CN109338002B - SNP molecular marker related to watermelon peel background color and application thereof - Google Patents
SNP molecular marker related to watermelon peel background color and application thereof Download PDFInfo
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- CN109338002B CN109338002B CN201811338549.6A CN201811338549A CN109338002B CN 109338002 B CN109338002 B CN 109338002B CN 201811338549 A CN201811338549 A CN 201811338549A CN 109338002 B CN109338002 B CN 109338002B
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- 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
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- 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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- 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
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses an SNP molecular marker related to the bottom color of watermelon peel and application thereof. The SNP locus is located at 29880100 th nucleotide of No. 8 chromosome of watermelon genome, and the nucleotide is C or G. The nucleotide polymorphism marker locus obtained by the invention can be used for identifying the bottom color of watermelon peel. The SNP molecular marker is an SNP molecular marker which is developed based on candidate genes and is closely linked with the bottom color of the watermelon peel. The SNP molecular marker is used for identifying the peel background color of 105 watermelon germplasm resources, and the result shows that the accuracy of identifying the watermelon with the dark green peel background color and the green peel background color is 100%. The method does not need to identify the mature period of the watermelon fruit, can accurately and quickly identify the bottom color of the watermelon peel in the seedling period, can improve the breeding efficiency and shorten the identification time, and is suitable for genetic identification and breeding of the watermelon.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an SNP molecular marker related to the bottom color of watermelon peel and application thereof.
Technical Field
In conventional selective breeding, because it is difficult to determine the genotype of progeny, the selection is usually based on the phenotype of the plant rather than the genotype, the selection time is long, and the phenotype is susceptible to environmental factors, resulting in inaccurate selection and low efficiency. The molecular marker assisted breeding screens target characters by using a molecular marker which is tightly linked with a target shape as a tool, and screens germplasm resources by using genotypes through the molecular marker, so that the method has the advantages of accuracy, rapidness and no interference from environmental conditions, avoids blindness of character selection in the traditional breeding process, and improves breeding efficiency.
SNP molecular markers (single nucleotide polymorphisms) refer to changes in the DNA sequence at the genomic level due to mutations of individual nucleotides. The molecular marker is widely existed on a genome, has the characteristics of high throughput, simplicity, stability, high sensitivity and the like, and is a molecular marker with great development prospect in the current molecular marker-assisted breeding work. Watermelon is an important horticultural crop, China is the first major watermelon producing and consuming country in the world, however, in the breeding process of watermelon, available molecular markers are few, the traditional selective breeding is mainly used, the efficiency is low, and a new variety meeting the market demand is difficult to obtain quickly. The watermelon peel background color is an important appearance and commodity character of watermelon, researchers can research the character in the third and fourth decades, however, the most common green related characters (green and dark green) exist in the watermelon peel background color, and the research report of molecular markers closely linked with the characters is not seen yet, so that the research on the watermelon peel background color needs to be carried out, the molecular markers capable of being used for watermelon breeding are developed, and the breeding process is shortened and the breeding efficiency is improved.
Disclosure of Invention
In order to solve the problems in the breeding process of the watermelon, the invention provides an SNP molecular marker site related to the bottom color of watermelon peel. The SNP molecular marker which is developed based on candidate genes and is closely linked with the bottom color of the watermelon peel can be used for accurately and quickly identifying the bottom color of the watermelon peel in the seedling stage, has the advantages of high flux, stable amplification product and quick detection, and can improve the efficiency of selective breeding of the bottom color of the watermelon peel.
The technical scheme for solving the technical problems is as follows:
an SNP molecular marker related to the background color of watermelon peel, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 1, the 340 th base from the 5' end of the sequence is an SNP locus, and the SNP locus is 29880100 th nucleotide of No. 8 chromosome of a watermelon genome.
Further, the peel background color of the watermelon with the base G of the SNP site is dark green, and the peel background color of the watermelon with the base C of the SNP site is green.
The invention also provides application of the SNP molecular marker related to the watermelon peel background color in watermelon molecular marker-assisted selective breeding.
The invention also provides a PCR amplification primer pair for detecting the SNP molecular marker related to the bottom color of the watermelon peel, wherein an upstream primer of the primer pair is designed according to 29880100 th nucleotides and an upstream sequence of 8 th dyeing of a watermelon genome, and a downstream primer of the primer pair is designed according to 29880100 th nucleotides and a downstream sequence of 8 th chromosome of the watermelon genome.
Furthermore, the primer pair consists of two single-stranded DNAs, the upstream primer is shown as SEQ ID NO. 2, and the downstream primer is shown as SEQ ID NO. 3.
The PCR amplification primer pair of the SNP molecular marker related to the watermelon peel background color can be used for the auxiliary selection breeding of the watermelon molecular marker.
The invention also provides a kit for identifying the bottom color of the watermelon peel, which comprises a PCR amplification primer pair for detecting the SNP molecular marker related to the bottom color of the watermelon peel.
The invention also provides a method for identifying the background color of the watermelon peel, which comprises the following steps:
step 1: DNA extraction: extracting watermelon genome DNA;
step 2: and (3) PCR amplification: taking watermelon genome DNA to be detected as a template, and carrying out PCR amplification on a sample to be detected by using the primer pair;
and step 3: connection transformation: after the PCR product is cut and recovered, the PCR product is connected and transformed to competent cells, and after plating, the grown bacterial colony is cultured in an LB liquid culture medium;
and 4, step 4: genotyping: sequencing the bacterial liquid, and judging the bottom color of the peel according to the base of the SNP locus, wherein the bottom color of the watermelon peel with the base of the SNP locus G is dark green, and the bottom color of the watermelon peel with the base of the SNP locus C is green.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention constructs a watermelon high-density genetic map by utilizing a whole genome re-sequencing technology (WGR), locates the genetic region related to the peel background color (dark green/green), realizes fine location by an F2 population, obtains an SNP molecular marker closely linked with the peel background color of the watermelon based on a candidate gene, can quickly identify the peel background color of the watermelon in a plant seedling stage by the SNP molecular marker, is convenient to detect and high in accuracy, and greatly improves the breeding efficiency.
2. The PCR amplification primer pair provided by the invention according to the 29880100 th single nucleotide polymorphism of the No. 8 chromosome of the watermelon genome has strong specificity and stable amplification effect, and can be accurately used for identifying the background color (dark green/green) of the watermelon peel.
3. The method for identifying the bottom color of the watermelon peel can be used for identifying the bottom color of the watermelon peel in each period, is not limited by the development period, and is convenient and fast to detect.
4. The SNP molecular marker is used for verifying 105 watermelon germplasm resources with different peel bottom colors, the result proves that the accuracy of the SNP molecular marker in identifying the watermelon germplasm resources with the dark green and green peel bottom colors is 100%, and the identification result shows that the SNP molecular marker and the identification method provided by the invention have the advantages of high efficiency, rapidness and high accuracy, can be used for breeding work and improve the breeding efficiency.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise specified.
EXAMPLE 1 acquisition of SNP molecular marker sites
1.1 construction of high Density genetic map
A female parent (9904) of the bottom color of the dark green peel and a male parent (Handel) of the bottom color of the green peel are utilized to construct a recombinant inbred line group containing 126 single plants through continuous multi-generation inbreeding, the parents and the recombinant inbred line group are sequenced through whole genome re-sequencing, high-density SNP is developed by utilizing HighMap software to construct a watermelon high-density genetic map, and 178762 SNP molecular markers which are suitable for the recombinant inbred line and have the depth of not less than 4X are detected among the parents. Through bioinformatics analysis, the positioning interval related to the bottom color of the watermelon peel is located in a 2.07Mb region at the tail end of No. 8 chromosome of the watermelon genome.
1.2 acquisition of SNP sites
Designing CAPS markers by using single nucleic acid polymorphism in a positioning interval, carrying out genotype analysis on a segregating population F2, screening a recombinant individual plant according to the phenotype (dark green peel background color or green) of the corresponding F2 individual plant, and finally obtaining a candidate gene for controlling the background color of the watermelon peel, wherein the candidate gene is positioned between 29869645 nucleotide and 29901009 nucleotide of No. 8 chromosome of a watermelon genome. In this interval, the 29880100 site was identified as the SNP site.
Example 2 method for identifying watermelon peel background color by using SNP molecular marker
2.1 extraction of watermelon genomic DNA
Extracting the tissue DNA of the watermelon sample to be detected by adopting a conventional CTAB method, and removing RNA pollution, wherein the volume of the DNA sample is 100 mu L. Determination of OD of DNA sample by ultraviolet spectrophotometer260/280The ratio, the value should be between 1.8 and 2.0 to obtain high quality DNA sample, and the concentration is diluted to 100 ng/muL.
2.2 primer design
Primers are designed according to 400bp sequences of the watermelon genome No. 8 chromosome about 29880100 th base. The specific nucleotide sequence is shown in a sequence table SEQ ID NO. 1, and the primer sequence is shown in a table 1.
TABLE 1 primer sequences
2.3 PCR reaction System
The PCR reaction system is shown in Table 2.
TABLE 2 PCR reaction System
| Reagent | Volume (μ L) |
| 2×PCR Mix | 25 |
| Upstream primer | 2 |
| Downstream primer | 2 |
| DNA template | 2 |
| Deionized water | 19 |
| Total volume | 50 |
The PCR amplification procedure was: 94 ℃ for 1.5min, 94 ℃ for 20sec, 57 ℃ for 20sec, 72 ℃ for 50sec, 35 cycles; 5min at 72 ℃; storing at 4 ℃.
2.4 ligation transformation
The PCR amplification product was recovered from the agarose gel, and the recovered PCR product was ligated to pTOPO vector as described in Table 3 below.
TABLE 3 glue recovery product linking System
| Reagent | Add volume (μ L) |
| 10×Enhancer | 0.5 |
| ptopO | 0.5 |
| PCR recovery of products | 4.0 |
| Total volume | 5.0 |
Then the samples are mixed evenly and connected for 5 minutes at room temperature (20 ℃ -30 ℃).
And transforming the obtained connecting product into a competent cell, coating a bacterial liquid on a plate (a culture medium contains ampicillin 50-100 mu g/ml), culturing overnight at 37 ℃, culturing the grown bacterial colony in a liquid LB culture medium, sequencing the bacterial liquid to obtain a base sequence of the SNP molecular marker, and judging the background color of the peel according to the genotype of the SNP locus. The basic group of the SNP locus is G, and the peel bottom color is dark green; and if the base of the SNP site is C, the bottom color of the pericarp is green.
Example 3 identification of 105 watermelon germplasm resources by SNP molecular markers
The method takes 105 watermelon germplasm resources with different peel bottom colors in a national watermelon mid-term bank as materials, and utilizes the SNP molecular marker of the invention to identify the peel bottom colors. The specific identification method was as described in example 2 above. The identification result shows that the phenotype and genotype results of the 105 watermelon materials completely accord with each other, and the SNP molecular marker can accurately identify the dark green color and the green peel background color of the watermelon, and the accuracy rate is 100%.
31 parts of watermelon with dark green peel background color and 74 parts of watermelon with green peel background color in 105 parts of germplasm resources, wherein specific varieties and phenotypes are shown in Table 4, wherein ink represents dark green, and green represents green.
TABLE 4105 Peel ground color of watermelon germplasm resources
In conclusion, the SNP molecular marker provided by the invention can accurately and quickly identify the bottom color of the watermelon peel, can improve the breeding efficiency and shorten the identification time, and is suitable for genetic identification and breeding of the watermelon.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences
<120> SNP molecular marker related to watermelon peel background color and application thereof
<130> do not
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 667
<212> DNA
<213> watermelon (Citrullus lanatus)
<220>
<221> variation
<222> (340)..(340)
<223> n = g or c
<400> 1
aaggccataa ccagatgcga ctgggacaac gatgttgttg atcatccatt ccttgcatag 60
aggaagttca ggagaaacaa gcaaaataag taggggcttt agaggtaaac actgagtaga 120
gactctgcta aatttgaagt gtgcaccaca cataaaaaat ttaggccttc acacaaaagt 180
acctgaatag cagcagtagt cggttgtgta gtcttgttaa agtcgagaat tgatacctta 240
ctacctggaa agaccacttg aattttgtaa ctgaatgacc atgttaacaa tcaagaagga 300
ccaaaaaaaa aaaacaaaaa ccttctttta gtactctacn catctcctct aatgctctcc 360
gtttatccac cacatttcgt agaccgtagc ccattgtaat ggcatcgaaa gagccgtcag 420
gaaatggcaa attgagtgca tcaccctcca cccaccttca ctttggcagc agcgttatta 480
ggttgttaat ttcacaaatc aattaccaat tacaattctc actgaagcaa aactgtatcg 540
tgaaatacac agaggaatgc ttcaatattt tcaaaatagt agaggccaac acattaattg 600
ttcaagttta gtaatgctca ctcaatgttg tcatagcagg agttggagag ggaacgttgg 660
cgagaag 667
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaggccataa ccagatgcga 20
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cttctcgcca acgttccct 19
Claims (6)
1. An SNP molecular marker related to the background color of watermelon peel is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 1, the 340 th base from the 5' end of the sequence is an SNP locus, and the SNP locus is 29880100 th nucleotide of No. 8 chromosome of a watermelon genome;
the peel background color of the watermelon with the base G of the SNP locus is dark green, and the peel background color of the watermelon with the base C of the SNP locus is green.
2. The use of the SNP molecular markers of claim 1 for assisted selection breeding of watermelon molecular markers identifying the greenish black color and the bottom color of the watermelon peel.
3. The PCR amplification primer pair for detecting the SNP molecular marker related to the bottom color of the watermelon peel of claim 1, wherein an upstream primer of the primer pair is designed according to 29880100 th nucleotides and an upstream sequence of No. 8 staining of the watermelon genome, and a downstream primer of the primer pair is designed according to 29880100 th nucleotides and a downstream sequence of No. 8 chromosome of the watermelon genome;
the primer pair consists of two single-stranded DNAs, the upstream primer is shown as SEQ ID NO. 2, and the downstream primer is shown as SEQ ID NO. 3.
4. The application of the PCR amplification primer pair for detecting the SNP molecular marker related to the bottom color of the watermelon peel in the molecular marker-assisted selective breeding of the watermelon as claimed in claim 3, wherein the primer pair is used for identifying the dark green color and the bottom color of the green peel of the watermelon.
5. A kit for identifying the background color of watermelon peel, comprising the primer pair of claim 3.
6. A method for identifying the background color of watermelon peel is characterized by comprising the following steps:
step 1: DNA extraction: extracting watermelon genome DNA;
step 2: and (3) PCR amplification: carrying out PCR amplification on a sample to be detected by using the primer pair of claim 3 by using watermelon genome DNA to be detected as a template;
and step 3: connection transformation: after the PCR product is cut and recovered, the PCR product is connected and transformed to competent cells, and after plating, the grown bacterial colony is cultured in an LB liquid culture medium; and 4, step 4: genotyping: sequencing the bacterial liquid, and judging the bottom color of the peel according to the base of the SNP locus, wherein the bottom color of the watermelon peel with the base of the SNP locus G is dark green, and the bottom color of the watermelon peel with the base of the SNP locus C is green; the nucleotide sequence of the PCR product is shown as SEQ ID NO. 1, and the 340 th base from the 5' end of the PCR product is an SNP site.
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| CN111549172B (en) * | 2020-06-12 | 2023-02-28 | 中国农业科学院郑州果树研究所 | Watermelon leaf posterior green gene linkage site and CAPS marker |
| CN111647666B (en) * | 2020-06-28 | 2023-09-22 | 云南中烟工业有限责任公司 | Primer group, application, kit and method for detecting SNP locus related to human watermelon preference |
| CN114457182B (en) * | 2022-02-21 | 2022-08-12 | 广东省农业科学院蔬菜研究所 | SNP molecular marker related to color of towel gourd peel and application thereof |
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| CN108192990A (en) * | 2018-02-01 | 2018-06-22 | 中国农业科学院郑州果树研究所 | SNP marker relevant with watermelon pericarp background color and its application |
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2018
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| CN108192990A (en) * | 2018-02-01 | 2018-06-22 | 中国农业科学院郑州果树研究所 | SNP marker relevant with watermelon pericarp background color and its application |
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| Title |
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