherein the sequencing samples are subjected to DNA map analysis, and the results are shown in the accompanying figures 2-4;
FIG. 2. The detection result in experiment 1 is the result of DNA sequencing of the watermelon sample represented by blue dots;
FIG. 3. The detection result in experiment 1 is the result of DNA sequencing of the watermelon sample represented by red dots;
FIG. 4 shows the result of the test in experiment 1, which is the result of DNA sequencing of the watermelon sample represented by the green dots.
The sequencing results of fig. 2-4 show that the sample DNA represented by the blue dot and the red dot are homozygous, the sample DNA represented by the green dot is heterozygous, the watermelon represented by the blue dot is a non-dwarf plant, the watermelon represented by the red dot is a dwarf plant, the watermelon represented by the green dot is a non-dwarf plant, and the sequencing result is consistent with the phenotype result. Therefore, the molecular marker can be used for assisting in selecting the dwarf variety of the watermelon, and has high accuracy.
Example 3A method of growing a New plant with watermelon dwarf Property
The method comprises the following steps:
step 1, preparing F1 generation hybrid combination by using a receptor parent P1 and a donor parent P2; and backcrossing by taking the receptor parent P1 as a recurrent parent to obtain a BC1F1 generation backcrossing segregation population.
The parent P1: a plant of a watermelon variety Arka Manik with excellent quality and a non-dwarfing character;
the parent P2: the watermelon variety Bush Sugar Baby with general quality has dwarf characters.
And 2, carrying out genotype detection on the BC1F1 generation backcross segregating colony by adopting the KASP marking primer combination of the embodiment 1 and carrying out PCR detection on the dwarf trait of the watermelon by adopting the KASP marking primer combination of the embodiment 2, and selecting a single plant with the genotype of AC to continuously backcross to obtain the BC2F1 generation backcross segregating colony.
Step 3, performing genotype detection on the BC2F1 generation backcross segregation population by adopting the KASP marking primer combination of the embodiment 1 and performing PCR detection on the dwarf trait of the watermelon by adopting the KASP marking primer combination of the embodiment 2, selecting a single plant with the genotype of AC, and continuously backcross n generations to obtain a BCnF1 generation backcross segregation population; and n is 4.
And 4, carrying out genotype detection on the BCnF1 generation backcross segregating population by continuously adopting the KASP marking primer combination of the embodiment 1, carrying out PCR detection on the dwarf trait of the watermelon by adopting the KASP marking primer combination of the embodiment 2, and selecting a single plant with the genotype of AC for selfing to obtain the homozygous dwarf strain of the Arka Manik variety.
Except for special descriptions, the percentages in the invention are mass percentages, and the proportions are mass ratios.
Finally, it should be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are also conceivable. All modifications and variations therein may occur to those skilled in the art from the present disclosure and are intended to be included herein within the scope of this invention.

Claims (9)

1. A KASP marker primer combination for detecting the dwarf trait of watermelons, characterized in that: the control gene of the Dwarf trait of the watermelon is Dsurf gene; the KASP labeled primer combination comprises the following three primers:
core sequence of the upstream primer 1:
5'-tatgcttatactctcactggactta-3'；
core sequence of the upstream primer 2:
5'-tatgcttatactctcactggacttc-3'；
sequence of the downstream primer:
5'-gcaattatgccagcattggcataggattc-3'。
2. the KASP marker primer combination for detecting the dwarf trait of watermelon according to claim 1, wherein:
the KASP labeled primer combination comprises the following three primers:
sequence of the upstream primer 1:
5'-gaaggtgaccaagttcatgcttatgcttatactctcactggactta-3'；
sequence of the upstream primer 2:
5'-gaaggtcggagtcaacggatttatgcttatactctcactggacttc-3'；
sequence of the downstream primer:
5'-gcaattatgccagcattggcataggattc-3'。
3. the application of a KASP (kaSP) marked primer combination for detecting the dwarf trait of watermelons is characterized in that: the application is as follows: PCR detection of the dwarf trait of watermelons is carried out by adopting a KASP marked primer combination, and the method comprises the following steps:
(1) Extracting genome DNA of a watermelon variety to be detected;
(2) PCR amplification of watermelon genomic DNA using KASP-labeled primer combinations;
(3) According to the difference of PCR fluorescent signals, the genotype of each watermelon to be detected is identified by utilizing software, namely the watermelon variety belonging to homozygous AA, CC and heterozygous AC genotypes is identified.
4. Use of a KASP marker primer combination for detecting the dwarf trait of watermelon according to claim 3, wherein:
the PCR amplification: the PCR reaction system adopted is as follows: 20-100 ng/. Mu.L of watermelon genomic DNA 2.5. Mu.L, KASP Master Mix 2.5. Mu.L, primer Mix 0.075. Mu.L, and 5.075. Mu.L in total.
5. Use of a KASP marker primer combination for detecting the dwarf trait of watermelon according to claim 3, wherein: the Primer mix: the upstream primer 1, the upstream primer 2 and the downstream primer are respectively subjected to ddH 2 After diluting O to 50. Mu.M, the upstream primer 1, the upstream primer 2 and the downstream primer were mixed in a molar concentration ratio of 1:1:3.
6. Use of a KASP marker primer combination for detecting the dwarf trait of watermelon according to claim 3, wherein:
the PCR amplification: the reaction procedure adopted is: pre-denaturation at 95℃for 10min; 15s at 95 ℃ and 45s at 61 ℃ for 10 cycles, each cycle being reduced by 0.6 ℃; 15s at 95℃and 1min at 55℃for 34 cycles.
7. The application of a KASP (kaSP) marked primer combination for detecting the dwarf trait of watermelons is characterized in that: the application is as follows: and (5) cultivating a new plant with the dwarf trait of the watermelon.
8. The use of a KASP marker primer combination for detecting the dwarf trait of watermelon according to claim 7, wherein: the method comprises the following steps:
step 1, preparing F1 generation hybrid combination by using a receptor parent P1 and a donor parent P2; then backcrossing is carried out by taking the receptor parent P1 as a recurrent parent to obtain a BC1F1 generation backcrossing segregation population;
step 2, carrying out genotype detection on the BC1F1 generation backcross segregating colony by adopting a method for carrying out PCR detection on the dwarf trait of the watermelon by adopting a KASP (KASP-labeled primer) combination, and selecting a single plant with the genotype of AC to continuously backcross to obtain a BC2F1 generation backcross segregating colony;
step 3, carrying out genotype detection on the BC2F1 generation backcross segregating colony by adopting a method for carrying out PCR detection on the dwarf trait of the watermelon by adopting a KASP (sequence-specific amplified fragment) marked primer combination, selecting a single plant with the genotype of AC, and continuously backcrossing for n generations to obtain a BCnF1 generation backcross segregating colony; n is an integer from 3 to 8;
and 4, carrying out genotype detection on the BCnF1 generation backcross segregating population by adopting a method for PCR detection of the dwarf trait of the watermelon by adopting KASP (KASP-related sequence) labeled primer combination, and selecting a single plant with the genotype of AC for selfing to obtain a homozygous dwarf strain of the parent P1 variety.
9. The use of a KASP marker primer combination for detecting the dwarf trait of watermelon according to claim 8, wherein: in the step 3, n is 4.
CN202211357421.0A 2022-11-01 2022-11-01 KASP (KASP-labeled primer combination for detecting dwarf trait of watermelons and application of KASP-labeled primer combination Pending CN116179742A (en)

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