CN112280883A - SNP (Single nucleotide polymorphism) site with watermelon band peel grain covering characteristic, dCAPS (dendritic cell activating protein) molecular marker, primer and application thereof - Google Patents

SNP (Single nucleotide polymorphism) site with watermelon band peel grain covering characteristic, dCAPS (dendritic cell activating protein) molecular marker, primer and application thereof Download PDF

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CN112280883A
CN112280883A CN202011140291.6A CN202011140291A CN112280883A CN 112280883 A CN112280883 A CN 112280883A CN 202011140291 A CN202011140291 A CN 202011140291A CN 112280883 A CN112280883 A CN 112280883A
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李娜
马双武
尚建立
周丹
王吉明
李楠楠
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Zhengzhou Fruit Research Institute CAAS
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Abstract

The invention relates to an SNP (single nucleotide polymorphism) site with a watermelon band peel grain covering characteristic, a dCAPS (dendritic cell activating protein) molecular marker, a primer and application thereof. The SNP locus of the watermelon strip pericarp covering stripe characteristic is 25949518 th nucleotide G → A on No. 6 chromosome of watermelon genome; the dCAPs labeled primer set comprises a forward primer 5'-TTGAATGGATTAAAAGACCT-3' and a reverse primer 5'-GTTGGTGGACCCAACGACT-3'. The SNP locus or dCAPS molecular marker and the primer thereof can be applied to identification of the watermelon peel grain covering characteristic and auxiliary screening breeding. The SNP locus and dCAPS marker identified by the invention have the characteristics of rapidness, directness, high specificity and the like, can complete the screening and predicting work of mass watermelon stripe peel grain-covering characteristic materials in a short time, and can effectively serve the breeding work of the watermelon peel grain-covering characteristic.

Description

SNP (Single nucleotide polymorphism) site with watermelon band peel grain covering characteristic, dCAPS (dendritic cell activating protein) molecular marker, primer and application thereof
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SNP (single nucleotide polymorphism) site for covering grain characteristics of watermelon band pericarp, a dCAPS molecular marker, a primer and application thereof.
Background
The characteristic of covering the grain on the peel is one of the most important appearance quality traits of the watermelon, and the purchase requirements of consumers can be influenced to a certain extent, so that the market sales volume is influenced. Genes related to the peel pattern are reviewed by the Xiaoguanghui (2012), the mottled peel gene m generates medium-dark green peel with green and white spots (Weetman., 1937), the gene p controls narrow lines on the natural color peel, and intermittent stripe genes ins (Gusmini et al, 2006) are found and illustrate the complexity of the characteristic character of the peel grain covering. With the completion of sequencing of the whole genome of watermelon (Luo et al, 2013), Gama et al (2014) found two SSR markers linked to the watermelon pericarp refined or direct trait, both located on chromosome 6. Wesamamine et al (2016) developed an In Del molecular marker that identifies the streak character of the rack and cross-hatched peel.
However, watermelon has rich phenotypic variation, common characteristics of peel grain covering include strips, reticulate patterns, racks, radial strips, black peel (see fig. 1) and the like, and effective molecular markers are lacked in the breeding process of the peel grain covering characteristics.
Disclosure of Invention
In order to solve the technical problem of lack of effective molecular markers in the breeding process of the watermelon peel grain-covering characteristic, the invention provides a primer and application for detecting an SNP (single nucleotide polymorphism) site of the watermelon strip peel grain-covering characteristic and a dCAPS (direct nucleotide polymorphism) marker.
The invention provides an SNP locus for detecting the peel and grain covering characteristics of watermelon strips, which is 25949518 th nucleotide G → A on No. 6 chromosome of watermelon genome (http:// cucurbitangencis. org/organissm/1).
In a second aspect of the present invention, there is provided a dCAPS marker for detecting the characteristic of the skin-covered streak of a watermelon strip, wherein the nucleotide sequence of the primer is as follows:
an upstream primer: 5'-TTGAATGGATTAAAAGACCT-3'
A downstream primer: 5'-GTTGGTGGACCCAACGACT-3' are provided.
Further, dCAPS molecular marked restriction enzyme of the SNP site, and the restriction enzyme used for the primer digestion reaction is HinfI.
In a third aspect of the invention, a method for identifying the characteristics of the covering lines of the watermelon band peel by a dCAPS marker is provided:
extracting the whole genome DNA of the watermelon leaves;
performing PCR amplification on watermelon genomic DNA by using the dCAPS labeled primer;
carrying out enzyme digestion on a PCR amplification product;
and (3) carrying out electrophoresis on the enzyme digestion product, and judging the phenotype of the watermelon peel grain covering characteristic according to the band type of the enzyme digestion product, wherein the band peel grain covering characteristic of the band shows a band type of 156bp, and other peel grain covering characteristics show a band type of 137bp or two bands of 137bp and 156 bp.
A kit for identifying the peel and grain covering characteristics of watermelon strips is designed, and comprises the primers and a restriction enzyme HinfI.
The invention has the positive and beneficial technical effects that:
the SNP locus and dCAPS marker identified by the invention have the characteristics of rapidness, directness, high specificity and the like, can complete the screening and predicting work of mass watermelon stripe peel grain-covering characteristic materials in a short time, and can effectively serve the watermelon breeding related to the peel grain-covering characteristic.
Drawings
FIG. 1 is a photograph showing the comparison of the various peel grain features of watermelon.
FIG. 2 is a genome-wide association analysis map of the fruit peel grain-covering characteristics (whether or not the bands are present).
FIG. 3 is a photograph showing the amplification result of primers marked by dCAPS with the characteristic of covering the fruit peel with bands in the box as the target band in partial genomic DNA of watermelon resources.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The raw materials and reagents used in the following examples are commercially available products unless otherwise specified, and the detection methods used therein are conventional methods unless otherwise specified.
Example 1 acquisition of SNP site for watermelon band pericarp grain covering feature
The method takes 1022 watermelon germplasm resources selected by 'national watermelon and melon middle-stage library' of Zhengzhou fruit tree institute of Chinese academy of agricultural sciences as samples, utilizes an Illumina HiSeq4000 sequencer to perform simplified genome sequencing to obtain 142.19Gb data, the average coverage degree of the reference genome is 3.54%, and the average sequencing depth of the covered region is 6.3X. 121405 SNP marker sites were finally obtained by identification screening. And carrying out GWAS (global genome association analysis) on 1022 watermelon resources according to whether the peel coating line characteristics are bands or not as phenotypes, and obtaining a candidate region related to the peel coating line characteristics of the watermelon, wherein the candidate region is located between 25115921 th nucleotide and 25950735 th nucleotide of No. 6 chromosome of the watermelon genome (figure 2).
Example 2 design and identification of dCAPS marker primer for stripe peel coating line characteristic
And (2) excavating SNP and InDel sites near a peak value in a candidate region by combining deep re-sequencing results of partial resources, developing a co-dominant molecular marker for verification in the resources, and finally obtaining the dCAPS marker for detecting the characteristics of the watermelon strip cortex striation, wherein the primer sequences are as follows (see sequence tables SEQ ID NO.1 and SEQ ID NO. 2):
an upstream primer: 5'-TTGAATGGATTAAAAGACCT-3'
A downstream primer: 5'-GTTGGTGGACCCAACGACT-3' are provided.
The method for identifying the peel grain covering characteristics of watermelon strips by using the molecular marker mainly comprises the following steps:
(1) the CTAB method is used for extracting the total DNA of the leaves, and the specific steps are as follows:
Figure DEST_PATH_IMAGE001
putting 1 g of fresh leaves into a mortar, adding liquid nitrogen, grinding into powder, then transferring into a centrifuge tube added with 1 ml of CTAB extraction liquid, fully mixing the two, then placing in a constant temperature water bath at 65 ℃ for 60 min, and reversely mixing for 2-3 times;
Figure 653179DEST_PATH_IMAGE002
taking out from the water bath, and centrifuging at 8000 rpm for 1 min;
Figure DEST_PATH_IMAGE003
taking the supernatant, placing the supernatant into another centrifuge tube, adding equal volume of chloroform: isoamyl alcohol (24: 1, V/V), and slightly inverting to fully mix;
Figure 711265DEST_PATH_IMAGE004
centrifuging at 10000 rpm for 5 min, and collecting supernatant (not collecting middle layer precipitate as much as possible);
Figure DEST_PATH_IMAGE005
adding 0.7 times volume of isopropanol (precooling for 30 min in advance), uniformly mixing, and freezing at-20 ℃ for no more than 30 min to precipitate DNA; taking out, centrifuging at 10000 rpm for 5 min, and leaving precipitate and removing supernatant;
Figure 897527DEST_PATH_IMAGE006
washing the precipitate with anhydrous ethanol for several times, pouring off the soaking solution, opening the cover of the centrifugal tube, and air drying;
Figure DEST_PATH_IMAGE007
adding 200 mul of distilled water to dissolve the DNA; the DNA concentration was measured with an ultraviolet spectrophotometer and stored in a freezer at-20 ℃ until use.
(2) PCR reaction system and procedure
The reaction system is as follows: 1. mu.l of total DNA of watermelon leaves (100 ng/. mu.l), 1. mu.l of Forward primer (10. mu. mol/L), 1. mu.l of Reverse primer (10. mu. mol/L), 12.5. mu.l of 2 XPower Taq PCR MasterMix, ddH2O 9.5 μl。
The PCR reaction program is: 94 ℃ for 5 minutes, 35 cycles of 94 ℃ for 20 seconds, 55 ℃ for 1 minute, 72 ℃ for 30 seconds, and 72 ℃ for 5 minutes.
(3) Enzyme digestion reaction and procedure
The reaction system is as follows: PCR product 5. mu.l, EcoRII restriction enzyme 0.5. mu.l, 10 XBuffer 1.5. mu.l, ddH2O 8 μl；
The reaction procedure is as follows: constant temperature treatment at 65 ℃ for 10 hours;
(4) and (3) carrying out 8% polyacrylamide gel electrophoresis, developing, dyeing and band type interpretation on the enzyme digestion product.
Figure 433682DEST_PATH_IMAGE001
Preparing a reagent:
A．5×TBE ：
tris-base 53.9 g
EDTA 3.72 g
Boric acid 27.5 g
The volume was adjusted to 1 liter with distilled water.
B. 40% polyacrylamide solution:
polyacrylamide 193.34 g
Methylene bisacrylamide 6.66 g
The volume is adjusted to 500 ml with distilled water.
C. 8% polyacrylamide gel (ready for use):
10 ml of 40% polyacrylamide solution
5 XTBE 5 ml
200 microliter of 10% Ammonium Persulfate (APS)
Tetramethylethylenediamine (TEMED) 80. mu.l
22 ml of distilled water
D. Silver staining solution:
silver nitrate 1 g
Glacial acetic acid 5 ml
50 ml of absolute ethyl alcohol
The volume was adjusted to 500 ml with deionized water.
E. Developing solution:
sodium hydroxide 15 g
Formaldehyde (37%) 2.5 ml
The volume was adjusted to 500 ml with deionized water.
Figure 451316DEST_PATH_IMAGE002
Preparing a gel plate: the glass plate was washed with distilled water, air-dried, wiped with absorbent cotton balls soaked with absolute ethanol, and air-dried. The concave plate and the flat plate are tightly overlapped and then put into a glue maker to be tightly pressed and fastened with clips at two sides (one glue maker can make two-plate gel). Preparing 8% polyacrylamide gel solution (two parts) in a washing bottle, uniformly mixing, quickly injecting into a gap between the two plates, and inserting into a comb with teeth (no air bubbles are generated below the teeth of the comb) after the gap is filled; if the liquid level is lowered, a liquid transfer device can be used for sucking the unset solution for supplement; wait for the solution to solidify sufficiently.
Figure 211462DEST_PATH_IMAGE003
Electrophoresis: taking down the support of the gel maker from the base, directly putting the support into a matched electrophoresis tank, and pouring a proper amount of 1 xTBE buffer solution into the bottom of the electrophoresis tank and the middle of two glass plates on the support. Adding 6 XDNA Loading Buffer with 0.2 times volume into the PCR product, mixing uniformly, adding 0.8 microliter into the sample application hole, and carrying out electrophoresis at 260V for 35 minutes.
Figure 416178DEST_PATH_IMAGE004
Dyeing and developing: after electrophoresis is finished, taking out the glass plate from the electrophoresis tank, prying off the concave plate, attaching the gel to the flat plate, putting the flat plate into the silver staining solution with the gel surface facing upwards, placing the flat plate on a decoloring shaking table, and shaking gently for 15 minutes to ensure that the gel can automatically fall off; after silver staining is finished, taking out the gel, and putting the gel into deionized water for washing for 10 seconds; and after the washing is finished, transferring the gel into a developing solution, slightly shaking a shaking table, taking out the gel after the strips are clear, placing the gel on a film reader, observing the position difference of the strips by naked eyes, and taking a picture for storage.
Figure 552762DEST_PATH_IMAGE005
And (3) band type interpretation: and (3) placing the developed and naturally dried glass plate on a reading table, and observing the position difference of the two parent strips by naked eyes.
Example 3 identification and validation of dCAPS markers in watermelon germplasm
The application of the dCAPS marker for the stripe peel grain covering characteristic in the identification of the stripe peel grain covering characteristic of the watermelon comprises the following application steps:
(1) 62 watermelon germplasms with known pericarp grain-covering characteristics were genotyped using the dCAPs molecular markers and method steps described in example 2.
(2) According to the enzyme digestion product electrophoresis result, if only a strip of 156bp is obtained, the material to be detected is characterized by strip peel grain covering; if only 137bp band type is obtained, the material to be detected shows the non-band peel grain covering characteristic; if two bands of 137bp and 156bp are obtained, the material to be detected shows the non-band peel grain-covering characteristic; see fig. 3.
(3) Identification results
The detection results of 62 watermelon germplasms show that 7 watermelon germplasms show a strip genotype (only one strip of 156 bp) and 55 watermelon germplasms show a non-strip genotype (only one strip of 137bp or two strips of 137bp and 156 bp). The phenotype results of germplasm show that 7 resources expressing the banding genotypes are all banding, 13 resources expressing the non-banding genotypes are expressed as racks, 40 resources expressing reticulate patterns, 1 resource expressing radial stripes and 1 resource expressing black skin (see table 1); the coincidence rate of the 62 watermelon germplasm phenotypes and the genotypes reaches 100 percent.
The above results are enough to show that the stripe-peel-grain-covering characteristic dCAPS marker is used for predicting the stripe-peel-grain-covering characteristic of the watermelon peel, so that the selection efficiency of the breeding of the stripe-peel-grain-covering characteristic of the watermelon peel can be obviously improved, the breeding process is accelerated, and the oriented genetic improvement of the stripe-peel-grain-covering characteristic of the watermelon peel is realized.
TABLE 1 identification and validation of dCAPS markers in watermelon germplasm
Figure DEST_PATH_IMAGE009
TABLE 1 identification and validation of dCAPS markers in watermelon germplasm
Figure DEST_PATH_IMAGE010
SEQUENCE LISTING
<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences
<120> SNP (Single nucleotide polymorphism) site with watermelon band peel grain covering characteristic, dCAPS (dendritic cell activating protein) molecular marker, primer and application thereof
<130> /
<160> 2
<170> PatentIn version 3.2
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
ttgaatggat taaaagacct 20
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
gttggtggac ccaacgact 19

Claims (6)

1. The SNP locus is characterized in that the 25949518 th nucleotide on No. 6 chromosome of a watermelon genome is mutated from G to A.
2. A dCAPS marker for detecting the peel streak characteristic of a watermelon band based on the SNP locus, which is characterized in that the primer sequence is as follows:
an upstream primer: 5'-TTGAATGGATTAAAAGACCT-3'
A downstream primer: 5'-GTTGGTGGACCCAACGACT-3' are provided.
3. The dCAPS molecular marker of claim 2, wherein the restriction enzyme used in conjunction with said primer is HinfI.
4. The method for identifying the peel and grain covering characteristics of the watermelon strips is characterized by comprising the following steps of:
a. DNA extraction: extracting total DNA of two true-leaf-stage leaves of the watermelon by using a CTAB method;
b. and (3) PCR amplification:
the reaction system is as follows: 100 ng/mu L total DNA of watermelon leaves 1 mu L, 1 mu L upstream primer of claim 2, 1 mu L downstream primer of claim 2, 2 XPower Taq PCR MasterMix 12.5 mu L ddH2O 9.5μl；
The reaction procedure is as follows: 94 ℃ for 5 min, 35 cycles of 94 ℃ for 20 s, 55 ℃ for 1 min, 72 ℃ for 30 s, and 72 ℃ for 5 min;
c. enzyme digestion reaction system and procedure:
the reaction system is as follows: PCR product 5. mu.l, EcoRII restriction enzyme 0.5. mu.l, 10 XBuffer 1.5. mu.l, ddH2O 8μl；
The reaction procedure is as follows: constant temperature treatment at 65 ℃ for 10 hours;
d. electrophoretic map analysis: and (3) performing polyacrylamide gel electrophoresis, development, dyeing and banding pattern interpretation on the enzyme digestion product to find a target band, determining the genotype according to the size and the position relationship of the band of the amplification product, wherein the banding pattern of the peel of the band shows 156p, and the banding pattern of 137bp or two bands of 137bp and 156bp is shown in the other peel banding characteristics.
5. The SNP locus of claim 1 or the dCAPS molecular marker of claim 2 and the application of the primers thereof in identification and auxiliary screening of watermelon peel grain covering characteristic breeding.
6. A kit for identifying the peel and grain covering characteristics of watermelon strips, which comprises the primer and the restriction enzyme HinfI in claim 2.
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Publication number Priority date Publication date Assignee Title
CN113322345A (en) * 2021-07-01 2021-08-31 河南农业大学 Molecular marker co-separated from watermelon peel grain covering gene ClGS and application

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