Application of Molecular Markers Linked to Rice Seed Storage-Tolerance Gene OsNBS2 in Identification of Rice Varieties with Storage-Tolerance

Abstract

The present invention relates to the field of gene engineering technology, and in particular to the application of a rice seed storage gene OsNBS2 linked molecular marker in identifying storage-resistant rice varieties. The rice seed storage gene OsNBS2 linked molecular marker is used in identifying storage-resistant rice varieties. The rice seed storage gene OsNBS2 linked molecular marker is located at Chr 6:10438484 in the exon region of the rice OsNBS2 gene, and the storage-intolerant rice variety lacks the base G. The rice seed storage gene obtained by the present invention can be stably expressed in different genetic generations and has high reliability. The molecular marker of the present invention can be used for the identification and selection of rice seedling genotypes, and individuals carrying excellent natural allele variations of seed storage genes can be obtained, which can overcome the shortcomings of conventional breeding methods such as a long time period, difficulty in phenotypic identification, and poor repeatability, and accelerate the breeding process of new varieties of highly storage-resistant rice.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to the application of rice seed storage-resistant gene OsNBS2 linked molecular markers in identifying storage-resistant rice varieties. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world. More than half of the world's population relies on rice for food (Amudha et al., 2011). Its stable production is directly related to global food security. Food security is a major issue for a country, and basic self-sufficiency in food must be ensured. With the increase in rice planting area in my country and the utilization of rice hybrid advantages, my country's grain output has been greatly improved. However, due to the complexity and unpredictability of rice seed production and its supply and demand market, seeds often need to be stored for short and long periods of time, and the problem of rice storage has become increasingly prominent. Studies have found that hybrid rice seeds have poor storage resistance, and the germination rate usually drops to 70% or even lower after one year of conventional storage (Deng Jiuying et al., 2008). Every year, my country suffers losses of billions of kilograms of rice seeds due to aging, deterioration, insect pests, and mildew during storage, with economic losses as high as 20 billion yuan (National Bureau of Statistics, 2017; Fei Yuexin et al., 2018), which seriously affects food security and food safety. The loss of vitality of rice seeds during storage leads to a decrease in germination rate, which will also have an adverse effect on rice production safety and breeding process (Mao et al., 2018), resulting in a decrease in seed value. Although measures such as gas conditioning and temperature regulation can extend the storage period of rice, they also increase the cost of storage. Therefore, it is of great theoretical and practical significance to breed storage-resistant rice varieties to reduce losses during storage and achieve green, low-carbon, high-quality and efficient production of rice.

[0003] Rice storage tolerance is an important trait closely related to seed value, seed provenance and food security, and is controlled by multiple genes. With the development of biotechnology and bioinformatics, research on seed storage QTL and genetic mechanisms has made great progress (Yan Yuntao et al., 2022). So far, more than 70 rice storage tolerance QTLs have been detected by natural storage or artificial aging, and they are widely distributed on 12 chromosomes, among which the most QTLs are detected on chromosome 9 (Liu et al., 2022; Zheng et al., 2024; Yan Yuntao et al., 2022; Dong et al., 2017). The major effect QTLqLG-9 affecting seed life was first detected on chromosome 9 (Miura et al., 2002), and it has been finely positioned. The major storability QTLs RC9-2, qRGR-9, qMT-SGC9.1, and qDT-SGC9.1 identified in subsequent studies all overlap with qLG-9 (Sasaki et al., 2005; Xue et al., 2008; Jiang et al., 2011). Although a large number of rice storability QTLs have been identified, only three storability QTLs, qLG-9 / qSS-9 (Lin et al., 2015; Yuan et al., 2019; Liu et al., 2015), qSS1, and qSS3.1 (Yuan et al., 2019; Yuan et al., 2021), have been finely mapped. In the above-mentioned studies on QTLs for storage tolerance, most of them focused on constructing genetic populations of cultivated rice or rice landraces, while fewer used wild rice / cultivated rice genetic populations (Jin et al., 2018; Zhao et al., 2021). This shows that the discovery of favorable alleles for storage tolerance in wild rice needs to be strengthened.

[0004] So far, four storage-resistant genes have been cloned based on QTL, namely, OsFAH2 encoding fatty acid hydroxylase (Yuan et al., 2019), indole-3-acetic acid (IAA)-amido synthetase gene OsGH3-2 (Yuan et al., 2021), OsCSD2 (copper / zinc superoxide dismutase 2) and OsCSD3 (Zheng et al., 2024). Superoxide dismutase (SOD) genes OsCSD2 (copper / zinc superoxide dismutase 2) and OsCSD3 are candidate genes for two storage-resistant QTLs (qSL3 and qSL7.2), respectively, and play a positive regulatory role in promoting rice storage resistance (Zheng et al., 2024). It can be seen that the above storage-resistant QTL / gene research also lays the foundation for rice storage-resistant molecular breeding. Therefore, studying the molecular mechanism of rice storage resistance has important theoretical and application value for rice storage-resistant breeding.

[0005] References

[0006] 1. Deng Jiuying, Liu Jun, Chen Guanghui. Research progress on the viability maintenance of hybrid rice seeds during storage. Crop Research, 2008, 22(S1): 401-404.

[0007] 2. Fei Yuexin, Cao Yujie, Wu Min, et al. Research progress and prospects of the mechanism of rice quality deterioration during storage. Chinese Rice, 2018, 24(5): 22-26.

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[0009] 4. Liu Xi, Lin Qiuyun, Sun Ailing, et al. Fine mapping of QTL qSS-9 for rice seed storability. Journal of Nanjing Agricultural University, 2015, 38(6): 877-882.

[0010] 5. Yan Yuntao, He Xi, Zhang Haiqing, et al. Research progress on rice seed storability. Chinese Agricultural Science Bulletin, 2022, 38(5): 1-8.

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[0012] 7. Dong XY, Fan SX, Liu J, et al. Identification of QTLs for seedstorability in rice under natural aging conditions using two RILs with the same parent Shennong 265. Journal of Integrative Agriculture, 2017, 16(5): 1084-1092.

[0013] 8. JiangW, Lee J, JinTM, et al. Identification of QTLs for seedgerminationcapability after various storage periods using two RIL populations in rice. Molecules and Cells, 2011, 31(4): 385-392.

[0014] 9.Jin J,Long W X,Wang L T,et al.QTL mapping ofseed vigor ofbackcrossinbred lines derived from Oryza longistaminataunderartificial aging.FrontiersinPlant Science,2018,9:1909.

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[0019] (Oryza Sativa L.).Theoretical andAppliedGenetics,2002,104(6):981-986.

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[0021] (Oryza sativa L.)aftervarious periods ofseed storage.PlantBreeding,2005,124(4):361-366.15.Xue Y,Zhang S Q,Yao Q H,et al.Identificationofquantitative trait loci for seed storability in rice(Oryza sativa L.).Euphytica,2008,164(3):739-744.

[0022] 16.Yuan ZY,Fan K,Xia LF,et al.Genetic dissection ofseed storabilityand validation ofcandidate gene associated with antioxidant capability inrice(Oryza sativa L.).Int J Mol Sci,2019,20(18):4442.

[0023] 17.Yuan ZY,Fan K,WangYT,et al.OsGRETCHENHAGEN3-2 modulates rice seedstorability via accumulation ofabscisic acidandprotective substances.PlantPhysiology,2021,186(1):469-482.18.Zhao MM,Hu BL,Fan YW,etal.Identification,analysis,and confirmation of seed storability-relatedlociin Dongxiangwildrice(Oryza rufifipogon Griff.).genes,2021,12:1831.19.Zheng XH, Yuan ZY, Yu YY, et al.OsCSD2 and OsCSD3 enhance seedstorability by modulating antioxidant enzymes and abscisic acid inrice.Plants,2024,13,310. Summary of the invention

[0024] In order to solve the above problems, the present invention provides the use of rice seed storage gene OsNBS2 linkage molecular markers in identifying storage-resistant rice varieties. The molecular markers provided by the present invention can be used to identify whether rice belongs to a storage-resistant rice variety.

[0025] In order to achieve the above object, the present invention provides the following technical solutions:

[0026] The invention provides an application of a rice seed storage-resistant gene OsNBS2 linked molecular marker in identifying storage-resistant rice varieties. The rice seed storage-resistant gene OsNBS2 linked molecular marker is located at Chr 6:10438484 of the exon region of the rice OsNBS2 gene, and the storage-intolerant rice varieties lack the base G.

[0027] Preferably, the storage-resistant rice variety is a rice variety with a germination rate higher than 70% after aging treatment at 42° C. and 80% humidity for 28 days.

[0028] The present invention also provides a primer pair for amplifying the rice seed storage-resistant gene OsNBS2 linked molecular marker, wherein the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No.2.

[0029] The present invention also provides a method for identifying storage-resistant rice varieties, comprising the following steps:

[0030] 1) extracting genomic DNA from rice, and using the genomic DNA as a template to perform PCR amplification using the primer pair described in the above technical solution to obtain an amplified product;

[0031] 2) using restriction endonuclease BseRI to digest the amplified product obtained in step 1), and performing electrophoresis and staining on the digested product. When a band of 183 bp is obtained, the rice is a storage-resistant rice variety.

[0032] Preferably, the system for PCR amplification in step 1) is: 5.0 μL of 2×Tolo FastTaq Premix, 1 μL of each of the upstream and downstream primers with a concentration of 10 pmol / μL, 1 μL of rice genomic DNA with a concentration of 300-500 ng / μL, and sterilized ultrapure water to 10 μL.

[0033] Preferably, the PCR amplification procedure is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, 35 cycles; final extension at 72°C for 10 min; and storage at 4°C.

[0034] Preferably, the electrophoresis in step 2) uses polyacrylamide gel electrophoresis, and the staining uses silver nitrate staining.

[0035] The present invention also provides a kit for identifying storage-resistant rice varieties, comprising the primer pair and reagents described in the above technical solution.

[0036] Preferably, the reagent comprises 2×Tolo FastTaq Premix.

[0037] The present invention uses the strain 19H19 of the Xieqingzao B / Dongxiang wild rice BC4F2 backcross recombinant inbred lines (BILs) population to backcross once and self-pollinate once with the recurrent parent Xieqingzao B, develops the Xieqingzao B / Dongxiang wild rice BC5F2 generation BILs, and constructs a high-density genetic map containing 2059 Bin markers by high-throughput resequencing. The storage resistance of 120 Xieqingzao B / Dongxiang wild rice BC5F2 BILs seeds was identified by treating them at 42°C / 80% humidity for 28 days, and the results show that the germination rate of the BILs seeds is between 0-100%. The cim() function of R / qtl was used to perform composite interval mapping of seed storage traits, with a scanning step of 1 cM and LOD = 2.5 as the threshold for screening QTLs. A new major QTL qSS6 controlling rice seed storage resistance was detected at the interval 7652428-11096971 bp on chromosome 6 of rice. DXWR ( Figure 1 The LOD value of this QTL is 3.18, and the phenotypic contribution rate is 21.84%. Its synergistic allele comes from Dongxiang wild rice, which provides a new storage-resistant gene source for the storage-resistant breeding and improvement of cultivated rice. The whole genome association analysis (GWAS) was used to detect the genetic variation polymorphism of BC5F2 BILs, and the p value of the marker interval of chromosome 6 6_7652428-6_11096971 was less than 0.00001 ( Figure 2 ), indicating that the variation in this interval is closely related to the storage resistance of rice seeds. Further BSA-seq analysis of 20 extremely storage-resistant materials and 20 extremely storage-intolerant materials in BC5F2 BILs narrowed the interval to 10000296-10439625bp ( Figure 3 ). DXWR Fourteen candidate genes related to seed storage stability were identified in the interval, and transcriptome analysis found that 9 genes were differentially expressed under artificial aging treatment ( Figure 4 ). Sequencing analysis of the 9 genes revealed that the exon region of the OsNBS2 gene of Xieqingzao B was missing one base compared to 19H19 ( Figure 5 ). OsNBS2 genotypes were detected in 120 rice samples from the BC5F2 population ( Figure 6). There are 14 missing copies, and the rest are 31 Xieqingzao B homozygous, 26 Dongxiang wild rice homozygous, and 49 parental heterozygous. The Xieqingzao B homozygous, heterozygous, and Dongxiang wild rice homozygous types in the BC5F2 population meet the 1:2:1 separation ratio (Table 1). The BIP (biparental population QTL positioning) method in QTL IciMapping V4.2 was used to detect this site in the BC5F2 population. The LOD value of this site is 3.70, and the phenotypic contribution rate is 19.57% (Table 2). The association analysis of the gene frequency of this site and the germination rate after artificial aging is shown in Table 1 below; the t-test was used to compare the differences in the mean germination rates of each genotype, and the significance level was set at P<0.05. It was found that the mean germination rates of Xieqingzao B homozygous, heterozygous, and Dongxiang wild rice homozygous were significantly different. It is shown that the dCAPS molecular marker designed in the present invention can accurately determine the Xieqingzao B homozygous type, heterozygous type and Dongxiang wild rice homozygous type of the OsNBS2 gene of individual rice, and is suitable for molecular marker-assisted breeding of the OsNBS2 gene.

[0038] Compared with the prior art, the present invention has the following advantages and effects:

[0039] 1. The rice seed storage-resistant gene obtained by the present invention can be stably expressed in different genetic generations and has high reliability.

[0040] 2. By screening molecular markers that are closely linked to rice seed storage-resistance genes, rice materials or varieties with strong seed storage resistance can be identified.

[0041] 3. The molecular markers of the present invention can be used for the identification and selection of rice seedling genotypes to obtain individuals carrying excellent natural allelic variations of seed storage-resistant genes. This can overcome the shortcomings of conventional breeding methods, such as the long time period required, the difficulty in phenotypic identification, and the poor repeatability, and accelerate the breeding process of new varieties of rice with strong storage resistance.

[0042] The strain 19H19 of the Xieqingzao B / Dongxiang wild rice BC4F2 backcross recombinant inbred line population of the present invention was backcrossed once and selfed once with the recurrent parent Xieqingzao B to develop the Xieqingzao B / Dongxiang wild rice BC5F2 generation BILs, and a high-density genetic map containing 2059 Bin markers was constructed by high-throughput resequencing. 120 Xieqingzao B / Dongxiang wild rice BC5F2 BILs seeds were treated at 42°C / 80% for 28 days, and the results showed that the germination rate of the BILs seeds was between 0-100%. The cim() function of R / qtl was used to perform composite interval mapping on the seed storage trait, and a new major effect QTL qSS6 for controlling rice seed storage resistance was detected at the interval 6_6964302-6_10977254 of chromosome 6 of rice. DXWR ( Figure 1). The LOD value of this QTL is 3.18, and the phenotypic contribution rate is 21.84%. Its synergistic allele comes from Dongxiang wild rice, providing a new storage-resistant gene source for the storage-resistant breeding and improvement of cultivated rice. GWAS detected the genetic variation polymorphism of BC5F2 BILs and found that the p value of the marker interval of chromosome 6 6_7652428-6_11096971 was less than 0.00001 ( Figure 2 ), indicating that the interval variation is closely related to the storage characteristics of rice seeds ( Figure 2 ).

[0043] At the same time, BSA-seq analysis was performed on 20 extremely storable materials (germination rate after aging greater than 80%) and 20 extremely non-storable materials (germination rate after aging less than 20%) in BC5F2 BILs. A total of 2496 single nucleotide polymorphism (SNP) sites and 400 insertion or deletion (InDel) sites were obtained for subsequent gene positioning. At a confidence level of 99%, the window of 10000296-10439625bp on chromosome 6 was greater than the threshold value through SNP-index and InDel-index analysis, which can be used as a candidate interval ( Figure 3 ). Based on the annotation results of ANNOVAR, 14 candidate genes with insertion or substitution in the coding region and non-synonymous mutation types were selected. Transcriptome analysis found that 9 of them were differentially expressed under cold stress ( Figure 4 ). Sequencing analysis of these 14 genes revealed that Forward (SEQ ID No.1): 5'-GTTATGAGGACATAAGAG-3'; Reverse (SEQ ID No.2): 5'-GTGTAATTACTCTAGTC-3'. Among them, the exon region of the OsNBS2 gene of Xieqingzao B lacked one base compared to 19H19 ( Figure 5 ) Based on the sequence differences, a storage-resistant linked dCAPS molecular marker SS6 was developed ( Figure 5 , 6), laying a foundation for molecular marker-assisted selection breeding of rice with storage tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0045] Figure 1 For qSS6 DXWR Location on rice chromosomes;

[0046] Figure 2 Analyze rice seed storage tolerance loci for GWAS;

[0047] Figure 3 Analysis of candidate genomic regions for rice seed storage tolerance;

[0048] Figure 4 The differential gene expression patterns of Xieqingzao B and 19H19 transcriptomes;

[0049] Figure 5 Schematic diagram of partial sequence alignment and dCAPS marker design for Xieqingzao B, 19H19 and heterozygous offspring;

[0050] Figure 6 This is the electrophoresis diagram of using dCAPS marker to detect the OsNBS2 genotype of Xieqingzao B, 19H19 and 48 BC5F2 strains. DETAILED DESCRIPTION

[0051] The present invention provides an application of a rice seed storage-resistant gene OsNBS2 linked molecular marker in identifying storage-resistant rice varieties, wherein the rice seed storage-resistant gene OsNBS2 linked molecular marker is located at Chr 6:10438484 of the exon region of the rice OsNBS2 gene, and the base G is missing in the storage-intolerant rice variety. In the present invention, the storage-resistant rice variety is preferably: a rice variety with a germination rate higher than 70% after aging treatment at 42°C and 80% humidity for 28 days. In the present invention, the rice variety with weak seed storage resistance is preferably: a rice variety with a germination rate lower than 30% after aging treatment at 42°C and 80% humidity for 28 days.

[0052] The present invention also provides a primer pair for amplifying the rice seed storage-resistant gene OsNBS2 linked molecular marker, wherein the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No.2.

[0053] SEQ ID No.1: 5'-GTTATGAGGACATAAGAG-3';

[0054] SEQ ID No. 2: 5'-GTGTAATTACTCTAGTC-3'.

[0055] The present invention also provides a method for identifying storage-resistant rice varieties, comprising the following steps:

[0056] 1) Extracting rice genomic DNA, using the genomic DNA as a template to perform PCR amplification using the primer pair described in the above technical solution to obtain an amplified product; and digesting the amplified product with restriction endonuclease BseRI.

[0057] 2) The enzyme digestion product obtained in step 1) is subjected to electrophoresis and staining. When a band of 183 bp is obtained, the rice is a storage-resistant rice variety.

[0058] The present invention does not specifically limit the method for extracting rice genomic DNA, and those skilled in the art can use conventional methods, such as:

[0059] (1) Cut 0.05 g of rice leaves and place them in a 2 L centrifuge tube filled with small steel balls and containing 400 μL of 1.5× CTAB solution (1.5% CTAB, 75 mM Tris-HCl, 15 mM EDTA, 1.05 M NaCl, pH = 8.0);

[0060] (2) Oscillate for 60 seconds, add 400 μL of chloroform, vortex to mix, and centrifuge at 12,000 rpm for 5 minutes;

[0061] (3) Pipette 400 μL of supernatant into a new 1.5 mL centrifuge tube and add 800 μL of anhydrous ethanol;

[0062] (4) Place at -20°C for 30 min, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and dry at 65°C for 1 h;

[0063] (5) Add 100 μL of double distilled water.

[0064] In the present invention, the system of PCR amplification is preferably: 2×Tolo FastTaqPremix 5.0μL, 1μL of upstream and downstream primers at a concentration of 10pmol / μL, 1μL of rice genomic DNA at a concentration of 300-500ng / μL, and sterilized ultrapure water to 10μL. In the present invention, the procedure of PCR amplification is preferably: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 45s, and 35 cycles; finally extension at 72°C for 10min; storage at 4°C. In the present invention, the electrophoresis is preferably polyacrylamide gel electrophoresis, and the staining is preferably silver nitrate staining. The present invention does not specifically limit the electrophoresis and staining, and those skilled in the art can follow the routine.

[0065] The present invention also provides a kit for identifying storage-resistant rice varieties, comprising the primer pair and reagents described in the above technical solution. In the present invention, the reagents preferably include 2×Tolo Fast Taq Premix.

[0066] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] Rice seed storage gene qSS6 DXWR Decomposition verification and development of dCAPS molecular marker SS6

[0069] 1. Construction of test materials

[0070] In the early stage, 237 BC1F were developed by hybridizing and backcrossing the wild Dongxiang rice with the storage-resistant indica rice variety Xieqingzao B bred by the Rice Research Institute of Anhui Academy of Agricultural Sciences. 10 The seed storage resistance of the BILs was evaluated by treating them at 42℃ and 80% humidity for 28 days. The recurrent parent Xieqingzao B was backcrossed with the extreme storage-resistant strain 5339 three times and selfed once to obtain the BC4F2 high-generation backcross population. The storage-resistant introgression line 19H19 was screened by combining the storage-resistant phenotype and genotype. The BC5F2 population was further constructed by backcrossing 19H19 with Xieqingzao B.

[0071] 2. Development of dCAPS molecular marker SS6

[0072] The line 19H19 of the Xieqingzao B / Dongxiang wild rice BC4F2 population was backcrossed once and selfed once with the recurrent parent Xieqingzao B to develop the Xieqingzao B / Dongxiang wild rice BC5F2 generation BILs. A high-density genetic map containing 2059 Bin markers was constructed by high-throughput resequencing. The cim() function of R / qtl was used to perform composite interval mapping of seed storage traits, and a new major QTL qSS6 controlling rice seed storage resistance was detected at the interval 7652428-11096971bp on chromosome 6 of rice. DXWR ( Figure 1 ). The LOD value of this QTL is 3.18, and the phenotypic contribution rate is 21.84%. Its synergistic allele comes from Dongxiang wild rice. GWAS detected the genetic variation polymorphism of BC5F2BILs and found that the p value of the marker interval of chromosome 6 6_7652428-6_11096971 was less than 0.00001 ( Figure 2 ), indicating that the variation in this interval is closely related to the storage resistance of rice seeds. Further BSA-seq analysis of 20 extremely storage-resistant materials and 20 extremely storage-intolerant materials in BC5F2 BILs narrowed the interval to 10000296-10439625bp ( Figure 3 ). DXWR Fourteen candidate genes related to seed storage stability were identified in the interval, and transcriptome analysis found that 9 genes were differentially expressed under artificial aging treatment ( Figure 4 ). Sequencing analysis of the 9 genes revealed that the exon region of the OsNBS2 gene of Xieqingzao B was missing one base compared to 19H19 ( Figure 5 Based on the sequence differences, the OsNBS2 gene-linked dCAPS molecular marker SS6 was developed and primer pair SS6 was used to identify 19H19, Xieqingzao B and their derived 120 BC5F2 populations. S Genotype identification of the NBS2 gene.

[0073] Upstream primer SEQ ID No. 1: 5′-GTTATGAGGACATAAGAG-3′;

[0074] Downstream primer SEQ ID No. 2: 5'-GTGTAATTACTCTAGTC-3'.

[0075] Example 2

[0076] Rice seed storage gene qSS6 DXWR Detection of linked molecular marker SS6

[0077] The test materials were planted at the Nanchang Experimental Base of the Rice Research Institute of Jiangxi Academy of Agricultural Sciences, and young rice leaves were cut to extract the DNA of the whole genome using the CTAB method. The linked marker SS6 was used to detect the genotypes of Xieqingzao B, 19H19, and 120 BC5F2 population lines. The PCR reaction system was as follows: 2×Tolo FastTaq Premix 5.0μl, 10pmol / μl primer pair SS6 1μl, 300-500ng / μl rice genomic template DNA 1μl, and sterilized ultrapure water to 10μl.

[0078] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, 35 cycles; final extension at 72°C for 10 min; the amplified product was stored at 4°C.

[0079] The amplified product was digested with restriction endonuclease BseRI and separated by 6% polyacrylamide denaturing electrophoresis. The band type of each molecular marker was detected by 260V constant voltage electrophoresis. The band size of Dongxiang wild rice genotype was 183bp, and the band size of Xieqing Zao B genotype was 204bp. The amplified band types of individual plants of 19H19 and its derivative BC5F2 population were compared with those of Dongxiang wild rice and Xieqing Zao B. The band type that was the same as the parent Xieqing Zao B type was recorded as 1, the same as the parent Dongxiang wild rice type was recorded as 2, heterozygous was recorded as 3, and missing was recorded as "-". OsNBS2 genotype detection was performed on 120 rice samples of the BC5F2 population ( Figure 6 ). There are 14 deletions, and the rest are 31 Xieqingzao B homozygous types, 26 Dongxiang wild rice homozygous types, and 49 parental heterozygous types (Table 1). The average germination rate of type 2 strains after aging seeds is 71.13%, which is significantly higher than the average germination rate of type 1 strains after aging seeds of 30.55%. The average germination rate of type 3 strains after aging seeds is between the two (Table 1). This shows that the molecular marker SS6 is linked to the storage resistance of rice seeds and can accurately assist in the selection of rice seed storage resistance ( Figure 5), the molecular marker of this gene can be used for assisted selection breeding improvement.

[0080] Example 3

[0081] Expression analysis and validation of storage-resistant candidate genes

[0082] Total RNA from rice seeds was extracted using a plant universal RNA extraction kit (Kangwei Century Company, Jiangsu). RT-qPCR analysis was performed using QuantiNova SYBR Green PCR Kit (QIAGEN), and the gene Osactin was used as an internal reference. The instrument used was the real-time fluorescence quantitative PCR instrument 7500 from Applied Biosystems, USA. Xieqingzao B and 19H19 seeds were treated at 42°C and 80% humidity for 28 days. RT-qPCR and transcriptome analysis were performed on rice seed samples after untreated and aged for 28 days. The results showed that the results of RT-qPCR and transcriptome analysis were consistent. Under aging treatment, qSS6 DXWR The candidate gene OsNBS2 was down-regulated in 19H19 after aging, but up-regulated in Xieqingzao B after aging ( Figure 4 ). The expression of this gene changes in opposite ways after aging in rice materials with different seed storage tolerance, which may be one of the key reasons for the difference in seed storage tolerance. DXWR The primers for RT-qPCR of candidate gene OsNBS2 are:

[0083] RT-F(SEQ ID No.3):5'-GAAGATTGCCTCGCTGAGTT-3';

[0084] RT-R (SEQ ID No. 4): 5'-GCGAAATTCATCCCTCAAGG-3'.

[0085] Table 1 Association analysis between gene frequency of deletion site and germination rate trait

[0086]

[0087] Table 2BC5F 2:3 Validation of OsNBS2 markers in populations

[0088] peak.marker LOD PVE ADD SS6 3.70 19.5671 19.3721

[0089] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of a rice seed storage-tolerant gene OsNBS2 linked molecular marker in identifying storage-tolerant rice varieties, wherein the rice seed storage-tolerant gene OsNBS2 linked molecular marker is located in the exon region of the rice OsNBS2 gene, and the nucleotide sequence of the linked molecular marker is shown in SEQ ID No.

5. The 19th nucleotide from the 5' end of the sequence has a - / G polymorphism, and the base G is missing in storage-intolerant rice varieties.

2. The use according to claim 1, characterized in that: The storage-resistant rice variety is a rice variety with a germination rate higher than 80% after aging treatment at 42° C. and 80% humidity for 28 days.

3. A primer pair for amplifying the rice seed storage gene OsNBS2 linked molecular marker as claimed in claim 1, characterized in that: The nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID No. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No.

2.

4. A method for identifying storage-resistant rice varieties, characterized in that: The following steps are involved: 1) extracting genomic DNA from rice, using the genomic DNA as a template, and performing PCR amplification using the primer pair of claim 3 to obtain an amplified product; 2) The amplified product obtained in step 1) is digested with BseRI and then subjected to electrophoresis and staining. When a band of 183 bp is obtained, the rice is a storage-resistant rice variety.

5. The method according to claim 4, characterized in that The system for PCR amplification in step 1) is: 5.0 μL of 2×ToloFastTaq Premix, 1 μL of each of the upstream and downstream primers with a concentration of 10 pmol / μL, 1 μL of rice genomic DNA with a concentration of 300-500 ng / μL, and sterilized ultrapure water to 10 μL.

6. The method according to claim 4 or 5, characterized in that: The PCR amplification procedure is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, 35 cycles; final extension at 72°C for 10 min; and storage at 4°C.

7. The method according to claim 4, characterized in that The electrophoresis in step 2) uses polyacrylamide gel electrophoresis, and the staining uses silver nitrate staining.

8. A kit for identifying storage-resistant rice varieties, characterized in that: Comprising the primer pair and reagents described in claim 3.

9. The kit according to claim 8, characterized in that The reagents included 2×Tolo Fast Taq Premix.

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