Primer set closely linked to a major gene for sterility in indica-japonica subspecies hybridization of rice

Abstract

The application provides a primer set of a molecular marker closely linked to a major sterile locus of indica-japonica subspecies hybridization, including a primer set of kasp2-14 and a primer set of kasp2-15, the primer set of kasp2-14 includes kasp2-14 forward primer 1, kasp2-14 forward primer 2 and kasp2-14 common reverse primer, and the nucleotide sequences are SEQ ID NO. 1-3 in sequence, the primer set of kasp2-15 includes kasp2-15 forward primer 1, kasp2-15 forward primer 2 and kasp2-15 common reverse primer, and the nucleotide sequences are SEQ ID NO. 4-6 in sequence, and a detection method is also provided, and the primer set is applied to identification of a sterile locus S69 of indica-japonica subspecies hybridization. The primer set can improve the accuracy and efficiency of detection of the sterile locus S69 in indica-japonica subspecies hybridization combinations.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics technology, specifically relating to a primer set of molecular markers closely linked to the major site of hybrid sterility between indica and japonica rice subspecies, as well as its detection method and application. Background Technology

[0002] Asian cultivated rice (Oryza sativa L.) is one of the world's most important food crops. It is one of the two cultivated species in the genus *Oryza*. Through long-term natural selection and domestication, Asian cultivated rice has evolved into two main subspecies: indica and japonica. Indica rice is mainly distributed in low-latitude tropical and subtropical regions, while japonica rice is mainly distributed in temperate and high-altitude regions. Significant differences exist between the two subspecies in morphology, physiology, and genetics, which result in strong heterosis in the F1 generation of indica-japonica hybrids. Utilizing the strong heterosis between indica and japonica subspecies to cultivate intersubspecies hybrids has gradually become an important approach to increasing rice yield. However, hybrid sterility, primarily due to pollen sterility or spikelet sterility, leads to a generally low seed setting rate in the F1 generation of indica-japonica hybrids, limiting the effective utilization of heterosis. Therefore, it is urgent to gain a deeper understanding of the genetic laws governing hybrid sterility between indica and japonica subspecies and to find effective methods to overcome hybrid sterility between indica and japonica subspecies.

[0003] Hybrid sterility is a common form of postzygotic reproductive isolation and a primary driving force behind speciation and evolution. Since the first report of hybrid sterility between indica and japonica subspecies by Japanese scholars Kato et al. (1928), more than 40 QTLs or genes related to hybrid sterility between indica and japonica subspecies of cultivated rice in Asia have been reported (Zhang et al., 2022), of which 7 hybrid sterility genes / locuses have been cloned. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a primer set of molecular markers closely linked to the major heterosis locus between indica and japonica rice, as well as its detection method and application. This primer set can improve the accuracy and efficiency of detecting the heterosis locus S69 in hybrid combinations between indica and japonica rice.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a primer set of molecular markers closely linked to the major site of hybrid sterility between indica and japonica rice subspecies, wherein the primer set includes the primer set of kasp2-14 and the primer set of kasp2-15.

[0006] The primer set of kasp2-14 includes kasp2-14 forward primer 1, kasp2-14 forward primer 2 and kasp2-14 shared reverse primer;

[0007] The nucleotide sequence of the kasp2-14 forward primer 1 is shown in SEQ ID NO.1, the nucleotide sequence of the kasp2-14 forward primer 2 is shown in SEQ ID NO.2, and the nucleotide sequence of the kasp2-14 common reverse primer is shown in SEQ ID NO.3.

[0008] The primer set of kasp2-15 includes kasp2-15 forward primer 1, kasp2-15 forward primer 2 and kasp2-15 shared reverse primer;

[0009] The nucleotide sequence of the kasp2-15 forward primer 1 is shown in SEQ ID NO.4, the nucleotide sequence of the kasp2-15 forward primer 2 is shown in SEQ ID NO.5, and the nucleotide sequence of the kasp2-15 common reverse primer is shown in SEQ ID NO.6.

[0010] This invention locates a major locus controlling hybrid sterility between indica and temperate japonica rice subspecies on the short arm of chromosome 2, and names it S69. Simultaneously, two kasp molecular markers, kasp2-14 and kasp2-15, closely linked to S69, were developed, with corresponding physical locations of 2,135,390-2,414,268. These molecular markers, kasp2-14 and kasp2-15, which are closely linked to the hybrid sterility locus S69 in rice interspecific hybrids, exhibit high specificity and significant differences between parents. The two molecular markers are located on the short arm of rice chromosome 2, with a physical distance of approximately 278.9 kb between them. Using eight publicly available SSR molecular markers and two sets of SNP molecular markers, kasp2-14 and kasp2-15, S69 was located within a 3.01 cM region between RM7033 and kasp2-15. The molecular marker kasp2-14 co-segregated with S69 and can be used to detect the presence of this heterosis locus in interspecific hybrids of indica and japonica rice. This invention improves the accuracy and efficiency of detecting the heterosis locus S69 in interspecific hybrids of indica and japonica rice.

[0011] This invention also provides a method for detection using primers containing molecular markers closely linked to the major site of hybrid sterility between indica and japonica rice subspecies. The method is as follows:

[0012] Rice genomic DNA was extracted, and PCR amplification was performed on the extracted rice genomic DNA using primer sets kasp2-14 and kasp2-15 to obtain PCR products.

[0013] The PCR amplification reaction system was as follows: 37.5–62.5 ng of rice genomic DNA, 1.31 μL of kasp primer working solution, 0.07 μL of KASP master mix, and sterile water to a final volume of 5 μL.

[0014] 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℃.

[0015] 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.

[0016] The present invention also provides the application of the primers for the molecular markers closely linked to the major heterosis locus between indica and japonica rice subspecies, and the application of the primer set in the identification of the S69 heterosis locus between indica and temperate japonica rice subspecies.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. Reproductive isolation, primarily characterized by hybrid sterility, is a major obstacle to utilizing the strong heterosis between indica and japonica subspecies. A novel hybrid male sterility locus, S69, was detected in the hybrid offspring of indica rice Pokkali and temperate japonica rice Dianjingyou 1. No other hybrid sterility loci have been reported to have been located between indica and japonica subspecies in this region, providing important clues for studying the genetics and patterns of hybrid sterility between indica and japonica subspecies.

[0019] 2. The molecular markers kasp2-14 and kasp2-15 of this invention, which are closely linked to the hybrid sterility locus S69 between indica and japonica rice subspecies, exhibit high specificity and significant differences between parents. Both markers are located on the short arm of rice chromosome 2, with a physical distance of approximately 278.9 kb between them. Marker kasp2-14 co-segregates with S69 and can be used to detect the presence of this hybrid sterility locus in indica-japonica rice hybrids. This invention improves the accuracy and efficiency of detecting the hybrid sterility locus S69 in indica-japonica rice hybrids.

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1This describes the pollen fertility phenotypes of the recurrent parents, the S69 near-isogenic line, and the heterozygote in Example 2 of this invention. AC represents the pollen fertility of the recurrent parents DJY1, NIL-S69, and their hybrid F1, respectively, with a scale bar of 200 μm; D represents the quantitative analysis results of pollen fertility and spikelet fertility of the recurrent parents DJY1, NIL-S69, and their hybrid F1.

[0022] Figure 2 This is a pollen fertility frequency distribution diagram of the F2 population of the recurrent parent and the S69 near-isogenic hybrid of Embodiment 2 of the present invention.

[0023] Figure 3 This is the S69 genetic linkage diagram of Embodiment 3 of the present invention.

[0024] Figure 4 This is the genotyping diagram of the kasp2-14 marker partial genetic segregating population in Example 4 of the present invention.

[0025] Figure 5 This is the genotyping diagram of the kasp2-15 marker partial genetic segregating population in Example 4 of the present invention. Detailed Implementation

[0026] Example 1

[0027] This example describes the construction of a near-isogenic line containing the S69 locus.

[0028] This invention locates a novel locus on chromosome 2 that controls the semi-sterility of pollen in hybrids of the Asian cultivated indica rice variety Pokkali and the temperate japonica rice variety Dianjingyou 1, and names it S69. Simultaneously, a kasp molecular marker closely linked to S69 was developed.

[0029] Using the temperate japonica rice variety Dianjingyou 1 (DJY1), bred by the Yunnan Academy of Agricultural Sciences, as the female parent and the indica rice variety Pokkali as the male parent, a hybrid F1 was obtained. Then, using the F1 as the female parent and DJY1 as the male parent, a backcross was performed to obtain the BC1F1 population. The inventors used I2-KI staining to detect the pollen fertility of each individual plant. The method is as follows: 5-10 spikelets that were to bloom on the same day were collected from the upper part of the main panicle of each plant before flowering and fixed in a 70% ethanol solution. Staining was performed with 1% I2-KI solution, and pollen fertility was classified into four categories under a 160× ordinary optical microscope: typical abortion, round abortion, stained abortion, and fertile. The number of pollen in each category was recorded. Three fields of view were observed per microscope, and the percentage of each category of pollen was calculated for each plant. Starting with BC1F1, plants with pollen fertility below 70% were repeatedly backcrossed into BC6F1. Then, semi-sterile pollen plants from the BC6F1 population were self-crossed to form the BC6F2 population. Genotyping of the BC6F2 population was performed using a rice 1K liquid phase chip (Huazhi Biotechnology Co., Ltd.). Almost all semi-sterile pollen plants showed heterozygous introgression on the short arm of chromosome 2, while all fertile pollen plants at this location were of the donor parent Pokkali genotype. This indicates that the S69 locus controlling semi-sterility is located on the short arm of chromosome 2. Single plants with normal pollen fertility, homozygous Pokkali genome on the short arm of chromosome 2, and a genetic background of DJY1 were selected as near-isogenic lines for S69.

[0030] Example 2

[0031] This example is a genetic analysis of the S69 locus.

[0032] The F1 hybrid was obtained by crossing NIL-S69 as the female parent and Dianjingyou 1 as the recurrent parent. The spikelets of NIL-S69, Dianjingyou 1, and the F1 hybrid are all fertile. Figure 1 (D). Pollen from both parents, NIL-S69 and Dianjingyou 1, showed normal fertility, while in the pollen of hybrid F1, about half of the pollen grains showed chromosomal abortion (D). Figure 1 (AD). This indicates that the hybrid sterility locus S69 only affects pollen fertility and does not affect spikelet fertility.

[0033] To further investigate the genetic patterns of S69, the inventors analyzed the pollen fertility of individual plants in the F2 population formed by F1 self-pollination. The distribution of pollen fertility is as follows: Figure 2 As shown in the figure. The F2 population consisted of 943 plants, with pollen fertility exhibiting a bimodal distribution. Of these, 492 were fertile and 451 were semi-sterile, maintaining a 1:1 ratio (χ²). 2 =1.783, P=0.18), indicating that S69 is a single Mendelian hereditary factor.

[0034] Example 3

[0035] This embodiment describes the molecular localization of S69.

[0036] To locate the S69 locus, the inventors selected 25 SSR markers from the infiltrated fragment region and screened between DJY1 and NIL-S69, identifying 8 pairs of polymorphic markers. The genotypes of each individual plant in the population were analyzed using these 9 marker pairs. Combined with the phenotypes of each individual plant, the S69 locus was initially located within a 3.89 cM range between molecular markers RM7033 and RM12460. Figure 3 In this section, no loci have been reported that control hybrid sterility between indica and japonica rice subspecies. Therefore, S69 is a novel locus that controls semi-sterile pollen in hybrids within the indica and japonica subspecies.

[0037] Table 1. Primer sequences used for preliminary localization of the S69 heterosterilization locus.

[0038]

[0039] Example 4

[0040] This embodiment is an encryption of the S69 interval marker for the interspecific hybrid sterility locus between indica and japonica subspecies.

[0041] Based on the genotyping of DJY1 and Pokkali on a rice 6K microarray (Ilumina) and the chromosomal location of the S69 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 were obtained within the S69 region, with one pair of kasp markers co-segregating with S69.

[0042] (1) The primer set of the molecular marker kasp2-14 includes kasp2-14 forward primer 1, kasp2-14 forward primer 2 and kasp2-14 shared reverse primer;

[0043] The nucleotide sequence of the kasp2-14 forward primer 1 is shown in SEQ ID NO.1, the nucleotide sequence of the kasp2-14 forward primer 2 is shown in SEQ ID NO.2, and the nucleotide sequence of the kasp2-14 common reverse primer is shown in SEQ ID NO.3.

[0044] The kasp2-14 forward primers 1 and 2 have FAM and HEX adapter sequences attached to their 5' ends, respectively, and are used as kasp forward primers (SEQ ID NO.7-SEQ ID NO.8) to detect the polymorphism of the S69 tightly linked marker and the kasp2-14 single nucleotide polymorphism. The DJY1 allele is attached to the FAM fluorescent group, and the Pokkali allele is attached to the HEX fluorescent group (Figure 4).

[0045] (2) The primer set of the molecular marker kasp2-15 includes kasp2-15 forward primer 1, kasp2-15 forward primer 2 and kasp2-15 shared reverse primer;

[0046] The nucleotide sequence of the kasp2-15 forward primer 1 is shown in SEQ ID NO.4, the nucleotide sequence of the kasp2-15 forward primer 2 is shown in SEQ ID NO.5, and the nucleotide sequence of the kasp2-15 common reverse primer is shown in SEQ ID NO.6.

[0047] The kasp2-15 forward primers 1 and 2 have FAM and HEX adapter sequences attached to their 5' ends, respectively, and are used as kasp forward primers (SEQ ID NO.9-SEQ ID NO.10) to detect the polymorphism of single nucleotide (SNP) sites of the major S69 tightly linked marker kasp2-15. The DJY1 type allele is attached to the HEX fluorescent group, and the Pokkali type allele is attached to the FAM fluorescent group (Figure 5).

[0048] (3) Implementation of KASP markup

[0049] ① Primer preparation: The KASP primer working solution includes: forward primer 1, forward primer 2, 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 shared reverse primer is 30 μmol / mL;

[0050] ②The PCR amplification reaction system is as follows: 37.5-62.5 ng of rice genomic DNA, 1.31 μL of kasp primer working solution, 0.07 μL of KASP master mix, and sterile water to a final volume of 5 μL;

[0051] ③PCR amplification reaction program: 94℃ pre-denaturation for 15 min; 94℃ denaturation 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; 94℃ denaturation for 20 s, annealing and extension at 55℃ for 60 s, for a total of 28~34 cycles; store at 12℃;

[0052] After the PCR amplification cycle was completed, the reaction plate was first dried and cooled to room temperature. Then, the fluorescence signal values ​​were read using an LGC genotyping instrument. The specific genotyping criteria were as follows: if the SNP locus was homozygous, only one type of fluorescence signal would be generated; HEX type fluorescence would appear orange, and FAM type fluorescence would appear blue. If the SNP locus was heterozygous, the result would be a green fluorescence signal; the blank control (NTC) fluorescence would appear black.

[0053] In summary, the tightly linked molecular markers kasp2-14 and kasp2-15 of this invention for detecting the heterosis site S69 in interspecific hybrid rice combinations (indica-japonica), have a physical distance of approximately 278.9 kb. These molecular markers, kasp2-14 and kasp2-15, exhibit high specificity and significant differences between parents, making them highly effective for detecting the heterosis site S69 in interspecific hybrid rice combinations.

[0054] PCR amplification products were analyzed and genotyped to identify the interspecific hybrid sterility locus S69 between indica and temperate japonica subspecies. The specific genotyping criteria were as follows: if the SNP locus was homozygous, only one fluorescent signal would be generated, with HEX type fluorescence appearing as orange and FAM type fluorescence appearing as blue. If the SNP locus was heterozygous, the result would be a green fluorescent signal. The blank control (NTC) fluorescence appeared as black.

[0055] 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 of a molecular marker closely linked to a major gene locus for hybrid sterility between indica and japonica subspecies of rice, characterized in that, The primer set comprises a primer set of kasp2-14 and a primer set of kasp2-15; The primer set of kasp2-14 comprises a kasp2-14 forward primer 1, a kasp2-14 forward primer 2 and a kasp2-14 common reverse primer; The nucleotide sequence of the kasp2-14 forward primer 1 is shown as SEQ ID NO. 1, the nucleotide sequence of the kasp2-14 forward primer 2 is shown as SEQ ID NO. 2, and the nucleotide sequence of the kasp2-14 common reverse primer is shown as SEQ ID NO. 3; The primer set of kasp2-15 comprises a kasp2-15 forward primer 1, a kasp2-15 forward primer 2 and a kasp2-15 common reverse primer; The nucleotide sequence of the kasp2-15 forward primer 1 is shown as SEQ ID NO. 4, the nucleotide sequence of the kasp2-15 forward primer 2 is shown as SEQ ID NO. 5, and the nucleotide sequence of the kasp2-15 common reverse primer is shown as SEQ ID NO.

6.

2. A method for detecting the molecular marker primer set linked to the major gene for sterility of indica-japonica subspecies hybridization of rice according to claim 1, characterized in that, The method is: rice genomic DNA is extracted, and the extracted rice genomic DNA is subjected to PCR amplification by using the primer set of kasp2-14 and the primer set of kasp2-15 to obtain a PCR product; The reaction system of the PCR amplification is as follows: 37.5-62.5 ng of rice genomic DNA, 1.31 μL of kasp primer working solution, 0.07 μL of KASP master Mix, and sterile water supplemented to 5 μL; The reaction procedure of the PCR amplification is as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 60℃-55℃ annealing / elongation for 60 s, wherein, 60℃-55℃ gradient PCR, 0.5℃ decrease per cycle, a total of 10 cycles; 94℃ denaturation for 20 s, 55℃ recombination and elongation for 60 s, a total of 28-34 cycles; 12℃ storage; The kasp primer working solution comprises a forward primer 1, a 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.

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