KASP molecular marker closely linked with sterile site S66 of temperate zone japonica and tropical zone japonica hybrid

Abstract

The invention provides a KASP molecular marker closely linked with a sterile site S66 of a hybrid of temperate zone japonica and tropical zone japonica. The KASP molecular marker comprises KASP 9-28 and KASP 9-29; a primer group of the molecular marker KASP9-28 comprises a forward primer 1 of the KASP9-28, a forward primer 2 of the KASP9-28 and a common reverse primer of the KASP9-28, and nucleotide sequences of the forward primer 1, the forward primer 2 and the common reverse primer of the KASP9-28 are SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in sequence; a primer group of the molecular marker KASP9-29 comprises a forward primer 1 of the KASP9-29, a forward primer 2 of the KASP9-29 and a reverse primer shared by the KASP9-29, and the nucleotide sequences of the forward primer 1, the forward primer 2 and the reverse primer shared by the KASP9-29 are SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 in sequence. The invention also provides a detection method of the molecular marker and application of the molecular marker in identification of a sterile site S66 of a hybrid of temperate zone japonica and tropical zone japonica. The KASP molecular marker disclosed by the invention can be used for improving the accuracy and the efficiency of detecting the sterile site S66 of the hybrid of temperate zone japonica and tropical zone japonica.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics, and particularly relates to a KASP molecular marker tightly linked to the sterility locus S66 of hybrids of temperate japonica and tropical japonica, as well as a detection method and application thereof. Background Art

[0002] Fully utilizing heterosis is one of the most important approaches to increasing rice yield. Utilizing heterosis has played a significant role in increasing yields in my country over the past 70 years of rice breeding. However, since the beginning of this century, rice yield growth has entered a bottleneck period. Due to the influence of hybrid sterility, parent selection has remained primarily based on indica-indica hybrid combinations. This narrow genetic base has led to severe varietal homogeneity, and distant heterosis between subspecies remains underutilized. Asian cultivated rice, the most important cultivated species in the AA genome of the genus Oryza, was initially divided into two subgroups: indica and japonica. However, with in-depth research in biochemistry, molecular biology, and genomics, it is generally believed that Asian cultivated rice can be further divided into five subgroups: indica, temperate japonica, tropical japonica, aus, and basmati. The rich genetic variation among these subgroups is a valuable source of intraspecific heterosis within cultivated rice. Temperate japonica rice is primarily distributed in temperate regions and high-altitude areas of East Asia, characterized by short, rounded seeds, short plants, and numerous tillers. Tropical japonica rice, on the other hand, is primarily found in South Asia, Southeast Asia, Africa, and the Americas. It exhibits tall plants, few tillers, large ears, and wide, flat seeds. It also exhibits good disease and drought resistance. Significant differences between the two subpopulations at the morphological, physiological, and genetic levels result in strong heterosis in the F1 hybrids between the two subpopulations. Exploiting and utilizing this heterosis to cultivate strong hybrid varieties is a key approach to achieving super-high-yield rice breeding. However, hybrid sterility, primarily due to pollen sterility or spikelet sterility, generally results in low seed set in F1 hybrids, limiting the effective utilization of heterosis. Therefore, large-scale and systematic identification of hybrid sterility loci between temperate japonica and tropical japonica rices, as well as in-depth investigation of their genetic patterns and molecular mechanisms, are urgently needed to overcome hybrid sterility.

[0003] Rice hybrid sterility is a quantitative trait controlled by multiple genes, and has been studied for more than 100 years. However, only about 40 QTLs or genes related to hybrid sterility of Asian cultivated rice have been reported (Ouyang et al. 2010; Xie et al. 2019; Wang et al. 2024), and only 7 hybrid sterility genes / loci have been cloned (Li Shunmei et al. 2024). The allelic and non-allelic interactions make hybrid sterility extremely complex. In addition, hybrid sterility is easily affected by the environment, which increases the difficulty of its research. With the in-depth understanding of hybrid sterility and the rapid development of SNP chip typing technology, hybrid sterility sites can be efficiently identified in a lower backcross generation, greatly improving the detection efficiency of sterile sites. Therefore, identifying hybrid sterility sites between temperate japonica and tropical japonica, and developing molecular markers closely linked to them, have important theoretical significance and application value for understanding the nature of hybrid sterility between subgroups and overcoming hybrid sterility. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a KASP molecular marker closely linked to the hybrid sterility site S66 between temperate japonica and tropical japonica, and a detection method and application thereof, which has high specificity and significant differences between parents, and can be efficiently used for detecting the hybrid sterility site S66 in hybrid combinations between temperate japonica and tropical japonica.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is: a KASP molecular marker closely linked to the hybrid sterility site S66 between temperate japonica and tropical japonica, the KASP molecular marker comprising KASP9-28 and KASP9-29; the primer set of the molecular marker KASP9-28 comprises KASP9-28 forward primer 1, KASP9-28 forward primer 2 and KASP9-28 common reverse primer; the nucleotide sequence of the KASP9-28 forward primer 1 is shown as SEQ ID NO. 1, the nucleotide sequence of the KASP9-28 forward primer 2 is shown as SEQ ID NO. 2, and the nucleotide sequence of the KASP9-28 common reverse primer is shown as SEQ ID NO. 3; the primer set of the molecular marker KASP9-29 comprises KASP9-29 forward primer 1, KASP9-29 forward primer 2 and KASP9-29 common reverse primer; the nucleotide sequence of the KASP9-29 forward primer 1 is shown as SEQ ID NO. 4, the nucleotide sequence of the KASP9-29 forward primer 2 is shown as SEQ ID NO. 5, and the nucleotide sequence of the KASP9-29 common reverse primer is shown as SEQ ID NO. 6.

[0006] The present invention also provides a method for detecting the KASP molecular marker tightly linked to the sterility locus S66 of the hybrid of temperate japonica and tropical japonica, which is: Extracting rice genomic DNA, performing PCR amplification on the extracted rice genomic DNA using a primer set of KASP9-28 and a primer set of KASP9-29 to obtain a PCR product; The PCR amplification reaction system is as follows: 50 ng of rice genomic DNA, 1.31 μL of kasp primer working solution, 0.07 μL of KASPmaster Mix, and sterile water to 5 μL; The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing / extension at 60°C to 55°C for 60 seconds, wherein gradient PCR at 60°C to 55°C is performed with the temperature decreasing by 0.5°C per cycle, for a total of 10 cycles; denaturation at 94°C for 20 seconds, annealing and extension at 55°C for 60 seconds, for a total of 28 to 34 cycles; and storage at 12°C. The kasp primer working solution includes: forward primer 1, forward primer 2, a common reverse primer and sterile water; the concentration of the forward primer 1 is 12 μmol / mL, the concentration of the forward primer 2 is 12 μmol / mL, and the concentration of the common reverse primer is 30 μmol / mL.

[0007] The present invention also provides the use of the KASP molecular marker tightly linked to the sterile locus S66 of the hybrid of temperate japonica and tropical japonica, and the use of the KASP molecular marker in identifying the sterile locus S66 of the hybrid of temperate japonica and tropical japonica.

[0008] Compared with the prior art, the present invention has the following advantages: Hybrid sterility is the root cause of the constraints on the utilization of hybrid vigor between temperate and tropical japonica rices. This study detected a new hybrid female sterility locus, S66, in the hybrid offspring of tropical japonica Moroberkan and temperate japonica Dianjing You 1. No other hybrid sterility loci have been reported in this region, providing important clues for studying the genetic patterns of hybrid sterility between temperate and tropical japonica rices.

[0009] 2. The present invention provides tightly linked KASP markers KASP9-28 (6796166 bp) and KASP9-29 (7965694 bp) for detecting the hybrid sterility locus S66 in hybrid combinations between temperate japonica and tropical japonica. KASP9-28 and KASP9-29 have high specificity and significant differences between their parents, and can be effectively used to detect the hybrid sterility locus S66 in hybrid combinations between temperate japonica and tropical japonica.

[0010] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the pollen and spikelet fertility distribution of the Dianjing You No. 1 / Moroberekan / 3 / Dianjing You No. 1 BC2F2 population in Example 1 of the present invention.

[0012] Figure 2 This is the introgression situation of the gene fragment of the Dianjing You No. 1 / Moroberekan / 3 / Dianjing You No. 1 BC2F2 population in Example 1 of the present invention.

[0013] Figure 3 This is the genetic linkage map of S66 in Example 1 of the present invention.

[0014] Figure 4 This is the genotyping diagram of the genetic segregation population of the KASP9-28 marker in Example 1 of the present invention.

[0015] Figure 5 This is the genotyping diagram of the genetic segregation population of the KASP9-29 marker in Example 1 of the present invention. DETAILED DESCRIPTION

[0016] Example 1 This example is the phenotypic analysis of the genetic population and the molecular mapping of S66.

[0017] The BC2F2 population of Dianjingyou 1 / Moroberekan / 3 / Dianjingyou 1 consisted of 232 plants. Pollen fertility segregated, but the majority of the plants were fertile, with 199 fertile plants and 33 semi-sterile plants. Spikelet fertility showed a continuous bimodal distribution, with peaks at 65% and 95% ( Figure 1 ), the ratio of spikelet fertile plants to semi-sterile plants conformed to the 1:2 segregation ratio (χ 2 = 0.26, P = 0.61), suggesting that spikelet fertility in this population is controlled by a single gene.

[0018] Using Illumina 6K gene chip for Bulked Segregant Analysis (BSA), it was found that the heterozygous introgression fragment of 5970596bp-6667280bp on chromosome 9 was associated with spikelet fertility ( Figure 2 ), Figure 2 a is the introgression of gene fragments in normal spikelet plants in the population (i.e., plants without S66 gene; Figure 2 b is the gene fragment introgression situation of spikelet semi-sterile plants in the population (that is, plants with S66), Figure 2The colors represent genotypes: green represents the homozygous band of Dianjing You No. 1; red represents the heterozygous band of the hybrid of the two parents; black represents the homozygous band of Moeroberekan. Based on the SNP information of the parents Dianjing You No. 1 and Moeroberekan in this interval, polymorphic SNP markers were developed. The developed SNP markers were then used to perform genotyping on the BC2F2 population using the KASP typing technology. Combining the phenotype and genotype of individual plants, genetic linkage analysis was used to find that spikelet fertility was closely linked to the SNP marker KASP9-2. The gene was eventually located at 6.3kb. The hybrid sterility locus between temperate japonica and tropical japonica was located on the long arm of rice chromosome 9 and was named S66 ( Figure 3 In this region, no loci controlling hybrid sterility between temperate japonica and tropical japonica have been reported. Therefore, S66 is a new locus controlling spikelet sterility in hybrids between temperate japonica and tropical japonica.

[0019] Example 2 Genotyping of hybrid sterility locus S66 by KASP molecular markers Based on the genotyping results of Moroberkan and Dianjing You 1 using the Rice 40K Microarray (Wuhan Shuanglvyuan Chuangxin Technology Research Institute Co., Ltd.) and the chromosomal location of the S66 locus, SNP information was converted into KASP (Kompetitive allele-specific PCR) markers based on the parental sequences. After interparental PCR amplification and population validation, two pairs of polymorphic KASP markers within the S66 region were identified: KASP9-28 and KASP9-29, corresponding to physical locations 6796166 bp to 7965694 bp. KASP markers KASP9-28 and KASP9-29 are closely linked to the S66 sterility locus in hybrids of temperate and tropical japonica rice.

[0020] (1) The primer set for the molecular marker KASP9-28 includes KASP9-28 forward primer 1, KASP9-28 forward primer 2, and KASP9-28 common reverse primer; The nucleotide sequence of the molecular marker KASP9-28 forward primer 1 is shown in SEQ ID NO. 1, the nucleotide sequence of the KASP9-28 forward primer 2 is shown in SEQ ID NO. 2, and the nucleotide sequence of the KASP9-28 common reverse primer is shown in SEQ ID NO. 3; KASP9-28 forward primer 1 and KASP9-28 forward primer 2 were connected to the FAM and HEX adapter sequences at the 5' end, respectively, and used as KASP forward primers to detect the polymorphism of the S66 tightly linked marker and the single nucleotide marker (SNP) of KASP9-28, respectively. Among them, the allele of Dianjingyou No. 1 type was connected to the FAM fluorescent group, and the allele of Moroberkan type was connected to the HEX fluorescent group.

[0021] (2) The primer set for the molecular marker KASP9-29 includes KASP9-29 forward primer 1, KASP9-29 forward primer 2, and KASP9-29 common reverse primer; The nucleotide sequence of the KASP9-29 forward primer 1 is shown in SEQ ID NO.4, the nucleotide sequence of the KASP9-29 forward primer 2 is shown in SEQ ID NO.5, and the nucleotide sequence of the KASP9-29 common reverse primer is shown in SEQ ID NO.6.

[0022] KASP9-29 forward primer 1 and KASP9-29 forward primer 2 were connected to the FAM and HEX adapter sequences at the 5' end, respectively, and used as KASP forward primers to detect the major effect S66 tightly linked marker, the polymorphism of the single nucleotide (SNP) site of S66, among which the allele of Dianjingyou No. 1 type was connected to the FAM fluorescent group, and the allele of Moroberkan type was connected to the HEX fluorescent group.

[0023] (3) Implementation of KASP marking The method for detecting the hybrid sterility site S66 of temperate japonica and tropical japonica using the KASP molecular marker closely linked to the above-mentioned site is as follows: Extracting rice genomic DNA, performing PCR amplification on the extracted rice genomic DNA using a primer set of KASP9-28 and a primer set of KASP9-29 to obtain a PCR product; The PCR amplification reaction system is as follows: 50 ng of rice genomic DNA, 1.31 μL of kasp primer working solution, 0.07 μL of KASPmaster Mix, and sterile water to 5 μL; The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing / extension at 60°C to 55°C for 60 seconds, wherein gradient PCR at 60°C to 55°C is performed with the temperature decreasing by 0.5°C per cycle, for a total of 10 cycles; denaturation at 94°C for 20 seconds, annealing and extension at 55°C for 60 seconds, for a total of 28 to 34 cycles; and storage at 12°C. The kasp primer working solution includes: forward primer 1, forward primer 2, a common reverse primer and sterile water; the concentration of the forward primer 1 is 12 μmol / mL, the concentration of the forward primer 2 is 12 μmol / mL, and the concentration of the common reverse primer is 30 μmol / mL.

[0024] After the PCR amplification cycle is complete, the reaction plate is dried and cooled to room temperature. The fluorescence signal is then read using an LGC instrument at 37°C for 1 minute.

[0025] The PCR amplification products were analyzed and genotyped to identify the hybrid sterility locus S66 between temperate japonica and tropical japonica. The specific typing standard is: if the genotype of the SNP locus is homozygous, only one fluorescent signal will be generated, HEX type fluorescence will be displayed as red (Moroberekan), FAM type fluorescence will be displayed as blue (Yunnan Jingyou No. 1), and if the SNP locus is heterozygous, the result will be a green fluorescent signal (genotype heterozygous); the blank control (NTC) fluorescence will be displayed as black ( Figure 4-5 ).

[0026] In summary, the KASP molecular markers KASP9-28 (6796166 bp) and KASP9-29 (7965694 bp) for detecting the tightly linked sterility locus S66 in hybrids of temperate japonica and tropical japonica of the present invention have high specificity, obvious differences between the parents, and can be effectively used for the detection of the sterility locus S66 in hybrids of temperate japonica and tropical japonica.

[0027] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A KASP molecular marker tightly linked to the sterility locus S66 in hybrids of temperate japonica and tropical japonica, characterized in that: The KASP molecular markers include KASP9-28 and KASP9-29; the primer set for the molecular marker KASP9-28 includes KASP9-28 forward primer 1, KASP9-28 forward primer 2 and KASP9-28 common reverse primer; The nucleotide sequence of the KASP9-28 forward primer 1 is shown in SEQ ID NO. 1, the nucleotide sequence of the KASP9-28 forward primer 2 is shown in SEQ ID NO. 2, and the nucleotide sequence of the KASP9-28 common reverse primer is shown in SEQ ID NO. 3; The primer set of the molecular marker KASP9-29 includes KASP9-29 forward primer 1, KASP9-29 forward primer 2 and KASP9-29 common reverse primer; The nucleotide sequence of the KASP9-29 forward primer 1 is shown in SEQ ID NO.4, the nucleotide sequence of the KASP9-29 forward primer 2 is shown in SEQ ID NO.5, and the nucleotide sequence of the KASP9-29 common reverse primer is shown in SEQ ID NO.

6.

2. A method for detecting the hybrid sterility locus S66 of temperate japonica and tropical japonica using the KASP molecular marker tightly linked to the hybrid sterility locus S66 of temperate japonica and tropical japonica according to claim 1, characterized in that: The method is: Extracting rice genomic DNA, performing PCR amplification on the extracted rice genomic DNA using a primer set of KASP9-28 and a primer set of KASP9-29 to obtain a PCR product; The PCR amplification reaction system is as follows: 50 ng of rice genomic DNA, 1.31 μL of kasp primer working solution, 0.07 μL of KASPmaster Mix, and sterile water to 5 μL; The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing / extension at 60°C to 55°C for 60 seconds, wherein gradient PCR at 60°C to 55°C is performed with the temperature decreasing by 0.5°C per cycle, for a total of 10 cycles; denaturation at 94°C for 20 seconds, annealing and extension at 55°C for 60 seconds, for a total of 28 to 34 cycles; and storage at 12°C. The kasp primer working solution includes: forward primer 1, forward primer 2, a common reverse primer and sterile water; the concentration of the forward primer 1 is 12 μmol / mL, the concentration of the forward primer 2 is 12 μmol / mL, and the concentration of the common reverse primer is 30 μmol / mL.

3. A use of the KASP molecular marker tightly linked to the sterility locus S66 in hybrids of temperate japonica and tropical japonica as claimed in claim 1, characterized in that: The application of the KASP molecular marker in the identification of the sterility locus S66 in hybrids of temperate japonica and tropical japonica.

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