SNP molecular marker closely linked with crisp character of lotus powder, KASP detection primer group and application

Abstract

The invention discloses an SNP molecular marker closely linked with the crisp character of lotus powder, a KASP detection primer group and application. According to the invention, a large number of SNP (Single Nucleotide Polymorphism) markers are developed by utilizing SLAF-seq to obtain a main effect QTL (Quantitative Trait Loci) for controlling the crisp character of the lotus powder, the main effect QTL is closely linked with an SNP molecular marker, the contribution rate of the main effect QTL to the crisp character of the lotus powder is high, and the main effect QTL is used for map-based cloning mining of functional genes for controlling the crisp morning and evening and molecular marker-assisted selection; the invention provides an SNP molecular marker closely linked with two major QTL loci for controlling the crisp character of lotus powder and designs and obtains a KASP detection primer group with strong specificity and high accuracy, and the KASP detection primer group or a detection kit containing the detection primer group can specifically and accurately identify the powder or crisp character or genotype of lotus powder. The molecular marker has application prospects in the aspects of selecting or identifying powder or crisp lotus root varieties, lotus powder crisp molecular marker assisted breeding and the like.

Description

Technical Field

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

[0002] Lotus can be divided into two types according to quality: soup-type powdery lotus root and stir-fried (fresh) type crispy lotus root. Soup-type powdery lotus root is in great demand in southern China and is highly favored. In particular, the development of the processing industry in recent years has put an urgent demand on powdery lotus varieties. At present, lotus breeding is still mainly based on traditional methods, which are labor-intensive, long-term and inefficient. No molecular markers closely linked to the lotus powdery and crispy traits 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 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] Conventional hybrid breeding technology is used to select varieties with relative traits as parents, and after hybridization, F1 is obtained. After sowing and maturity, the swollen underground stems are manually dug, and then washed, peeled, sliced, cooked, and then sensory evaluation is performed to screen out powdery or brittle varieties. This trait investigation cycle is long, the process is repetitive and cumbersome, it takes up a lot of cultivation space, and the efficiency is low.

[0005] If the major QTL locus controlling the lotus powder brittle trait is obtained, and primers are then designed within the positioning segment to develop co-dominant fluorescent molecular markers with strong specificity and high accuracy, it will not only be able to effectively overcome the existing problems in identifying lotus powder brittleness, but also provide a feasible method for lotus powder brittleness molecular marker-assisted breeding. Summary of the invention

[0006] One of the purposes of the present invention is to provide a SNP molecular marker that is closely linked to the major effect QTL controlling the lotus powder brittle trait;

[0007] The second object of the present invention is to provide a KASP detection primer set for detecting SNP molecular markers closely linked to the major effect QTL controlling the lotus powder brittle trait;

[0008] The third object of the present invention is to apply the SNP molecular marker or KASP detection primer set to identify the powdery or brittle traits of lotus powder or to lotus molecular marker-assisted breeding.

[0009] To achieve the above purpose, the main technical solutions adopted by the present invention include:

[0010] One aspect of the present invention is to provide four SNP molecular markers that are closely linked to two major QTL loci controlling the lotus powder brittle trait;

[0011] Among them, the first SNP molecular marker is named Fc1.890, and the allele variant base is G / A. If the alleles of the SNP site are all G, it is very likely to be brittle; if the alleles of the SNP site are all A, it is likely to be powdery.

[0012] The second SNP molecular marker is named Fc1.255, and the allele variant base is A / G. If the alleles of the SNP site are all A, it is very likely to be powdery; if the alleles of the SNP site are all G, it is likely to be brittle.

[0013] The third SNP molecular marker is named Fc1.880, and the allele variant base is C / G. If the alleles of the SNP site are all C, it is very likely to be brittle; if the alleles of the SNP site are all G, it is likely to be powdery.

[0014] The fourth SNP molecular marker is named Fc1.252, and the allele variant base is G / A. If the alleles of the SNP site are all G, it is very likely to be brittle; if the alleles of the SNP site are all A, it is likely to be powdery.

[0015] The main QTL loci controlling the lotus powder brittleness trait are two main QTL loci, one of which is located at 139.5cM to 144.5cM of the first linkage group; and the other is located at 112.5cM to 115.5cM of the first linkage group.

[0016] 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'; the KASP primer set is selected from any one of the following KASP primer sets (1)-(4):

[0017] (1) A KASP primer set for detecting the SNP molecular marker Fc1.890, 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;

[0018] (2) A KASP primer set for detecting the SNP molecular marker Fc1.255, wherein 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;

[0019] (3) A KASP primer set for detecting the SNP molecular marker Fc1.880, wherein the nucleotide sequences of the forward primer 1 and the forward primer 2 are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 9;

[0020] (4) A KASP primer set for detecting the SNP molecular marker Fc1.252, wherein the nucleotide sequences of forward primer 1 and forward primer 2 are shown in SEQ ID No. 10 and SEQ ID No. 11, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 12.

[0021] In a preferred specific embodiment of the present invention, during detection, the 5' ends of the two forward primers are respectively connected with fluorescent label sequences, wherein the 5' end of forward primer 1 is connected with a FAM sequence, the nucleotide sequence of which is shown in SEQ ID No.13; and the 5' end of forward primer 2 is connected with a HEX sequence, the nucleotide sequence of which is shown in SEQ ID No.14.

[0022] Another aspect of the present invention provides a PCR detection kit for identifying lotus powder brittleness or powdery traits, comprising: a KASP detection primer set, a KASP master mix and ddH2O; wherein the KASP detection primer set is selected from any one of the following KASP primer sets (1) to (4):

[0023] (1) A KASP primer set for detecting the SNP molecular marker Fc1.890, 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;

[0024] (2) A KASP primer set for detecting the SNP molecular marker Fc1.255, wherein 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;

[0025] (3) A KASP primer set for detecting the SNP molecular marker Fc1.880, wherein the nucleotide sequences of the forward primer 1 and the forward primer 2 are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 9;

[0026] (4) A KASP primer set for detecting the SNP molecular marker Fc1.252, wherein the nucleotide sequences of forward primer 1 and forward primer 2 are shown in SEQ ID No. 10 and SEQ ID No. 11, respectively, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 12.

[0027] The present invention further provides an application of the KASP detection primer set in identifying lotus powdery or brittle traits, including:

[0028] (1) extracting genomic DNA of the lotus to be tested;

[0029] (2) Using the extracted genomic DNA of the lotus to be tested as an amplification template, adding the KASP primer set for detecting SNP molecular markers and the universal KASP master mix to establish a PCR amplification system for PCR amplification;

[0030] (3) Detecting the PCR amplification products in a fluorescence quantitative PCR instrument, reading the fluorescence intensity signal value, scanning the PCR amplification products, and typing the amplification products based on the different excitation wavelengths and emission wavelengths of FAM fluorescence and HEX fluorescence;

[0031] Among them, when the KASP primer set for detecting SNP molecular markers Fc1.890 or Fc1.252 was used for detection, the samples aggregated on the X-axis were brittle genotypes, the samples aggregated on the Y-axis were powdery genotypes, and the samples in the middle were heterozygous genotypes;

[0032] When the KASP primer set for detecting the SNP molecular marker Fc1.255 or Fc1.880 is used for detection, the samples aggregated on the X-axis are powdery genotypes, the samples aggregated on the Y-axis are brittle genotypes, and the samples in the middle are heterozygous genotypes.

[0033] 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.

[0034] As a preferred specific embodiment of the present invention, in step (2), when performing detection, the 5' ends of the two forward primers are respectively connected to fluorescent label sequences, wherein the 5' end of forward primer 1 is connected to a FAM sequence, the nucleotide sequence of which is shown in SEQ ID No. 13; and the 5' end of forward primer 2 is connected to a HEX sequence, the nucleotide sequence of which is shown in SEQ ID No. 14.

[0035] 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.

[0036] As a preferred specific embodiment of the present invention, in step (3), the PCR amplification product is scanned using a QuantStudio6Flex machine.

[0037] The present invention uses SLAF-seq to develop a large number of SNP polymorphic markers to obtain two major QTL sites controlling the lotus powdery brittle trait, which are closely linked to the SNP molecular markers, have a high contribution rate to the lotus powdery brittleness, and can be used for map-based cloning to mine functional genes controlling the early and late powdery brittleness and molecular marker-assisted selection; the present invention further provides four SNP molecular markers closely linked to the two major QTL sites controlling the lotus powdery brittleness trait, and a KASP detection primer set with strong specificity and high accuracy is designed for the four SNP molecular markers. The KASP detection primer set or a detection kit containing the KASP detection primer set can specifically and accurately identify the lotus powdery or brittle traits or genotypes, and has application prospects in selecting or identifying early or late maturing lotus powdery or brittle lotus varieties and lotus powdery brittle molecular marker-assisted breeding.

[0038] Detailed description of the overall technical solution of the present invention

[0039] Identification of major QTL candidate regions controlling lotus powder crispness and development of linked markers using SLAF-seq

[0040] The present invention uses lotus "Eolian No. 8" as the female parent and lotus "Eolian No. 10" as the female parent to construct an F2 genetic population. The female parent is powdery and the male parent is brittle. The powdery and brittle traits of the parents and each F2 plant are detected and counted. 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 samples are used for SLAF-seq sequencing to construct a SLAF library.

[0041] 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.

[0042] 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 to 2.5 or 2.0. Finally, two major QTL loci controlling the lotus powder crispness trait were obtained, one of which was located at 139.5cM to 144.5cM on the first linkage group; the other major QTL loci were located at 112.5cM to 115.5cM on the first linkage group.

[0043] Based on the marker sequence of the above positioning interval, four sets of competitive allele-specific PCR primer pairs were designed according to the mutation characteristics of SNP, including forward primer 1, forward primer 2 and reverse primer. The end of the forward primer is the allele variant base, which is G / A, A / G, C / G or G / A.

[0044] The KASP detection primer set designed for SNP molecular markers was used to detect the F2 population and the two parents.

[0045] 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 Fc1.890 was used for detection, the samples aggregated on the X-axis were brittle genotypes, the samples aggregated on the Y-axis were powdery genotypes, and the samples in the middle were heterozygous genotypes; when the KASP primer set for detecting the SNP molecular marker Fc1.255 was used for detection, the samples aggregated on the X-axis were powdery genotypes, the samples aggregated on the Y-axis were brittle genotypes, and the samples in the middle were heterozygous genotypes. When the KASP primer set for detecting the SNP molecular marker Fc1.880 was used for detection, the samples aggregated on the X-axis were powdery genotypes, the samples aggregated on the Y-axis were brittle genotypes, and the samples in the middle were heterozygous genotypes; when the KASP primer set for detecting the SNP molecular marker Fc1.252 was used for detection, the samples aggregated on the X-axis were brittle genotypes, the samples aggregated on the Y-axis were powdery genotypes, and the samples in the middle were heterozygous genotypes. Definitions of abbreviations and key terms in this invention

[0046] QTL: quantitative trait locus.

[0047] SNP: single nucleotide polymorphism.

[0048] CTAB: cetyltrimethylammonium bromide.

[0049] KASP: competitive allele-specific PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a high-density genetic linkage map of Lotus.

[0051] Figure 2 Linkage map for the location of QTLs controlling the lotus powder brittle trait.

[0052] Figure 3 The results of the amplification of the samples by the KASP primer set for detecting the SNP molecular marker Fc1.890 were obtained by scanning the PCR amplification products using the QuantStudio6Flex machine; the samples aggregated on the X-axis are the brittle genotype, the samples aggregated on the Y-axis are the powdery genotype, and the samples in the middle are the heterozygous genotype.

[0053] Figure 4The results of the amplification of the samples by the KASP primer set for detecting the SNP molecular marker Fc1.255 were obtained by scanning the PCR amplification products using the QuantStudio6Flex machine; the samples aggregated on the X-axis are powdery genotypes, the samples aggregated on the Y-axis are brittle genotypes, and the samples in the middle are heterozygous genotypes.

[0054] Figure 5 The results of the amplification of the samples by the KASP primer set for detecting the SNP molecular marker Fc1.880 were obtained by scanning the PCR amplification products using the QuantStudio6Flex machine; the samples aggregated on the X-axis are the powdery genotype, the samples aggregated on the Y-axis are the brittle genotype, and the samples in the middle are the heterozygous genotype.

[0055] Figure 6 The results of the amplification of the samples by the KASP primer set for detecting the SNP molecular marker Fc1.252 were obtained by scanning the PCR amplification products using the QuantStudio6Flex machine; the samples aggregated on the X-axis are the brittle genotype, the samples aggregated on the Y-axis are the powdery genotype, and the samples in the middle are the heterozygous genotype. DETAILED DESCRIPTION

[0056] 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.

[0057] Example 1 Determination of major QTL candidate regions controlling lotus powder crispness and development of linked markers using SLAF-seq method

[0058] 1. Construction of a population segregating for the brittle trait of lotus

[0059] In this embodiment, the F2 genetic population was constructed with lotus "Elian 8" as the female parent and lotus "Elian 10" as the male parent, wherein the female parent showed powdery texture and the male parent showed brittle texture.

[0060] 2. Field identification of the pink-brittle phenotype of lotus

[0061] The pinky brittleness traits of the parents and each F2 plant were tested and counted.

[0062] 3. Extraction of genomic DNA and construction of SLAF library

[0063] 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.

[0064] The construction process of the SLAF library is as follows:

[0065] (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;

[0066] (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;

[0067] (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).

[0068] 4. Construction of a high-density genetic linkage map of Lotus

[0069] 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.

[0070] 5. QTL Mapping of Lotus Fencui

[0071] 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 lotus powder crispness trait were obtained, one of which was located at 139.5cM to 144.5cM on the first linkage group; the other major QTL loci were located at 112.5cM to 115.5cM on the first linkage group ( Figure 2 ).

[0072] 6. Development of QTL interval markers for brittleness

[0073] Based on the marker sequences in the above-mentioned positioning interval, four SNP molecular markers closely linked to the two major QTL loci controlling the trait of lotus powder brittleness were identified; among them, the first SNP molecular marker was named Fc1.890, and the allelic variation base was G / A. If the alleles of the SNP locus are all G, it is very likely to show brittleness; if the alleles of the SNP locus are all A, it is likely to show powdery quality. The second SNP molecular marker is named Fc1.255, and the allele variation base is A / G. If the alleles of the SNP site are all A, it is very likely to be powdery; if the alleles of the SNP site are all G, it is likely to be brittle; the third SNP molecular marker is named Fc1.880, and the allele variation base is C / G. If the alleles of the SNP site are all C, it is very likely to be brittle; if the alleles of the SNP site are all G, it is likely to be powdery; the fourth SNP molecular marker is named Fc1.252, and the allele variation base is G / A. If the alleles of the SNP site are all G, it is very likely to be brittle; if the alleles of the SNP site are all A, it is likely to be powdery.

[0074] According to the mutation characteristics of SNP, this experiment designed four sets of competitive allele-specific PCR primer pairs, including forward primer 1, forward primer 2 and reverse primer; among them, the ends of the forward primers were allelic variant bases G / A, A / G, C / G and G / A respectively.

[0075] Among them, the nucleotide sequences of forward primer 1, forward primer 2 and reverse primer used to detect SNP molecular marker Fc1.890 are as follows:

[0076] Fc1.890F_G (forward primer 1):

[0077] 5'-AGTTAGTGAGGGTGTGACGAGG-3'(SEQ ID No.1)

[0078] Fc1.890F_A (forward primer 2):

[0079] 5'-AAGTTAGTGAGGGTGTGACGAGA-3'(SEQ ID No.2)

[0080] Fc1.890R (reverse primer):

[0081] 5'-TGGCATCATCTGAGCACTCT-3' (SEQ ID No. 3);

[0082] The nucleotide sequences of forward primer 1, forward primer 2 and reverse primer used to detect SNP molecular marker Fc1.255 are as follows:

[0083] Fc1.255F_A (forward primer 1):

[0084] 5'-ACACGACACAGGGTACTCCAA-3'(SEQ ID No.4)

[0085] Fc1.255F_G (forward primer 2):

[0086] 5'-ACACGACACAGGGTACTCCAG-3'(SEQ ID No.5)

[0087] Fc1.255R:

[0088] 5'-GAAGAAGCTCTCGCCACTGT-3' (SEQ ID No. 6).

[0089] The nucleotide sequences of forward primer 1, forward primer 2 and reverse primer used to detect SNP molecular marker Fc1.8801 are as follows:

[0090] Fc1.880F_G (forward primer 1):

[0091] 5'-GCCAAATTGTCCAAGCCAAG-3'(SEQ ID No.7)

[0092] Fc1.880F_C (forward primer 2):

[0093] 5'-GCCAAATTGTCCAAGCCAAC-3'(SEQ ID No.8)

[0094] Fc1.880R:

[0095] 5'-AAAAGGGAAACAGACAGGACA-3' (SEQ ID No. 9).

[0096] The nucleotide sequences of forward primer 1, forward primer 2 and reverse primer used to detect SNP molecular marker Fc1.252 are as follows:

[0097] Fc1.252F_G (forward primer 1):

[0098] 5'-TATGTTTTGTCTGCAAGTGAGG-3'(SEQ ID No.10)

[0099] Fc1.252F_A (forward primer 2):

[0100] 5'-GTATGTTTTGTCTGCAAGTGAGA-3'(SEQ ID No.11)

[0101] Fc1.252R:

[0102] 5'-AACGTGCCCAAGACAAGAAT-3' (SEQ ID No. 12).

[0103] 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. 13); 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. 14).

[0104] Experimental Example 1: Experiment on detecting lotus F2 population and two parents using KASP detection primers

[0105] 1. Extraction of Lotus Genomic DNA

[0106] An F2 genetic population was constructed with the lotus variety "Eolian 8" as the female parent and the lotus variety "Eolian 10" as the male parent. The female parent was powdery and the male parent was brittle.

[0107] The marker types and phenotypes of the brittle trait of 194 F2 progeny were tested, and the results are shown in Table 1.

[0108] Table 1 Marker types and crumbly texture traits of 194 F2 progeny strains

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] Young leaves of the 194 progeny were taken and genomic DNA of the lotus plants was obtained by CTAB extraction method.

[0116] 2. Preparation of KASP Primer Mix

[0117] Table 2 Ingredients of KASP Primer mix

[0118]

[0119] Note: The KASP primer set is the KASP primer set consisting of forward primer 1, forward primer 2 and reverse primer selected in Example 1 for detecting SNP molecular markers.

[0120] 3. PCR Amplification

[0121] 3.1 PCR reaction system:

[0122] Table 3 PCR reaction system

[0123]

[0124] 3.2PCR reaction procedure is:

[0125] 94℃, 15min; 94℃, 20sec, 61-55℃, 60sec, decrease 0.6℃ each cycle, 10 cycles; 94℃, 20sec, 55℃, 60sec, 26 cycles.

[0126] 4. Genotyping

[0127] 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).

[0128] 5. Test results

[0129] When the KASP primer set for detecting the SNP molecular marker Fc1.890 was used for detection, the samples aggregated on the X-axis were brittle genotypes (their powdery and brittle phenotypes were mainly brittle), the samples aggregated on the Y-axis were powdery genotypes (their powdery and brittle phenotypes were mainly powdery), and the samples in the middle were heterozygous genotypes ( Figure 3 ); when the KASP primer set for detecting the SNP molecular marker Fc1.255 was used for detection, the samples aggregated on the X-axis were powdery genotypes (the powdery and brittle phenotypes were mainly powdery), the samples aggregated on the Y-axis were brittle genotypes (the powdery and brittle phenotypes were mainly brittle), and the samples in the middle were heterozygous genotypes ( Figure 4 ); when the KASP primer set for detecting the SNP molecular marker Fc1.880 was used for detection, the samples aggregated on the X-axis were powdery genotypes (the powdery and brittle phenotypes were mainly powdery), the samples aggregated on the Y-axis were brittle genotypes (the powdery and brittle phenotypes were mainly brittle), and the samples in the middle were heterozygous genotypes ( Figure 5); when the KASP primer set for detecting the SNP molecular marker Fc1.252 was used for detection, the samples aggregated on the X-axis were brittle genotypes (the powdery and crispy phenotypes were mainly brittle), the samples aggregated on the Y-axis were powdery genotypes (the powdery and crispy phenotypes were mainly powdery), and the samples in the middle were heterozygous genotypes ( Figure 6 ).

[0130] According to the test results, the detection results of the pink and crisp genotypes of 194 F2 progeny strains using the four KASP primer sets provided by the present invention are basically consistent with the pink and crisp genotypes or their phenotypes of the 194 F2 progeny strains, proving that the four KASP primer sets provided by the present invention can effectively detect the lotus pink and crisp genotype or its phenotype.

Claims

1. A SNP molecular marker closely linked to the main QTL locus controlling the lotus powder brittle trait, characterized in that: The SNP molecular markers are four SNP molecular markers; among them, the allele variation base of the first SNP molecular marker is G / A; the allele variation base of the second SNP molecular marker is A / G; the allele variation base of the third SNP molecular marker is C / G; and the allele variation base of the fourth SNP molecular marker is G / A; there are two main effect QTL sites controlling the lotus powder brittleness trait, one of which is located at 139.5cM to 144.5cM of the first linkage group; and the other main effect QTL site is located at 112.5cM to 115.5cM of the first linkage group.

2. A KASP primer set for detecting the SNP molecular marker according to claim 1, wherein each KASP primer set comprises a forward primer 1, a forward primer 2 and a reverse primer, and the nucleotide sequence is 5'-3'; characterized in that: The KASP primer set is selected from any one of the following primer sets (1) to (4): (1) KASP primer set 1 consisting of forward primer 1 and forward primer 2 shown in SEQ ID No. 1 and SEQ ID No. 2 and reverse primer shown in SEQ ID No. 3; (2) KASP primer set 2 consisting of forward primer 1 and forward primer 2 shown in SEQ ID No. 4 and SEQ ID No. 5 and reverse primer shown in SEQ ID No. 6; (3) KASP primer set 3 consisting of forward primer 1 and forward primer 2 shown in SEQ ID No. 7 and SEQ ID No. 8 and reverse primer shown in SEQ ID No. 9; (4) KASP primer set 4 consisting of forward primer 1 and forward primer 2 represented by SEQ ID No. 10 and SEQ ID No. 11 and reverse primer represented by SEQ ID No.

12.

3. The KASP primer set according to claim 2, characterized in that During detection, a FAM sequence is connected to the 5' end of the forward primer 1, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No.13; a HEX sequence is connected to the 5' end of the forward primer 2, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No.

14.

4. A PCR detection kit for identifying lotus powder brittleness, 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.

5. Use of the SNP molecular marker according to claim 1 in detecting or identifying lotus powdery or brittle traits or genes, or in lotus molecular marker-assisted breeding.

6. Use of the KASP detection primer set according to claim 2 or 3 in detecting or identifying lotus powdery or brittle traits or genes, or in lotus molecular marker-assisted breeding.

7. Use of the PCR detection kit according to claim 4 in detecting or identifying lotus powdery or brittle traits or genes, or in lotus molecular marker-assisted breeding.

8. The use according to claim 6, 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 the amplification template, adding the KASP primer set and the universal KASPmaster mix 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 KASP primer set 1 is used for detection, the samples aggregated on the X-axis are brittle genotypes, the samples aggregated on the Y-axis are powdery genotypes, and the samples in the middle are heterozygous genotypes; when KASP primer set 2 is used for detection, the samples aggregated on the X-axis are powdery genotypes, the samples aggregated on the Y-axis are brittle genotypes, and the samples in the middle are heterozygous genotypes; when KASP primer set 3 is used for detection, the samples aggregated on the X-axis are powdery genotypes, the samples aggregated on the Y-axis are brittle genotypes, and the samples in the middle are heterozygous genotypes; when KASP primer set 3 is used for detection, the samples aggregated on the X-axis are powdery genotypes, the samples aggregated on the Y-axis are brittle genotypes, and the samples in the middle are heterozygous genotypes.

9. The use according to claim 8, characterized in that: In step (1), the 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), 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. 13; 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. 14; The reaction procedure of the PCR in step (2) is: 94°C, 15 min; 94°C, 20 sec, 61-55°C, 60 sec, decreasing 0.6°C in each cycle, 10 cycles; 94°C, 20 sec, 55°C, 60 sec, 26 cycles.

10. The use according to claim 8, characterized in that: In step (3), the PCR amplification products were scanned using the QuantStudio6Flex machine.

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