A KASP molecular marker method for identifying superior alleles of rice nitrogen efficient gene NRT2.3

Abstract

The present invention provides a KASP molecular marker method for identifying excellent alleles of rice nitrogen high-efficiency gene NRT2.3, which relates to the field of bioengineering technology. According to the base variation of A-T (adenine-thymine) at 82bp upstream of the NRT2.3 start codon between Nanjing 5818 and sequenced indica and japonica varieties, KASP molecular marker primers are designed to perform fluorescent quantitative PCR amplification on DNA of different rice materials and determine whether they have NRT2.3 excellent genotypes. The method of the present invention can quickly and accurately identify whether there is NRT2.3 excellent genotype in rice germplasm resources or breeding populations, and can simultaneously perform high-throughput detection on more samples, which is faster and more efficient. Therefore, the molecular marker method can further improve the selection efficiency of NRT2.3 excellent genotypes in breeding and accelerate the breeding process of nitrogen high-efficiency rice varieties.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a KASP molecular marker method for identifying superior alleles of rice nitrogen efficient gene NRT2.3. Background Art

[0002] Rice is the largest food crop in my country. Nitrogen is one of the most demanded nutrients in the growth and development of rice, and is also the most sensitive factor affecting rice growth. It plays an important role in the growth and development of rice and the formation of yield. The large-scale use of nitrogen fertilizer has made an important contribution to the increase in rice yield in my country since the last century. However, the pursuit of high yield has led to a rapid increase in the application of nitrogen fertilizer in my country's rice production. The nitrogen fertilizer consumed by my country's rice production accounts for about 37% of the world's total rice nitrogen fertilizer consumption. China has become the world's largest nitrogen fertilizer consumer, but the nitrogen fertilizer absorption and utilization efficiency of rice is significantly lower than the world average (Ma Qun et al. Current status and countermeasures of rice nitrogen fertilizer utilization in my country. Guangdong Agricultural Science, 2010, 37(11):126-129). Excessive nitrogen application has led to serious air, soil and water pollution. Improving the nitrogen use efficiency of rice is of great significance for reducing nitrogen fertilizer pollution to the ecosystem, promoting sustainable agricultural development and ensuring national food security (Du Wenting et al. Effect of reduced nitrogen fertilizer application on the yield of my country's three major grain crops. Chinese Agricultural Science, 2022, 55(24):4863-4878).

[0003] There are significant differences in nitrogen absorption and utilization among different rice varieties. Nitrogen-efficient varieties can achieve higher yields and nitrogen fertilizer utilization rates at lower nitrogen application rates. It is a key issue that needs to be addressed at present to study the molecular mechanism of nitrogen absorption and utilization in rice, identify the excellent genotypes of rice nitrogen utilization efficiency, and improve rice varieties with nitrogen utilization efficiency by screening genotypes.

[0004] There are two main forms of nitrogen in the soil: inorganic nitrogen and organic nitrogen. Among them, inorganic nitrogen mainly comes in two forms: ammonium nitrogen and nitrate nitrogen, which is the main source of nitrogen for plants in the soil. The efficiency of nitrate nitrogen absorption and assimilation is a key factor in rice productivity (Wang S et al. Functional analysis of the OsNPF4.5 nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants. Proc Natl Acad Sci USA, 2020, 117: 16649-16659.). The absorption of nitrate nitrogen by rice is mainly responsible for high-affinity nitrate transport proteins, among which the NRT2.3 gene (LOC_Os01g50820, Os01g0704100) encoding high-affinity nitrate transport proteins is a key gene affecting rice nitrogen utilization efficiency. OsNRT2.3 has two transcripts, OsNRT2.3a is mainly expressed in roots and in NO3 – OsNRT2.3b plays an important role in long-distance transport from underground to aboveground (Tang et al. Knockdown of a rice stelar nitrate transporteralters long-distance translocation but not root influx. Plant Physiology, 2012, 160(4): 2052-2063). OsNRT2.3b is mainly expressed in the aboveground part and is not affected by changes in nitrogen form and concentration. It can enhance the pH buffering capacity of rice, improve the plant's absorption of nitrogen, iron and phosphorus, and improve the effective utilization rate of nitrogen. It is very important for plants to adapt to different forms of nitrogen sources (Fan et al. Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields. Proc Natl Acad Sci USA, 2016, 113(26): 7118-7123).

[0005] Xueya Guo found that 239 rice genetic materials had differences in nitrogen absorption, utilization and yield under different nitrogen treatment conditions (Xueya Guo, Association analysis of natural variation of OsNRT2.3 gene and nitrogen use efficiency, Nanjing Agricultural University, 2020). OsNRT2.3 was further divided into three main haplotypes, among which the HapB type had linked SNPs at two sites 111 and 7bp before the ATG of the NRT2.3 gene. The plant height, yield, nitrogen use efficiency and other nitrogen absorption-related indicators of the material with this allele variation were significantly improved, and at the same time, it led to an increase in NRT2.3b protein levels and higher yields during high temperature stress (Zhang et al. High-temperature adaptation of an OsNRT2.3 allele is thermos regulated by small RNAs. Science Advances, 2022, 8(47): eadc9785). Identification of superior alleles of the NRT2.3 gene is a feasible method for breeding nitrogen-efficient rice varieties.

[0006] Nanjing 5818 is a good-tasting japonica rice variety approved by Jiangsu Province in 2023. Previous studies have found that Nanjing 5818 is a dual-high-efficiency variety with high nitrogen utilization efficiency, and has high nitrogen utilization efficiency at different nitrogen application levels (Zhao Ling et al. Preliminary study on nitrogen utilization efficiency of Nanjing series varieties. Journal of Jiangsu Agricultural Sciences, 2022, 38(05): 1153-1161). By sequencing the whole genome of Nanjing 5818 and its three sister lines with large differences in nitrogen utilization efficiency, analyzing the sequences and SNP sites of 40 known nitrogen utilization-related genes, it was determined that there was a new specific SNP site in the 5'UTR region (-82bp) of the NRT2.3 gene that was closely related to nitrogen utilization efficiency and closely linked. Nanjing 5818 was T, and the other three sister lines were A, which has great application prospects in improving nitrogen utilization efficiency.

[0007] The identification of traditional nitrogen use efficiency requires the selection of phenotypes such as plant yield and nitrogen content under different nitrogen levels, which not only takes a lot of energy and time, but is also easily disturbed by the environment. The best way to improve the selection efficiency of nitrogen-efficient rice is to develop specific molecular markers for auxiliary selection by utilizing the SNP differences in target genes closely related to nitrogen use efficiency. At present, there are no reports on molecular markers for the selection of excellent genotypes of NRT2.3. Since the SNP variation site of its excellent allele is located in the 5'UTR region (-82bp) and has a high GC content (62.34%), it is difficult to develop traditional PCR markers. In order to improve the identification of germplasm resources containing NRT2.3 excellent alleles and the efficiency of variety breeding, it is necessary to establish an efficient genotyping method to identify the excellent genotypes of NRT2.3.

[0008] Competitive allele-specific PCR (Kompetitive Allele-Specific PCR, KASP) molecular marker is a new type of marker based on high-sensitivity fluorescence detection. Its principle is to design two forward primers and a universal reverse primer for the SNP site of the allele. Each forward primer carries a different fluorescent marker and combines with the universal reverse primer to PCR amplify the DNA of the sample to be tested. The allelic variation is reflected by different fluorescent signals (He CL, etal. SNP genotyping: the KASP assay. MethodsMolBiol, 2014, 1145, 75-86). Compared with traditional SSR markers, KASP molecular markers have high throughput and good stability, and are suitable for identifying various SNP sites that cannot be amplified and detected by traditional PCR (Zhao Yue et al. Application progress of KASP marker technology in crop gene positioning. Northern Horticulture, 2023, (19): 122-127; Rosas JE, et al., One-step, codominant detection of imidazolinoneresistance mutations in weedy rice ( Oryza sativa L). ElectronicJBiotechnol,2014, 17(2): 95-101).

[0009] With the decrease in genome sequencing costs and the increase in accuracy, KASP markers are increasingly used in the mining and identification of excellent alleles of important rice traits and the screening of target genotypes (Zhao Chuanchao et al. Development and application of KASP markers for the rice blast resistance gene Pi2. Journal of South China Agricultural University, 2023, 44(05):725-734; Deng Zhao et al. Development and verification of tightly linked KASP markers for the rice brown planthopper resistance gene Bph43. Hubei Agricultural Sciences, 2023, 62(06):169-174). The development of KASP markers targeting the 5'UTR region of the NRT2.3 gene has very important breeding value for the identification and utilization of excellent alleles of the NRT2.3 gene. Summary of the invention

[0010] In view of the above-mentioned prior art, the present invention proposes a KASP molecular marker method for identifying superior alleles of rice nitrogen-efficient gene NRT2.3. In view of the problem that the breeding of nitrogen-efficient rice containing NRT2.3 superior allele is time-consuming and labor-intensive, is extremely susceptible to environmental interference and has low accuracy, a new specific site located 82 bp upstream of the promoter is used to design KASP molecular marker primers to quickly and accurately identify the superior genotype of rice NRT2.3, so as to achieve the purpose of improving the nitrogen utilization efficiency of varieties and rapid selection by utilizing the NRT2.3 superior positioning gene.

[0011] The present invention provides a KASP molecular marker method for identifying superior alleles of rice nitrogen efficient gene NRT2.3, and the KASP molecular marker primers of NRT2.3 gene include:

[0012] The forward primer NRT2.3-FT has the following sequence:

[0013] 5'-GAAGGTGACCAAGTTCATGCTGCGCTTTCCGCTATGCTATT-3';

[0014] The forward primer NRT2.3-FA has the following sequence:

[0015] 5'-GAAGGTCGGAGTCAACGGATTGCGCTTTCCGCTATGCTATA-3';

[0016] The reverse universal primer NRT2.3-R has the following sequence:

[0017] 5'-AGCAGCTTAGCTAGTGACTGWGTGTA-3'.

[0018] Preferably, when synthesizing the NRT2.3 gene KASP molecular marker primers, the 5' end of the forward primer NRT2.3-FT is added with a fluorescent signal label of carboxyfluorescein FAM, and the 5' end of the forward primer NRT2.3-FA is added with a fluorescent signal label of 6-carboxy 3,6-diamino-9-phenoxy-2,7-dimethyl-fluorenyl ammonium chloride VIC.

[0019] Preferably, the method specifically comprises the following steps:

[0020] S1. Extracting genomic DNA from rice plants;

[0021] S2, adding the NRT2.3 gene KASP molecular marker primers to the same PCR reaction system, setting up two blank controls with double distilled water instead of sample template DNA, and amplifying the DNA of rice germplasm resources or breeding population plants on a fluorescent quantitative PCR instrument;

[0022] S3. Data analysis was performed using Step One Software v2.2.2.

[0023] Preferably, the PCR reaction system is 5 μl, including: 1.25 μl of sample template DNA with 10 ng / μl, fluorescent primers mixed in a ratio of upstream typing primer 1: upstream typing primer 2: downstream universal primer = 1:1:3, 1.25 μl of primer mixture, 2.5 μl of 2×KASP Master mix;

[0024] PCR reaction conditions included: activation at 95°C for 10 min; denaturation at 95°C for 20 sec, annealing at 61-55°C for 60 sec, decreasing by 0.6°C in each cycle, for 10 cycles; denaturation at 95°C for 20 sec, annealing at 55°C for 60 sec, for 27 cycles.

[0025] Preferably, the judgment is made according to the color and position of the data analysis graph of Step One Software v2.2.2, wherein the blue square close to the Y-axis indicates the homozygous NRT2.3 gene carrying the A allele variation; the yellow dot close to the X-axis indicates the homozygous NRT2.3 gene carrying the T allele variation; the green triangle between the X and Y axes indicates the heterozygous NRT2.3 gene carrying both the A / T allele variation; the black dot indicates the blank control in which double distilled water is used instead of the sample template DNA.

[0026] The nitrogen utilization efficiency of the present invention is determined by the following method: 4 pure nitrogen application rates are set in the field, which are 0, 150, 300 and 450 kg / hm 2Different treatments were separated by cement ridges and irrigated separately. Each material was planted in 9 rows, 9 plants in each row, and the row-plant spacing was 25.0 cm × 13.3 cm. This was repeated 3 times. Sowing was done on May 10 and transplanting was done on June 15. The compound fertilizer was used uniformly as base fertilizer, and the application rate was 45 kg / hm2 of phosphorus fertilizer (P2O5) 2 , Potassium fertilizer (K2O) 60kg / hm 2 . Seedlings were raised in plastic trays and blankets, and the seedlings were 25 days old. They were transplanted by artificial simulator. The nitrogen fertilizer management mode was basal fertilizer: tillering fertilizer: ear fertilizer = 2:4:4, and other management measures were the same as conventional field cultivation. After maturity, all the crops were harvested after removing two rows around the four weeks in each section, and the weight was weighed after drying to a moisture content of 15%. The agronomic nitrogen utilization efficiency was calculated = (grain yield per unit area in the area with high nitrogen fertilizer application - grain yield per unit area in the area with low nitrogen fertilizer application) / the difference in pure nitrogen application per unit area of ​​farmland in the two areas in the season)

[0027] In the present invention, OsNRT2.3 is 2116bp in length and produces two transcripts by selective splicing. OsNRT2.3a expression is induced by nitrate. Under extremely low external nitrate concentration conditions, OsNRT2.3a participates in the long-distance transport of nitrate from the root to the aerial part (Tang Z et al. Knockdown of a rice, stelar nitrate transporteralters long-distance translocation but not rootinflux. Plant Physiol, 2012, 160: 2052-2063). Expression of OsNRT2.3a and its chaperone can significantly improve rice yield and nitrogen use efficiency (Chen J G et al. Co-overexpression of OsNAR2.1 and OsNRT2.3a increased agronomic nitrogen use efficiency in transgenic rice plants. Front Plant Sci, 2020, 11: 1245). The expression of OsNRT2.3b is insensitive to nitrate and plays a role in sensing the cytoplasmic pH of phloem cells to balance NO3 – and NH4 +OsNRT2.3b plays a key role in rice uptake. Overexpression of OsNRT2.3b promotes rice growth, nitrogen use efficiency and final grain yield (Lu Haiyan, et al. Overexpression of OsNRT2.3b promotes rice Wuyujing 7 growth and increases grain yield. Molecular Plant Breeding 2015, 13: 497-504; Fan XR et al. Overexpression of a pH-sensitive nitratetransporter in rice increases crop yields. Proc Natl Acad Sci USA, 2016, 113, 7118-7123).

[0028] The NRT2.3 gene is highly conserved in rice. By querying the 3k database, it was found that compared with the Nipponbare genome sequence, 3024 resources had 4 synonymous mutations in the NRT2.3 gene coding region, and 4 and 2 SNPs in the 5' and 3' UTR regions, respectively. The SNPs in the 5' region were located at 73, 100, 135, and 216 bp, respectively, and 1654, 145, 2259, and 240 materials had mutated SNP types. The SNPs in the 3' region were located at 1931 and 2051 bp, respectively, and 2255 and 2252 materials had mutated SNP genotypes. NRT2.3 The gene promoter region has a TATA-box element located at 325-328bp. Zhang et al. showed that TATA-box plays a key regulatory role in the transcription of OsNRT2.3. After the T at -83bp was mutated to C, the TATA-box mutated to TACA-box, which promoted the transcription of NRT2.3b and inhibited the expression of NRT2.3a, thereby improving the nitrogen utilization efficiency and yield of rice (Yong Zhang, et al. OsTBP2.1, a TATA-Binding Protein, Alters the Ratio of OsNRT2 . 3b to OsNRT2.3a and Improves Rice Grain Yield. IntJMolSci . 2022, 23(18):10795).

[0029] We found that the variety Nanjing 5818 has the characteristic of high nitrogen efficiency. We sequenced the genome of Nanjing 5818 and its sister lines with the same parental origin and low nitrogen utilization efficiency. After analyzing the sequences of 40 nitrogen utilization-related genes, we found that an AT base mutation occurred at the 328bp (-82bp) of the NRT2.3 gene. This mutation led to the promotion of NRT2.3b transcription and the inhibition of NRT2.3a expression, thereby improving the nitrogen utilization rate and yield of rice. The present invention uses NRT2.3 at the specific site of -82bp to design a specific marker for the excellent genotype selection of the offspring with genetic improvement of nitrogen utilization efficiency using Nanjing 5818. The provided KASP molecular marker method for identifying the excellent allele of the rice nitrogen efficiency gene NRT2.3 has the following advantages:

[0030] 1. The molecular marker provided by the present invention is a specific marker designed based on the specific nucleic acid variation site of the NRT2.3 gene in the 5'UTR region of Nanjing 5818. It can directly reflect the genotype of the plant NRT2.3 without errors in genotype identification caused by genetic exchange between markers and genes.

[0031] 2. The molecular marker method provided by the present invention can detect the single base variation (A / T) at 82 bp upstream of the start codon of the NRT2.3 gene in Nanjing 5818, without the need to determine its allele through sequencing and a large number of complex genetic tests.

[0032] 3. The molecular marker method provided by the present invention can be effectively used for auxiliary breeding of nitrogen-efficient rice varieties containing the NRT2.3 superior allele. Since the identification of nitrogen efficiency in traditional breeding is mainly through field planting of rice materials, different nitrogen treatments are set and the parameters of nitrogen utilization efficiency are calculated according to the grain yield after maturity. This method is not only affected by environmental factors such as temperature and moisture during growth and development, but also time-consuming and labor-intensive, easily affected by environmental interference, and has low accuracy. Using the KASP molecular marker method of the present invention, DNA can be extracted after seed germination to identify and select the NRT2.3 genotype, eliminate a large number of individual plants containing non-target genotypes, effectively improve the selection efficiency of nitrogen-efficient rice varieties containing the NRT2.3 superior genotype, shorten the breeding cycle and reduce breeding costs.

[0033] 4. Since the SNP of the excellent genotype of NRT2.3 is located in the 5'UTR region and has a high GC content, it is difficult to develop traditional markers for PCR amplification. The present invention uses the KASP molecular marker method to identify different genotypes of the NRT2.3 gene mutation site, which can not only effectively distinguish its homozygous and heterozygous genotypes, but also omit the electrophoresis detection process, which is more efficient and fast, and is suitable for high-throughput identification and selection of a large number of rice resources and intermediate materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a diagram showing the DNA sequencing results of the TATA-box region of the Nanjing 5818 and sister line NRT2.3 genes in the examples of the present invention.

[0035] Figure 2 It is a diagram of the KASP molecular marker design strategy for detecting the NRT2.3 superior genotype in an embodiment of the present invention. Figure 2 In the figure, numbers indicate the relative position of bases in the ATG of NRT2.3 genome; shaded areas indicate bases that differ among Nanjing 5818, Nipponbare and Nanjing 5818; bold letters indicate the KASP primer sequences of NRT2.3 gene.

[0036] Figure 3 This is a diagram showing the detection of the NRT2.3 genotype of different rice varieties (lines) by the KASP molecular marker in the embodiment of the present invention. Figure 3 In the figure, the black origin represents the blank control without template DNA; the yellow dots and blue squares represent the homozygous NRT2.3 gene carrying the T allele - Nanjing 5818, and the homozygous NRT2.3 gene carrying the A allele, Nipponbare, Nanjing 5718, Sidao 301, Nanjing 0145, Nanjing 9108, Yuejing 2, Zaoxiangyu, Xudao 9, etc.

[0037] Figure 4 This is a diagram showing the detection of the NRT2.3 genotype of some individual plants in the Nanjing 5818 / F2 population by the KASP molecular marker in the embodiment of the present invention. Figure 4 In the figure, blue and red represent the homozygous NRT2.3 gene carrying A and T allele variations, respectively, and green represents the heterozygous NRT2.3 gene carrying both A and T allele variations.

[0038] Figure 5 This is a technical roadmap for using the NRT2.3 gene to improve the nitrogen efficiency of the new japonica rice variety Nanjing 9108 in an embodiment of the present invention.

[0039] Figure 6 It is a comparison diagram of the nitrogen utilization efficiency improved lines of Nanjing 9108 and Nanjing 9108 containing the NRT2.3 gene in the examples of the present invention. Figure 6 Medium, A: without NRT2.3 5818 Plants of the homozygous Nanjing 9108 under four nitrogen levels; B: NRT2.3 5818 Plants of the homozygous improved line of Nanjing 9108 under four nitrogen levels. DETAILED DESCRIPTION

[0040] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0041] The sequence of the NRT2.3 gene is relatively conservative in different rice varieties. By querying the genomic information of rice resources in the 3k database (https: / / snp-seek.irri.org / ), it was found that compared with the Nipponbare genome sequence, the NRT2.3 gene of 3024 rice resources only had 4 synonymous mutations in the coding region, and 4 and 2 SNPs in the 5' and 3' UTR regions, respectively. The promoter region of the NRT2.3 gene has a TATA box element, located 82 to 85bp before the promoter. Zhang et al. created mutants and confirmed that the mutation of -83bp T to C promoted the transcription of NRT2.3b and inhibited the expression of NRT2.3a, thereby improving the nitrogen utilization efficiency and yield of rice (Yong Zhang et al. High-temperature adaptation of an OsNRT2.3 allele is thermos-regulated by small RNAs. Science Advances , 2022, 8(47): eadc9785).

[0042] The present invention studies the nitrogen-efficient variety Nanjing 5818, and clarifies that there is an AT mutation in the NRT2.3 gene in the 82 bp before the promoter and in the TATA-box element. The genome sequences of Nanjing 5818 and 3024 rice resources in the 3k database are compared, but the SNP site cannot be found, and it is speculated that the site is a new SNP site unique to Nanjing 5818. KASP molecular markers are designed based on the specific nucleotide differences. The use of this marker method can not only quickly and accurately identify whether the rice breeding population carries the excellent genotype of NRT2.3, but also realize high-throughput detection of more samples to be tested, which is faster than conventional molecular marker technology. In breeding, the selection efficiency of the excellent homozygous genotype containing NRT2.3 can be further improved, and the breeding process of nitrogen-efficient rice varieties can be accelerated.

[0043] Example: A KASP molecular marker method for identifying superior alleles of rice nitrogen efficient gene NRT2.3, the specific implementation steps are as follows:

[0044] 1. Experimental materials (all of which are publicly known and used, and can be provided by the Jiangsu Agricultural Germplasm Resources Medium-term Bank)

[0045] Homozygous genotype material containing NRT2.3 superior allele: conventional japonica rice variety Nanjing 5818 (Institute of Grain Crops, Jiangsu Academy of Agricultural Sciences).

[0046] The homozygous genotype materials of excellent alleles not resistant to NRT2.3: conventional japonica rice varieties Nipponbare (Agricultural Experiment Station in Aichi Prefecture, Japan), Koshihikari (Agricultural Experiment Station in Fukui Prefecture, Japan), Nanjing 5818 (Institute of Food Crops, Jiangsu Academy of Agricultural Sciences), Nanjing 5718 (Institute of Food Crops, Jiangsu Academy of Agricultural Sciences), Sidao 301 (Suqian Agricultural Science Institute, Jiangsu Academy of Agricultural Sciences), Nanjing 0145 (Institute of Food Crops, Jiangsu Academy of Agricultural Sciences), Nanjing 9108 (Institute of Food Crops, Jiangsu Academy of Agricultural Sciences), Yuexiangjing No. 2 (Jiangsu Yuefeng Seed Technology Co., Ltd.), Zaoxiangyu (Guangming Seed Co., Ltd.), Xudao No. 9 (Xuzhou Agricultural Science Institute, Xuhuai District, Jiangsu Province) (Institute of Agricultural Sciences, Nanjing Agricultural University), Ningxiangjing 9 (Rice Research Institute, Nanjing Agricultural University), Huaidao 9 (Huaiyin Agricultural Science Research Institute, Xuhuai District, Jiangsu), Wukejing 7375 (Jiangsu (Wujin) Rice Research Institute, etc.), Wankenjing 516 (Anhui Wanken Seed Co., Ltd., etc.), Jianongjing 6 (Shanghai Jiading District Agricultural Technology Extension Service Center, etc.), Huzaoxiangruan 1 (Shanghai Academy of Agricultural Sciences, etc.), Chujing 39 (Chuxiong Prefecture Agricultural Science Research Institute, Yunnan), Yanfeng 47 (Liaoning Salt-alkali Land Utilization Research Institute), indica rice restorer line material Zhenhui 084 (Zhenjiang Agricultural Science Research Institute, Jiangsu Hilly Area), 93-11 (Agricultural Science Research Institute, Lixiahe Area, Jiangsu).

[0047] Other plant materials included F1 plants obtained by hybridization with Nanjing 9108 as the female parent and Nanjing 5818 as the male parent, as well as 179 F2 plants.

[0048] 2. Nucleic acid sequence analysis

[0049] The NRT2.3 genome sequence of Nanjing 5818 is 2116 bp in length, with a coding region of 1997 bp. Two transcripts are generated by alternative splicing during transcription: OsNRT2.3a (AK109776) and OsNRT2.3b (AK072215). NRT2.3a encodes a 516-amino acid plasma membrane protein that plays a role in the long-distance transport of nitrate from roots to the aerial parts in the xylem, while NRT2.3b encodes a shorter 486-amino acid membrane protein that is moderately expressed in the aerial phloem and weakly expressed in the roots. OsNRT2.3a and OsNRT2.3b differ in amino acid sequence by 30 amino acids, with a similarity of 94.2% (Feng HM et al. Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status. J Experimental Botany, 2011, 62(7): 2319-2332).

[0050] OsNRT2.3a and OsNRT2.3b have different lengths of 5'UTR. The 5'UTR of OsNRT2.3a is 47 bp and has G-box and ABRE elements.

[0051] (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The 5'UTR of OsNRT2.3b is 247 bp, and contains ABRE, TCT-motif, TGACG-motif, CGTCA-motif, G-box, LTR, MYB and other elements.

[0052] The genome sequences of Minghui 63, Zhenshan 97 and 93-11 in the genome database of Huazhong Agricultural University were used for re-comparison (http: / / rice.hzau.edu.cn / ). It was found that only the genome sequences of Nanjing 5818 NRT2.3 There is a T at -82 bp upstream of the gene start codon, and this site is A in all four sequenced varieties. This indicates that the specific site of the single nucleotide variation exists in the NRT2.3 genome sequence of Nanjing 5818.

[0053] 3. Development of Molecular Markers

[0054] The full-length sequence of the Nipponbare NRT2.3 gene was downloaded, and the 150 bp sequence upstream and downstream of the specific single nucleotide variant site was selected. The labeled primer sequence was designed using oligo7.57, and the KASP primers with FAM and VIC were synthesized by Nanjing Jisi Huiyuan Biotechnology Co., Ltd. ( Figure 1 ).

[0055] 4. Mutual verification of genotype and phenotype by molecular marker detection

[0056] (1) DNA extraction

[0057] Thirty days after rice transplanting, fresh young leaves of rice varieties (lines), F1 hybrids, and F2 populations were collected and genomic DNA was extracted using the CTAB method (Murray MG, et al.Rapid isolation of high molecular weight plant DNA, Nucleic Acids Res, 1980, 8(19): 4321-4325). The specific steps are as follows: take 2-3cm rice tender leaves and put them into a 2ml centrifuge tube, freeze them in liquid nitrogen and grind them into powder; after grinding, add 700μl of DNA extraction solution with a mass concentration of 2% CTAB preheated in a 65℃ constant temperature water bath for 30min, mix well and put them into a 35℃ constant temperature water bath for 30min, shaking every 10min during the period. Take out the centrifuge tube, add 700μl of 24:1 (chloroform: isoamyl alcohol = 24:1) to each tube in a fume hood, mix well and put them into a centrifuge at 1,2000rpm for 15min. Take 400μl of the supernatant and put it into another 1.5ml centrifuge tube, add 400μl of isopropanol precooled at -20℃, mix slowly, and put them into a -20℃ refrigerator to settle freely for 30min. Then centrifuge at 10000rpm for 10min, discard the supernatant, add 400μl of 70% ethanol for washing, discard the supernatant and air-dry. Add 100-200μl of TE buffer, place at room temperature for 1-2 days to dissolve into DNA solution, and store at -20℃.

[0058] (2) Molecular marker amplification and genotyping

[0059] The KASP molecular marker primers of the NRT2.3 gene were added to the same PCR reaction system, and two blank controls were set up with double distilled water instead of template DNA. The DNA of rice germplasm resources or breeding population plants was amplified on a fluorescent quantitative PCR instrument. The 5μl reaction system included 1.25μl DNA (10ng / ul), 1.25μl fluorescent primer mixture, and 2×KASP Master mix 2.5L. PCR reaction conditions: 95℃ activation for 10min; 95℃ denaturation for 20sec, 61-55℃ annealing for 60sec, 0.6℃ reduction for each cycle, 10 cycles; 95℃ denaturation for 20sec, 55℃ annealing for 60sec, 27 cycles.

[0060] The PCR results were analyzed using Step One Software v2.2.2.

[0061] (3) Results analysis

[0062] ① KASP molecular markers for NRT2.3 genotyping of different varieties (lines)

[0063] KASP molecular marker primers were used to amplify and genotype 68 indica and japonica rice varieties (lines) on a real-time fluorescence quantitative PCR instrument. The results showed that the molecular marker primers can clearly separate the two genotypes, and the blue squares close to the Y axis represent NRT2.3 carrying the A allele variant. A Homozygous for the gene; the yellow dots close to the X-axis represent NRT2.3 carrying the T allele variant T Gene homozygotes; the green triangle in the middle of the X and Y axes represents the heterozygotes of the NRT2.3 gene carrying both A / T allele variations; the black dots represent the blank controls in which double distilled water is used instead of sample template DNA.

[0064] ② Genotyping of NRT2.3 in Huaidao 9 / Nanjing 5818 F2 Population by KASP Molecular Markers

[0065] To verify the detection effect of KASP molecular markers on heterozygous genotypes, DNA of 179 individuals in the F2 population of Huaidao No. 9 / Nanjing 5818 was genotyped. KASP primers can effectively distinguish the three genotypes ( Figure 4 ). The blue dots close to the Y axis are NRT2.3 carrying the A allele variant. A The red dots close to the X-axis are homozygous for the gene, and they carry the T allele. NRT2.3 T Gene homozygotes, the green dot in the middle of the X and Y axes is a heterozygote carrying the A / T allele variation of the NRT2.3 gene. A Homozygous for the gene, containing NRT2.3 A / T Heterozygous for NRT2.3 T The number of plants homozygous for the gene was 62, 166, and 72, respectively. Statistical analysis was consistent with the segregation ratio of 1:2:1, which was consistent with the segregation law of a pair of genes (χ 2 =4.08<χ 2 0.05, 2 , 0.10< P <0.25).

[0066] (V) The molecular marker method of the present invention is used for breeding nitrogen-efficient rice containing the NRT2.3 gene

[0067] Nanjing 5818 with an excellent NRT2.3 genotype was selected as a donor, and Nanjing 9108 with a low nitrogen utilization efficiency was selected as a recipient. The strategy of backcross breeding was adopted to improve the nitrogen utilization efficiency of Nanjing 9108. In the process of continuous backcrossing and selfing, the KASP marker of the present invention was used to track and detect the NRT2.3 genotype, and combined with the systematic selection of agronomic traits, an improved strain with high nitrogen efficiency and other agronomic traits basically consistent with Nanjing 9108 was obtained in the BC3F4 generation ( Figure 5, Figure 6 ). This indicates that the molecular marker method of the present invention can be used to select the NRT2.3 excellent genotype of the rice breeding population, thereby achieving efficient breeding of nitrogen-efficient rice varieties.

[0068] The above are only implementation modes of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure made using the contents of the present invention specification, directly or indirectly used in other related technical fields, is also within the patent protection scope of the present invention.

Claims

1. A KASP molecular marker method for identifying superior alleles of rice nitrogen efficient gene NRT2.3, characterized in that: NRT2.3 gene KASP molecular marker primers include: The forward primer NRT2.3-FT has the following sequence: 5'-GAAGGTGACCAAGTTCATGCTGCGCTTTCCGCTATGCTATT-3'; The forward primer NRT2.3-FA has the following sequence: 5'-GAAGGTCGGAGTCAACGGATTGCGCTTTCCGCTATGCTATA-3'; Reverse universal primer NRT2.3 - R, whose sequence is: 5'-AGCAGCTTAGCTAGTGACTGWGTGTA-3'.

2. The KASP molecular labeling method according to claim 1, characterized in that: When synthesizing the NRT2.3 gene KASP molecular marker primers, the 5' end of the forward primer NRT2.3-FT is added with a fluorescent signal label of carboxyfluorescein FAM, and the 5' end of the forward primer NRT2.3-FA is added with a fluorescent signal label of 6-carboxyl 3,6-diamino-9-phenoxy-2,7-dimethyl-fluorenyl ammonium chloride VIC.

3. The KASP molecular labeling method according to claim 2, characterized in that: The specific steps include: S1. Extracting genomic DNA from rice plants; S2, adding the NRT2.3 gene KASP molecular marker primers to the same PCR reaction system, setting up two blank controls with double distilled water instead of sample template DNA, and amplifying the DNA of rice germplasm resources or breeding population plants on a fluorescent quantitative PCR instrument; S3. Data analysis was performed using Step One Software v2.2.

2.

4. The KASP molecular labeling method according to claim 3, characterized in that: The PCR reaction system is 5 μl, including: 1.25 μl of sample template DNA (10 ng / μl), fluorescent primers according to the forward primer NRT2.3-FT: forward primer NRT2.3-FA: reverse universal primer NRT2.3 - R = 1:1:3 ratio, take 1.25μl of primer mixture and 2×KASP Master mix 2.5μl; PCR reaction conditions included: activation at 95°C for 10 min; denaturation at 95°C for 20 sec, annealing at 61-55°C for 60 sec, decreasing by 0.6°C in each cycle, for 10 cycles; denaturation at 95°C for 20 sec, annealing at 55°C for 60 sec, for 27 cycles.

5. The KASP molecular labeling method according to claim 3 or 4, characterized in that: The judgment was made based on the color and position of the data analysis graph of Step One Softwarev2.2.2 software. The blue squares close to the Y-axis represent the homozygous NRT2.3 gene carrying the A allele variation; the yellow dots close to the X-axis represent the homozygous NRT2.3 gene carrying the T allele variation; the green triangle between the X and Y axes represents the heterozygous NRT2.3 gene carrying both the A / T allele variation; the black dots represent the blank control in which double distilled water was used instead of the sample template DNA.

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