KASP molecular markers tightly linked to the S66 site of hybrid sterility in temperate and tropical japonica rice

Abstract

This invention provides a KASP molecular marker tightly linked to the S66 hybrid sterility site in temperate and tropical japonica rice, comprising KASP9-28 and KASP9-29. The primer set for molecular marker KASP9-28 includes KASP9-28 forward primer 1, KASP9-28 forward primer 2, and a shared reverse primer, with nucleotide sequences of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The primer set for molecular marker KASP9-29 includes KASP9-29 forward primer 1, KASP9-29 forward primer 2, and a shared reverse primer, with nucleotide sequences of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. This invention also provides a method for detecting this molecular marker and its application in identifying the S66 hybrid sterility site in temperate and tropical japonica rice. The KASP molecular marker of this invention can improve the accuracy and efficiency of detecting the S66 sterility locus in temperate and tropical japonica hybrids.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics technology, specifically relating to a KASP molecular marker tightly linked to the S66 sterility locus in temperate and tropical japonica hybrids, and its detection method and application. Background Technology

[0002] Making full use of heterosis is one of the most important ways to increase rice yield. Due to the influence of hybrid sterility, the selection of parents still mainly relies on indica-indica hybrid combinations. The narrow genetic base leads to serious homogenization of varieties, and the heterosis of distant relatives among subspecies has not yet been fully utilized. Asian cultivated rice is the most important cultivated species in the AA genome of the Oryzae genus. In the early days, it was mainly 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 heterosis within cultivated rice varieties. Temperate japonica rice is mainly distributed in temperate and high-altitude regions of East Asia, characterized by short, round grains, stunted plants, and abundant tillering. Tropical japonica rice, on the other hand, is mainly distributed in South Asia, Southeast Asia, Africa, and the Americas, characterized by tall plants, fewer tillers, large ears, and flat, wide grains, while also exhibiting better disease and drought resistance. The significant differences between the two subgroups at the morphological, physiological, and genetic levels result in strong heterosis in the F1 generation of hybrids from these two subgroups. Fully utilizing the heterosis between these two subgroups to cultivate highly vigorous hybrid varieties is one of the important ways to achieve ultra-high-yield breeding. However, hybrid sterility, primarily due to pollen sterility or spikelet sterility, leads to a generally low seed setting rate in the F1 generation, limiting the effective utilization of heterosis. Therefore, there is an urgent need for large-scale, systematic identification of heterosis loci between temperate and tropical japonica rice, and in-depth research into its genetic laws and molecular mechanisms, ultimately overcoming hybrid sterility.

[0003] Rice hybrid sterility is a quantitative trait controlled by multiple genes, and its study has been ongoing for over 100 years. However, only about 40 QTLs or genes related to hybrid sterility in Asian cultivated rice have been reported so far (Ouyang et al. 2010; Xie et al. 2019; Wang et al. 2024), of which only 7 hybrid sterility genes / locuses have been cloned (Li et al. 2024). Allelic and non-allelic interactions make hybrid sterility itself extremely complex. Furthermore, hybrid sterility is highly susceptible to environmental influences, further increasing the difficulty of its research. With a deeper understanding of hybrid sterility and the rapid development of SNP-based microarray genotyping technology, hybrid sterility loci can be efficiently identified at lower backcross generations, greatly improving the detection efficiency of sterility loci. Therefore, identifying hybrid sterility loci between temperate and tropical japonica rice and developing closely linked molecular markers is of significant theoretical and practical value for understanding the nature of hybrid sterility between subpopulations and overcoming hybrid sterility. Summary of the Invention

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

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a KASP molecular marker tightly linked to the S66 sterility site of temperate japonica and tropical japonica hybrids, wherein the KASP molecular marker includes KASP9-28 and KASP9-29; the primer set of the molecular marker KASP9-28 includes KASP9-28 forward primer 1, KASP9-28 forward primer 2 and KASP9-28 shared reverse primer;

[0006] 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.

[0007] The primer set for the molecular marker KASP9-29 includes KASP9-29 forward primer 1, KASP9-29 forward primer 2, and a common reverse primer for KASP9-29;

[0008] 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.

[0009] This invention also provides a method for detecting the KASP molecular marker tightly linked to the S66 sterility locus in temperate and tropical japonica hybrids, the method being as follows:

[0010] Rice genomic DNA was extracted, and PCR amplification was performed on the extracted rice genomic DNA using primer sets KASP9-28 and KASP9-29 to obtain PCR products.

[0011] The PCR amplification reaction system was 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 a final volume of 5 μL.

[0012] The PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing / extension at 60℃~55℃ for 60 s, wherein the 60℃~55℃ gradient PCR is performed, decreasing by 0.5℃ per cycle, for a total of 10 cycles; denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 60 s, for a total of 28~34 cycles; storage at 12℃.

[0013] The KASP primer working solution includes: forward primer 1, forward primer 2, a shared reverse primer, and sterile water; the concentration of forward primer 1 is 12 μmol / mL, the concentration of forward primer 2 is 12 μmol / mL, and the concentration of the shared reverse primer is 30 μmol / mL.

[0014] The present invention also provides the application of the KASP molecular marker closely linked to the above-mentioned hybrid sterility locus S66 in temperate and tropical japonica rice, and the application of the KASP molecular marker in the identification of the hybrid sterility locus S66 in temperate and tropical japonica rice.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. Hybrid sterility is the root cause restricting the utilization of heterosis between temperate and tropical japonica rice varieties. This invention detected a novel hybrid female sterility locus, S66, in the hybrid offspring of tropical japonica Moroberkan and temperate japonica Dianjingyou 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 rice varieties.

[0017] 2. This invention provides closely linked KASP markers KASP9-28 (6796166bp) and KASP9-29 (7965694bp) for detecting the heterosis locus S66 in temperate and tropical japonica hybrid combinations. KASP9-28 and KASP9-29 have high specificity and significant differences between parents, and can be efficiently used for detecting the heterosis locus S66 in temperate and tropical japonica hybrid combinations.

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This refers to the pollen and spikelet fertility distribution of the Yunnan Jingyou No. 1 / Moroberekan / 3 / Yunnan Jingyou No. 1 BC2F2 population in Example 1 of this invention.

[0020] Figure 2 This describes the infiltration of gene fragments from the Yunnan Jingyou 1 / Moroberekan / 3 / Yunnan Jingyou 1 BC2F2 population in Example 1 of this invention.

[0021] Figure 3 This is the genetic linkage map of S66 in Embodiment 1 of the present invention.

[0022] Figure 4 This is a genotyping diagram of the KASP9-28 marker-segregated population in Example 1 of the present invention.

[0023] Figure 5 This is a genotyping diagram of the KASP9-29 marker-segregated population in Example 1 of the present invention. Detailed Implementation

[0024] Example 1

[0025] This embodiment describes the phenotypic analysis of the genetic population and the molecular localization of S66.

[0026] The population of Dianjingyou 1 / Moroberekan / 3 / Dianjingyou 1 BC2F2 consisted of 232 plants. Pollen fertility segregated, but fertile plants were predominant, 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 fertile spikelets to semi-sterile spikelets conforms to a 1:2 segregation ratio (χ²). 2 = (0.26, P=0.61), suggesting that spikelet fertility in this population is controlled by a single gene.

[0027] Using Illumina 6K gene chip for bulked segregant analysis (BSA), a heterozygous introgression fragment of chromosome 9 from 5970596bp to 6667280bp was found to be associated with spikelet fertility. Figure 2 ), Figure 2 a represents the introgression of gene fragments into normal spikelets in the population (i.e., plants that do not carry the S66 gene). Figure 2 b represents the introgression of gene fragments into the semi-sterile spikelets in the population (i.e., plants carrying S66). Figure 2 The colors represent genotypes: green represents the homozygous band of Dianjingyou 1; red represents the heterozygous band of the cross between the two parents; and black represents the homozygous band of Moeroberekan. Based on the SNP information of the parents Dianjingyou 1 and Moeroberekan in this interval, polymorphic SNP markers were developed. Using the developed SNP markers, the BC2F2 population was genotyped using KASP typing technology. Combining the phenotype and genotype of individual plants, genetic linkage analysis revealed that spikelet fertility was closely linked to the SNP marker KASP9-2. This gene was ultimately mapped to 6.3 kb. This interbreeding sterility locus between temperate and tropical japonica rice was located on the long arm of chromosome 9 of rice and named S66. Figure 3 In this region, no sites controlling hybrid sterility between temperate and tropical japonica rice have been reported. Therefore, S66 is a novel site for controlling spikelet sterility in hybrids between temperate and tropical japonica rice.

[0028] Example 2

[0029] KASP molecular marker genotyping of the hybrid sterility locus S66

[0030] Based on the genotyping of Moroberkan and Dianjingyou 1 on the rice 40K chip (Wuhan Shuanglvyuan Chuangxin Technology Research Institute Co., Ltd.) and the chromosomal location of the S66 locus, SNP information from the parental sequences was used to convert them into KASP (Kompetitive allele-specific PCR) markers. After PCR amplification between parents and population validation, two pairs of polymorphic KASP markers within the S66 region were obtained: KASP9-28 and KASP9-29, corresponding to physical locations of 6796166 bp-7965694 bp. The KASP molecular markers closely linked to the S66 hybrid sterility locus in temperate and tropical japonica rice include KASP9-28 and KASP9-29.

[0031] (1) The primer set for the molecular marker KASP9-28 includes KASP9-28 forward primer 1, KASP9-28 forward primer 2 and KASP9-28 shared reverse primer;

[0032] 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.

[0033] KASP9-28 forward primers 1 and 2 have FAM and HEX adapter sequences attached to their 5' ends, respectively, to detect the polymorphism of S66 tightly linked markers and KASP9-28 single nucleotide markers (SNPs). The alleles of the Dianjingyou 1 type are attached with the FAM fluorescent group, and the alleles of the Moroberkan type are attached with the HEX fluorescent group.

[0034] (2) The primer set for the molecular marker KASP9-29 includes KASP9-29 forward primer 1, KASP9-29 forward primer 2 and KASP9-29 shared reverse primer;

[0035] 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.

[0036] KASP9-29 forward primers 1 and 2 have FAM and HEX adapter sequences attached to their 5' ends, respectively, to detect the polymorphism of major S66 tightly linked markers and S66 single nucleotide polymorphisms (SNPs). The alleles of the Dianjingyou 1 type are attached with FAM fluorescent groups, and the alleles of the Moroberkan type are attached with HEX fluorescent groups.

[0037] (3) Implementation of KASP markup

[0038] The method for detecting the S66 hybrid sterility locus in temperate and tropical japonica rice using the aforementioned KASP molecular markers is as follows:

[0039] Rice genomic DNA was extracted, and PCR amplification was performed on the extracted rice genomic DNA using primer sets KASP9-28 and KASP9-29 to obtain PCR products.

[0040] The PCR amplification reaction system was 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 a final volume of 5 μL.

[0041] The PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing / extension at 60℃~55℃ for 60 s, wherein the 60℃~55℃ gradient PCR is performed, decreasing by 0.5℃ per cycle, for a total of 10 cycles; denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 60 s, for a total of 28~34 cycles; storage at 12℃.

[0042] The KASP primer working solution includes: forward primer 1, forward primer 2, a shared reverse primer, and sterile water; the concentration of forward primer 1 is 12 μmol / mL, the concentration of forward primer 2 is 12 μmol / mL, and the concentration of the shared reverse primer is 30 μmol / mL.

[0043] After the PCR amplification cycle was completed, the reaction plate was first dried and cooled to room temperature. Then, the fluorescence signal value was read using an LGC instrument at 37°C for 1 min.

[0044] PCR amplification products were analyzed and genotyped to identify the S66 heterosis-inhibiting locus between temperate and tropical japonica rice varieties. The specific genotyping criteria were as follows: if the SNP genotype was homozygous, only one type of fluorescent signal would be generated; HEX type fluorescence would appear red (Moroberekan), and FAM type fluorescence would appear blue (Yunnan Japonica Variety 1). If the SNP was heterozygous, the result would be a green fluorescent signal (heterozygous genotype). The blank control (NTC) would appear black (…). Figures 4-5 ).

[0045] In summary, the KASP molecular markers KASP9-28 (6796166bp) and KASP9-29 (7965694bp) of the present invention, which are closely linked to the S66 hybrid sterility locus in temperate and tropical japonica rice, have high specificity and significant differences between the parents. They can be efficiently used for the detection of the S66 hybrid sterility locus in temperate and tropical japonica rice.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A primer set for a KASP molecular marker tightly linked to the sterility site of a hybrid of temperate and tropical japonica rice, characterized in that, The primer set includes the primer set for KASP9-28 and the primer set for KASP9-29; the primer set for KASP9-28 includes KASP9-28 forward primer 1, KASP9-28 forward primer 2 and the common reverse primer for KASP9-28; 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 for KASP9-29 includes KASP9-29 forward primer 1, KASP9-29 forward primer 2, and a common reverse primer for KASP9-29; 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 detection using a primer set of KASP molecular markers tightly linked to the sterility site of temperate japonica and tropical japonica hybrids as described in claim 1, characterized in that, The method is as follows: Rice genomic DNA was extracted, and PCR amplification was performed on the extracted rice genomic DNA using primer sets KASP9-28 and KASP9-29 to obtain PCR products. The PCR amplification reaction system was 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 a final volume of 5 μL. The PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing / extension at 60℃~55℃ for 60 s, wherein the 60℃~55℃ gradient PCR is performed, decreasing by 0.5℃ per cycle, for a total of 10 cycles; denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 60 s, for a total of 28~34 cycles; storage at 12℃. The KASP primer working solution includes: forward primer 1, forward primer 2, a shared reverse primer, and sterile water; the concentration of forward primer 1 is 12 μmol / mL, the concentration of forward primer 2 is 12 μmol / mL, and the concentration of the shared reverse primer is 30 μmol / mL.

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