Main-effect QTL for controlling maturity of lotus, SNP molecular marker, KASP detection primer group and application

Abstract

The invention discloses a major QTL (Quantitative Trait Loci), an SNP (Single Nucleotide Polymorphism) molecular marker and a KASP detection primer group for controlling maturity of lotus root and lotus and application. According to the invention, SLAF-seq is utilized to develop an SNP (Single Nucleotide Polymorphism) marker to obtain a major QTL (Quantitative Trait Loci) for controlling the maturity character of lotus root and lotus root, and then a primer is designed in a positioning section to obtain a codominant fluorescent molecular marker with strong specificity and high accuracy. The invention also provides a KASP primer group for detecting the SNP molecular marker and a PCR (Polymerase Chain Reaction) detection kit for identifying the maturity of the lotus. The major QTL site for controlling the maturity of the lotus, provided by the invention, is closely linked with an SNP molecular marker, has a relatively high contribution rate to the maturity of the lotus, can be used for map-based cloning mining of functional genes for controlling the maturity and molecular marker-assisted selection, and is suitable for large-scale popularization and application. According to the SNP molecular marker of the major QTL site of lotus root maturity, early-maturing or late-maturing lotus root varieties can be efficiently selected, and the lotus seed breeding process is accelerated.

Description

Technical Field

[0001] The present invention relates to QTLs, molecular markers and detection primers for controlling lotus maturity traits, and in particular to major effect QTLs, SNP molecular markers, KASP detection primer sets for controlling lotus maturity traits and their applications in identifying lotus maturity traits or in lotus molecular marker-assisted breeding, belonging to the field of major effect QTLs, SNP molecular markers and their applications for controlling lotus maturity traits. Background Art

[0002] Lotus (Nelumbonucifera) belongs to the genus Nelumbo, family Nelumbo. It is one of the crops with the largest cultivated area among aquatic vegetables in China. It is mainly concentrated in the Yangtze River Basin and the areas to its south. There are three cultivated types: seed lotus, lotus root and flower lotus. In recent years, the lotus root industry in China has been developing rapidly. The lotus root is mainly eaten for underground stems. As an important aquatic vegetable, the demand for lotus root in the market has been increasing year by year. Therefore, the development of lotus root industry has significant economic and social benefits. The harvest period of conventional lotus root cultivation in the middle and lower reaches of the Yangtze River in China is generally from late June to April of the following year. Cultivating early-maturing or late-maturing lotus root varieties is of great significance to meet the market demand for lotus root supply at different times. At present, lotus root breeding is still mainly based on traditional methods, which has a large workload, a long cycle and low efficiency. No molecular markers closely linked to the maturity traits of lotus root have been developed and used in breeding work.

[0003] With the in-depth development of molecular biology, the sequencing of plant genomes has been completed one after another, and breakthroughs have been made in the development of molecular markers and bioinformatics, which has greatly shortened the breeding period and laid the foundation for revealing the genetic mechanism and molecular mechanism of related traits. Simplified genome sequencing (Specific Length Amplified Fragment Sequencing, SLAF-seq) technology is a highly automated high-throughput sequencing technology based on bioinformatics. It has high repeatability, short sequencing time, large amount of information, and can use many polymorphic SNP markers. It has been widely used in genetic map construction and molecular marker development. Compared with SSR, ALFP, etc., SNP-based molecular marker technology has the advantages of rapid, efficient, automated batch detection, and is easier to perform genotyping.

[0004] Using conventional hybrid breeding technology, varieties with relative traits are selected as parents, and F1 is obtained after hybridization. After sowing and maturity, early-maturing or late-maturing varieties are selected according to the number of lotus roots in the underground part of lotus roots at different periods, and the varieties are planted in asexual reproduction. This process is to count the lotus roots in the underground part in the field, which requires a lot of manpower to dig, and there are problems such as long cycle, repeated and cumbersome process, large amount of cultivation space occupied and low efficiency. If the main effect QTL site controlling the lotus maturity trait is obtained, and then primers are designed in the positioning section to develop a co-dominant fluorescent molecular marker with strong specificity and high accuracy, it will not only effectively overcome the existing problems in identifying lotus maturity, but also provide a feasible method for lotus maturity molecular marker-assisted breeding. Summary of the invention

[0005] One of the purposes of the present invention is to provide a major QTL locus controlling the maturity trait of lotus root;

[0006] The second object of the present invention is to provide a SNP molecular marker that is closely linked to the major QTL controlling the maturity trait of lotus root;

[0007] The third object of the present invention is to provide a KASP detection primer set for the SNP molecular marker;

[0008] The fourth purpose of the present invention is to use the SNP molecular marker or KASP detection primer pair to identify the maturity traits of lotus or to use it in lotus molecular marker-assisted breeding.

[0009] The above object of the present invention is achieved through the following technical solutions:

[0010] One aspect of the present invention provides two major QTL loci controlling the maturity trait of lotus root, wherein one major QTL locus is located at 146.311cM to 146.827cM of the first linkage group, and the other major QTL locus is located at 38.345cM to 41.653cM of the sixth linkage group.

[0011] Another aspect of the present invention provides two SNP molecular markers that are closely linked to the two main effect QTL sites of the trait controlling lotus root maturity; one of the SNP molecular markers is named Marker14-594, the allelic variation base is T / C, and the alleles of the SNP site are all C, which is very likely to show early maturity; if the alleles of the SNP site are all T, it is likely to show late maturity; the other SNP molecular marker is named Marker20-601, the allelic variation base is C / T, and the alleles of the SNP site are all C, which is very likely to show early maturity; if the alleles of the SNP site are all T, it is likely to show late maturity.

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

[0013] Among them, the nucleotide sequences of forward primer 1 and forward primer 2 of the KASP primer set for detecting 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; when performing detection, the 5' ends of the two forward primers are respectively connected with fluorescent label sequences, wherein the 5' end of the forward primer 1 is connected with a FAM sequence, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No.7; the 5' end of the forward primer 2 is connected with a HEX sequence, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No.8.

[0014] Among them, the nucleotide sequences of forward primer 1 and forward primer 2 of the KASP primer set for detecting SNP molecular marker Marker 20-601 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; when performing detection, the 5' ends of the two forward primers are respectively connected with fluorescent label sequences, wherein the 5' end of the forward primer 1 is connected with a FAM sequence, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No. 7; the 5' end of the forward primer 2 is connected with a HEX sequence, and the nucleotide sequence of the HEX sequence 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 above-mentioned 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] The present invention further provides an application of the KASP detection primer set in identifying the maturity traits of lotus root, comprising:

[0017] (1) extracting genomic DNA of the lotus to be tested;

[0018] (2) Using the extracted genomic DNA of the lotus to be tested as an amplification template, adding the KASP primer set for detecting the SNP molecular marker Marker 14-594 or the KASP primer set for detecting the SNP molecular marker Marker 20-601 and the universal KASPmaster mix to establish a PCR amplification system for PCR amplification;

[0019] (3) The PCR amplification products are detected in a fluorescence quantitative PCR instrument, the fluorescence intensity signal value is read, the PCR amplification products are scanned, and the amplification products are typed based on the different excitation wavelengths and emission wavelengths of FAM fluorescence and HEX fluorescence; wherein, when the KASP primer set for detecting the SNP molecular marker Marker14-594 is used for detection, the samples aggregated on the X-axis are late-maturing genotypes, the samples aggregated on the Y-axis are early-maturing genotypes, and the samples in the middle are heterozygous genotypes; when the KASP primer set for detecting the SNP molecular marker Marker20-601 is used for detection, the samples aggregated on the X-axis are early-maturing genotypes, the samples aggregated on the Y-axis are late-maturing genotypes, and the samples in the middle are heterozygous genotypes.

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

[0021] As a preferred specific embodiment of the present invention, the KASP primer set used for detecting the SNP molecular marker Marker14-594 in step (2) consists of a forward primer 1, a forward primer 2 and a reaction primer, wherein the nucleotide sequences of the forward primer 1 and the 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; when performing the detection, the 5' ends of the two forward primers are respectively connected with fluorescent label sequences, wherein the 5' end of the forward primer 1 is connected with a FAM sequence, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No.7; the 5' end of the forward primer 2 is connected with a HEX sequence, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No.8;

[0022] The KASP primer set for detecting the SNP molecular marker Marker 20-601 consists of a forward primer 1, a forward primer 2 and a reaction primer, the nucleotide sequences of the forward primer 1 and the 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; when performing the detection, the 5' ends of the two forward primers are respectively connected with fluorescent label sequences, wherein the 5' end of the forward primer 1 is connected with a FAM sequence, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No. 7; the 5' end of the forward primer 2 is connected with a HEX sequence, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No. 8.

[0023] As a preferred specific embodiment of the present invention, the reaction procedure of the PCR in step (2) is: 94°C, 15 min; 94°C, 20 sec, 61-55°C, 60 sec, reducing 0.6°C in each cycle, 10 cycles; 94°C, 20 sec, 55°C, 60 sec, 26 cycles.

[0024] As a preferred specific embodiment of the present invention, in step (3), the PCR amplification product is scanned using a QuantStudio6Flex machine.

[0025] The present invention uses SLAF-seq to develop a large number of SNP polymorphic markers to obtain the main effect QTL site controlling the lotus root maturity trait, and then designs primers in the positioning section to develop a batch of co-dominant fluorescent molecular markers with strong specificity and high accuracy. The main effect QTL site controlling the maturity of lotus root provided by the present invention is closely linked to the SNP molecular marker, has a high contribution rate to the maturity of lotus root, can be used for map-based cloning to mine functional genes that control the early and late maturity and molecular marker-assisted selection, and is suitable for large-scale promotion and application. The SNP molecular markers of the main effect QTL site of lotus root maturity provided by the present invention can efficiently select early-maturing or late-maturing lotus root varieties and accelerate the breeding process of lotus.

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

[0027] Identification of major QTL candidate regions controlling lotus maturity and development of linked markers using SLAF-seq

[0028] The present invention uses lotus "Elian No. 8" as the female parent and lotus "Elian No. 10" as the female parent to construct an F2 genetic population. The female parent is late-maturing and the male parent is early-maturing. The maturity of the parents and each F2 plant is identified and counted in the field. One lotus seed is sown for each sample of two parents and 194 offspring in the F2 genetic segregation population, and young leaves are taken after germination, and genomic DNA is extracted by the CTAB method. Agarose gel electrophoresis and NanoDrop 2000 are used to detect DNA quality and concentration; the DNA sample is used for SLAF-seq sequencing. A SLAF library is constructed.

[0029] Taking linkage groups as units, HighMap software was used to analyze the linear arrangement of markers within linkage groups and estimate the genetic distances between adjacent markers. 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, and the minimum was 0.29 cM. They were located on the 3rd and 4th linkage groups, respectively. The shortest linkage group was 21.15 cM and the longest was 147.60 cM.

[0030] Using QTL-IciMapping4.1 software, ICIM-ADD was used to analyze and calculate the phenotypic data and genetic map information of the population, and QTL detection was performed on 8 linkage groups, with the LOD value set at 2.5 or 2.0. Finally, two major QTL loci controlling the maturity trait of lotus root were obtained, located at 146.311cM to 146.827cM on the first linkage group and 38.345cM to 41.653cM on the sixth linkage group.

[0031] Based on the marker sequence of the above positioning interval and according to the mutation characteristics of SNP, a competitive allele-specific PCR primer pair is designed, including forward primer 1, forward primer 2 and reverse primer. The ends of the forward primers are T / C and C / T for allele variant bases respectively.

[0032] Using molecular markers to detect the F2 population and the two parents

[0033] Young leaves of 194 randomly selected progeny from the F2 population were taken, and the genomic DNA of the lotus plants was obtained by CTAB extraction. KASP Primer mix was prepared and PCR amplification was performed. The QuantStudio6Flex machine was used to scan the PCR amplification products, and the amplification products were typed based on the different excitation wavelengths and emission wavelengths of the two fluorescences (FAM fluorescence and HEX fluorescence). According to the amplification results, when the KASP primer set for detecting the SNP molecular marker Marker14-594 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 the SNP molecular marker Marker20-601 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. The present invention relates to and definitions of abbreviations and key terms

[0034] QTL: quantitative trait locus.

[0035] SNP: single nucleotide polymorphism.

[0036] CTAB: cetyltrimethylammonium bromide.

[0037] KASP: competitive allele-specific PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a high-density genetic linkage map of Lotus.

[0039] Figure 2 Linkage map for the location of QTLs controlling lotus maturity traits.

[0040] Figure 3 The results of the amplification of the samples by the KASP primer set for detecting the SNP molecular marker Marker14-594 were obtained by scanning the PCR amplification products using the QuantStudio6Flex machine; the samples aggregated on the X-axis are late-maturing genotypes, the samples aggregated on the Y-axis are early-maturing genotypes, and the samples in the middle are heterozygous genotypes.

[0041] Figure 4 The results of the amplification of the samples by the KASP primer set for detecting the SNP molecular marker Marker20-601 were obtained by scanning the PCR amplification products using the QuantStudio6Flex machine; the samples aggregated on the X-axis are early-maturing genotypes, the samples aggregated on the Y-axis are late-maturing genotypes, and the samples in the middle are heterozygous genotypes. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0043] Example 1 Determination of major QTL candidate regions controlling lotus maturity and development of linked markers using the SLAF-seq method

[0044] 1. Construction of a segregating population for lotus maturity traits

[0045] In this embodiment, the F2 genetic population was constructed with the lotus "Elian 8" as the female parent and the lotus "Elian 10" as the male parent, wherein the female parent exhibits late maturity and the male parent exhibits early maturity.

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

[0047] The maturity of the parents and each F2 plant was identified and counted in the field.

[0048] 3. Extraction of genomic DNA and construction of SLAF library

[0049] One lotus seed was sown for each sample of the two parents and 194 progeny in the F2 genetic segregation population, and young leaves were taken after germination, and genomic DNA was extracted using the CTAB method. Agarose gel electrophoresis and NanoDrop 2000 were used to detect DNA quality and concentration; DNA samples were used for SLAF-seq sequencing.

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

[0051] (1) First, the reference genome of lotus was electronically digested to screen the double enzyme digestion scheme, and the enzyme finally selected was RsaI+HaeIII enzyme;

[0052] (2) Use the enzyme digestion scheme to digest the sample DNA and build a library; use the Illumina platform Hiseq2500 for double-end sequencing, with a sequencing length of PE150;

[0053] (3) After the initial filtering, SLAF marker development was performed using SLAF technology. GATK and samtools were used to mine and screen high-quality homozygous SNP sites (for the specific process, please refer to the GATK official website).

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

[0055] Taking linkage groups as units, HighMap software was used to analyze the linear arrangement of markers within linkage groups and estimate the genetic distances between adjacent markers, ultimately obtaining a high-density genetic linkage map of lotus ( Figure 1 ), 27454 SNP markers were integrated into 1310 bin markers, and 8 linkage groups were constructed, with a total map distance of 497.80cM and an average map distance of 0.38cM between markers. The maximum average distance between markers was 0.82cM and the minimum was 0.29cM, which were located on the third and fourth linkage groups, respectively. The shortest linkage group was 21.15cM and the longest was 147.60cM.

[0056] 5. QTL Mapping of Lotus Root Maturity

[0057] The phenotypic data and genetic map information of the population were analyzed and calculated using QTL-IciMapping4.1 software and the additive-complete composite interval mapping method (ICIM-ADD). QTL detection was performed on 8 linkage groups, and the LOD value was set to 2.5 or 2.0. Finally, two major QTL loci controlling the maturity trait of lotus root were obtained, located at 146.311cM to 146.827cM on the first linkage group and 38.345cM to 41.653cM on the sixth linkage group ( Figure 2 ).

[0058] 6. Development of QTL interval markers for maturity

[0059] Based on the marker sequences in the above-mentioned positioning interval, two SNP molecular markers closely linked to the two major QTL loci controlling the maturity of lotus roots were identified; one of the SNP molecular markers was named Marker14-594, with an allelic variation base of T / C. If the alleles at the SNP site are all C, it is very likely to show early maturity; if the alleles at the SNP site are all T, it is likely to show late maturity; the other SNP molecular marker was named Marker20-601, with an allelic variation base of C / T. If the alleles at the SNP site are all C, it is very likely to show early maturity; if the alleles at the SNP site are all T, it is likely to show late maturity.

[0060] According to the mutation characteristics of SNP, the present invention designs a competitive allele-specific PCR primer pair, including forward primer 1, forward primer 2 and reverse primer. The ends of the forward primers are allele variant bases T / C and C / T respectively.

[0061] The nucleotide sequences of forward primer 1, forward primer 2 and reverse primer used to detect 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 forward primer 1, forward primer 2 and reverse primer used to detect SNP molecular marker Marker 20-601 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 label sequence is connected to the 5' end of the forward primer. Specifically, a FAM sequence is connected to the 5' end of the forward primer 1, and the nucleotide sequence of the FAM sequence is: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID No. 7); a HEX sequence is connected to the 5' end of the 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 primer 1 and forward primer 2 used to detect SNP molecular marker Marker14-594 after the fluorescent label sequence is connected at the 5' end are as follows:

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

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

[0076] 594CNF_C (the 5' end of forward primer 2 is connected to the HEX sequence):

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

[0078] The nucleotide sequences of forward primer 1 and forward primer 2 used to detect SNP molecular marker Marker14-594 after the fluorescent label sequence is connected at the 5' end are as follows:

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

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

[0081] 601CNF_T (the 5' end of forward primer 2 is connected to the HEX sequence):

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

[0083] Experimental Example 1: Experiment on detecting lotus F2 population and two parents using molecular marker KASP detection primers

[0084] 1. Extraction of Lotus Genomic DNA

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

[0086] 2. Preparation of KASP Primer Mix

[0087] Table 1 Composition of KASP Primer mix

[0088]

[0089] Note: The KASP primer set is the forward primer 1, forward primer 2 and reverse primer for detecting the SNP molecular marker Marker14-594 screened in Example 1 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 PCR reaction system:

[0092] Table 2 PCR reaction system

[0093]

[0094] 3.2PCR reaction procedure is:

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

[0096] 4. Genotyping

[0097] The QuantStudio6Flex machine is used to scan the PCR amplification products, and the amplification products are typed based on the different excitation wavelengths and emission wavelengths of the two fluorescences (FAM fluorescence and HEX fluorescence).

[0098] 5. Test results

[0099] The amplification results of the KASP primer set for detecting the SNP molecular marker Marker14-594 were as follows: Figure 3 As shown in the figure, the samples aggregated on the X-axis are late-maturing genotypes, the samples aggregated on the Y-axis are early-maturing genotypes, and the samples in the middle are heterozygous genotypes; the amplification results of the samples by the KASP primer set for detecting the SNP molecular marker Marker20-601 are shown in the figure after scanning the PCR amplification products using the QuantStudio6Flex machine. Figure 4 As shown, the samples aggregated on the X-axis are early-maturing genotypes, the samples aggregated on the Y-axis are late-maturing genotypes, and the samples in the middle are heterozygous genotypes.

Claims

1. Two major QTL loci controlling the maturity of lotus root, characterized by: One of the major QTL loci was located at 146.311cM to 146.827cM on linkage group 1; the other major QTL locus was located at 38.345cM to 41.653cM on linkage group 6.

2. Two SNP molecular markers closely linked to the two major effect QTL loci of claim 1, characterized in that: One of the SNP molecular markers is named Marker14-594, and the allele variant base is T / C; the other SNP molecular marker is named Marker20-601, and the allele variant base is C / T.

3. The KASP primer set for detecting the two SNP molecular markers according to claim 2, characterized in that: The KASP primer set includes forward primer 1, forward primer 2 and reverse primer, and the nucleotide sequence is 5'-3'; The nucleotide sequences of forward primer 1 and forward primer 2 of the KASP primer set for detecting 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; The nucleotide sequences of forward primer 1 and forward primer 2 of the KASP primer set for detecting the SNP molecular marker Marker 20-601 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.

4. The KASP primer set according to claim 3, characterized in that: During detection, a FAM sequence is connected 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 connected to the 5' end of forward primer 2, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No.

8.

5. A PCR detection kit for identifying lotus maturity, comprising: KASP detection primer set, KASP master mix and ddH2O; characterized in that the KASP detection primer set is the KASP primer set according to claim 3 or 4.

6. Use of the two major QTL loci according to claim 1 in detecting or identifying lotus maturity traits or in lotus molecular marker-assisted breeding.

7. Use of the two SNP molecular markers according to claim 2 in detecting or identifying lotus maturity traits or in lotus molecular marker-assisted breeding.

8. Use of the KASP detection primer set according to claim 3 or 4 in identifying lotus maturity traits.

9. The use according to claim 8, characterized in that: include: (1) extracting genomic DNA of the lotus to be tested; (2) Using the extracted genomic DNA of the lotus to be tested as an amplification template, adding a KASP primer set for detecting SNP molecular marker Marker 14-594 or a KASP primer set for detecting SNP molecular marker Marker 20-601 and a universal KASP mastermix to establish a PCR amplification system for PCR amplification; (3) The PCR amplification products are detected in a fluorescence quantitative PCR instrument, the fluorescence intensity signal value is read, the PCR amplification products are scanned, and the amplification products are typed based on the different excitation wavelengths and emission wavelengths of FAM fluorescence and HEX fluorescence; wherein, when the KASP primer set for detecting the SNP molecular marker Marker14-594 is used for detection, the samples aggregated on the X-axis are late-maturing genotypes, the samples aggregated on the Y-axis are early-maturing genotypes, and the samples in the middle are heterozygous genotypes; when the KASP primer set for detecting the SNP molecular marker Marker20-601 is used for detection, the samples aggregated on the X-axis are early-maturing genotypes, the samples aggregated on the Y-axis are late-maturing genotypes, and the samples in the middle are heterozygous genotypes.

10. The use according to claim 9, characterized in that: In step (1), the extracted genomic DNA of the lotus to be tested is diluted to 18-22 ng / μL and then used as a template for PCR amplification; in step (2), the reaction procedure of the PCR is: 94°C, 15 min; 94°C, 20 sec, 61-55°C, 60 sec, reducing 0.6°C in each cycle, 10 cycles; 94°C, 20 sec, 55°C, 60 sec, 26 cycles; in step (3), the PCR amplification product is scanned using a QuantStudio6Flex machine.

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