Functional marker of rice flood-resistant gene OsUGT75A and application thereof

Abstract

The invention provides a functional marker of a rice flood-tolerant gene OsUGT75A and application of the functional marker, specific primers based on different haplotypes are designed aiming at the rice flood-tolerant gene OsUGT75A binding linkage SNP and functional marker SNP sites, two sets of functional markers 75AFM and 75AFMB are successfully developed, both the two sets of markers can accurately identify the homozygous type and the heterozygous type of the OsUGT75A, and the application of the functional marker to the rice flood-tolerant gene OsUGT75A is wide. A foundation is laid for breeding application of the gene, and a new thought is provided for developing functional markers. The functional marker of the rice flooding-resistant gene OsUGT75A can be used for identifying the genotype of the rice flooding-resistant gene OsUGT75A, identifying the flooding-resistant character of the rice, identifying and screening flooding-resistant rice germplasm resources and molecular marker-assisted breeding of rice flooding-resistant varieties; theoretical basis and technical support are provided for excavating high-quality flooding-resistant germplasm resources, analyzing flooding-resistant hereditary basis and molecular breeding practice of new rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a functional marker of a rice flooding tolerance gene OsUGT75A and an application thereof. Background Art

[0002] Rice is one of the most important food crops in the world, and flooding stress is an important factor affecting rice growth and yield. In rice field management, rice germination is particularly sensitive to flooding. Excessive flooding will prevent seeds from germinating normally, thus affecting the final yield and quality. The aging of the agricultural population and the reduction of the rural labor force have made drone live broadcasting an important rice planting method with its advantages of cost saving, labor saving, and high efficiency. However, this method has high requirements for the flatness of the field. Rice seeds are prone to rot and root rot in low-lying and waterlogged areas, resulting in a lack of seedlings and a low seedling rate, which in turn affects the direct broadcast yield. Therefore, screening and cultivating germination-resistant flooded rice varieties is an effective means to reduce the loss of direct broadcast yield.

[0003] In recent years, the cloning and research of a series of flooding tolerance genes have made molecular breeding of flooding-tolerant rice possible. These flooding tolerance genes resist flooding through two different strategies. One is the submerged dormancy strategy, in which rice reduces its metabolic rate to conserve energy under flooding stress to cope with short-term flooding. The other is the water-breaking and rapid sprouting strategy, in which rice elongates its coleoptile under flooding conditions so that the leaves extend out of the water for gas exchange and photosynthesis. Under current production conditions in my country, the second water-breaking and rapid sprouting strategy has greater potential for application in production. Studies have found that multiple genes use the water-breaking and rapid sprouting strategy to participate in regulating the flooding tolerance of rice. Among them, the OsUGT75A gene can accelerate the elongation of the coleoptile by mediating the interaction between the JASMONATE ZIM-DOMAIN (OsJAZ) protein and the ABA INSENSITIVE (OsABI) protein. However, the effect of this gene in different genetic backgrounds is still controversial, and its potential application value in variety breeding needs further study. Therefore, combining genotype analysis with phenotypic evaluation, exploring the functional effects of flooding tolerance genes and developing reliable molecular markers can provide important technical support for improving flooding tolerance traits in rice.

[0004] There are two SNPs at LOC_Os11g25990 of the OsUGT75A gene, which are the reason for the differences in flooding tolerance among different rice varieties. The functional SNP1 "G" is at vg1114850366 on chromosome 11, and the functional SNP2 "A" is at vg1114849038 on chromosome 11. The CG content in the upstream and downstream regions of the functional SNP1 "G" (vg1114850366) of the OsUGT75A gene is too high, resulting in a high TM value, which is prone to nonspecific amplification (Deng J, Li Y, Shi W, Liu R, Ma C, Shi C. Primer design strategy for denaturation bubble-mediated strand exchange amplification. Analytical Biochemistry, 2020, 593: 113593). The CG content in the functional SNP2 "A" (vg1114849038) is too low, resulting in a low TM value, which in turn leads to low primer expansion efficiency at high temperature. The typing method represented by Tetra-primer ARMS-PCR technology requires appropriate high temperature to inhibit nonspecific amplification (Chiapparino E, Lee D, Donini P. Genotyping single nucleotide polymorphisms inbarley by tetra-primer ARMS–PCR, 2004, 47(2): 414-420), but Tetra-primer Due to the relatively fixed position of the inner primers in ARMS-PCR technology, the differences in melting temperature values ​​between primers are difficult to eliminate, resulting in a contradiction between specificity and amplification efficiency. Therefore, it is difficult to directly design effective functional markers based on these two functional SNPs using conventional Tetra-primer ARMS-PCR technology. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a functional marker of the rice flooding tolerance gene OsUGT75A and its application in view of the deficiencies of the above-mentioned prior art. OsUGT75A has two haplotypes. The present invention names the haplotype that can significantly improve the flooding tolerance of rice as OsUGT75A-T, and the flood-intolerant haplotype as OsUGT75A-I. The present invention finds that there are some highly reliable SNPs with linkage relationships near the functional SNP of the OsUGT75A gene. When the frequencies of these linkage SNPs remain stable in each subpopulation of the 3K genome (https: / / ricevarmap.ncpgr.cn / ), it indicates that the linkage relationship is very stable, and the genetic linkage disequilibrium (LD) is high and can be applied on a large scale. Based on this, the present invention combines linkage SNPs and functional marker SNP sites to design specific primers based on different haplotypes, and successfully develops two sets of functional markers-75AFM and 75AFMB. Both sets of markers can accurately identify homozygous and heterozygous OsUGT75A.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A functional marker of a rice flood tolerance gene OsUGT75A, the functional marker comprising 75AFM and 75AFMB,

[0008] The 75AFM functional marker primers include specific primers 75AFM-SI, 75AFM-DI and non-specific primers 75AFM-SO, 75AFM-DO; the nucleotide sequence of the primer 75AFM-SI is shown in SEQ ID NO.1, the nucleotide sequence of 75AFM-DI is shown in SEQ ID NO.2, the nucleotide sequence of 75AFM-SO is shown in SEQ ID NO.3, and the nucleotide sequence of 75AFM-DO is shown in SEQ ID NO.4;

[0009] The 75AFMB functional marker primers include specific primers 75AFMB-SF, 75AFMB-SR and non-specific primers 75AFMB-DF, 75AFMB-CR; the nucleotide sequence of the primer 75AFMB-SF is shown in SEQ ID NO.5, the nucleotide sequence of 75AFMB-SR is shown in SEQ ID NO.6, the nucleotide sequence of 75AFMB-DF is shown in SEQ ID NO.7, and the nucleotide sequence of 75AFMB-CR is shown in SEQ ID NO.8.

[0010] The present invention also provides an application of the functional label, including the following:

[0011] (1) Application in genotyping of the rice flooding tolerance gene OsUGT75A;

[0012] (2) Application in identifying flood tolerance traits in rice;

[0013] (3) Application in identification and screening of flood-tolerant rice germplasm resources;

[0014] (4) Application in molecular marker-assisted breeding of flood-tolerant rice varieties.

[0015] The present invention also provides a method for genotyping rice flooding tolerance gene OsUGT75A using the functional marker, comprising the following steps:

[0016] S1, extracting the rice genomic DNA to be tested;

[0017] S2, using the genomic DNA obtained in step S1 as a template, and using the 75AFM or 75AFMB functional marker primers to perform PCR amplification to obtain a PCR product;

[0018] S3. Separate the PCR products obtained in S2 by polyacrylamide gel electrophoresis and then perform silver staining to observe the band pattern.

[0019] Preferably, the PCR amplification system of the functional marker 75AFM is: 2×Taq PCR Mix 7.5 μL, 10 μM primers 75AFM-SO, 75AFM-SI, 75AFM-DI, 75AFM-DO 0.7 μL each, 1 μL of rice genomic DNA to be tested, and ddH2O is added to 15 μL; the PCR amplification reaction conditions are: 94°C 5 min, 94°C 30 s, 61°C 30 s, 72°C 30 s, 35 cycles, and finally 72°C extension for 10 min; if the amplified product shows two bands of 205 bp and 410 bp, it is the OsUGT75A-T homozygous genotype, if the amplified product shows three bands of 160 bp, 210 bp and 410 bp, it is the OsUGT75A-I homozygous genotype, and if the amplified product shows three bands of 160 bp, 205 bp and 410 bp, it is the heterozygous type.

[0020] Preferably, the PCR amplification system of the functional marker 75AFMB is: 2×Taq PCR Mix 7.5 μL, 10 μM primer 75AFM-DF 1 μL, 10 μM primer 75AFM-CR 0.5 μL, 10 μM primer 75AFM-SF 0.75 μL, 10 μM primer 75AFM-SR 0.75 μL, 1 μL of rice genomic DNA to be tested, and ddH2O is added to 15 μL; the PCR amplification reaction conditions are: 94°C 5 min, 94°C 30 s, 62°C 30 s, 72°C 30 s, 35 cycles, and finally 72°C extension for 10 min; if the amplified product shows two bands of 220 bp and 450 bp, it is the OsUGT75A-T homozygous genotype; if the amplified product shows 367 bp If there is only one band, it is the homozygous genotype of OsUGT75A-I. If the amplified product shows two bands of 220 bp and 367 bp, it is the heterozygous type.

[0021] Preferably, the OsUGT75A-T genotype is a flood-tolerant haplotype, and the OsUGT75A-I genotype is a flood-intolerant haplotype.

[0022] The present invention has the following significant technical effects:

[0023] 1. The present invention designs specific primers based on different haplotypes for the rice flood tolerance gene OsUGT75A in combination with linked SNP and functional marker SNP sites, and successfully develops two sets of functional markers 75AFM and 75AFMB. Both sets of markers can accurately identify the homozygous and heterozygous types of OsUGT75A, laying the foundation for the identification of the gene breeding application. The present invention provides a new idea for developing functional markers by using linked SNP sites near functional SNPs to develop functional markers.

[0024] 2. The functional marker of the rice flood tolerance gene OsUGT75A of the present invention can be used to identify the genotype of the rice flood tolerance gene OsUGT75A, identify the flood tolerance trait of rice, identify and screen flood-tolerant rice germplasm resources, and molecular marker-assisted breeding of rice flood-tolerant varieties, providing a theoretical basis and technical support for exploring high-quality flood-tolerant germplasm resources, analyzing the genetic basis of flood tolerance, and molecular breeding practice of new rice varieties.

[0025] 3. The present invention screened 21 rice materials with strong germination flooding resistance from 288 rice materials through phenotypic identification and genotypic analysis, of which 7 were japonica rice materials and 14 were indica rice materials. The germination flooding resistance traits were systematically identified, and germplasm resources with excellent performance were screened out. It was also proved that OsUGT75A-T has the effect of significantly enhancing rice flooding resistance under different genetic backgrounds, which provides a reliable basis for the feasibility and potential in large-scale breeding practice, and is helpful to promote the selection and popularization of new flood-tolerant rice varieties.

[0026] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the allele distribution of SNPs involved in the functional markers 75AFM and 75AFMB of Example 1 of the present invention, A: SNP distribution frequency of vg1114850366 and vg1114850439 among different rice populations, wherein vg1114850366 is a functional SNP site; B: SNP distribution frequency of vg1114849038, vg1114849137, and vg1114849222 among different rice populations, wherein vg1114849038 is a functional SNP site;

[0028] Figure 2 This is the functional marker primer design strategy of Example 1 of the present invention, A: 75AFM functional marker primer design strategy; B: 75AFMB functional marker primer design strategy, the red bases are SNP sites, and * indicates base mismatch;

[0029] Figure 3 It is the amplification result of the 75AFM functional marker in Example 2 of the present invention in rice materials with known genotypes, wherein lane M is 'Marker DL1000', and lanes 1-7 are 'Nipponbare', 'Zhejing 100', 'Zhehujing 25', 'Jiahe 218', '9311', 'Huanghuazhan', and 'kasalash';

[0030] Figure 4 The 75AFM functional marker of Example 2 of the present invention was sequenced and compared with the amplified fragments of 'Nipponbare' and '9311'. SNP1 and SNP2 were vg1114850366 and vg1114850439, of which vg1114850366 was a functional SNP site;

[0031] Figure 5 The results of genotyping 30 individual plants of the hybrid F2 generation of 'Jiahe 218' and 'Zhejing 100' by using the 75AFM functional marker of Example 2 of the present invention, wherein lane M is the marker DL1000, and lanes 1 to 30 are the individual plants of the hybrid F2 generation of 'Jiahe 218' and 'Zhejing 100';

[0032] Figure 6It is the amplification result of the 75AFMB functional marker in Example 2 of the present invention in rice materials with known genotypes, wherein lane M is 'Marker DL1000', and lanes 1-7 are 'Nipponbare', 'Zhejing 100', 'Zhehujing 25', 'Jiahe 218', '9311', 'Huanghuazhan', and 'kasalash';

[0033] Figure 7 The results of genotyping 30 individual plants of the hybrid F2 generation of 'Jiahe 218' and 'Zhejing 100' by using the 75AFMB functional marker of Example 2 of the present invention, wherein lane M is the marker DL1000, and lanes 1 to 30 are the individual plants of the hybrid F2 generation of 'Jiahe 218' and 'Zhejing 100';

[0034] Figure 8 The distribution of OsUGT75A-T gene in 288 test rice materials in Example 3 of the present invention;

[0035] Fig. 9 is a frequency distribution histogram of coleoptile length in Example 3 of the present invention;

[0036] Fig.10 is a box plot of the coleoptile lengths of indica rice and japonica rice according to Example 3 of the present invention;

[0037] Fig.11 This is a comparison of the coleoptile lengths of rice materials with the OsUGT75A allele of Example 3 of the present invention. DETAILED DESCRIPTION

[0038] Example 1

[0039] This example is the development of a functional marker for the rice flooding tolerance gene OsUGT75A.

[0040] The OsUGT75A gene has two haplotypes. The present invention named the haplotype that can significantly improve the flooding tolerance of rice as OsUGT75A-T, and the flood-intolerant haplotype as OsUGT75A-I. Referring to the principle of ARMS-PCR molecular markers, according to the presence of two functional SNP sites at LOC_Os11g25990 of the OsUGT75A gene (functional SNP1 "G" at vg1114850366 on chromosome 11, functional SNP2 "A" at vg1114849038 on chromosome 11), two different sets of functional markers were developed for the OsUGT75A gene, and primers were designed using Primer Premier5 software, and the primers were synthesized and sequenced by Shanghai Bioengineering.

[0041] By querying the RiceVarMap V2.0 database (https: / / ricevarmap.ncpgr.cn / ), it was found that there was a SNP (vg1114850439) upstream of the gene, which had a highly reliable linkage relationship with the functional SNP1 "G" (vg1114850366) of the gene ( Figure 1 A), therefore, the first set of functional markers 75AFM of OsUGT75A gene was developed based on these two linked SNP sites, and 4 primers were designed. First, non-specific primers 75AFM-SO and 75AFM-DO were designed, then specific primer 75AFM-SI was designed on functional SNP1 "G" (vg1114850366) and matched with OsUGT75A-I gene, and finally, specific primer 75AFM-DI was designed on linked SNP "G" (vg1114850439), and a base C was added at the 7th base at the 3' end of the primer, and a 1 base mismatch was set at the second base ( Figure 2 A). Theoretically, the functional marker 75AFM can amplify two bands of 175 bp and 340 bp in the homozygous OsUGT75A-T flooding tolerance gene, two bands of 139 bp and 340 bp in the homozygous OsUGT75A-I flooding intolerant gene, and three bands of 340 bp, 175 bp and 139 bp in the heterozygous OsUGT75A-I flooding intolerant gene.

[0042] By querying the RiceVarMap V2.0 database (https: / / ricevarmap.ncpgr.cn / ), it was found that there were two SNPs upstream of the gene, namely SNP "A" (vg1114849137) and SNP "T" (vg1114849222), which had a highly reliable linkage relationship with the functional SNP2 "A" (vg1114849038) of the gene ( Figure 1 B), therefore, the second set of functional markers 75AFMB of OsUGT75A gene was developed based on these three nearly completely linked SNP sites, and a total of 4 primers were designed. First, according to the functional SNP2 "A" (vg1114849038) and the linked marker SNP "T" (vg1114849222), specific primers 75AFMB-SF and 75AFMB-SR were designed to match the OsUGT75A-T gene; then, according to the linked SNP "A" (vg1114849137), a non-specific primer 75AFMB-DF was designed to match the OsUGT75A-I gene, and a base mismatch was set at the 4th base at the 3' end of the primer; finally, a non-specific primer 75AFMB-CR was designed as the downstream primer of 75AFMB-DF ( Figure 2B). The functional marker 75AFMB can amplify two bands in the OsUGT75A-T submergence tolerance homozygote, with sizes of 220 bp and 450 bp, and one band in the OsUGT75A-I submergence intolerance homozygote, with a size of 367 bp, and two bands in the heterozygote, with sizes of 220 bp and 367 bp, respectively.

[0043] The primer sequences for OsUGT75A functional marker are shown in Table 1.

[0044] Table 1 Primers and sequences of OsUGT75A functional marker

[0045]

[0046]

[0047] Note: The nucleotides with bold underline indicate mismatched nucleotides or inserted mismatched nucleotides, and the nucleotides with bold underlined wavy lines indicate the nucleotides at the functional SNP site.

[0048] Example 2

[0049] This example is the verification of the functional marker of the rice flooding tolerance gene OsUGT75A.

[0050] 1. 75AFM functional marking verification

[0051] The 75AFM functional markers were used to verify the functional markers of seven varieties with known genotypes ('Nipponbare', 'Zhejing 100', 'Zhehujing 25', 'Jiahe 218', '9311', 'Huanghuazhan', and 'kasalash'). Figure 3 It is the amplification result of 75AFM functional marker in rice materials with known genotypes, where lane M is 'Marker DL1000', and lanes 1-7 are 'Nipponbare', 'Zhejing 100', 'Zhehujing 25', 'Jiahe 218', '9311', 'Huanghuazhan', and 'kasalash', respectively. The results showed that the functional marker 75AFM amplified two bands in 'Nipponbare', 'Zhejing 100', and 'Zhehujing 25', with fragment sizes of about 205bp and 410bp, respectively, which were OsUGT75A-T genotypes; three bands were amplified in 'Jiahe 218', '9311', 'Huanghuazhan', and 'kasalash', with sizes of about 160bp, 210bp, and 410bp, respectively, of which one band was slightly larger than 205bp, and the specific size was unknown, which may be a secondary conformation of the heterozygous chain band, which was OsUGT75A-I genotype. There was a migration lag between the band size of the amplified fragment and the theoretical size.

[0052] To this end, this example uses agarose electrophoresis to cut the gel and recover and use 75AFM primers to sequence and verify the 410bp band of 'Nipponbare' and '9311'. The specific method is: the genomic DNA of 'Nipponbare' and '9311' is amplified by 75AFM primers, the PCR product is subjected to 1% agarose gel electrophoresis, the target band of about 410bp is observed and cut, and the target DNA is recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0. The recovered DNA fragments are sent to Shanghai Shenggong Company for Sanger sequencing, and bidirectional sequencing is performed using 75AFM external primers. The sequence results are analyzed by the software Chromas. The sequencing results show that the sequences of the amplified fragments of the two varieties are consistent with the target fragments, and the sequences of the two varieties are different in both the functional SNP sites and the linked SNP sites, indicating that the amplified main band fragment is the target fragment. We speculate that there are too many continuous A / T bases in the functional marker amplification interval, which leads to the generation of a Poly structure in the target fragment, affecting the charge effect of electrophoresis, thus causing its molecular migration rate to be slow in polyacrylamide electrophoresis. The sequencing results show that the electrophoresis test results are consistent with the known variety genotype ( Figure 4 ), therefore, this functional marker can be used to accurately type the OsUGT75A homozygous gene.

[0053] To further verify the accuracy of 75AFM functional markers for hybrid populations, genotype identification was performed on 30 F2 hybrids of 'Jiahe 218' and 'Zhejing 100'. Figure 5 As shown, lane M is a marker (TakaraDL1000), and lanes 1-30 are F2 hybrids of 'Jiahe 218' and 'Zhejing 100'. The results showed that 10 individual plants in lanes 1, 4, 7, 9, 11, 20, 22, 23, 28, and 29 amplified 2 bands, with sizes of about 205bp and 410bp, which were OsUGT75A-T; 7 individual plants in lanes 2, 3, 12, 13, 14, 19, and 25 amplified 3 bands, of which two fragments were about 160bp, 210bp, and 410bp, respectively, and one of them was The band is slightly larger than 205bp, which is OsUGT75A-I; 13 individual strains in lanes numbered 5, 6, 8, 10, 15, 16, 17, 18, 20, 24, 26, 27, and 30 amplified 3 bands, with sizes of about 160bp, 205bp, and 410bp, respectively, which are heterozygous. Compared with OsUGT75A-I, the fragment of about 200bp of this band type is slightly larger than that of OsUGT75A-T.

[0054] 2. 75AFMB functional marker verification

[0055] Since the amplified fragment of functional marker 75AFMB was different from the target fragment, we designed a second set of functional markers 75AFMB and used this marker to verify 7 varieties with known genotypes. The results are as follows: Figure 6 As shown, lane M is 'Marker DL1000', and lanes 1-7 are 'Nipponbare', 'Zhejing 100', 'Zhehujing 25', 'Jiahe 218', '9311', 'Huanghuazhan', and 'kasalash', respectively. The results showed that two bands were amplified in 'Nipponbare', 'Zhejing 100' and 'Zhehujing 25', with fragment sizes of 220bp and 450bp respectively, belonging to the OsUGT75A-T genotype; one band was amplified in 'Jiahe 218', '9311', 'Huanghuazhan' and 'kasalash', with a size of 367bp, belonging to the OsUGT75A-I genotype. However, because the CG content in the primer amplification region was too high, both band types produced some lighter non-specific amplification bands, but the main band sizes of the amplified fragments of different genotypes were consistent with the theoretical sizes, and the typing results were consistent with the functional marker 75AFM.

[0056] In order to verify the accuracy of the 75AFMB marker in the hybrid population, 30 F2 hybrids of 'Jiahe 218' and 'Zhejing 100' were tested for typing. Figure 7 As shown, lane M is a marker (Takara DL1000), and lanes 1-30 are F2 hybrids of 'Jiahe 218' and 'Zhejing 100'. The results showed that 10 individual plants in lanes 1, 4, 7, 9, 11, 20, 22, 23, 28, and 29 amplified 2 bands with sizes of 220 bp and 450 bp, which were OsUGT75A-T; 7 individual plants in lanes 2, 3, 12, 13, 14, 19, and 25 amplified 1 band with sizes of 367 bp, which were OsUGT75A-I; 13 individual plants in lanes 5, 6, 8, 10, 15, 16, 17, 18, 20, 24, 26, 27, and 30 amplified 2 bands with sizes of about 220 bp and 367 bp, which were heterozygous. The 450bp fragment and the 220bp fragment shared the same primer 75AFMB-CR during the PCR process. Since the short fragment was easier to complete the denaturation, annealing and extension steps, the amplification efficiency of the 450bp fragment was usually higher than that of the long fragment, resulting in the inhibition of the amplification of the 450bp fragment. The typing results of the functional marker 75AFMB and the functional marker 75AFM were consistent, proving that the functional marker 75AFMB can be used to accurately type the hybrid offspring of OsUGT75A.

[0057] Example 3

[0058] This example is an application of the rice flood tolerance gene OsUGT75A functional marker, using the rice flood tolerance gene OsUGT75A functional marker to identify the genotype of 288 rice materials and screen rice flood tolerance germplasm resources, the method is as follows:

[0059] 1. Experimental Materials

[0060] The materials included 'Nipponbare', 'Zhejing 100', 'Zhehujing 25', 'Jiahe 218', '9311', 'Huanghuazhan', 'kasalash' and other 288 collected germplasm resources from different sources, including 37 japonica rice varieties and 251 indica rice materials. In order to reduce the impact of seed dormancy, all tested materials were treated at 45℃ for 7 days.

[0061] 2. DNA extraction and genotype identification of germplasm resources

[0062] The total genomic DNA of rice seedling leaves was extracted by SDS method; the target fragment of the test material was amplified by 15 μL PCR amplification system, the PCR product was separated and electrophoresed on 4% polyacrylamide gel and then silver-stained, and the band pattern was photographed and preserved on a film observation light.

[0063] The PCR amplification system for the functional marker 75AFM was as follows: 2×Taq PCR Mix 7.5 μL, 10 μM specific primers and non-specific primers 2.8 μL (0.7 μL each of 75AFM-SO, 75AFM-SI, 75AFM-DI, and 75AFM-DO), 1 μL of rice genomic DNA to be tested, and ddH2O was added to 15 μL; the PCR amplification reaction conditions were: 94°C for 5 min, 94°C for 30 s, 61°C for 30 s, 72°C for 30 s, 35 cycles, and finally extension at 72°C for 10 min. If the amplified product shows two bands of 205bp and 410bp, it is the homozygous genotype of OsUGT75A-T. If the amplified product shows three bands of 160bp, 210bp and 410bp, it is the homozygous genotype of OsUGT75A-I. If the amplified product shows three bands of 160bp, 205bp and 410bp, it is the heterozygous type.

[0064] The PCR amplification system of the functional marker 75AFMB was as follows: 2×Taq PCR Mix 7.5μL, 10μM specific primers and 3μL non-specific primers (75AFM-DF 1μL, 75AFM-CR 0.5μL, 75AFM-SF 0.75μL, 75AFM-SR 0.75μL), 1μL of rice genomic DNA to be tested, and ddH2O was added to 15μL; the PCR amplification reaction conditions were: 94℃5min, 94℃30s, 62℃30s, 72℃30s, 35 cycles, and finally extension at 72℃ for 10min. If the amplified product shows two bands of 220bp and 450bp, it is the homozygous genotype of OsUGT75A-T. If the amplified product shows one band of 367bp, it is the homozygous genotype of OsUGT75A-I. If the amplified product shows two bands of 220bp and 367bp, it is the heterozygous type.

[0065] 3. Phenotypic identification of coleoptile length of germplasm resources

[0066] For each of the 288 rice varieties, 20 seeds with full grains and no obvious damage were selected, treated in 1.5% sodium hypochlorite solution for 12 minutes, rinsed with pure water, placed in a transparent plastic cup, and then pure water was carefully poured into the cup to ensure that the seeds sank to the bottom of the cup. The water depth in the cup was kept at 15 cm, and the cup was placed in a light and temperature incubator (12 h light / 12 h dark), and water was added every day to a water depth of 15 cm, and the temperature was set at 30°C. The length of the coleoptile was measured on the 11th day after flooding.

[0067] 4. Statistical analysis

[0068] SPSS20.0 software was used to input data on genotype, coleoptile length, rice varieties, and indica / japonica types. The correlation between coleoptile length and flooding tolerance genes was tested using regression analysis. Tukey's method was used to conduct multiple comparison tests on coleoptile length among rice varieties. Non-parametric tests were used to analyze the subspecies differences between indica and japonica rice. SPSS software or Excel 2016 software was used for graphical or tabular processing.

[0069] The genotypes and coleoptile lengths of the submergence tolerance gene OsUGT75A of 288 rice varieties are shown in Table 2 .

[0070] Table 2 Genotypes and coleoptile lengths of submergence tolerance gene OsUGT75A in 2288 rice varieties

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Note: A represents the OsUGT75A-I flood-intolerant genotype, B represents the OsUGT75A-T flood-tolerant genotype, and H represents the heterozygous type.

[0084] The results showed that 25 materials carried OsUGT75A-T, accounting for 8.7% of the total materials. Among them, 21 japonica materials and 4 indica materials, including IRAT144, MESBAB, and BG1370, contained OsUGT75A-T. The proportion of OsUGT75A-T in indica rice was low, but high in japonica rice ( Figure 8 ).

[0085] The results of the coleoptile length measurement of 288 rice varieties are as follows: Fig. 9 As shown, its length is continuously distributed, ranging from 4.08 to 57.83 mm. The frequency distribution of coleoptile length showed skewed distribution characteristics, with a coefficient of variation of 13.65%. This result shows that there are obvious genotypic differences in the elongation of rice coleoptiles under flooding conditions. By comparing the elongation length of coleoptiles of rice varieties under flooding conditions, varieties with flooding tolerance traits can be effectively screened. Among them, 21 varieties including Yue 24, R651, Nongxiangzhan, P469, P393, Ye T14878, Yongjing 108, Runxiangnuo 2, Jingjiai, Yangnongjing 1030, CH58, R1607, Jinyouzhan, Yujing 91, ZH-20-58, NY48, Yuzhenxiang, Yuekangxinzhan, R2172, ZH-19-58 and Xinhejing 49 showed strong flooding tolerance (Table 3). Further analysis showed that the average coleoptile lengths of indica and japonica rice varieties were 37.82 mm and 53.30 mm, respectively.

[0086] Table 3 Multiple comparisons of the top 21 materials with the longest coleoptile length

[0087]

[0088]

[0089] Since the data distribution did not conform to normality, the Mann-Whitney U test was used to analyze the difference between the two. The results showed that p = 0.006 < 0.05, and the Z value was -7.392, indicating that there was a statistically significant difference in the coleoptile length between indica and japonica rice varieties. The coefficient of variation of japonica rice was 19.32%, and the coefficient of variation of indica rice was 12.81%. Fig.10 As shown in the figure, the median length of the coleoptile of japonica rice is significantly higher than that of indica rice, indicating that the length of the coleoptile of japonica rice is significantly longer than that of indica rice as a whole. In addition, the bin range of japonica rice is wider, the data is more discrete, and the individual differences are more significant.

[0090] Regression analysis was performed on the coleoptile length of 288 varieties and the distribution of the OsUGT75A gene. It was found that OsUGT75A was significantly correlated with the coleoptile length (p=0.007<0.05) (Table 4).

[0091] Table 4 Regression coefficient and significance analysis of flood tolerance genes

[0092] name Regression coefficient B Standard error Standard coefficient T-value p-value constant 19.368 0.278 / 69.572 0 OsUGT75A 2.158 0.801 0.243 2.693 0.007

[0093] In this example, it was also found that the coleoptile length of rice materials carrying OsUGT75A-T was significantly longer than that of materials carrying OsUGT75A-I gene (p=0.006<0.05) ( Fig.11 ).

[0094] In this experiment, the genotype of OsUGT75A of 288 rice materials was identified by 75AFM and 75AFMB functional markers, and regression analysis was performed in combination with the distribution of coleoptile length and genotype. 21 rice materials with strong germination flooding tolerance were screened from 288 rice materials, of which 7 were japonica rice materials and 14 were indica rice materials. This example further verified the role of OsUGT75A-T in significantly enhancing rice flooding resistance under different genetic backgrounds, indicating that OsUGT75A-T has the potential to be developed as a molecular breeding tool. Therefore, it is particularly important to introduce the function of OsUGT75A-T into a wide range of genetic background materials that are close to production needs for verification. Breeders can use the two functional markers 75AFM and 75AFMB developed by the present invention to verify the effect of OsUGT75A-T in a more complex field environment. This verification can not only evaluate its actual breeding value, but also provide a reliable basis for its feasibility and potential in large-scale breeding practice, thereby promoting the selection and promotion of new flood-tolerant rice varieties.

[0095] The present invention designs specific primers based on different haplotypes for OsUGT75A in combination with linked SNP and functional marker SNP sites, and successfully develops two sets of functional markers, 75AFM and 75AFMB. Both sets of markers can accurately identify the homozygous and heterozygous types of OsUGT75A, and provide a basis for the identification of the application of this gene in breeding. In addition, the present invention provides a new idea for breeding and developing functional markers by using linked SNP sites near the functional SNP. However, in practical applications, these markers each have certain limitations. The continuous T / A content of the amplified fragment of the functional marker 75AFM is too high, and the adjacent continuous short poly A and poly T formally form an SSR-like structure, which affects its migration behavior in electrophoresis, resulting in the actual observed fragment length being inconsistent with the designed length. This problem can be solved by single-stranded nucleic acid electrophoresis. The CG content in the amplification region of the functional marker 75AFMB is too high, and the base pairing stability is strong, which easily leads to the formation of erroneous amplification products. In addition, the excessively high CG content makes it difficult for the primers to effectively anneal to the template, thereby affecting the specificity and efficiency of the amplification. When using the functional marker 75AFMB, it is necessary to pay attention to the primer ratio and control the annealing temperature to ensure the reliability and accuracy of the amplification results.

[0096] The above 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 change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Functional marker of rice flooding tolerance gene OsUGT75A, characterized in that: The functional markers include 75AFM and 75AFMB, The 75AFM functional marker primers include specific primers 75AFM-SI, 75AFM-DI and non-specific primers 75AFM-SO, 75AFM-DO; the nucleotide sequence of the primer 75AFM-SI is shown in SEQ ID NO.1, the nucleotide sequence of 75AFM-DI is shown in SEQ ID NO.2, the nucleotide sequence of 75AFM-SO is shown in SEQ ID NO.3, and the nucleotide sequence of 75AFM-DO is shown in SEQ ID NO.4; The 75AFMB functional marker primers include specific primers 75AFMB-SF, 75AFMB-SR and non-specific primers 75AFMB-DF, 75AFMB-CR; the nucleotide sequence of the primer 75AFMB-SF is shown in SEQ ID NO.5, the nucleotide sequence of 75AFMB-SR is shown in SEQ ID NO.6, the nucleotide sequence of 75AFMB-DF is shown in SEQ ID NO.7, and the nucleotide sequence of 75AFMB-CR is shown in SEQ ID NO.

8.

2. The use of the functional label according to claim 1, characterized in that: Includes the following: (1) Application in genotyping of the rice flooding tolerance gene OsUGT75A; (2) Application in identifying flood tolerance traits in rice; (3) Application in identification and screening of flood-tolerant rice germplasm resources; (4) Application in molecular marker-assisted breeding of flood-tolerant rice varieties.

3. A method for genotyping rice flooding tolerance gene OsUGT75A using the functional marker of claim 1, characterized in that: The steps include: S1, extracting the rice genomic DNA to be tested; S2, using the genomic DNA obtained in step S1 as a template, and using the 75AFM or 75AFMB functional marker primers to perform PCR amplification to obtain a PCR product; S3. Separate the PCR products obtained in S2 by polyacrylamide gel electrophoresis and then perform silver staining to observe the band pattern.

4. The method according to claim 3, characterized in that The PCR amplification system of the functional marker 75AFM is as follows: 7.5 μL of 2×Taq PCR Mix, 0.7 μL of each of 10 μM primers 75AFM-SO, 75AFM-SI, 75AFM-DI and 75AFM-DO, 1 μL of rice genomic DNA to be tested, and ddH2O is added to 15 μL; the PCR amplification reaction conditions are as follows: 94°C for 5 min, 94°C for 30 s, 61°C for 30 s, 72°C for 30 s, 35 cycles, and finally extension at 72°C for 10 min; if the amplified product shows two bands of 205 bp and 410 bp, it is an OsUGT75A-T homozygous genotype; if the amplified product shows three bands of 160 bp, 210 bp and 410 bp, it is an OsUGT75A-I homozygous genotype; if the amplified product shows three bands of 160 bp, 205 bp and 410 bp, it is a heterozygous type.

5. The method according to claim 3, characterized in that: The PCR amplification system of the functional marker 75AFMB is: 2×Taq PCR Mix 7.5 μL, 10 μM primer 75AFM-DF 1 μL, 10 μM primer 75AFM-CR 0.5 μL, 10 μM primer 75AFM-SF 0.75 μL, 10 μM primer 75AFM-SR 0.75 μL, 1 μL of rice genomic DNA to be tested, and ddH2O is added to 15 μL; the PCR amplification reaction conditions are: 94°C for 5 min, 94°C for 30 s, 62°C for 30 s, 72°C for 30 s, 35 cycles, and finally 72°C for 10 min; if the amplified product has two bands of 220 bp and 450 bp, it is the homozygous genotype of OsUGT75A-T, and if the amplified product has 367 bp, it is the homozygous genotype of OsUGT75A-T. If there is only one band, it is the homozygous genotype of OsUGT75A-I. If the amplified product shows two bands of 220 bp and 367 bp, it is the heterozygous type.

6. The method according to claim 4 or 5, characterized in that: The OsUGT75A-T genotype is a flood-tolerant haplotype, and the OsUGT75A-I genotype is a flood-intolerant haplotype.

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