Major QTLs, SNP molecular markers, and KASP detection primer sets for controlling lotus ripening and their applications

Abstract

This invention discloses major-effect QTLs, SNP molecular markers, KASP detection primer sets, and their applications for controlling lotus maturity. This invention utilizes SLAF-seq to develop SNP polymorphic markers to obtain major-effect QTL loci controlling lotus maturity traits, and then designs primers within the localized regions to obtain highly specific and accurate co-dominant fluorescent molecular markers. This invention also provides KASP primer sets for detecting SNP molecular markers and a PCR detection kit for identifying lotus maturity. The major-effect QTL loci controlling lotus maturity provided by this invention are tightly linked to SNP molecular markers and contribute significantly to lotus maturity. They can be used for map-based cloning to discover functional genes controlling early or late maturity and for marker-assisted selection, making them suitable for large-scale application. The SNP molecular markers for major-effect QTL loci of lotus maturity provided by this invention can efficiently select early- or late-maturing lotus varieties, accelerating the lotus breeding process.

Description

Technical Field

[0001] This invention relates to QTLs, molecular markers, and detection primers for controlling lotus maturity traits, and particularly to major QTLs, SNPs, KASP detection primer sets for controlling lotus maturity traits and their applications in identifying lotus maturity traits or in marker-assisted breeding of lotus. It belongs to the field of major QTLs, SNPs, and their applications for controlling lotus maturity traits. Background Technology

[0002] Lotus (Nelumbonucifera), belonging to the genus Nelumbo of the family Nelumbo, is one of the most widely cultivated aquatic vegetables in China, mainly concentrated in the Yangtze River basin and areas south of it. There are three cultivation types: seed lotus, rhizome lotus, and flowering lotus. In recent years, the lotus root industry in China has been booming. Lotus roots are primarily harvested for their underground stems, and as an important aquatic vegetable, the market demand for lotus roots is increasing year by year. Therefore, developing the lotus root industry has significant economic and social benefits. In the middle and lower reaches of the Yangtze River in China, the harvest period for conventional lotus root cultivation is generally from late June to April of the following year. Developing early-maturing or late-maturing lotus root varieties is of great significance for meeting the market demand for lotus roots at different times. Currently, lotus root breeding still mainly relies on traditional methods, which are labor-intensive, time-consuming, and inefficient. No molecular markers closely linked to the maturity traits of lotus roots have been developed and utilized in breeding work.

[0003] With the in-depth development of molecular biology, the successive completion of plant whole-genome sequencing, and breakthroughs in molecular marker development and bioinformatics, breeding time has been greatly shortened, laying the foundation for revealing the genetic mechanisms and molecular mechanisms of related traits. Specific Length Amplified Fragment Sequencing (SLAF-seq) technology is a highly automated, high-throughput sequencing technology based on bioinformatics. It boasts high reproducibility, short sequencing time, large information content, and a large number of available polymorphic SNP markers, and has been widely used in genetic map construction and molecular marker development. Compared with SSR, ALFP, and other methods, SNP-based molecular marker technologies offer advantages such as speed, efficiency, automated batch detection, and easier genotyping.

[0004] Using conventional hybridization breeding techniques, varieties with contrasting traits are selected as parents. After hybridization, F1 plants are obtained. Early-maturing or late-maturing varieties are selected based on the number of rhizomes formed in the underground parts of the lotus root at different stages. These varieties are then planted asexually. This process involves statistically analyzing the rhizome formation in the field, requiring significant manual labor for harvesting. It also suffers from problems such as a long cycle, repetitive and tedious processes, large space requirements, and low efficiency. If the major-effect QTL locus controlling lotus maturity is obtained, and primers are designed within the target region to develop highly specific and accurate co-dominant fluorescent molecular markers, it will not only effectively overcome the existing problems in identifying lotus maturity but also provide a feasible method for marker-assisted breeding of lotus maturity. Summary of the Invention

[0005] One objective of this invention is to provide major-effect QTL sites that control the ripening trait of lotus root;

[0006] The second objective of this invention is to identify SNP molecular markers that are closely linked to the major QTL controlling the ripening trait of lotus root.

[0007] A third objective of this invention is to provide a KASP detection primer set for the aforementioned SNP molecular marker;

[0008] The fourth objective of this invention is to apply the aforementioned SNP molecular markers or KASP detection primer pairs to identify the maturity trait of lotus or to use them for marker-assisted breeding of lotus.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] One aspect of the present invention provides two major QTL loci controlling the ripening trait of lotus root, one major QTL locus being located at 146.311 cM to 146.827 cM in linkage group 1, and the other major QTL locus being located at 38.345 cM to 41.653 cM in linkage group 6.

[0011] Another aspect of the present invention provides two SNP molecular markers closely linked to two major QTL loci controlling the maturity of lotus roots; one SNP molecular marker is named Marker14-594, with an allelic variation of T / C. If all alleles at the SNP locus are C, it is highly likely to indicate early maturity; if all alleles at the SNP locus are T, it is highly likely to indicate late maturity. The other SNP molecular marker is named Marker20-601, with an allelic variation of C / T. If all alleles at the SNP locus are C, it is highly likely to indicate early maturity; if all alleles at the SNP locus are T, it is highly likely to indicate late maturity.

[0012] Another aspect of the present invention provides a KASP primer set for detecting the SNP marker, comprising forward primer 1, forward primer 2 and reverse primer, with a nucleotide sequence of 5'-3'.

[0013] The nucleotide sequences of the forward primers 1 and 2 of the KASP primer set used to detect the SNP molecular marker Marker14-594 are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3. During detection, fluorescent tag sequences are attached to the 5' ends of the two forward primers. Specifically, the 5' end of the forward primer 1 is attached to the FAM sequence, the nucleotide sequence of which is shown in SEQ ID No. 7; and the 5' end of the forward primer 2 is attached to the HEX sequence, the nucleotide sequence of which is shown in SEQ ID No. 8.

[0014] The nucleotide sequences of the forward primers 1 and 2 of the KASP primer set used to detect the SNP molecular marker Marker20-601 are shown in SEQ ID No. 4 and SEQ ID No. 5, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 6. During detection, fluorescent tag sequences are attached to the 5' ends of the two forward primers. Specifically, the 5' end of the forward primer 1 is attached to the FAM sequence, the nucleotide sequence of which is shown in SEQ ID No. 7; and the 5' end of the forward primer 2 is attached to the HEX sequence, the nucleotide sequence of which is shown in SEQ ID No. 8.

[0015] Another aspect of the present invention provides a PCR detection kit for identifying lotus maturity, comprising: a KASP detection primer set, a KASP master mix, and ddH2O; wherein the aforementioned KASP detection primer set is a KASP primer set for detecting SNP molecular marker Marker14-594 or a KASP primer set for detecting SNP molecular marker Marker20-601.

[0016] This invention further provides an application of the KASP detection primer set in identifying the ripening trait of lotus root, including:

[0017] (1) Extract genomic DNA from the lotus to be tested;

[0018] (2) Using the extracted genomic DNA of lotus to be tested as an amplification template, add the KASP primer set for detecting SNP molecular marker Marker14-594 or the KASP primer set for detecting SNP molecular marker Marker20-601 and the universal KASPmaster mix to establish a PCR amplification system for PCR amplification.

[0019] (3) The PCR amplification products were detected in a real-time PCR instrument, and the fluorescence intensity signal value was read. The PCR amplification products were scanned. Based on the different excitation and emission wavelengths of FAM fluorescence and HEX fluorescence, the amplification products were classified. When the KASP primer set for detecting SNP molecular marker Marker 14-594 was used for detection, the samples aggregated on the X-axis were late-maturing genotypes, the samples aggregated on the Y-axis were early-maturing genotypes, and the samples in the middle were heterozygous genotypes. When the KASP primer set for detecting SNP molecular marker Marker 20-601 was used for detection, the samples aggregated on the X-axis were early-maturing genotypes, the samples aggregated on the Y-axis were late-maturing genotypes, and the samples in the middle were heterozygous genotypes.

[0020] As a preferred embodiment of the present invention, in step (1), the extracted genomic DNA of lotus to be tested is diluted to 18-22 ng / μL and then used as a template for PCR amplification.

[0021] In a preferred embodiment of the present invention, the KASP primer set used to detect the SNP molecular marker Marker14-594 in step (2) consists of forward primer 1, forward primer 2, and reaction primer. The nucleotide sequences of forward primer 1 and forward primer 2 are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3. During detection, fluorescent tag sequences are attached to the 5' ends of the two forward primers. Specifically, a FAM sequence is attached to the 5' end of forward primer 1, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No. 7. A HEX sequence is attached to the 5' end of forward primer 2, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No. 8.

[0022] The KASP primer set used to detect the SNP molecular marker Marker20-601 consists of forward primer 1, forward primer 2, and a reaction primer. The nucleotide sequences of forward primer 1 and forward primer 2 are shown in SEQ ID No. 4 and SEQ ID No. 5, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 6. During detection, fluorescent tag sequences are attached to the 5' ends of the two forward primers. Specifically, a FAM sequence is attached to the 5' end of forward primer 1, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No. 7. A HEX sequence is attached to the 5' end of forward primer 2, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No. 8.

[0023] As a preferred embodiment of the present invention, the PCR reaction program in step (2) is as follows: 94℃, 15min; 94℃, 20sec, 61-55℃, 60sec, decreasing by 0.6℃ per cycle, 10 cycles; 94℃, 20sec, 55℃, 60sec, 26 cycles.

[0024] In a preferred embodiment of the present invention, the PCR amplification products are scanned using a QuantStudio6Flex machine in step (3).

[0025] This invention utilizes SLAF-seq to develop a large number of SNP polymorphic markers, obtaining major-effect QTL loci controlling the maturity trait of lotus. Subsequently, primers were designed within the localized regions to develop a batch of highly specific and accurate co-dominant fluorescent molecular markers. The major-effect QTL loci controlling lotus maturity provided by this invention are tightly linked to SNP molecular markers and contribute significantly to lotus maturity. They can be used for map-based cloning to discover functional genes controlling early or late maturity and for marker-assisted selection, making them suitable for large-scale application. The SNP molecular markers for the major-effect QTL loci of lotus maturity provided by this invention can efficiently select early- or late-maturing lotus varieties, accelerating the lotus breeding process.

[0026] Detailed description of the overall technical solution of the present invention

[0027] The SLAF-seq method was used to identify candidate regions of major-effect QTLs controlling lotus ripeness and to develop linkage markers.

[0028] This invention constructed an F2 genetic population using two lotus varieties, 'Elian 8' and 'Elian 10', as the female parent. The female parent exhibited late maturity, while the male parent exhibited early maturity. The maturity of the parents and each F2 individual plant was determined and statistically analyzed in the field. One lotus seed was sown from each sample of the two parents and 194 offspring in the F2 genetic segregating population. After germination, young leaves were collected, and genomic DNA was extracted using the CTAB method. DNA quality and concentration were detected using agarose gel electrophoresis and NanoDrop 2000; the DNA samples were then used for SLAF-seq sequencing. A SLAF library was constructed.

[0029] Using linkage groups as units, the linear arrangement of markers within linkage groups was analyzed using HighMap software, and the genetic distance between adjacent markers was estimated. Finally, a high-density genetic linkage map of lotus was obtained. 27,454 SNP markers were integrated into 1,310 bin markers, and a total of 8 linkage groups were constructed. The total map distance was 497.80 cM, the average map distance between markers was 0.38 cM, the maximum average distance between markers was 0.82 cM, the minimum average distance between markers was 0.29 cM, and they were located on linkage groups 3 and 4, respectively. The shortest linkage group was 21.15 cM, and the longest was 147.60 cM.

[0030] Using QTL-IciMapping 4.1 software, the additive-complete composite partitioning (ICIM-ADD) method was employed to analyze and calculate the phenotypic data and genetic map information of the population. QTL detection was performed on eight linkage groups, with LOD values ​​set at 2.5 or 2.0. Two major QTL loci controlling the maturity trait of lotus root were ultimately obtained, located at 146.311 cM to 146.827 cM in linkage group 1 and 38.345 cM to 41.653 cM in linkage group 6, respectively.

[0031] Based on the marker sequences of the aforementioned location intervals, and according to the mutation characteristics of SNPs, competitive allele-specific PCR primer pairs were designed, including forward primer 1, forward primer 2, and reverse primer. The forward primers have allelic variants at their ends, namely T / C and C / T.

[0032] Detection of F2 population and two parents using molecular markers

[0033] Young leaves were randomly selected from 194 offspring plants in the F2 population, and genomic DNA was obtained from the lotus plants using the CTAB extraction method. KASP Primer mix was prepared and PCR amplification was performed. The PCR amplification products were scanned using a QuantStudio 6Flex machine. Based on the different excitation and emission wavelengths of the two fluorescence spectra (FAM fluorescence and HEX fluorescence), the amplification products were genotyped. According to the amplification results, when using the KASP primer set for detecting the SNP molecular marker Marker 14-594, samples aggregated on the X-axis were late-maturing genotypes, samples aggregated on the Y-axis were early-maturing genotypes, and samples in between were heterozygous genotypes. When using the KASP primer set for detecting the SNP molecular marker Marker 20-601, samples aggregated on the X-axis were early-maturing genotypes, samples aggregated on the Y-axis were late-maturing genotypes, and samples in between were heterozygous genotypes. This invention relates to Definitions of abbreviations and key terms

[0034] QTL: Quantitative Trait Loci.

[0035] SNP: Single nucleotide polymorphism.

[0036] CTAB: Hexadecyltrimethylammonium bromide.

[0037] KASP: Competitive allele-specific PCR. Attached Figure Description

[0038] Figure 1 A high-density genetic linkage map of lotus.

[0039] Figure 2 Linkage map for QTL mapping of lotus ripening trait.

[0040] Figure 3 To detect the amplification results of the SNP molecular marker Marker 14-594 using the KASP primer set, the PCR amplification products were scanned using a QuantStudio 6Flex machine. The samples aggregated on the X-axis were late-maturing genotypes, those aggregated on the Y-axis were early-maturing genotypes, and those in the middle were heterozygous genotypes.

[0041] Figure 4 To detect the amplification results of the SNP molecular marker Marker20-601 using the KASP primer set, the PCR amplification products were scanned using a QuantStudio6Flex machine. The samples aggregated on the X-axis were early-maturing genotypes, those aggregated on the Y-axis were late-maturing genotypes, and those in the middle were heterozygous genotypes. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0043] Example 1: Identification of major QTL candidate regions controlling lotus ripeness and development of linkage markers using the SLAF-seq method.

[0044] 1. Constructing a segregating population of lotus for the ripening trait

[0045] In this embodiment, the lotus variety "Elian 8" was used as the female parent and the lotus variety "Elian 10" was used as the male parent to construct the F2 genetic population. The female parent showed late maturity, while the male parent showed early maturity.

[0046] 2. Field identification of maturity phenotype in lotus root

[0047] The maturity of the parent plants and each F2 individual plant was assessed and statistically analyzed in the field.

[0048] 3. Genomic DNA extraction and SLAF library construction

[0049] One lotus seed was sown in each sample from the two parents and 194 progeny in the F2 genetic segregating population. After germination, young leaves were collected, and genomic DNA was extracted using the CTAB method. DNA quality and concentration were detected by agarose gel electrophoresis and NanoDrop 2000. The DNA samples were then used for SLAF-seq sequencing.

[0050] The construction process of the SLAF library is as follows:

[0051] (1) First, the lotus reference genome was subjected to electronic enzyme digestion to screen for double enzyme digestion schemes. The enzyme finally selected was RsaI+HaeIII enzyme.

[0052] (2) The sample DNA was digested and library constructed using an enzyme digestion scheme; paired-end sequencing was performed using the Illumina platform HiSeq 2500 with a sequencing length of PE150.

[0053] (3) After initial filtering, SLAF markers are developed using SLAF technology. High-quality homozygous SNP sites are mined and screened using GATK and samtools (for specific procedures, please refer to the official GATK website).

[0054] 4. Construction of a high-density genetic linkage map of lotus

[0055] Using linkage groups as units, HighMap software was used to analyze and obtain the linear arrangement of markers within the linkage groups, and the genetic distance between adjacent markers was estimated, ultimately yielding a high-density genetic linkage map of lotus. Figure 1 The study integrated 27,454 SNP markers into 1,310 bins, constructing 8 linkage groups with a total map distance of 497.80 cM and an average map distance of 0.38 cM between markers. The maximum average distance between markers was 0.82 cM, and the minimum was 0.29 cM, located in linkage groups 3 and 4, respectively. The shortest linkage group had a distance of 21.15 cM, and the longest had a distance of 147.60 cM.

[0056] 5. QTL mapping of lotus root ripeness

[0057] Using QTL-IciMapping 4.1 software, the additive-complete composite partitioning (ICIM-ADD) method was employed to analyze and calculate the phenotypic data and genetic map information of the population. QTL detection was performed on 8 linkage groups, with LOD values ​​set at 2.5 or 2.0. Two major QTL loci controlling the maturity trait of lotus root were ultimately obtained, located at 146.311 cM to 146.827 cM in linkage group 1 and 38.345 cM to 41.653 cM in linkage group 6, respectively. Figure 2 ).

[0058] 6. Development of mature QTL range tags

[0059] Based on the marker sequences of the above-mentioned location intervals, two SNP molecular markers closely linked to two major QTL sites controlling the maturity of lotus roots were identified. One SNP molecular marker was named Marker14-594, with an allelic variation of T / C. If all alleles at the SNP site are C, it is highly likely to indicate early maturity; if all alleles at the SNP site are T, it is highly likely to indicate late maturity. The other SNP molecular marker was named Marker20-601, with an allelic variation of C / T. If all alleles at the SNP site are C, it is highly likely to indicate early maturity; if all alleles at the SNP site are T, it is highly likely to indicate late maturity.

[0060] Based on the mutation characteristics of SNPs, this invention designs competitive allele-specific PCR primer pairs, including forward primer 1, forward primer 2, and reverse primer. The forward primers have allelic variant bases at their ends, namely T / C and C / T.

[0061] The nucleotide sequences of the forward primer 1, forward primer 2, and reverse primer used to detect the SNP molecular marker Marker14-594 are as follows:

[0062] 594CNF_T (forward primer 1): 5'-TAACTAGAGGGTTGGAACGTGTTT-3' (SEQ ID No. 1)

[0063] 594CNF_C (forward primer 2): 5'-TAACTAGAGGGTTGGAACGTGTTC-3' (SEQ ID No. 2)

[0064] 594CNR (reverse primer): 5'-ATCTGGTCTCTGTTTGACGTGTTA-3' (SEQ ID No. 3);

[0065] The nucleotide sequences of the forward primer 1, forward primer 2, and reverse primer used to detect the SNP molecular marker Marker20-601 are as follows:

[0066] 601CNF_C (forward primer 1):

[0067] 5'-TTAGTTCAAAACAAAGATTCTCTATTCAC-3'(SEQ ID No.4)

[0068] 601CNF_T (forward primer 2):

[0069] 5'-TTAGTTCAAAACAAAGATTCTCTATTCAT-3'(SEQ ID No.5)

[0070] 601CNR:

[0071] 5'-TGGGTGAAGAGAATTGCTAACTCA-3' (SEQ ID No. 6).

[0072] A fluorescent tag sequence is attached to the 5' end of the aforementioned forward primers. Specifically, a FAM sequence is attached to the 5' end of forward primer 1, and the nucleotide sequence of the FAM sequence is: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID No. 7); a HEX sequence is attached to the 5' end of forward primer 2, and the nucleotide sequence of the HEX sequence is: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID No. 8).

[0073] The nucleotide sequences of forward primers 1 and 2, used for detecting the SNP molecular marker Marker14-594, after the fluorescent tag sequence is attached to the 5' end, are as follows:

[0074] 594CNF_T (FAM sequence linked to the 5' end of forward primer 1):

[0075] 5'-GAAGGTGACCAAGTTCATGCTTAACTAGAGGGTTGGAACGTG TTT-3'(SEQ ID No.9)

[0076] 594CNF_C (5' end of forward primer 2 ligated with HEX sequence):

[0077] 5'-GAAGGTCGGAGTCAACGGATTTAACTAGAGGGTTGGAACGTG TTC-3'(SEQ ID No.10)

[0078] The nucleotide sequences of forward primers 1 and 2, used for detecting the SNP molecular marker Marker14-594, after the fluorescent tag sequence is attached to the 5' end, are as follows:

[0079] 601CNF_C (FAM sequence attached to the 5' end of forward primer 1):

[0080] 5'-GAAGGTGACCAAGTTCATGCTTTAGTTCAAAACAAAGATTCTC TATTCAC-3'(SEQ IDNo.11)

[0081] 601CNF_T (5' end of forward primer 2 ligated with HEX sequence):

[0082] 5'-GAAGGTCGGAGTCAACGGATTTTAGTTCAAAACAAAGATTCTC TATTCAT-3'(SEQ IDNo.12)

[0083] Experiment Example 1: Detection of lotus F2 population and two parents using KASP molecular marker primers.

[0084] 1. Extracting lotus genomic DNA

[0085] Young leaves were taken from 194 randomly selected progeny plants in the F2 population, and genomic DNA of the lotus plants was obtained by CTAB extraction.

[0086] 2. Preparation of KASP Primer mix

[0087] Table 1. Ingredients of KASP Primer mix

[0088]

[0089] Note: The KASP primer set mentioned above is the forward primer 1, forward primer 2 and reverse primer selected in Example 1 for detecting the SNP molecular marker Marker14-594, or the forward primer 1, forward primer 2 and reverse primer for detecting the SNP molecular marker Marker14-594.

[0090] 3. PCR amplification

[0091] 3.1 The PCR reaction system is as follows:

[0092] Table 2 PCR reaction system

[0093]

[0094] 3.2 The PCR reaction procedure is as follows:

[0095] 94℃, 15min; 94℃, 20sec, 61-55℃, 60sec, decreasing by 0.6℃ per cycle, 10 cycles; 94℃, 20sec, 55℃, 60sec, 26 cycles.

[0096] 4. Genotyping

[0097] The QuantStudio 6Flex machine was used to scan the PCR amplification products. Based on the different excitation and emission wavelengths of the two fluorescences (FAM fluorescence and HEX fluorescence), the amplification products were genotyped.

[0098] 5. Test Results

[0099] The amplification results of the KASP primer pair for detecting the SNP molecular marker Marker 14-594 were obtained by scanning the PCR amplification products using a QuantStudio 6Flex machine, as shown below. Figure 3 As shown, samples aggregated on the X-axis represent late-maturing genotypes, those aggregated on the Y-axis represent early-maturing genotypes, and samples in the middle represent heterozygous genotypes. The amplification results of the samples using the KASP primer set for the SNP molecular marker Marker 20-601 were obtained by scanning the PCR amplification products using a QuantStudio 6Flex machine, as shown below. Figure 4 As shown, the samples aggregated on the X-axis are precocious genotypes, the samples aggregated on the Y-axis are late-maturing genotypes, and the samples in the middle are heterozygous genotypes.

Claims

1. A KASP primer set for detecting and controlling two major QTL sites closely linked to two SNP molecular markers in lotus root maturation traits, characterized in that, Each KASP primer set includes forward primer 1, forward primer 2, and reverse primer, with a nucleotide sequence of 5'-3'; the KASP primer set is selected from either of the following two sets of KASP primer sets (1) or (2): (1) The KASP primer set used to detect the SNP molecular marker Marker14-594 has the nucleotide sequences of forward primer 1 and forward primer 2 as shown in SEQ ID No.1 and SEQ ID No.2, respectively, and the nucleotide sequence of its reverse primer is shown in SEQ ID No.3; (2) The KASP primer set used to detect the SNP molecular marker Marker20-601 has the nucleotide sequences of forward primer 1 and forward primer 2 as shown in SEQ ID No.4 and SEQ ID No.5, respectively, and the nucleotide sequence of its reverse primer is shown in SEQ ID No.6; During detection, a FAM sequence is attached to the 5' end of forward primer 1, the nucleotide sequence of which is shown in SEQ ID No. 7; and a HEX sequence is attached to the 5' end of forward primer 2, the nucleotide sequence of which is shown in SEQ ID No.

8.

2. A PCR detection kit for identifying lotus maturity, comprising: The KASP detection primer set, KASP master mix, and ddH2O are characterized in that the KASP detection primer set is the KASP primer set as described in claim 1.

3. The application of the KASP detection primer set according to claim 1 in identifying the ripening trait of lotus, comprising: (1) Extract genomic DNA from the lotus to be tested; (2) Using the extracted lotus genomic DNA as the amplification template, add the KASP primer set for detecting SNP molecular marker Marker14-594 or the KASP primer set for detecting SNP molecular marker Marker20-601 and the universal KASP mastermix to establish a PCR amplification system for PCR amplification. (3) The PCR amplification products were detected in a real-time PCR instrument, and the fluorescence intensity signal value was read. The PCR amplification products were scanned. Based on the different excitation and emission wavelengths of FAM fluorescence and HEX fluorescence, the amplification products were classified. When the KASP primer set for detecting SNP molecular marker Marker 14-594 was used for detection, if HEX fluorescence signal was obtained, it was a precocious genotype; if FAM fluorescence signal was obtained, it was a late-maturing genotype; if both HEX and FAM signals were present, it was a heterozygous genotype. When the KASP primer set for detecting SNP molecular marker Marker 20-601 was used for detection, if HEX fluorescence signal was obtained, it was a late-maturing genotype; if FAM fluorescence signal was obtained, it was a precocious genotype; if both HEX and FAM signals were present, it was a heterozygous genotype.

4. The application according to claim 3, characterized in that, In step (1), the extracted genomic DNA of lotus to be tested is diluted to 18-22 ng / μL and then used as a template for PCR amplification. The PCR reaction program in step (2) is as follows: 94℃, 15 min; 94℃, 20 sec, 61-55℃, 60 sec, decreasing by 0.6℃ for each cycle, 10 cycles; 94℃, 20 sec, 55℃, 60 sec, 26 cycles. In step (3), the PCR amplification products are scanned using a QuantStudio6Flex machine.

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