CN113528702A - KASP marker closely linked with main effect QTL of lycopene in carrot and primer and application thereof - Google Patents
KASP marker closely linked with main effect QTL of lycopene in carrot and primer and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to KASP markers closely linked with a main effect QTL of lycopene in carrots, primers and application thereof, wherein a group of KASP markers, namely DC _ Chr1.02725 and DC _ Chr3.10274 are developed, the group of markers are closely linked with 2 main effect QTL sites, namely Lyco1.1 and Lyco3.1 for controlling lycopene respectively, and when the 31078407 th base of DC _ Chr1.02725 is C and the 4222837 th base of DC _ Chr3.10274 is T, the markers are closely linked with the character of lycopene. The marker combination can be used for screening the red high-lycopene character in the carrot, can realize high-flux rapid identification of the red carrot type in the seedling stage, provides effective technical support for breeding of the red carrot rich in lycopene, and has important application value.
Description
Technical Field
The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to a KASP marker closely linked with a main effect QTL of lycopene in carrots, and primers and application thereof.
Background
Carrot is one of ten vegetable crops in the world, the cultivation area in 2019 reaches 114.7 million hectares all over the world, the planting area in China is 40.5 million hectares, and accounts for 35 percent of the world, so that the carrot is the largest carrot producing country and the major export country in the world. During the long-term cultivation and domestication process, the color of the fleshy root of the carrot is subjected to abundant variation, namely, white, yellow, orange, red and the like, and the formation of the color is mainly related to the type and the content of carotenoid in the fleshy root. The red carrot is originated from China and India, is one of the main types of carrot, and the fleshy root of the carrot mainly contains a red carotenoid, namely lycopene (which can be as high as 1000 mug/g), so that the color of the fleshy root is red, and the content of the lycopene in the fleshy roots of other colors is extremely low or no. Lycopene has strong antioxidant activity, the effect of scavenging free radicals is far better than that of other carotenoids and vitamin E, the rate constant of quenching singlet oxygen is 100 times of that of vitamin E, the lycopene is one of the strongest antioxidants found in nature so far, and the lycopene has obvious cancer prevention and anticancer effects. Therefore, the lycopene content is an important nutritional quality character of the carrot and is one of the target characters of carrot variety breeding.
At present, the breeding of red carrot varieties enriched in lycopene mainly depends on observing the color of fleshy roots in a mature period to carry out phenotype identification, the method is long in time consumption, the accuracy of phenotype identification is influenced when the fleshy roots of carrots meet high temperature in a formation period, and the problems can be better solved by utilizing molecular marker-assisted selection. KASP, competitive Allele Specific PCR, allows for precise biallelic determination of SNPs and InDels at Specific sites in a wide range of genomic DNA samples. Because SNPline has high flexibility, the method is suitable for typing research of various genes and has the advantages of low cost, high flux, safe experimental operation, accurate fluorescence signal acquisition data and the like. Therefore, the KASP marker closely linked with the main effect QTL related to the content of the lycopene in the red carrot is developed, the red carrot type enriched with the lycopene can be rapidly identified at a high flux in the seedling stage, and a novel, efficient and practical molecular marker is provided for breeding the red carrot variety enriched with the lycopene.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the KASP marker which is closely linked with the main effect QTL of the lycopene in the carrot, and the marker can be used for identifying the high-lycopene-content material of the carrot and providing technical support for breeding the red carrot variety enriched with the lycopene.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides KASP markers closely linked to a lycopene major QTL in carrot, wherein the KASP markers comprise DC _ Chr1.02725 shown in SEQ ID NO.1 and DC _ Chr3.10274 shown in SEQ ID NO.2, the SNP variation site of the DC _ Chr1.02725 is A/C, is positioned at the 100nt base of the DC _ Chr1.02725, and is positioned at the 31078407 th position of the full-length sequence of No.1 chromosome; the SNP variation site of DC _ Chr3.10274 is A/T, is positioned at the 100nt base of DC _ Chr3.10274, and is positioned at the 4222832 th position of the full-length sequence of No.3 chromosome.
According to the invention, 2 major QTL (quantitative trait loci) Lyco1.1 and Lyco3.1 for controlling lycopene in a carrot Red inbred line 'Red 1' are found on chromosome 1 and chromosome 3 respectively, a KASP marker tightly linked with the Lyco1.1 is DC _ Chr1.02725, and a KSAP marker tightly linked with the Lyco3.1 is DC _ Chr 3.10274. The DC _ Chr1.02725 marker is located on carrot chromosome 1, the sequence is shown in SEQ ID NO.1, wherein the base at the 100nt position is A or C, the DC _ Chr3.10274 marker is located on carrot chromosome 3, the sequence is shown in SEQ ID NO.2, and the base at the 100nt position is A or T. The genotype of the parent material 'Red 1' at the test sites of DC _ Chr1.02725 and DC _ Chr3.10274 is C: C/T: T, which is Red in color and high in lycopene content. The test site genotype of 'FN 29' is A: A/A: A, and is orange and free of lycopene.
Through the analysis of the heavy sequencing data of 10 parts of high-lycopene material and 20 parts of low-lycopene material by using DC _ Chr1.02725 and DC _ Chr3.10274 markers, 1 SNP site variation A/C is found at the 31078407 position of No.1 chromosome, and 1 SNP site variation A/T is found at the 4222832 position of No.3 chromosome, and the mutations at the two sites show that the two sites are closely linked with high-lycopene when the two sites are C: C and T: T at the same time.
In addition, detection of 54 parts of carrot germplasm materials (28 parts of red high lycopene material and 26 parts of non-red low lycopene carrot germplasm materials) using DC _ chr1.02725 and DC _ chr3.10274 markers with genotype of C: C/T: T and 31 parts of C: C/T: T, wherein 28 parts of red high lycopene material, DC _ chr1.02725 and DC _ chr3.10274 markers with genotype of C: C/a: a or a: a/T: T, were found to be non-red low lycopene material, indicating that this group of KASP markers identified with accuracy of 94.4%. The group of KASP marker detection can be used for screening the high-lycopene character of the carrot, can realize high-flux rapid identification of the high-lycopene type of the carrot in the seedling stage, provides effective technical support for breeding the carrot quality, and has important application value.
Wherein the sequence of DC _ Chr1.02725 (SEQ ID NO.1) is:
AAAAAACAAGATTCTAGAGCCATAAGTCTTGATAATCCAAAGGAGCAAGGACAAGTTTCATCTACCCGGGTACGTCATGCTCTTGACATGGGAGATATGA[A/C]ATATGTCTCAGAGTTATTGGGTCGAAAACATCGTCTTATGTTGATGTGGAATGCTGGGGAAAAGTTTACTAGGGAAAATAATAGGATATCAGCTTCAAAG (the underlined part indicates the SNP variation site);
the sequence of DC _ chr3.10274 (seq id No.2) is:
TTTTGGTCCACATTGTAAGAATTCAAGAAGGAGCAGCTGAAAGTGCAAATTAGCAATTTAAGAGTTCTGAAAATATCAGGGGAAAGGCCTCTGAGTGCAG[A/T]GAAACAGAGTCGATTTTACAAGGAAGTAAAGGTGGCAAAGGAGTGCAATGCAAATGACATCCGTGCAAAGTTTGTTAATGGGCTTCTCTATGTTGTAATG (the underlined part indicates the SNP variation site).
The invention also provides a molecular marker primer for amplifying the KASP marker, wherein the molecular marker primer comprises two pairs of primer combinations corresponding to DC _ Chr1.02725 and DC _ Chr3.10274 respectively, each pair of primer combination comprises a specific primer F, a specific primer H and a universal primer C, the 5 'end of the specific primer F is connected with a fluorescent label sequence of a FAM group, and the 5' end of the specific primer H is connected with a fluorescent label sequence of a HEX group.
Specifically, the fluorescent tag sequence of the FAM group is 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO.9), and the fluorescent tag sequence of the HEX group is 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO. 10).
Preferably, the molecular marker primers comprise a primer combination of DC _ Chr1.02725 markers DC _ Chr1.02725F, DC _ Chr1.02725H and DC _ Chr1.02725C and a primer combination of DC _ Chr3.10274 markers DC _ Chr3.10274F, DC _ Chr3.10274H and DC _ Chr3.10274C, the nucleotide sequence of DC _ Chr1.02725F is shown in SEQ ID NO.3, the nucleotide sequence of DC _ Chr1.02725H is shown in SEQ ID NO.4, the nucleotide sequence of DC _ Chr1.02725C is shown in SEQ ID NO.5, the nucleotide sequence of DC _ Chr3.10274F is shown in SEQ ID NO.6, the nucleotide sequence of DC _ Chr3.10274H is shown in SEQ ID NO.7, and the nucleotide sequence of DC _ Chr3.10274C is shown in SEQ ID NO. 8.
The invention also provides a detection product containing the molecular marker primer, and the detection product comprises a detection kit and a detection reagent.
The invention also provides the application of the KASP marker or the molecular marker primer or the detection product in any one of the following aspects:
(1) detecting the high lycopene character of the carrot;
(2) carrot germplasm resources are improved;
(3) cultivating carrots with high lycopene;
(4) genotyping carrot germplasm material.
Preferably, the specific method of the above application comprises the steps of:
s1, extracting the carrot genome DNA to be detected;
s2, performing PCR amplification by using the molecular marker primer of claim 2 or 3, and reading a fluorescence signal by using an ABI QS3 fluorescence quantitative PCR instrument;
s3, red high lycopene genotype when DC _ Chr1.02725 polymerized on the X-axis and the fluorescence signal was red and DC _ Chr3.10274 aggregated on the X-axis and the fluorescence signal was also red; non-red low lycopene genotypes are when DC _ chr1.02725 is polymerized on the X-axis and the fluorescent signal is red and DC _ chr3.10274 is concentrated on the Y-axis and the fluorescent signal is blue, or DC _ chr1.02725 is polymerized on the Y-axis and the fluorescent signal is blue and DC _ chr3.10274 is concentrated on the X-axis and the fluorescent signal is red.
Further, the reaction system of PCR was 30. mu.L, containing 2 XKASP master mix 5. mu.L, specific primer F0.3. mu.L, specific primer H0.1. mu.L, universal primer C0.1. mu.L, DNA (10-100ng) 5. mu.L, DDH2O 5μL。
Further, the reaction procedure of PCR is: 10min at 95 ℃; 95 ℃ for 15sec, 65-55 ℃ for 60sec, and annealing extension temperature reduction of 1 ℃ per cycle for 10 cycles; 95 ℃ for 15sec, 55 ℃ for 60sec, for 36 cycles.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses two major QTLs for controlling lycopene content in carrots, namely Lyco1.1 of chromosome 1 and Lyco3.1 of chromosome 3. And simultaneously finding 1 SNP variation site A/C at the 31078407 position of the No.1 chromosome, finding 1 SNP variation site A/T at the 4222832 position of the No.3 chromosome, and closely linking with high lycopene when the two sites are mutated into C: C and T: T combination simultaneously.
The invention successfully develops a group of KASP markers, DC _ Chr1.02725 and DC _ Chr3.10274 based on the variation sites, which are molecular markers which are firstly reported on carrot and are closely linked with lycopene, wherein the molecular markers DC _ Chr1.02725 and DC _ Chr3.10274 are closely linked with 2 major QTL sites, namely Lyco1.1 and Lyco3.1, for controlling lycopene respectively, wherein DC _ Chr1.02725 is positioned on chromosome 1, DC _ Chr3.10274 is positioned on chromosome 3, and when 36732764 th base of DC _ Chr1.02725 is C and 4128452 th base of DC _ Chr3.10274 is T, the markers are closely linked with lycopene traits. The detection accuracy rate of the KASP marker combination in the natural germplasm of the carrot is 94.4%, which fully shows that the marker combination can be used for screening the red high-lycopene character in the carrot, the high-flux rapid identification of the red carrot type can be realized in the seedling stage, effective technical support is provided for breeding of the red carrot rich in lycopene, and the application value is important.
Drawings
FIG. 1 shows the parent material and F1A material;
FIG. 2 is a Manhattan plot of lycopene content based on segregating population association analysis;
FIG. 3 is a chart of the genotyping of DC _ Chr1.02725 and DC _ Chr3.10274 markers in 54 carrot material.
In FIG. 3, A represents the genotyping of the DC _ Chr1.02725 marker; b represents the genotyping of the DC _ Chr3.10274 marker; a blank control is indicated.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1 KASP marker closely linked to major QTL of lycopene in carrot and application thereof
1. Experimental Material
Red high lycopene carrot material 'Red 1' and orange low lycopene material 'FN 26' and 52 carrot germplasm materials for molecular marker applications (27 parts Red high lycopene material and 25 parts non-Red low lycopene carrot germplasm materials, as shown in table 1) were collected and created by the horticulture academy of the university of shanxi agriculture.
TABLE 154 carrot germplasm information
2. Experimental procedure and results
(1) Gene mapping of lycopene character in red carrot
Construction of F by hybridization of ` Red1 ` (female parent) and ` FN26 ` (male parent)1Generations (FIG. 1) and F2, F3 family was obtained by selfing a portion of F2 representative individuals (red root color), and by selfing a portion of representative F3The generation individual plants (red root color) are self-bred to obtain the F4 generation family. In 2017, 95F are paired through GLM model2The lycopene content trait of the population was subjected to association analysis (fig. 2A), and significantly related SNP sites (P) were detected on all chromosomes<1.33E-06), wherein 1697 and 1696 chromosomes are detected on chromosome 1 and chromosome 3, respectively, and 1-10 differences are detected on the rest chromosomes. Phenotypic variation rate (R) interpretable by a single SNP on chromosome 12) 25.6% -52.5%, a phenotypic variation rate (R) interpretable by a single SNP on chromosome 32) 25.2% -65.2%, and the SNP on the remaining chromosomes can explain the phenotypic variation rate (R)2) 25.5 to 47.1 percent. These two significantly associated regions on chromosome 1 and 3 were named Lyco1.1 and Lyco3.1, with the most significant regions within Lyco1.1The SNP position of (1) is 31845645bp, and the most significant SNP position in the Lyco3.1 is 4807080 bp. 2018, utilize MLM model to pair 285F2、F3And F4Association analysis is carried out on the content traits of lycopene in individual plants of the combined population, and obviously related SNP loci (P) are detected on chromosomes 1 and 3 respectively<7.47E-07)110 and 35, and the sites are respectively located in the regions of Lyco1.1 and Lyco3.1 (FIG. 2B), the most significant SNP position in Lyco1.1 is 31740562bp, the most significant SNP position in Lyco3.1 is 4842544bp, and the single SNP on chromosome 1 can explain the phenotypic variation rate (R)2) 8.8% -9.1%, a phenotypic variation rate (R) interpretable by a single SNP on chromosome 32) 10.0 to 12.8 percent. Two major QTL sites, namely Lyco1.1 and Lyco3.1, are detected in different years of the two populations, and the most significant SNP site is closer.
(2) Primer design for KASP tag in the region of Lyco1.1 and Lyco3.1
Whole genome re-sequencing of 2 parents, selected the mutation sites of the red high lycopene material coding region in the region of 35.6Mb-37.5Mb where Lyco1.1 contains the most significant SNP site and in the region of 3.9Mb-5.9Mb where Lyco3.1 contains the most significant SNP site, 6 sites of 5 gene coding regions in the region of Lyco1.1 (Table 2), and non-synonymous mutations were caused at 106 sites of 33 gene coding regions in the region of Lyco3.1 (Table 3). And 1 SNP locus variation A/C is found at the position of 31078407 # chromosome 1, 1 SNP locus variation A/T is found at the position of 4222832 # chromosome 3, and the mutation of the two loci shows that the two loci are closely linked with high lycopene when the two loci are simultaneously C: C and T: T. KASP primers were designed for each site using Primerpickerlite, each pair of primers consisting of specific primer F, specific primer H and universal primer C. Wherein, the 5 'end of the specific primer F is connected with a fluorescent tag sequence 5'-GAAGGTGACCAAGTTCATGCT-3'(SEQ ID NO.9) of FAM group, and the 5' end of the specific primer H is connected with a fluorescent tag sequence 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO.10) of HEX group.
TABLE 2 variation information in the Lyco1.1 interval (data of NCBI for Gene ID)
TABLE 3 variation information in the Lyco3.1 interval
(3) Screening of KASP markers closely linked to Lyco1.1 and Lyco3.1 and application thereof in natural population
Genotyping the KASP primers designed in step (2) in 28 parts red high lycopene material and 26 parts non-red low lycopene carrot germplasm material. The specific operation method comprises the following steps of;
54 young leaves of the germplasm material are taken, genomic DNA of carrot is obtained by a CTAB extraction method, and PCR amplification is carried out by using the KASP primer designed in the step (2), wherein the PCR reaction system is 30 mu L, and the PCR reaction system comprises 2 xKASP master mix 5 mu L, a forward primer C0.3 mu L, a reverse primer X0.1 mu L, a reverse primer Y0.1 mu L, DNA (10-100ng)5 mu L and DDH2O5. mu.L. The PCR reaction program is: 10min at 95 ℃; 95 ℃ for 15sec, 65-55 ℃ for 60sec, and annealing extension temperature reduction of 1 ℃ per cycle for 10 cycles; 95 ℃ for 15sec, 55 ℃ for 60sec, for 36 cycles. Reading the fluorescent signal by using an ABI QS3 fluorescent quantitative PCR instrument to obtain a clear and visual parting chart, and outputting a genotype detection result according to different colors. Wherein the KASP marker DC _ Chr1.02725 (shown in SEQ ID NO.1) in the Lyco1.1 interval and the marker DC _ Chr3.10274 (shown in SEQ ID NO.2) in the Lyco3.1 interval haveObvious genotyping effect. The sequence of the DC _ chr 1.02725-labeled primer is:
DC _ Chr1.02725F (specific primer F, SEQ ID NO. 3): GAAGGTGACCAAGTTCATGCTTCGACCCAATAACTCTGAGACATATT, respectively;
DC _ Chr1.02725H (specific primer H, SEQ ID NO. 4): GAAGGTCGGAGTCAACGGATTCGACCCAATAACTCTGAGACATATG, respectively;
DC _ Chr1.02725C (Universal primer C, SEQ ID NO. 5): CTACCCGGGTACGTCATGCTCTT, respectively;
the primer sequence for the DC _ chr3.10274 marker is:
DC _ Chr3.10274F (specific primer F, SEQ ID NO. 6): GAAGGTGACCAAGTTCATGCTGGAAAGGCCTCTGAGTGCAGA, respectively;
DC _ Chr3.10274H (specific primer H, SEQ ID NO. 7): GAAGGTCGGAGTCAACGGATTGGAAAGGCCTCTGAGTGCAGT, respectively;
DC _ Chr3.10274C (Universal primer C, SEQ ID NO. 8): TCCTTTGCCACCTTTACTTCCTTGTAAA are provided.
A red high lycopene genotype when DC _ chr1.02725 is polymerized on the X-axis and the fluorescent signal is red and DC _ chr3.10274 is concentrated on the X-axis and the fluorescent signal is also red (C: C/T: T genotype); non-red low lycopene genotype when DC _ chr1.02725 polymerized on the X-axis and the fluorescence signal was red and DC _ chr3.10274 was blue (C: C/a: a genotype) or DC _ chr1.02725 polymerized on the Y-axis and the fluorescence signal was blue and DC _ chr3.10274 was blue (a: a genotype) or DC _ chr1.02725 polymerized on the Y-axis and the fluorescence signal was blue and DC _ chr3.10274 was red (a: a/T: T genotype) when the fluorescence signal was red (a: a/T: T genotype), see in particular table 4 and fig. 3.
According to the analysis of the phenotype and the genotype of the detected material, only 3 carrot resources with different sources and types are non-red in genotype C/T and T, the phenotype is not consistent with the genotype, and the detection accuracy reaches 94.4%. Therefore, when the SNP mutation site on the chromosome 1 is C and the SNP mutation site on the chromosome 3 is T, the SNP mutation site is closely linked with the lycopene character, and the corresponding DC _ Chr1.02725 and DC _ Chr3.10274 are KASP markers closely linked with Lyco1.1 and Lyco3.1 respectively, and the marker combination can effectively detect the carrot high lycopene character.
TABLE 4 detection of DC _ Chr1.02725 and DC _ Chr3.10274 markers in the Natural population of carrots
(4) Association analysis of KASP marker combination genotype and phenotype
Pearson correlation analysis was performed on genotype and phenotype of DC _ Chr1.02725 and DC _ Chr3.10274 markers in the natural population of carrots with a correlation coefficient of 0.544(P <0.01), indicating that this marker combination is closely related to the carrot lycopene trait.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> Shanxi university of agriculture
<120> KASP marker closely linked with lycopene major QTL in carrot, and primer and application thereof
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<211> 28
<212> DNA
<213> DC_Chr3.10274C(Artificial Sequence)
<400> 8
tcctttgcca cctttacttc cttgtaaa 28
<210> 9
<211> 21
<212> DNA
<213> FAM(Artificial Sequence)
<400> 9
gaaggtgacc aagttcatgc t 21
<210> 10
<211> 21
<212> DNA
<213> HEX(Artificial Sequence)
<400> 10
gaaggtcgga gtcaacggat t 21
Claims (8)
1. A KASP marker closely linked to a major QTL of lycopene in carrot, wherein said KASP marker comprises DC _ chr1.02725 shown in SEQ ID No.1 and DC _ chr3.10274 shown in SEQ ID No.2, and the SNP variation site of DC _ chr1.02725 is a/C, is located at base 100nt of DC _ chr1.02725, and is located at position 31078407 of the full-length sequence of chromosome 1; the SNP variation site of DC _ Chr3.10274 is A/T, is positioned at the 100nt base of DC _ Chr3.10274, and is positioned at the 4222832 th position of the full-length sequence of No.3 chromosome.
2. The molecular marker primer for KASP labeling according to claim 1, wherein the molecular marker primer comprises two pairs of primer combinations corresponding to DC _ Chr1.02725 and DC _ Chr3.10274, respectively, each pair of primer combinations comprises a specific primer F, a specific primer H and a universal primer C, the 5 'end of the specific primer F is connected with the fluorescent tag sequence of FAM group, and the 5' end of the specific primer H is connected with the fluorescent tag sequence of HEX group.
3. The molecular marker primer according to claim 2, wherein the molecular marker primer comprises a primer combination of DC _ Chr1.02725, DC _ Chr1.02725F, DC _ Chr1.02725H and DC _ Chr1.02725C, and a primer combination of DC _ Chr3.10274, DC _ Chr3.10274F, DC _ Chr3.10274H and DC _ Chr3.10274C, the nucleotide sequence of DC _ Chr1.02725F is shown in SEQ ID NO.3, the nucleotide sequence of DC _ Chr1.02725H is shown in SEQ ID NO.4, the nucleotide sequence of DC _ Chr1.02725C is shown in SEQ ID NO.5, the nucleotide sequence of DC _ Chr3.10274F is shown in SEQ ID NO.6, the nucleotide sequence of DC _ Chr 3.74H is shown in SEQ ID NO.7, and the nucleotide sequence of DC _ Chr 3.102748 is shown in SEQ ID NO. 748.
4. The detection product containing the molecular marker primer according to claim 2 or 3, wherein the detection product comprises a detection kit and a detection reagent.
5. Use of a KASP marker according to claim 1 or a molecular marker primer according to claim 2 or 3 or a test product according to claim 4 in any one of:
(1) detecting the high lycopene character of the carrot;
(2) carrot germplasm resources are improved;
(3) cultivating carrots with high lycopene;
(4) genotyping carrot germplasm material.
6. Use according to claim 5, characterized in that it comprises the following steps:
s1, extracting the carrot genome DNA to be detected;
s2, performing PCR amplification by using the molecular marker primer of claim 2 or 3, and reading a fluorescence signal by using an ABI QS3 fluorescence quantitative PCR instrument;
s3, red high lycopene genotype when DC _ Chr1.02725 polymerized on the X-axis and the fluorescence signal was red and DC _ Chr3.10274 aggregated on the X-axis and the fluorescence signal was also red; non-red low lycopene genotypes are when DC _ chr1.02725 is polymerized on the X-axis and the fluorescent signal is red and DC _ chr3.10274 is concentrated on the Y-axis and the fluorescent signal is blue, or DC _ chr1.02725 is polymerized on the Y-axis and the fluorescent signal is blue and DC _ chr3.10274 is concentrated on the X-axis and the fluorescent signal is red.
7. The use according to claim 6, wherein the PCR reaction system is 30. mu.L, and comprises 2 XKASP master mix 5. mu.L, specific primer F0.3. mu.L, specific primer H0.1. mu.L, universal primer C0.1. mu.L, DNA (10-100ng) 5. mu.L, DDH2O 5μL。
8. The use of claim 6, wherein the PCR reaction is performed by: 10min at 95 ℃; 95 ℃ for 15sec, 65-55 ℃ for 60sec, and annealing extension temperature reduction of 1 ℃ per cycle for 10 cycles; 95 ℃ for 15sec, 55 ℃ for 60sec, for 36 cycles.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116391613A (en) * | 2023-03-27 | 2023-07-07 | 南充市农业科学院 | Method for creating new germplasm of radish color leaves |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146659A (en) * | 2013-02-06 | 2013-06-12 | 中国农业科学院蔬菜花卉研究所 | Cucumber fruit beta carotene content related SNP marker and its application |
| US20190021251A1 (en) * | 2015-07-06 | 2019-01-24 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Tomato plants having fruit with yellow and red segments |
-
2021
- 2021-08-09 CN CN202110908280.6A patent/CN113528702B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146659A (en) * | 2013-02-06 | 2013-06-12 | 中国农业科学院蔬菜花卉研究所 | Cucumber fruit beta carotene content related SNP marker and its application |
| US20190021251A1 (en) * | 2015-07-06 | 2019-01-24 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Tomato plants having fruit with yellow and red segments |
Non-Patent Citations (6)
| Title |
|---|
| B.J.JUST等: "Carotenoid biosynthesis structural genes in carrot (Daucus carota): isolation, sequence-characterization, single nucleotide polymorphism (SNP) markers and genome mapping", 《THEORETICAL AND APPLIED GENETICS》 * |
| JÉRÉMY CLOTAULT等: "Carotenoid biosynthesis genes provide evidence of geographical subdivision and extensive linkage disequilibrium in the carrot", 《THEORETICAL AND APPLIED GENETICS》 * |
| NCBI: "PREDICTED: Daucus carota subsp. sativus uncharacterized LOC108213849 (LOC108213849),mRNA", 《GENBANK》 * |
| NCBI: "PREDICTED:Daucus carota subsp.sativus FAD synthetase 2, chloroplastic(LOC108206533),mRNA", 《GENBANK》 * |
| 欧承刚等: "胡萝卜(Daucus carota L.)中主要胡萝卜素和番茄红素含量的QTL分析", 《遗传》 * |
| 欧承刚等: "胡萝卜根中主要类胡萝卜素含量相关QTL的精细定位", 《园艺学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116391613A (en) * | 2023-03-27 | 2023-07-07 | 南充市农业科学院 | Method for creating new germplasm of radish color leaves |
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