Core KASP molecular marker combination for sweet potato variety identification and application
By designing core KASP molecular marker combinations for sweet potato varieties and combining KASP technology with primer combinations, the problem of low identification rate in sweet potato variety identification has been solved, achieving efficient and accurate variety identification and management.
Patent Information
- Application Number
- CN202511500529.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-20
- Publication Date
- 2025-11-21
AI Technical Summary
Existing technologies have problems with low recognition accuracy and low identification efficiency in sweet potato variety identification. In particular, because sweet potatoes are hexaploid crops and have a large number of homologous sequences, DNA molecular marker identification is difficult.
A core KASP molecular marker combo for sweet potato variety identification was designed, including SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18, combined with supplementary molecular markers SpKASP2, 6, 9, 10, 11, 12 and 15. SNP sites were detected using KASP technology, and efficient identification was achieved using primer combos.
It has enabled efficient and accurate identification of sweet potato varieties, standardized variety management, enriched the application of molecular markers, and has both theoretical significance and practical application value.
Smart Images
Figure CN120989296A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the sweet potato breeding technical field, especially to a core KASP molecular marker combination for identifying sweet potato varieties and application thereof. BACKGROUND
[0002] Sweet potato is an important food, feed and energy crop, and is widely planted in more than 100 countries and regions in the world. Since sweet potato is a vegetative propagation crop, it is widely cultivated in various regions using vine cultivation. Especially since the middle of the last century, the speed of germplasm resource circulation has accelerated, which has brought the problems of "same name different species" and "same species different names" of sweet potato, increasing the difficulty of identifying the identity of sweet potato.
[0003] DNA molecular markers have been widely used in the authenticity identification of crop varieties. Sweet potato is a hexaploid crop and is an allohexaploid. The existence of a large number of homologous sequences brings great challenges to the DNA molecular marker identification of sweet potato. Commonly used DNA molecular markers mainly include RFLP, RAPD, AFLP, SSR, SNP and InDel, etc. In sweet potato, early SSR molecular markers were mainly used to identify sweet potato germplasm varieties, but the specificity was poor and the recognition degree was not high. Compared with traditional SSR markers, SNP markers have higher throughput, more accurate typing and are more convenient for automatic recognition, and have obvious advantages. There are many ways to identify SNP, including CAPS (cleaved amplified polymorphic sequences), AS-PCR (allele-specific PCR), genome sequencing, gene chip, etc. The competitive allele-specific polymerase chain reaction (KASP) technology is widely used for SNP identification due to its high efficiency, flexibility, accuracy and low cost, and is further used for the construction of molecular identity cards, which provides technical support for the identity identification and new variety protection of crops. Although previous studies have made some research on the development of KASP markers in sweet potato, there is still a lot of room for optimization and improvement of identification efficiency. SUMMARY
[0004] The purpose of the present application is to provide a core KASP molecular marker combination for identifying sweet potato varieties and application thereof, which can regulate the scientific and standardized management of sweet potato varieties and enrich the molecular markers for identifying sweet potato varieties.
[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a core KASP molecular marker combination for identifying sweet potato varieties, which comprises SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18; The SpKASP1 is located at position 23805905 of chromosome 2; The SpKASP3 is located at 15601719 of chromosome 4; The SpKASP4 is located at 27552209 of chromosome 6; The SpKASP5 is located at 27942438 of chromosome 6; The SpKASP7 is located at 21030365 of chromosome 7; The SpKASP8 is located at 24173507 of chromosome 7; The SpKASP13 is located at 8624205 of chromosome 8; The SpKASP14 is located at 4063242 of chromosome 9; The SpKASP16 is located at 29617800 of chromosome 11; The SpKASP17 is located at 5694324 of chromosome 12; The SpKASP18 is located at 3422717 of chromosome 13.
[0006] As preferred, the molecular marker combination further comprises a supplementary molecular marker; the supplementary molecular marker comprises SpKASP2, 6, 9, 10, 11, 12, 15; The SpKASP2 is located at 581024 of chromosome 3; The SpKASP6 is located at 12249986 of chromosome 7; The SpKASP9 is located at 24173584 of chromosome 7; The SpKASP10 is located at 3299441 of chromosome 8; The SpKASP11 is located at 5689829 of chromosome 8; The SpKASP12 is located at 7548404 of chromosome 8; The SpKASP15 is located at 28687214 of chromosome 11.
[0007] The application further provides application of the molecular marker combination in identification and distinction of sweet potato variety resources.
[0008] The application further provides a primer combination for detecting the molecular marker combination, wherein the primer combination comprises SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18 primer groups. The SpKASP1 primer group comprises SpKASP1-F1, SpKASP1-F2 and SpKASP1-R; the SpKASP3 primer set comprises SpKASP3-F1, SpKASP3-F2 and SpKASP3-R; the SpKASP4 primer set comprises SpKASP4-F1, SpKASP4-F2 and SpKASP4-R; the SpKASP5 primer set comprises SpKASP5-F1, SpKASP5-F2 and SpKASP5-R; the SpKASP7 primer set comprises SpKASP7-F1, SpKASP7-F2 and SpKASP7-R; the SpKASP8 primer set comprises SpKASP8-F1, SpKASP8-F2 and SpKASP8-R; the SpKASP13 primer set comprises SpKASP13-F1, SpKASP13-F2 and SpKASP13-R; the SpKASP14 primer set comprises SpKASP14-F1, SpKASP14-F2 and SpKASP14-R; the SpKASP16 primer set comprises SpKASP16-F1, SpKASP16-F2 and SpKASP16-R; the SpKASP17 primer set comprises SpKASP17-F1, SpKASP17-F2 and SpKASP17-R; the SpKASP18 primer set comprises SpKASP18-F1, SpKASP18-F2 and SpKASP18-R; the nucleotide sequence of SpKASP1-F1 is shown as SEQ ID NO. 1, the nucleotide sequence of SpKASP1-F2 is shown as SEQ ID NO. 2, and the nucleotide sequence of SpKASP1-R is shown as SEQ ID NO. 3; the nucleotide sequence of SpKASP3-F1 is shown as SEQ ID NO. 4, the nucleotide sequence of SpKASP3-F2 is shown as SEQ ID NO. 5, and the nucleotide sequence of SpKASP3-R is shown as SEQ ID NO. 6; the nucleotide sequence of SpKASP4-F1 is shown as SEQ ID NO. 7, the nucleotide sequence of SpKASP4-F2 is shown as SEQ ID NO. 8, and the nucleotide sequence of SpKASP4-R is shown as SEQ ID NO. 9; The nucleotide sequence of the SpKASP5-F1 is shown as SEQ ID NO. 10, the nucleotide sequence of the SpKASP5-F2 is shown as SEQ ID NO. 11, and the nucleotide sequence of the SpKASP5-R is shown as SEQ ID NO. 12; The nucleotide sequence of the SpKASP7-F1 is shown as SEQ ID NO. 13, the nucleotide sequence of the SpKASP7-F2 is shown as SEQ ID NO. 14, and the nucleotide sequence of the SpKASP7-R is shown as SEQ ID NO. 15; The nucleotide sequence of the SpKASP8-F1 is shown as SEQ ID NO. 16, the nucleotide sequence of the SpKASP8-F2 is shown as SEQ ID NO. 17, and the nucleotide sequence of the SpKASP8-R is shown as SEQ ID NO. 18; The nucleotide sequence of the SpKASP13-F1 is shown as SEQ ID NO. 19, the nucleotide sequence of the SpKASP13-F2 is shown as SEQ ID NO. 20, and the nucleotide sequence of the SpKASP13-R is shown as SEQ ID NO. 21; The nucleotide sequence of the SpKASP14-F1 is shown as SEQ ID NO. 22, the nucleotide sequence of the SpKASP14-F2 is shown as SEQ ID NO. 23, and the nucleotide sequence of the SpKASP14-R is shown as SEQ ID NO. 24; The nucleotide sequence of the SpKASP16-F1 is shown as SEQ ID NO. 25, the nucleotide sequence of the SpKASP16-F2 is shown as SEQ ID NO. 26, and the nucleotide sequence of the SpKASP16-R is shown as SEQ ID NO. 27; The nucleotide sequence of the SpKASP17-F1 is shown as SEQ ID NO. 28, the nucleotide sequence of the SpKASP17-F2 is shown as SEQ ID NO. 29, and the nucleotide sequence of the SpKASP17-R is shown as SEQ ID NO. 30; The nucleotide sequence of the SpKASP18-F1 is shown as SEQ ID NO. 31, the nucleotide sequence of the SpKASP18-F2 is shown as SEQ ID NO. 32, and the nucleotide sequence of the SpKASP18-R is shown as SEQ ID NO. 33.
[0009] The application also provides a primer combination for detecting the supplementary molecular marker, wherein the primer combination comprises SpKASP2, 6, 9, 10, 11, 12, 15 primer groups; The SpKASP2 primer group comprises SpKASP2-F1, SpKASP2-F2 and SpKASP2-R; The SpKASP6 primer group comprises SpKASP6-F1, SpKASP6-F3 and SpKASP6-R; The SpKASP9 primer group comprises SpKASP9-F1, SpKASP9-F2 and SpKASP9-R; The SpKASP10 primer group comprises SpKASP10-F1, SpKASP10-F2 and SpKASP15-R; The SpKASP11 primer group comprises SpKASP11-F1, SpKASP11-F2 and SpKASP11-R; The SpKASP12 primer group comprises SpKASP12-F1, SpKASP12-F2 and SpKASP12-R; The SpKASP15 primer group comprises SpKASP15-F1, SpKASP15-F2 and SpKASP15-R; The nucleotide sequence of the SpKASP2-F1 is shown as SEQ ID NO. 34, the nucleotide sequence of the SpKASP2-F2 is shown as SEQ ID NO. 35, and the nucleotide sequence of the SpKASP2-R is shown as SEQ ID NO. 36; The nucleotide sequence of the SpKASP6-F1 is shown as SEQ ID NO. 37, the nucleotide sequence of the SpKASP6-F2 is shown as SEQ ID NO. 38, and the nucleotide sequence of the SpKASP6-R is shown as SEQ ID NO. 39; The nucleotide sequence of the SpKASP9-F1 is shown as SEQ ID NO. 40, the nucleotide sequence of the SpKASP9-F2 is shown as SEQ ID NO. 41, and the nucleotide sequence of the SpKASP9-R is shown as SEQ ID NO. 42; The nucleotide sequence of the SpKASP10-F1 is shown as SEQ ID NO. 43, the nucleotide sequence of the SpKASP10-F2 is shown as SEQ ID NO. 44, and the nucleotide sequence of the SpKASP10-R is shown as SEQ ID NO. 45; The nucleotide sequence of the SpKASP11-F1 is shown as SEQ ID NO. 46, the nucleotide sequence of the SpKASP11-F2 is shown as SEQ ID NO. 47, and the nucleotide sequence of the SpKASP11-R is shown as SEQ ID NO. 48; The nucleotide sequence of the SpKASP12-F1 is shown as SEQ ID NO. 49, the nucleotide sequence of the SpKASP12-F2 is shown as SEQ ID NO. 50, and the nucleotide sequence of the SpKASP12-R is shown as SEQ ID NO. 51; The nucleotide sequence of the SpKASP15-F1 is shown as SEQ ID NO. 52, the nucleotide sequence of the SpKASP15-F2 is shown as SEQ ID NO. 53, and the nucleotide sequence of the SpKASP15-R is shown as SEQ ID NO. 54.
[0010] The present application provides a core KASP molecular marker combination for sweet potato variety identification and application, the molecular marker combination comprises SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18. The present application is based on the published resequencing data of sweet potato, and the SNP sites suitable for developing KASP markers are mined, the KASP marker combination is designed to distinguish different sweet potato varieties, and the least KASP markers capable of distinguishing most sweet potato varieties are selected as the core KASP markers for sweet potato variety identification, and the rest of the KASP markers are supplemented. The ultimate goal is to regulate the scientific and standardized management of sweet potato varieties on the one hand, and to enrich the molecular markers for sweet potato variety identification on the other hand, which has certain theoretical significance and application value in sweet potato variety identification, germplasm resource relationship analysis and population division. BRIEF DESCRIPTION OF DRAWINGS
[0011] Figure 1 It is the detection results of 18 KASP molecular markers on 195 sweet potato variety resources, in which red and blue dots represent two different homozygous genotypes respectively, green dots represent heterozygous genotypes, black represents control, and cross represents undetected.
[0012] Figure 2 It is the analysis results of 18 KASP molecular markers, and it is found that 11 KASP molecular markers can be used to distinguish all 195 sweet potato variety resources.
[0013] Figure 3 It is a molecular identity card constructed by using 11 KASP molecular markers for 195 sweet potato variety resources, the vertical coordinate is 11 KASP markers, the horizontal coordinate represents 195 sweet potato variety resources, and different colors represent different genotypes detected. DETAILED DESCRIPTION
[0014] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.
[0015] Example 1
[0016] Based on the SNP typing data of 314 sweet potato germplasm resources in the public database, a total of 4,599,509 SNPs were included. First, the SNPs in the 50 bp sequence upstream and downstream of the chromosome were preliminarily filtered; then the SNPs with an average sequencing depth of not less than 5, a quality value of not less than 30, a minimum integrity of not less than 0.9, a minimum allele frequency (MAF) of not less than 0.05, and being a double allele were reserved; then the 100 bp sequence upstream and downstream was intercepted and compared with the reference genome by BLAST, and the SNPs with multi-site comparison were removed; then the markers with a polymorphism information content (PIC) greater than 0.35 were screened. A total of 140 SNP sites were preliminarily selected for the next round of screening. Since SNP mining based on second-generation sequencing technology has certain false positives, in order to exclude false positives, the 200 bp sequence upstream and downstream of the above SNP sites was intercepted, and the amplification primers were designed. The DNA of 3 samples (Yushu 99, Yuhongxinshu 8 and Yuhongxinshu 4) was selected as the template for PCR amplification, and the non-specific amplification was excluded, leaving 79 SNP sites. The amplification products of the 79 pairs of primers were subjected to first-generation sequencing to verify the authenticity of the SNP sites. Finally, 18 SNP sites were obtained KASP markers (primer sequences are shown in Table 1).
[0017] Table 1
[0018] Further, the above 18 KASP molecular markers were used to detect 195 sweet potato variety resources, which can completely distinguish the 195 sweet potato variety resources (see Table 2).
[0019] Table 2
[0020] Example 2 Typing of 195 sweet potato variety resources using 18 KASP markers
[0021] Firstly, the DNA of 195 sweet potato germplasm resources was extracted by using cetyltrimethylammonium bromide (CTAB) method, and the quality and concentration of the DNA were determined by using ultraviolet spectrophotometer. Then, the DNA solution was diluted to 50 ng μL-1 by using ultrapure water and stored at -20℃ for use.
[0022] Further, the reaction system was prepared according to the following ratio: DNA template 1 μL (concentration of 30 ng / μL), 2×KASP master mix 2.5 μL, KASP Assay Mix (F1 primer concentration of 5 μmol / L, F2 primer concentration of 5 μmol / L, R primer of 15 μmol / L) 0.125 μL, and ddH2O to a total reaction system of 5 μL. The template added in the negative control (NTC) reaction system was ddH2O.
[0023] The above mixture was placed in a polymerase chain reaction (PCR) instrument, and the program was set as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 55℃ annealing for 1 min, a total of 10 cycles; third step 95℃ denaturation for 20 s, 55℃ for 1 min, a total of 35 cycles; 25℃ for 30 s. The PCR product was scanned and analyzed by QuantStudio Real Time PCR instrument. Finally, the genotype of each sweet potato germplasm resource at 18 SNP loci was obtained.
[0024] Example 3
[0025] Further analysis found that among the 18 KASP molecular markers, 11 of them could completely distinguish 195 sweet potato germplasm resources, i.e. using the 11 KASP molecular markers as a combination (SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18), a molecular identity card was constructed for 195 sweet potato germplasm resources. In summary, the 11 sweet potato KASP molecular markers can be used as core molecular markers for distinguishing and identifying sweet potato germplasm resources. When the 11 sweet potato KASP molecular markers cannot distinguish sweet potato germplasm resources, the other 7 molecular markers (SpKASP2, 6, 9, 10, 11, 12, 15) can be used as supplements.
[0026] From the above examples, the application provides a core KASP molecular marker combination for sweet potato variety identification and application, the molecular marker combination comprises SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18. The application is based on the published resequencing data of sweet potato, mines the SNP sites suitable for developing KASP markers, designs a KASP marker combination to distinguish different sweet potato varieties, and selects the least KASP markers capable of distinguishing most sweet potato varieties as the core KASP markers for sweet potato variety identification, and the rest of the KASP markers as supplements. The ultimate goal is to regulate the scientific and standardized management of sweet potato varieties on the one hand, and to enrich the molecular markers for sweet potato variety identification on the other hand, which has certain theoretical significance and application value in sweet potato variety identification, germplasm resource genetic relationship analysis and population division.
[0027] The above only describes the preferred embodiments of the application, and it should be noted that for those skilled in the art, without departing from the principles of the application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the application.
Claims
1. A core KASP molecular marker combination for sweet potato variety identification, characterized in that, The molecular marker combination comprises SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18; The SpKASP1 is located at position 23805905 of chromosome 2; The SpKASP3 is located at position 15601719 of chromosome 4; The SpKASP4 is located at position 27552209 of chromosome 6; The SpKASP5 is located at position 27942438 of chromosome 6; The SpKASP7 is located at position 21030365 of chromosome 7; The SpKASP8 is located at position 24173507 of chromosome 7; The SpKASP13 is located at position 8624205 of chromosome 8; The SpKASP14 is located at position 4063242 of chromosome 9; The SpKASP16 is located at position 29617800 of chromosome 11; The SpKASP17 is located at position 5694324 of chromosome 12; The SpKASP18 is located at position 3422717 of chromosome 13.
2. The KASP molecular marker combination according to claim 1, characterized in that, The molecular marker combination further comprises supplementary molecular markers; the supplementary molecular markers comprise SpKASP2, 6, 9, 10, 11, 12, 15; The SpKASP2 is located at position 581024 of chromosome 3; The SpKASP6 is located at position 12249986 of chromosome 7; The SpKASP9 is located at position 24173584 of chromosome 7; The SpKASP10 is located at position 3299441 of chromosome 8; The SpKASP11 is located at position 5689829 of chromosome 8; The SpKASP12 is located at position 7548404 of chromosome 8; The SpKASP15 is located at position 28687214 of chromosome 11.
3. The molecular marker combination of claim 1 or 2 in the identification and differentiation of sweet potato variety resources.
4. A primer combination for detecting the molecular marker combination of claim 1, characterized by, The primer combination comprises SpKASP1, 3, 4, 5, 7, 8, 13, 14, 16, 17 and 18 primer groups; The SpKASP1 primer group comprises SpKASP1-F1, SpKASP1-F2 and SpKASP1-R; The SpKASP3 primer group comprises SpKASP3-F1, SpKASP3-F2 and SpKASP3-R; The SpKASP4 primer group comprises SpKASP4-F1, SpKASP4-F2 and SpKASP4-R; The SpKASP5 primer group comprises SpKASP5-F1, SpKASP5-F2 and SpKASP5-R; The SpKASP7 primer group comprises SpKASP7-F1, SpKASP7-F2 and SpKASP7-R; The SpKASP8 primer group comprises SpKASP8-F1, SpKASP8-F2 and SpKASP8-R; The SpKASP13 primer set comprises SpKASP13-F1, SpKASP13-F2 and SpKASP13-R; The SpKASP14 primer set comprises SpKASP14-F1, SpKASP14-F2 and SpKASP14-R; The SpKASP16 primer set comprises SpKASP16-F1, SpKASP16-F2 and SpKASP16-R; The SpKASP17 primer set comprises SpKASP17-F1, SpKASP17-F2 and SpKASP17-R; The SpKASP18 primer set comprises SpKASP18-F1, SpKASP18-F2 and SpKASP18-R; The nucleotide sequence of SpKASP1-F1 is shown as SEQ ID NO. 1, the nucleotide sequence of SpKASP1-F2 is shown as SEQ ID NO. 2, and the nucleotide sequence of SpKASP1-R is shown as SEQ ID NO. 3; The nucleotide sequence of SpKASP3-F1 is shown as SEQ ID NO. 4, the nucleotide sequence of SpKASP3-F2 is shown as SEQ ID NO. 5, and the nucleotide sequence of SpKASP3-R is shown as SEQ ID NO. 6; The nucleotide sequence of SpKASP4-F1 is shown as SEQ ID NO. 7, the nucleotide sequence of SpKASP4-F2 is shown as SEQ ID NO. 8, and the nucleotide sequence of SpKASP4-R is shown as SEQ ID NO. 9; The nucleotide sequence of SpKASP5-F1 is shown as SEQ ID NO. 10, the nucleotide sequence of SpKASP5-F2 is shown as SEQ ID NO. 11, and the nucleotide sequence of SpKASP5-R is shown as SEQ ID NO. 12; The nucleotide sequence of SpKASP7-F1 is shown as SEQ ID NO. 13, the nucleotide sequence of SpKASP7-F2 is shown as SEQ ID NO. 14, and the nucleotide sequence of SpKASP7-R is shown as SEQ ID NO. 15; The nucleotide sequence of SpKASP8-F1 is shown as SEQ ID NO. 16, the nucleotide sequence of SpKASP8-F2 is shown as SEQ ID NO. 17, and the nucleotide sequence of SpKASP8-R is shown as SEQ ID NO. 18; The nucleotide sequence of SpKASP13-F1 is shown as SEQ ID NO. 19, the nucleotide sequence of SpKASP13-F2 is shown as SEQ ID NO. 20, and the nucleotide sequence of SpKASP13-R is shown as SEQ ID NO. 21; The nucleotide sequence of the SpKASP14-F1 is shown as SEQ ID NO. 22, the nucleotide sequence of the SpKASP14-F2 is shown as SEQ ID NO. 23, and the nucleotide sequence of the SpKASP14-R is shown as SEQ ID NO. 24; The nucleotide sequence of the SpKASP16-F1 is shown as SEQ ID NO. 25, the nucleotide sequence of the SpKASP16-F2 is shown as SEQ ID NO. 26, and the nucleotide sequence of the SpKASP16-R is shown as SEQ ID NO. 27; The nucleotide sequence of the SpKASP17-F1 is shown as SEQ ID NO. 28, the nucleotide sequence of the SpKASP17-F2 is shown as SEQ ID NO. 29, and the nucleotide sequence of the SpKASP17-R is shown as SEQ ID NO. 30; The nucleotide sequence of the SpKASP18-F1 is shown as SEQ ID NO. 31, the nucleotide sequence of the SpKASP18-F2 is shown as SEQ ID NO. 32, and the nucleotide sequence of the SpKASP18-R is shown as SEQ ID NO.
33.
5. A primer combination for detecting the supplementary molecular marker of claim 2, characterized in that, The primer combination comprises SpKASP2, 6, 9, 10, 11, 12, 15 primer groups; The SpKASP2 primer group comprises SpKASP2-F1, SpKASP2-F2 and SpKASP2-R; The SpKASP6 primer group comprises SpKASP6-F1, SpKASP6-F3 and SpKASP6-R; The SpKASP9 primer group comprises SpKASP9-F1, SpKASP9-F2 and SpKASP9-R; The SpKASP10 primer group comprises SpKASP10-F1, SpKASP10-F2 and SpKASP15-R; The SpKASP11 primer group comprises SpKASP11-F1, SpKASP11-F2 and SpKASP11-R; The SpKASP12 primer group comprises SpKASP12-F1, SpKASP12-F2 and SpKASP12-R; The SpKASP15 primer group comprises SpKASP15-F1, SpKASP15-F2 and SpKASP15-R; The nucleotide sequence of the SpKASP2-F1 is shown as SEQ ID NO. 34, the nucleotide sequence of the SpKASP2-F2 is shown as SEQ ID NO. 35, and the nucleotide sequence of the SpKASP2-R is shown as SEQ ID NO. 36; The nucleotide sequence of the SpKASP6-F1 is shown as SEQ ID NO. 37, the nucleotide sequence of the SpKASP6-F2 is shown as SEQ ID NO. 38, and the nucleotide sequence of the SpKASP6-R is shown as SEQ ID NO. 39; The nucleotide sequence of the SpKASP9-F1 is shown as SEQ ID NO. 40, the nucleotide sequence of the SpKASP9-F2 is shown as SEQ ID NO. 41, and the nucleotide sequence of the SpKASP9-R is shown as SEQ ID NO. 42; The nucleotide sequence of the SpKASP10-F1 is shown as SEQ ID NO. 43, the nucleotide sequence of the SpKASP10-F2 is shown as SEQ ID NO. 44, and the nucleotide sequence of the SpKASP10-R is shown as SEQ ID NO. 45; The nucleotide sequence of the SpKASP11-F1 is shown as SEQ ID NO. 46, the nucleotide sequence of the SpKASP11-F2 is shown as SEQ ID NO. 47, and the nucleotide sequence of the SpKASP11-R is shown as SEQ ID NO. 48; The nucleotide sequence of the SpKASP12-F1 is shown as SEQ ID NO. 49, the nucleotide sequence of the SpKASP12-F2 is shown as SEQ ID NO. 50, and the nucleotide sequence of the SpKASP12-R is shown as SEQ ID NO. 51; The nucleotide sequence of the SpKASP15-F1 is shown as SEQ ID NO. 52, the nucleotide sequence of the SpKASP15-F2 is shown as SEQ ID NO. 53, and the nucleotide sequence of the SpKASP15-R is shown as SEQ ID NO. 54.