SNP molecular marker closely linked to lotus powder brittleness trait, KASP detection primer set and application

Abstract

The application discloses SNP molecular markers closely linked to lotus powder brittle traits, a KASP detection primer group and application. A main QTL site for controlling lotus powder brittle traits is obtained by using SLAF-seq to develop a large number of SNP polymorphism markers, the main QTL site is closely linked to the SNP molecular markers, has a high contribution rate to lotus powder brittle traits, is used for map-based cloning to mine functional genes for controlling powder brittle early or late and molecular marker assisted selection, the application provides SNP molecular markers closely linked to two main QTL sites for controlling lotus powder brittle traits and designs a KASP detection primer group with high specificity and high accuracy, and the KASP detection primer group or a detection kit containing the detection primer group can be used for specifically and accurately identifying lotus powder or brittle traits or genotypes, and has application prospects in the selection or identification of lotus powder or brittle varieties and lotus powder brittle molecular marker assisted breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to QTLs, molecular markers and detection primers for controlling lotus powder brittleness, in particular to major QTLs for controlling lotus powder brittleness, SNP molecular markers, KASP detection primer sets and their application in identifying lotus powder brittleness or for lotus molecular marker assisted breeding, belonging to the field of major QTLs for controlling lotus powder brittleness, SNP molecular markers and their applications. BACKGROUND

[0002] Lotus can be divided into soup type powder quality lotus and stir-fried (fresh) type brittle quality lotus according to quality. Soup type powder quality lotus is in great demand in southern regions and is highly favored, especially in recent years, the development of the processing industry has a very urgent demand for powder quality lotus varieties. At present, lotus breeding is still mainly based on traditional methods, which is labor-intensive, time-consuming and inefficient, and no molecular markers closely linked to lotus powder brittleness have been developed and used in breeding work.

[0003] With the in-depth development of molecular biology, the completion of plant whole genome sequencing, the breakthrough of molecular marker development and bioinformatics, the breeding period has been greatly shortened, and the foundation for revealing the genetic mechanism and molecular mechanism of related traits has been laid. The Specific Length Amplified Fragment Sequencing (SLAF-seq) technology is a highly automated high-throughput sequencing technology based on bioinformatics, which has high repeatability, short sequencing time and large amount of information. It can use polymorphic SNP markers and has been widely used in genetic map construction and molecular marker development. Compared with SSR, ALFP and other markers, SNP-based molecular marker technology has the advantages of rapidness, efficiency, automation and batch detection, and is more suitable for genotyping.

[0004] Using conventional hybrid breeding techniques, selecting varieties with relative traits as parents, crossing to obtain F1, sowing maturely, then manually digging the enlarged underground stems, then washing, peeling, slicing, cooking and sensory evaluation to screen out powder or brittle varieties. This trait investigation has the problems of long cycle, repeated tedious process, large occupation of cultivation space and low efficiency.

[0005] If the major QTL site for controlling lotus powder brittleness is obtained, and specific and accurate co-dominant fluorescent molecular markers are designed in the positioning segment, it not only can effectively overcome the existing problems of identifying lotus powder brittleness, but also can provide a feasible method for lotus powder brittleness molecular marker assisted breeding. SUMMARY

[0006] One of the purposes of the present application is to provide SNP molecular markers closely linked to major QTLs for controlling lotus powder brittleness;

[0007] The second object of the present application is to provide a KASP detection primer set for detecting a SNP molecular marker closely linked to a major QTL for controlling the powder brittleness of lotus powder;

[0008] The third object of the present application is to apply the SNP molecular marker or the KASP detection primer set to identify the powder or brittleness of lotus powder or to lotus molecular marker assisted breeding.

[0009] To achieve the above object, the main technical solution adopted by the present application comprises:

[0010] An aspect of the present application provides four SNP molecular markers closely linked to two major QTL sites for controlling the powder brittleness of lotus powder;

[0011] The first SNP molecular marker is named Fc1.890, the allelic variation base is G / A, and if the alleles of the SNP site are all G, it is most likely to exhibit brittleness, and if the alleles of the SNP site are all A, it is most likely to exhibit powder;

[0012] The second SNP molecular marker is named Fc1.255, the allelic variation base is A / G, and if the alleles of the SNP site are all A, it is most likely to exhibit powder, and if the alleles of the SNP site are all G, it is most likely to exhibit brittleness;

[0013] The third SNP molecular marker is named Fc1.880, the allelic variation base is C / G, and if the alleles of the SNP site are all C, it is most likely to exhibit brittleness, and if the alleles of the SNP site are all G, it is most likely to exhibit powder;

[0014] The fourth SNP molecular marker is named Fc1.252, the allelic variation base is G / A, and if the alleles of the SNP site are all G, it is most likely to exhibit brittleness, and if the alleles of the SNP site are all A, it is most likely to exhibit powder;

[0015] The major QTL site for controlling the powder brittleness of lotus powder is two major QTL sites, 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 application 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 (1)-(4) KASP primer set:

[0017] (1) the KASP primer set for detecting the SNP molecular marker Fc1.890, the nucleotide sequences of the forward primer 1 and the forward primer 2 are respectively shown in SEQ ID No. 1 and SEQ ID No. 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3;

[0018] (2) the KASP primer set for detecting the SNP molecular marker Fc1.255, the nucleotide sequences of the forward primer 1 and the forward primer 2 are respectively shown in SEQ ID No. 4 and SEQ ID No. 5, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 6;

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

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

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

[0022] Another aspect of the present application provides a PCR detection kit for identifying the powdery or mealy trait of lotus, 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 (1)-(4) KASP primer sets:

[0023] (1) the KASP primer set for detecting the SNP molecular marker Fc1.890, the nucleotide sequences of the forward primer 1 and the forward primer 2 are respectively shown in SEQ ID No. 1 and SEQ ID No. 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3;

[0024] (2) the KASP primer set for detecting the SNP molecular marker Fc1.255, the nucleotide sequences of the forward primer 1 and the forward primer 2 are respectively shown in SEQ ID No. 4 and SEQ ID No. 5, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 6;

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

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

[0027] The application further provides the application of the KASP detection primer set in identifying the lotus powder or the brittle quality, comprising:

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

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

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

[0031] When the KASP primer set for detecting the SNP molecular marker Fc1.890 or Fc1.252 is used for detection, the sample aggregated on the X axis is the brittle genotype, the sample aggregated on the Y axis is the powder genotype, and the sample in the middle is the hybrid genotype;

[0032] When the KASP primer set for detecting the SNP molecular marker Fc1.255 or Fc1.880 is used for detection, the sample aggregated on the X axis is the powder genotype, the sample aggregated on the Y axis is the brittle genotype, and the sample in the middle is the hybrid genotype.

[0033] As a preferred specific embodiment of the application, the extracted genomic DNA of the lotus to be detected is diluted to 18-22 ng / μL in step (1), and then the PCR amplification is performed with the template.

[0034] As a preferred embodiment of the present application, in step (2), the 5' end of the two forward primers is respectively connected with a fluorescent label sequence, wherein the 5' end of forward primer 1 is connected with a FAM sequence, and the nucleotide sequence of the FAM sequence is shown in SEQ ID No. 13; the 5' end of forward primer 2 is connected with a HEX sequence, and the nucleotide sequence of the HEX sequence is shown in SEQ ID No. 14.

[0035] As a preferred embodiment of the present application, the reaction program of the PCR in step (2) is: 94℃, 15min; 94℃, 20sec, 61-55℃, 60sec, 10 cycles of decreasing 0.6℃ each time; 94℃, 20sec, 55℃, 60sec, 26 cycles.

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

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

[0038] Detailed description of the overall technical scheme of the present application

[0039] Determination of main QTL candidate region controlling lotus powder brittle and development of linkage marker by SLAF-seq method

[0040] The present application takes lotus "E lotus 8" as the female parent, and lotus "E lotus 10" as the female parent, and constructs an F2 genetic population. The female parent shows powder quality, and the male parent shows brittle quality. The powder and brittle traits of the male parent and each F2 single plant are detected and counted. Each sample of 2 parents and 194 offspring in the F2 genetic separation population is sowed with one lotus seed, and after germination, the young leaves are taken, and the genomic DNA is extracted by using the CTAB method. The DNA quality and concentration are detected by using agarose gel electrophoresis and NanoDrop 2000; the DNA sample is used for SLAF-seq sequencing, and a SLAF library is constructed.

[0041] In linkage group units, the linear arrangement of markers in the linkage group is obtained by using the HighMap software, and the genetic distance between adjacent markers is estimated, and finally a high-density genetic linkage map of lotus is obtained. 27454 SNP markers are integrated in 1310 bin markers, and 8 linkage groups are constructed, with a total map distance of 497.80 cM, an average marker distance of 0.38 cM, a maximum average distance between markers of 0.82 cM, a minimum average distance between markers of 0.29 cM, and respectively located on the 3rd and 4th linkage groups, the shortest linkage group is 21.15 cM, and the longest is 147.60 cM.

[0042] By using QTL-IciMapping4.1 software, the phenotype data and genetic map information of the population are analyzed and calculated by using the additive-perfect composite interval mapping method (ICIM-ADD), and QTL detection is carried out on 8 linkage groups, and the set LOD value is 2.5 or 2.0. Finally, two major QTL sites controlling the powder and brittle traits of lotus are obtained, one of which is located at 139.5 cM to 144.5 cM of the 1st linkage group, and the other is located at 112.5 cM to 115.5 cM of the 1st linkage group.

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

[0044] F2 population and two parents are detected by KASP detection primer group designed for SNP molecular marker

[0045] The young leaves of 194 offspring randomly selected from the F2 population were taken to obtain the genome DNA of lotus plants by CTAB extraction method. The KASP Primer mix was prepared and PCR amplification was carried out. The PCR amplification products were scanned by QuantStudio6Flex machine, and the amplification products were typed based on the different excitation and emission wavelengths of two fluorescence (FAM fluorescence and HEX fluorescence). According to the amplification results, when the KASP primer set for detecting SNP molecular marker Fc1.890 was used for detection, the samples aggregated on the X axis were the brittle genotype, the samples aggregated on the Y axis were the mealy genotype, and the samples in the middle were the heterozygous genotype; when the KASP primer set for detecting SNP molecular marker Fc1.255 was used for detection, the samples aggregated on the X axis were the mealy genotype, the samples aggregated on the Y axis were the brittle genotype, and the samples in the middle were the heterozygous genotype. When the KASP primer set for detecting SNP molecular marker Fc1.880 was used for detection, the samples aggregated on the X axis were the mealy genotype, the samples aggregated on the Y axis were the brittle genotype, and the samples in the middle were the heterozygous genotype; when the KASP primer set for detecting SNP molecular marker Fc1.252 was used for detection, the samples aggregated on the X axis were the brittle genotype, the samples aggregated on the Y axis were the mealy genotype, and the samples in the middle were the heterozygous genotype. The present application relates to acronym and key term definitions

[0046] QTL: quantitative trait locus.

[0047] SNP: single nucleotide polymorphism.

[0048] CTAB: cetyltrimethylammonium bromide.

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

[0050] Figure 1 A high-density genetic linkage map of lotus.

[0051] Figure 2 A linkage map for QTL positioning of lotus mealy-brittle trait control.

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

[0053] Figure 4The results of scanning the PCR amplification products by QuantStudio6 Flex machine for detecting the amplification results of the KASP primer set of SNP molecular marker Fc1.255 on samples; 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 hybrid genotypes.

[0054] Figure 5 The results of scanning the PCR amplification products by QuantStudio6 Flex machine for detecting the amplification results of the KASP primer set of SNP molecular marker Fc1.880 on samples; 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 hybrid genotypes.

[0055] Figure 6 The results of scanning the PCR amplification products by QuantStudio6 Flex machine for detecting the amplification results of the KASP primer set of SNP molecular marker Fc1.252 on samples; 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 hybrid genotypes. DETAILED DESCRIPTION

[0056] The advantages and features of the present application will become more apparent with the description of the specific embodiments. However, it should be understood that the described embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications or replacements all fall within the protection scope of the present application.

[0057] Example 1 Determination of main QTL candidate region controlling lotus powder brittleness and development of linkage markers by SLAF-seq method

[0058] 1. Construction of lotus powder brittleness segregation population

[0059] In this embodiment, the F2 genetic population was constructed with the lotus "E Lian 8" as the female parent and the lotus "E Lian 10" as the male parent. Among them, the female parent showed powdery, and the male parent showed brittle.

[0060] 2. Field identification of lotus powder brittleness phenotype

[0061] The powder brittleness traits of the parent and each F2 single plant were detected and counted.

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

[0063] 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.

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

[0065] (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.

[0066] (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.

[0067] (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).

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

[0069] 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.

[0070] 5. QTL positioning of lotus root crisps

[0071] 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 a LOD value set at 2.5 or 2.0. Two major QTL loci controlling the lotus pollen brittleness trait were ultimately obtained. One major QTL locus was located at 139.5 cM to 144.5 cM in linkage group 1; the other major QTL locus was located at 112.5 cM to 115.5 cM in linkage group 1. Figure 2 ).

[0072] 6. Development of a floury QTL interval marker

[0073] Based on the marker sequence of the above positioning interval, four SNP molecular markers closely linked to the two major QTL sites controlling the floury trait of lotus were determined; wherein the first SNP molecular marker is named Fc1.890, the allelic variation base is G / A, the alleles of the SNP site are all G, which is most likely to exhibit flouriness; the alleles of the SNP site are all A, which is most likely to exhibit flakiness. The second SNP molecular marker is named Fc1.255, the allelic variation base is A / G, the alleles of the SNP site are all A, which is most likely to exhibit flakiness; the alleles of the SNP site are all G, which is most likely to exhibit flouriness; the third SNP molecular marker is named Fc1.880, the allelic variation base is C / G, the alleles of the SNP site are all C, which is most likely to exhibit flakiness; the alleles of the SNP site are all G, which is most likely to exhibit flouriness; the fourth SNP molecular marker is named Fc1.252, the allelic variation base is G / A, the alleles of the SNP site are all G, which is most likely to exhibit flakiness; the alleles of the SNP site are all A, which is most likely to exhibit flouriness.

[0074] According to the mutation characteristics of the SNP, four sets of competitive allele-specific PCR primer pairs are designed, including forward primer 1, forward primer 2 and reverse primer; wherein the terminal of the forward primer is the allelic variation base G / A, A / G, C / G and G / A, respectively.

[0075] Among them, the nucleotide sequences of the forward primer 1, the forward primer 2 and the reverse primer for detecting the 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 for detecting 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 for detecting 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 for detecting 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] The above-mentioned forward primer is connected with a fluorescent label sequence at the 5' end, specifically, the FAM sequence is connected at the 5' end of the above-mentioned forward primer 1, the nucleotide sequence of the FAM sequence is: 5'- GAAGGTGACCAAGTTCATGCT-3' (SEQ ID No. 13); the HEX sequence is connected at the 5' end of the above-mentioned forward primer 2, the nucleotide sequence of the HEX sequence is: 5'- GAAGGTCGGAGTCAACGGATT-3' (SEQ ID No. 14).

[0104] Test Example 1: Detection of lotus F2 population and two parents by KASP detection primer

[0105] 1. Extraction of lotus genomic DNA

[0106] The F2 genetic population was constructed with the lotus "E lotus 8" as the female parent and the lotus "E lotus 10" as the male parent, wherein the female parent showed powdery and the male parent showed brittle.

[0107] The marker types and phenotypes of the 194 F2 offspring were detected, and the results are shown in Table 1.

[0108] Table 1: Marker types and powdery and brittle texture traits of 194 F2 offspring

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] The young leaves of the 194 offspring were taken to obtain the genomic DNA of the lotus plant 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 mentioned is the KASP primer set consisting of forward primer 1, forward primer 2 and reverse primer for detecting the SNP molecular marker screened in Example 1.

[0120] 3. PCR amplification

[0121] 3.1 The PCR reaction system is:

[0122] Table 3 PCR reaction system

[0123]

[0124] 3.2 The PCR reaction program is:

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

[0126] 4. Genotyping

[0127] The PCR amplification product is scanned by using QuantStudio6Flex machine, and the genotyping of the amplification product is realized based on the different excitation wavelength and emission wavelength of two fluorescence (FAM fluorescence and HEX fluorescence).

[0128] 5. Detection results

[0129] When the KASP primer set for detecting the SNP molecular marker Fc1.890 is used for detection, the samples aggregated on the X axis are the brittle genotype (the powder brittle phenotype is mainly brittle), the samples aggregated on the Y axis are the powdery genotype (the powder brittle phenotype is mainly powdery), and the samples in the middle are the heterozygous genotype (Fc1.890 is A / A); when the KASP primer set for detecting the SNP molecular marker Fc1.255 is used for detection, the samples aggregated on the X axis are the powdery genotype (the powder brittle phenotype is mainly powdery), the samples aggregated on the Y axis are the brittle genotype (the powder brittle phenotype is mainly brittle), and the samples in the middle are the heterozygous genotype (Fc1.255 is A / A); when the KASP primer set for detecting the SNP molecular marker Fc1.880 is used for detection, the samples aggregated on the X axis are the powdery genotype (the powder brittle phenotype is mainly powdery), the samples aggregated on the Y axis are the brittle genotype (the powder brittle phenotype is mainly brittle), and the samples in the middle are the heterozygous genotype (Fc1.880 is A / A). Figure 3 Figure 4 Figure 5 ​​); when the KASP primer set for detecting the SNP molecular marker Fc1.252 is used for detection, the samples aggregated on the X axis are the brittle genotype (the main brittle phenotype of which is brittle), the samples aggregated on the Y axis are the mealy genotype (the main mealy phenotype of which is mealy), and the samples in the middle are the hybrid genotype Figure 6

[0130] According to the detection results, it can be seen that the detection results of the four KASP primer sets provided by the application on the mealy-brittle genotypes of 194 F2 offspring are basically consistent with the mealy-brittle genotypes or phenotypes of the 194 F2 offspring, proving that the four KASP primer sets provided by the application can effectively detect the lotus mealy-brittle genotype or phenotype.​

Claims

1. A KASP primer set for detecting SNP molecular markers closely linked to a major QTL site for controlling the powder brittleness of lotus, each KASP primer set comprising a forward primer 1, a forward primer 2 and a reverse primer, and the nucleotide sequences are 5'-3'; characterized in that, The SNP molecular marker is four SNP molecular markers; wherein the allelic variation base of the first SNP molecular marker is G / A; the allelic variation base of the second SNP molecular marker is A / G; the allelic variation base of the third SNP molecular marker is C / G; and the allelic variation base of the fourth SNP molecular marker is G / A; the major QTL site for controlling the lotus powder brittleness trait is two, wherein one major QTL site is located at 139.5cM to 144.5cM of the first linkage group; and the other major QTL site is located at 112.5cM to 115.5cM of the first linkage group; The KASP primer group is selected from any one of the following (1)-(4) group primer groups: (1) the KASP primer group 1 consisting of the forward primer 1 and the forward primer 2 shown in SEQ ID No. 1, SEQ ID No. 2 and the reverse primer shown in SEQ ID No. 3; (2) the KASP primer group 2 consisting of the forward primer 1 and the forward primer 2 shown in SEQ ID No. 4, SEQ ID No. 5 and the reverse primer shown in SEQ ID No. 6; (3) the KASP primer group 3 consisting of the forward primer 1 and the forward primer 2 shown in SEQ ID No. 7, SEQ ID No. 8 and the reverse primer shown in SEQ ID No. 9; (4) the KASP primer group 4 consisting of the forward primer 1 and the forward primer 2 shown in SEQ ID No. 10, SEQ ID No. 11 and the reverse primer shown in SEQ ID No. 12; In the detection, the 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; the 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.

2. A PCR detection kit for identifying the Lotus powdery mildew trait, comprising: KASP detection primer group, KASP master mix and ddH2O; characterized in that the KASP detection primer group is the KASP primer group of claim 1.

3. The KASP detection primer group of claim 1 in the identification of lotus powder brittle genotype.

4. The PCR detection kit of claim 2 in the identification of lotus powder brittle genotype.

5. Use according to claim 3, characterized in that, including: (1) extracting the genomic DNA of the lotus to be detected; (2) taking the extracted genomic DNA of the lotus to be detected as the amplification template, adding the KASP primer group and the universal KASP master mix to establish the PCR amplification system for PCR amplification; (3) detecting the PCR amplification product in a fluorescence quantitative PCR instrument, reading the fluorescence intensity signal value, scanning the PCR amplification product, and typing the amplification product based on the different excitation wavelengths and emission wavelengths of FAM fluorescence and HEX fluorescence; wherein, when KASP primer group 1 is used for detection, if HEX fluorescence signal is obtained by scanning, the sample is the brittle genotype, if FAM fluorescence signal is obtained by scanning, the sample is the mealy genotype, and if the scanning result is that there are both HEX and FAM signals, the sample is the heterozygous genotype; when KASP primer group 2 is used for detection, if HEX fluorescence signal is obtained by scanning, the sample is the mealy genotype, if FAM fluorescence signal is obtained by scanning, the sample is the brittle genotype, and if the scanning result is that there are both HEX and FAM signals, the sample is the heterozygous genotype; when KASP primer group 3 is used for detection, if HEX fluorescence signal is obtained by scanning, the sample is the mealy genotype, if FAM fluorescence signal is obtained by scanning, the sample is the brittle genotype, and if the scanning result is that there are both HEX and FAM signals, the sample is the heterozygous genotype; when KASP primer group 4 is used for detection, if HEX fluorescence signal is obtained by scanning, the sample is the brittle genotype, if FAM fluorescence signal is obtained by scanning, the sample is the mealy genotype, and if the scanning result is that there are both HEX and FAM signals, the sample is the heterozygous genotype.

6. Use according to claim 5, characterized in that, In step (1), the extracted genome DNA of the lotus to be detected is diluted to 18-22 ng / μL, and then used as a template for PCR amplification; In step (2), the FAM sequence is connected to the 5' end of the forward primer 1, and the nucleotide sequence of the FAM sequence is represented by SEQ ID No. 13; the HEX sequence is connected to the 5' end of the forward primer 2, and the nucleotide sequence of the HEX sequence is represented by SEQ ID No. 14; In step (2), the reaction program of the PCR is: 94°C, 15 min; 94°C, 20 sec, 61-55°C, 60 sec, 10 cycles of decreasing 0.6°C each time; 94°C, 20 sec, 55°C, 60 sec, 26 cycles.

7. Use according to claim 5, characterized in that, In step (3), the PCR amplification product is scanned by using a QuantStudio6Flex machine.

Images