CN113969323B - SNP molecular marker related to luffa melon length and application thereof - Google Patents
SNP molecular marker related to luffa melon length and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biomolecular markers, and discloses an SNP molecular marker related to luffa melon growth and application thereof. The SNP molecular marker is positioned at the 151 th site of a nucleotide sequence shown as SEQ ID NO.1, the polymorphism is A/G, and the molecular marker is obviously related to the growth of towel gourd: the method can be used for identifying or assisting in identifying the melon length character of the to-be-detected towel gourd, identifying or assisting in identifying whether the fruit of the to-be-detected towel gourd is a long towel gourd or a short towel gourd, breeding the towel gourd with the long towel gourd fruit, breeding the towel gourd with the short towel gourd fruit and breeding the towel gourd.
Description
Technical Field
The invention belongs to the technical field of biomolecular markers, and particularly relates to an SNP molecular marker related to luffa melon growth and application thereof.
Background
Luffa cylindrica is an important multifunctional vegetable. The towel gourd fruit is rich in vitamins, amino acids, microelements and antioxidant substances, has the effects of clearing away summer heat, stopping bleeding and diminishing inflammation, and is a multifunctional vegetable used as medicine and food. In recent years, with the attention of people on nutrition and health, the sowing area of the towel gourd is increased year by year, and the towel gourd becomes an important daily consumption vegetable.
The fruit length of commercial luffa is an important appearance quality character. Due to different consumption habits and purposes, the requirements for the length of the towel gourd are different in various places. Aiming at the consumption habits in different areas, the loofah varieties with different melon lengths are cultivated, and the benefits of farmers can be obviously increased. However, complex environmental factors and genetic variation affect the breeding of different long varieties of luffa. However, the molecular marker can be used for quickly and simply performing auxiliary identification in the seedling stage, so that a large amount of manpower and material resources are saved.
Therefore, the method has important significance for the development of the towel gourd industry by deeply digging and utilizing the molecular markers related to the towel gourd melon growth.
Disclosure of Invention
In order to overcome the defects of the prior art, the first aspect of the invention aims to provide the SNP molecular marker related to the luffa melon length.
The second aspect of the present invention aims to provide a primer set for amplifying the SNP molecular marker related to the luffa melon length of the first aspect.
The third aspect of the invention aims to provide a kit for identifying or assisting in identifying the long character of luffa gourd.
The fourth aspect of the present invention is directed to providing the application of the SNP molecular marker related to luffa gourd growth in the first aspect.
The fifth aspect of the present invention is directed to the use of the primer set of the second aspect.
The sixth aspect of the present invention is directed to use of the kit of the third aspect.
The seventh aspect of the present invention is to provide a method for identifying or assisting in identifying the long trait of luffa.
An eighth aspect of the present invention is to provide a method for identifying or assisting in identifying whether a fruit of a luffa to be tested is a long melon or a short melon.
The ninth aspect of the invention aims to provide a method for breeding towel gourds with long-melon fruits.
The tenth aspect of the invention aims to provide a method for breeding the towel gourd with the fruit of short melon.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in the first aspect of the invention, an SNP molecular marker related to the luffa gourd length is provided, is positioned at the 151 th site of a nucleotide sequence shown as SEQ ID NO.1, and has polymorphism A/G.
In a second aspect of the present invention, a primer set for amplifying the SNP molecular marker related to luffa gourd growth in the first aspect of the present invention is provided.
Preferably, the primer set comprises a forward primer 1, a forward primer 2 and a reverse primer; the forward primer 1 is GAGAACACATTACTTGAATATTAAGCAA (SEQ ID NO. 5); the forward primer 2 is TGAGAACACATTACTTGAATATTAAGCAG (SEQ ID NO. 6); the reverse primer was TGTTGATGTATAGGTTAGGGTGGC (SEQ ID NO. 4).
In a third aspect of the invention, a kit for identifying or assisting in identifying the luffa melon growth trait is provided, which comprises the primer set of the second aspect of the invention.
Preferably, the 5' ends of the forward primer 1 and the forward primer 2 respectively carry a sequence tag A and a sequence tag B, and the nucleotide sequences of the sequence tag A and the sequence tag B are different from each other and are homologous with the genome sequence of the towel gourd.
Preferably, the sequence tag A is GAAGGTGACCAAGTTCATGCT (SEQ ID NO. 7).
Preferably, the sequence tag B is GAAGGTCGGAGTCAACGGAT (SEQ ID NO. 8).
Preferably, the kit further comprises a PCR premix containing a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B;
the nucleotide sequence of the fluorescent probe A is consistent with that of the sequence tag A, and the 5' end of the fluorescent probe A is connected with a fluorescent group A; the nucleotide sequence of the quenching probe A is reversely complementary with the nucleotide sequence of the sequence tag A, and the 3' end of the quenching probe A is connected with a quenching group;
the nucleotide sequence of the fluorescent probe B is consistent with that of the sequence label B, and the 5' end of the fluorescent probe B is connected with a fluorescent group B; the nucleotide sequence of the quenching probe B is reversely complementary with the nucleotide sequence of the sequence label B, and the 3' end of the quenching probe B is connected with a quenching group.
Preferably, the fluorophore A and the fluorophore B are different from each other.
Preferably, the fluorophore A and the fluorophore B are selected from FAM and HEX; further preferably, the fluorophore A is FAM and the fluorophore B is HEX.
Preferably, the quencher group is selected from BQH.
Preferably, the PCR master mix further comprises other reagents required for KASP amplification, such as polymerase, dntps and MgCl 2 And the like.
Preferably, a kit for identifying or assisting in identifying the luffa gourd growth trait, the kit comprising: a forward primer 1, a forward primer 2, a reverse primer and a PCR premix;
the forward primer 1 is GAAGGTGACCAAGTTCATGCTGAGAACACATTACTTGAATATTAAGCAA (SEQ ID NO. 2);
the forward primer 2 is GAAGGTCGGAGTCAACGGATTGAGAACACATTACTTGAATATTAAGCAG (SEQ ID NO. 3);
the reverse primer is TGTTGATGTATAGGTTAGGGTGGC (SEQ ID NO. 4);
the PCR premix was 2 × PARMS master mix.
In a fourth aspect of the invention, the invention provides the use of the SNP molecular marker related to luffa gourd length in any one of (1) - (10);
(1) identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(2) preparing a product for identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(3) identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(4) preparing a product for identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(5) selecting and breeding towel gourd with long melon fruits;
(6) preparing a product for breeding the towel gourd with the long melon fruit;
(7) selecting and breeding the towel gourd with short melon fruit;
(8) preparing a product for breeding the towel gourd with short melon fruits;
(9) breeding towel gourd;
(10) preparing a towel gourd breeding product.
In a fifth aspect of the present invention, there is provided a use of the primer set of the second aspect of the present invention in any one of (1) to (10);
(1) identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(2) preparing a product for identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(3) identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(4) preparing a product for identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(5) selecting and breeding towel gourd with long melon fruits;
(6) preparing a product for breeding the towel gourd with the long melon fruit;
(7) selecting and breeding the towel gourd with short melon fruit;
(8) preparing a product for breeding the towel gourd with short melon fruits;
(9) breeding towel gourd;
(10) preparing a towel gourd breeding product.
According to a sixth aspect of the invention, the application of the kit for identifying or assisting in identifying the luffa melon growth trait in the third aspect of the invention in any one of (1) to (10) is provided;
(1) identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(2) preparing a product for identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(3) identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(4) preparing a product for identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(5) selecting and breeding towel gourd with long melon fruits;
(6) preparing a product for breeding the towel gourd with the long melon fruit;
(7) selecting and breeding the towel gourd with short melon fruit;
(8) preparing a product for breeding the towel gourd with short melon fruits;
(9) breeding towel gourd;
(10) preparing a towel gourd breeding product.
The seventh aspect of the present invention provides a method for identifying or assisting in identifying the long character of luffa, which detects the genotype of the SNP molecular marker related to luffa length of the first aspect of the present invention in a luffa genome to be detected, and determines whether the luffa to be detected is a long luffa or a short luffa according to the genotype:
if the genotype of the SNP molecular marker related to the luffa growth in the luffa genome to be detected is AA, the luffa to be detected is a short luffa;
and if the genotype of the SNP molecular marker related to the luffa growth in the to-be-detected luffa genome is AG or GG, determining that the to-be-detected luffa is a long luffa.
Preferably, the method for detecting the genotype of the SNP molecular marker related to the luffa gourd length in the luffa genome to be detected comprises the following steps:
(1) mixing the DNA of the towel gourd to be detected with a primer group and a PCR premix in the kit of the third aspect of the invention, and carrying out KASP amplification;
(2) and detecting the PCR product by using a fluorescence detector, and determining the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected.
In an eighth aspect of the present invention, a method for identifying or assisting in identifying whether a fruit of a luffa to be tested is a long melon or a short melon is provided, the genotype of the SNP molecular marker related to the luffa length of the first aspect of the present invention in a luffa genome to be tested is detected, and whether the fruit of the luffa to be tested is a long melon or a short melon is determined according to the genotype:
if the genotype of the SNP molecular marker related to the luffa growth in the luffa genome to be detected is AA, the fruit of the luffa to be detected is short luffa;
and if the genotype of the SNP molecular marker related to the luffa growth in the luffa genome to be detected is AG or GG, the fruit of the luffa to be detected is a luffa.
Preferably, the method for detecting the genotype of the SNP molecular marker related to the luffa gourd length in the luffa genome to be detected comprises the following steps:
(1) mixing the DNA of the towel gourd to be detected with a primer group and a PCR premix in the kit of the third aspect of the invention, and carrying out KASP amplification;
(2) and detecting the PCR product by using a fluorescence detector, and determining the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected.
In the ninth aspect of the present invention, a method for breeding a luffa whose fruit is a long luffa is provided, which detects the genotype of the SNP molecular marker related to the luffa length of the first aspect of the present invention in the luffa genome to be tested, and selects a luffa whose genotype is AG or GG.
Preferably, the method for detecting the genotype of the SNP molecular marker related to the luffa gourd length in the luffa genome to be detected comprises the following steps:
(1) mixing the DNA of the towel gourd to be detected with a primer group and a PCR premix in the kit of the third aspect of the invention, and carrying out KASP amplification;
(2) and detecting the PCR product by using a fluorescence detector, and determining the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected.
In the tenth aspect of the present invention, a method for breeding a towel gourd with a fruit of short melon is provided, the genotype of the SNP molecular marker related to the towel gourd length of the first aspect of the present invention in the genome of the towel gourd to be tested is detected, and the towel gourd with the genotype of AA is selected.
Preferably, the method for detecting the genotype of the SNP molecular marker related to the luffa gourd length in the luffa genome to be detected comprises the following steps:
(1) mixing the DNA of the towel gourd to be detected with a primer group and a PCR premix in the kit of the third aspect of the invention, and carrying out KASP amplification;
(2) and detecting the PCR product by using a fluorescence detector, and determining the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected.
The invention has the beneficial effects that:
the invention discovers the SNP molecular marker related to the luffa melon growth for the first time, which is positioned at the 151 th site of the nucleotide sequence shown as SEQ ID NO.1 and has polymorphism of A/G, and the molecular marker is obviously related to the luffa melon growth: the method can be used for identifying or assisting in identifying the melon length character of the to-be-detected towel gourd, identifying or assisting in identifying whether the fruit of the to-be-detected towel gourd is a long towel gourd or a short towel gourd, breeding the towel gourd with the long towel gourd fruit, breeding the towel gourd with the short towel gourd fruit and breeding the towel gourd.
Drawings
Fig. 1 is an appearance diagram of short luffa 93075 and long luffa WJ55 in example 1.
FIG. 2 is a graph showing the frequency distribution of the length of the luffa fruit in the F2 population of example 1.
FIG. 3 is the mapping of the luffa gourd length gene in example 1: wherein a is a BSA-Seq positioning result graph; b is a molecular marker fine positioning result graph.
FIG. 4 is a graph showing the results of identifying the melon growth pattern of the M4962 primer set F2 in example 2.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments and accompanying drawings.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Example 1 mapping of genes associated with the growth of cucumis melo
1. Experimental Material
The chaulmoogra 93075 is derived from: a luffa inbred line bred by vegetable research institute of agricultural academy of Guangdong province.
The long chain melon WJ55 is derived from: a luffa inbred line bred by vegetable research institute of agricultural academy of Guangdong province.
The character patterns of the short luffa 93075 and the long luffa WJ55 are shown in figure 1.
2. Experimental procedure
2.1 hybridizing the short-thread melons 93075 and the long-thread melons WJ55 to obtain F1, selfing to obtain F2, planting the F2 in a greenhouse, investigating the length of each individual plant after 50 days, investigating 3 commercial melons for each individual plant, and taking the average number. The results are shown in fig. 2, the length frequency of the luffa fruits is normally distributed, which indicates that the luffa fruit is a typical quantitative trait and is controlled by a polygenic system.
2.2 respectively selecting 25 plants with the shortest melon length and 25 plants with the longest melon length by utilizing an F2 population, extracting DNA by utilizing a CTAB method, respectively mixing the DNA with an equimolar amount to form a long melon pool and a short melon pool, carrying out DNA library construction on the long melon pool and the short melon pool by utilizing TruSeq DNA LT Sample Prep Kit (Illumina), and sequencing by utilizing Illumina HiSeq 2000. The obtained raw data were used to extract polymorphic SNPs using QTG-Seq and mapped using deltaSNP-index (FIG. 3). Finally, the target gene is located in the 49.62-50.40 Mb interval of chromosome 4. DNA sequence variation of the two material genomes was analyzed to develop KASP markers (FIG. 3), and the recombinant individuals within the target segment were analyzed to finally localize the gene of interest between M4962 and M5004 (FIG. 3). Further, the SNP molecular marker (named M4962) is determined to be positioned at the 151 th site of the nucleotide sequence shown as SEQ ID NO.1, and the polymorphism is A/G. SEQ ID NO. 1: GAGAAAACTAAATAAAGAGAGGAGAAAAGATGAGAGATCTTAGCATAAGGTACTCTTTGGATTCAAGTCTCAGTCAAAAAAGCCCATAGATCTTAGCAATTTTCTTTTACCCTTCAAATCTTGGAGAACACATTACTTGAATATTAAGCAA CTCAAAACATTTATTGATTGCCACCCTAACCTATACATCAACACCCCCATTCCTAAAAGACTTCAAATCAGGCCTTAATCAACATCATATCATAAATAACCAAAAGAAACTTCCAAATTAGTACAACTTTAACACATTTTTCTATCTTGC are provided.
2.3 designing a primer group aiming at the SNP molecular marker M4962: m4962 Fa:GAAGGTGACCAAGTTCATGCTGAGAACACATTACTTGAATATTAAGCAA (SEQ ID NO.2, wherein the underlined part is the FAM fluorescent tag sequence); m4962 Fb:GAAGGTCGGAGTCAACGGATTGAGAACACATTACTTGAATATTAAGCAG (SEQ ID NO.3, wherein the underlined part is the HEX fluorescent tag sequence); M4962R: TGTTGATGTATAGGTTAGGGTGGC (SEQ ID NO. 4). Based on KASP reaction principle and single base difference design of long and short luffa, the marker can perform long and short luffa gene detection on the luffa sample at high fluxDetecting that if only FAM fluorescence (blue) is detected in the sample, the base of the sample is A; if only HEX fluorescence (green) is detected, the base of the sample is G; if two fluorescence (red) are detected simultaneously, the base at the site is in a heterozygous state.
The M4962 primer group is used for identifying the melon growth characteristics of the short-thread melons 93075, the long-thread melons WJ55 and 158 strain F2: extracting genome DNA of towel gourd, taking the genome DNA as a template, performing PCR amplification by adopting an M4962 primer group, wherein an amplification reaction system is shown in table 1, reaction conditions are shown in table 2, after the PCR is finished, reading a fluorescence signal by using a TECAN infinite M1000 microplate reader, analyzing and converting the fluorescence signal by using online software snpdecoder (http:// www.snpway.com/snpdecoder /), obtaining a clear and intuitive typing diagram, and outputting a genotype result according to different colors, wherein the results are as follows: the fluorescence signal of the amplification product of the short luffa 93075 is blue, the fluorescence signal of the amplification product of the long luffa WJ55 is green, and the fluorescence signal of 75.9% of the amplification products of the short luffa series in the F2 population is blue; the fluorescence signal of 73.5% long luffa amplification product was blue or red (fig. 4).
TABLE 1 amplification reaction System
TABLE 2 amplification reaction conditions
Example 2 application of M4962 molecular marker as luffa melon growth-like molecular marker
1. M4962 molecular marker and/or M4962 primer group for identifying or assisting in identifying growth of luffa melon
35 towel gourd strains were planted in the cloudburst lake cloudburst base in the cloudburst area of Guangzhou city, and water and fertilizer management was performed normally, and after 50 days, the melon length of each towel gourd variety was investigated (as shown in Table 3). And (3) carrying out melon growth shape identification on 35 towel gourd strains by utilizing an M4962 primer group: extracting genome DNA of towel gourd, taking the genome DNA as a template, performing PCR amplification by adopting an M4962 primer group, wherein an amplification reaction system is shown in Table 1, reaction conditions are shown in Table 2, after the PCR is finished, reading a fluorescence signal by using a TECAN infinite M1000 enzyme-labeled instrument, analyzing and converting the fluorescence signal by using online software snpdecoder (http:// www.snpway.com/snpdecoder /), obtaining a clear and intuitive typing map, outputting a genotype result according to different colors, and judging the melon length property of a sample according to a fluorescence color result of a PCR amplification product: the fluorescence signal of the amplification product is blue, and the sample is expressed as short melon; and the fluorescence signal of the amplification product is green or red, the sample shows long melon, and the results are shown in table 3: the fluorescence of the PCR amplification products of 17 strains is blue, the fluorescence of the PCR amplification products of 2 strains is red, and the fluorescence of the PCR amplification products of 16 strains is green: according to the melon long phenotype, 10 of 14 long melon lines are green fluorescence, 2 are blue fluorescence, 2 are red fluorescence, 6 of 21 short melon lines are green fluorescence, 15 are blue fluorescence, and the total accuracy is 77.14%.
Table 335 phenotype of Luffa cylindrica strain and fluorescence of amplification product
| Line of | Fluorescence | Melon growth table type | Line of | Fluorescence | Melon seed surface pattern |
| WN1 | Blue color | Short length | WN21 | Green colour | Long and long |
| WN3 | Green colour | Long and long | WN22 | Blue color | Is long and long |
| WN4 | Green colour | Long and long | WN23 | Blue colour | Short length |
| WN5 | Blue color | Short length | WN24 | Blue color | Short length |
| WN6 | Green colour | Short length | WN25 | Green colour | Long and long |
| WN7 | Red colour | Long and long | WN26 | Green colour | Long and long |
| WN8 | Blue color | Short length | WN27 | Red colour | Long and long |
| WN9 | Blue color | Short length | WN28 | Blue color | Short length |
| WN10 | Green colour | Short length | WN29 | Blue color | Short length |
| WN11 | Blue color | Short length | WN30 | Green colour | Long and long |
| WN12 | Green colour | Long and long | WN31 | Blue color | Short length |
| WN13 | Green colour | Long and long | WN32 | Green colour | Long and long |
| WN15 | Green colour | Short length | WN33 | Blue color | Is long and long |
| WN16 | Blue color | Short length | WN34 | Blue color | Short length |
| WN17 | Green colour | Long and long | WN35 | Green colour | Short length |
| WN18 | Blue color | Short length | WN36 | Blue color | Short length |
| WN19 | Green colour | Short length | WN37 | Green colour | Short length |
| WN20 | Blue color | Short length |
The experimental results show that the M4962 molecular marker can be used as a molecular marker for the melon length trait of the towel gourd and can be used for identifying or assisting in identifying the melon length trait of the towel gourd.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> vegetable research institute of academy of agricultural sciences of Guangdong province
<120> SNP molecular marker related to luffa gourd growth and application thereof
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Claims (13)
1. The SNP molecular marker related to the luffa melon length has a sequence shown in SEQ ID No.1, and the SNP locus is located at the 151 th position of the sequence and has polymorphism A/G.
2. A primer set for amplifying and typing the SNP molecular markers related to the luffa gourd fruit length, which is disclosed by claim 1, wherein the primer set comprises a forward primer 1, a forward primer 2 and a reverse primer; the forward primer 1 is GAGAACACATTACTTGAATATTAAGCAA; the forward primer 2 is TGAGAACACATTACTTGAATATTAAGCAG; the reverse primer is TGTTGATGTATAGGTTAGGGTGGC.
3. A kit comprising the primer set of claim 2.
4. The kit of claim 3, wherein: the 5' ends of the forward primer 1 and the forward primer 2 are respectively provided with a sequence tag A and a sequence tag B, and the nucleotide sequences of the sequence tag A and the sequence tag B are different from each other and are homologous with the genome sequence of the towel gourd.
5. The kit of claim 4, wherein:
the kit also comprises a PCR premix solution, wherein the PCR premix solution contains a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B;
the nucleotide sequence of the fluorescent probe A is consistent with that of the sequence tag A, and the 5' end of the fluorescent probe A is connected with a fluorescent group A; the nucleotide sequence of the quenching probe A is reversely complementary with the nucleotide sequence of the sequence label A, and the 3' end of the quenching probe A is connected with a quenching group;
the nucleotide sequence of the fluorescent probe B is consistent with that of the sequence label B, and the 5' end of the fluorescent probe B is connected with a fluorescent group B; the nucleotide sequence of the quenching probe B is reversely complementary with the nucleotide sequence of the sequence label B, and the 3' end of the quenching probe B is connected with a quenching group;
the fluorophore A and the fluorophore B are different from each other.
6. The application of the reagent for detecting the SNP molecular marker related to the towel gourd melon length in any one of (1) to (6) according to the parting claim 1;
(1) identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(2) preparing a product for identifying or assisting in identifying the melon growth character of the towel gourd to be detected;
(3) selecting and breeding towel gourd with long melon fruits;
(4) preparing a product for breeding the towel gourd with the long melon fruit;
(5) selecting and breeding the towel gourd with short melon fruit;
(6) preparing a product for breeding the towel gourd with short melon fruits;
if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AA, judging the luffa to be detected to be a short luffa;
and if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AG or GG, judging that the luffa to be detected is a long luffa.
Use of any one of (a 1) to (a 2) in any one of (1) to (6);
(a1) the primer set of claim 2;
(a2) the kit of any one of claims 3 to 5;
(1) identifying or assisting in identifying the melon length character of the to-be-detected towel gourd;
(2) preparing a product for identifying or assisting in identifying the melon growth character of the towel gourd to be detected;
(3) selecting and breeding towel gourd with long melon fruits;
(4) preparing a product for breeding the towel gourd with the long melon fruit;
(5) selecting and breeding the towel gourd with short melon fruit;
(6) preparing a product for breeding the towel gourd with the short melon fruit;
if the genotype of the SNP molecular marker related to the luffa length in the to-be-detected luffa genome is AA, judging that the to-be-detected luffa is a short luffa;
and if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AG or GG, judging that the luffa to be detected is a long luffa.
8. The application of the reagent for detecting the SNP molecular marker related to the towel gourd melon length in any one of (1) to (2) according to the parting claim 1;
(1) identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(2) preparing a product for identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long towel gourd or a short towel gourd;
if the genotype of the SNP molecular marker related to the luffa length in the to-be-detected luffa genome is AA, judging that the to-be-detected luffa is a short luffa;
and if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AG or GG, judging that the luffa to be detected is a long luffa.
Use of any one of (a 1) to (a 2) in any one of (1) to (2);
(a1) the primer set of claim 2;
(a2) the kit of any one of claims 3 to 5;
(1) identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long melon or a short melon;
(2) preparing a product for identifying or assisting in identifying whether the fruit of the towel gourd to be detected is a long towel gourd or a short towel gourd;
if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AA, judging the luffa to be detected to be a short luffa;
and if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AG or GG, judging that the luffa to be detected is a long luffa.
10. A method for identifying or assisting in identifying the long character of luffa melon, which comprises the steps of detecting the genotype of the SNP molecular marker related to luffa melon length in claim 1 in a luffa genome to be detected, and judging whether the luffa to be detected is long luffa or short luffa according to the genotype:
if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AA, judging the luffa to be detected to be a short luffa;
and if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AG or GG, judging that the luffa to be detected is a long luffa.
11. The method of claim 10, wherein:
the method for detecting the genotype of the SNP molecular marker related to the luffa gourd length in the luffa genome to be detected according to claim 1 comprises the following steps:
(1) mixing the DNA of the towel gourd to be detected with the primer group and the PCR premixed solution in the kit according to claim 5, and carrying out KASP amplification;
(2) and detecting the PCR product by using a fluorescence detector, and determining the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected.
12. The method as described in (b 1) or (b 2) or (b 3) below:
(b1) a method for identifying or assisting in identifying whether a fruit of a to-be-detected towel gourd is a long towel gourd or a short towel gourd, detects the genotype of the SNP molecular marker related to the towel gourd length in the to-be-detected towel gourd genome according to claim 1, and judges whether the fruit of the to-be-detected towel gourd is a long towel gourd or a short towel gourd according to the genotype:
if the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected is AA, judging that the fruit of the luffa to be detected is short luffa;
if the genotype of the SNP molecular marker related to the luffa growth in the to-be-detected luffa genome is AG or GG, judging that the fruit of the to-be-detected luffa is a long luffa;
(b2) a method for breeding towel gourd with long melon fruits, detecting the genotype of the SNP molecular marker related to the towel gourd length in claim 1 in the genome of the towel gourd to be detected, and selecting the towel gourd to be detected with the genotype AG or GG;
(b3) a method for breeding a towel gourd with short melon fruit, detecting the genotype of the SNP molecular marker related to the towel gourd length in claim 1 in the genome of the towel gourd to be detected, and selecting the towel gourd with the genotype AA to be detected.
13. The method of claim 12, wherein:
the method for detecting the genotype of the SNP molecular marker related to the luffa growth in claim 1 in a luffa genome to be detected comprises the following steps:
(1) mixing DNA of the towel gourd to be detected with a primer group and a PCR premix in the kit according to claim 5, and carrying out KASP amplification;
(2) and detecting the PCR product by using a fluorescence detector, and determining the genotype of the SNP molecular marker related to the luffa length in the luffa genome to be detected.
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| 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 |
| CN114457184A (en) * | 2022-02-21 | 2022-05-10 | 广东省农业科学院蔬菜研究所 | SNP molecular marker and KASP primer for identifying color of luffa seed coat and application thereof |
| CN115976054A (en) * | 2022-12-20 | 2023-04-18 | 广东省农业科学院蔬菜研究所 | Gene, protein and molecular marker related to stem thickness of towel gourd and application |
| CN118441086A (en) * | 2024-05-15 | 2024-08-06 | 湖南省蔬菜研究所 | Molecular marker linked with browning resistance of luffa fruits and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000046362A1 (en) * | 1999-02-03 | 2000-08-10 | Yeda Research And Development Co. Ltd. | Transgenic plants |
| CN104004833A (en) * | 2014-05-09 | 2014-08-27 | 广东省农业科学院蔬菜研究所 | EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof |
| CN108486128A (en) * | 2018-02-06 | 2018-09-04 | 华南农业大学 | Molecular labeling of the sponge gourd fruit picryl because of LaBt and for fruit bitter taste character assisted Selection |
| CN110055346A (en) * | 2019-04-11 | 2019-07-26 | 江苏省农业科学院 | A kind of KASP molecule labelling method of sponge gourd anti cucumber mosaic virus disease main effect QTL and its application |
| CN110950942A (en) * | 2019-12-11 | 2020-04-03 | 广东省农业科学院蔬菜研究所 | Gene related to luffa stem growth and application thereof |
| CN111996285A (en) * | 2020-09-27 | 2020-11-27 | 广东省农业科学院蔬菜研究所 | Molecular marker related to towel gourd powdery mildew and application thereof |
| CN112080515A (en) * | 2019-06-14 | 2020-12-15 | 中国科学院分子植物科学卓越创新中心 | UP gene and application thereof in plant improvement |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560964A (en) * | 2013-10-28 | 2015-04-29 | 常熟市董浜东盾蔬菜专业合作社 | Towel gourd virus resistance-related gene segment or gene marker and application |
| CN109880931A (en) * | 2019-04-11 | 2019-06-14 | 江苏省农业科学院 | A kind of SLAF-SNP molecule labelling method of sponge gourd anti cucumber mosaic virus CMV main effect QTL and application |
| CN111961747B (en) * | 2020-09-10 | 2022-09-06 | 北京市农林科学院 | Method for assisting in identifying sugar content of watermelon fruit, SNP marker and KASP marker |
-
2021
- 2021-10-14 CN CN202111195983.5A patent/CN113969323B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000046362A1 (en) * | 1999-02-03 | 2000-08-10 | Yeda Research And Development Co. Ltd. | Transgenic plants |
| CN104004833A (en) * | 2014-05-09 | 2014-08-27 | 广东省农业科学院蔬菜研究所 | EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof |
| CN108486128A (en) * | 2018-02-06 | 2018-09-04 | 华南农业大学 | Molecular labeling of the sponge gourd fruit picryl because of LaBt and for fruit bitter taste character assisted Selection |
| CN110055346A (en) * | 2019-04-11 | 2019-07-26 | 江苏省农业科学院 | A kind of KASP molecule labelling method of sponge gourd anti cucumber mosaic virus disease main effect QTL and its application |
| CN112080515A (en) * | 2019-06-14 | 2020-12-15 | 中国科学院分子植物科学卓越创新中心 | UP gene and application thereof in plant improvement |
| CN110950942A (en) * | 2019-12-11 | 2020-04-03 | 广东省农业科学院蔬菜研究所 | Gene related to luffa stem growth and application thereof |
| CN111996285A (en) * | 2020-09-27 | 2020-11-27 | 广东省农业科学院蔬菜研究所 | Molecular marker related to towel gourd powdery mildew and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| A high-quality sponge gourd (Luffa cylindrica) genome;Haibin Wu等;《nature horticulture research 》;20200801;第7卷(第1期);第1-10页 * |
| Genetic analysis of fruit shape traits at different maturation stages in sponge gourd;Zhang Sheng等;《Journal of Zhejiang University(Science B:An International Biomedicine & Biotechnology Journal)》;20070515(第05期);第52-58页 * |
| 三种不同类型的丝瓜果实香气物质解析与比较;李俊星等;《现代食品科技》;228-237;20210510;第37卷(第10期);第228-237页 * |
| 丝瓜种质资源的SRAP遗传多样性分析;吴海滨等;《西南农业学报》;20150908;第28卷(第4期);第1524-1529页 * |
| 丝瓜种质资源遗传多样性的SSR与SRAP分析;刘军等;《中国瓜菜》;20100305(第02期);第7-10页 * |
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