Rice head rice rate site qHRY4-10 as well as molecular marker and application thereof

Abstract

The invention belongs to agricultural biotechnology engineering, and particularly relates to a rice head rice rate site qHRY4-10 as well as a molecular marker and application thereof. The molecular marker is a site qHRY4-10 which is located at a chromosome 4 2480324bp and is remarkably related to the head rice rate, and the head rice rate of the site carrying A nucleotide allelic variation germplasm is remarkably higher than that of the site carrying T nucleotide allelic variation germplasm. The method for identifying the head rice rate of the rice is verified to be accurate and reliable.

Description

Technical Field

[0001] The invention belongs to agricultural biotechnology engineering, and specifically relates to a rice head rice rate locus qHRY4-10 and a molecular marker and application thereof. Background Art

[0002] Rice is one of the most important food crops in the world, and more than half of the world's population relies on rice as their staple food. On the basis of the current stable and increased rice production, further improving rice quality has become the main focus of rice breeding and industrial development. The polished rice rate is used to measure the processing quality and edible quality of rice, and is an important indicator of the commercial quality of rice. Rice with a high polished rice rate has a high rice yield, less broken rice, neat rice grains, and a high utilization rate of the edible part during processing. It has a high commodity economic value and great economic benefits for farmers. Therefore, improving the polished rice rate is of great significance to the economic value and edible quality of rice.

[0003] The head rice rate is a complex quantitative trait controlled by multiple genes and affected by environmental factors such as light, temperature, and humidity during grain filling, harvesting, and post-harvest. Therefore, conventional breeding based on phenotypic selection is difficult to effectively breed varieties with high head rice rates. With the development of molecular biology, analyzing the molecular genetic basis of controlling the head rice rate, using genes related to the head rice rate and molecular markers closely linked to them, and carrying out efficient and accurate molecular breeding can effectively solve this problem and make a breakthrough in the breeding of rice with high head rice rates. Summary of the invention

[0004] The present invention has been found through research that there is a locus qHRY4-10 significantly correlated with the polished rice rate at 2480324bp on chromosome 4 of rice, and that the polished rice rate of germplasm carrying the A nucleotide allele variation is significantly higher than that of germplasm carrying the T nucleotide allele variation, thereby completing the present invention.

[0005] The present invention provides a molecular marker for rice head milled rice rate, which is a locus qHRY4-10 located at 2480324bp on chromosome 4 and significantly correlated with the head milled rice rate, specifically a nucleotide difference at a physical position of 2480324bp, and the head milled rice rate of germplasm carrying A nucleotide allele variation at the locus is extremely significantly higher than that of germplasm carrying T nucleotide allele variation.

[0006] The present invention provides primers for detecting the molecular markers, preferably, they are KASP primers.

[0007] Specifically, it includes a high whole rice rate allele variation specific primer qHRY4-10-A for detecting the A nucleotide allele variation site, a low whole rice rate allele variation specific primer qHRY4-10-T for detecting the T nucleotide allele variation, and optionally also includes a universal primer qHRY4-10-C.

[0008] More specifically, the molecular marker qHRY4-10-A-) primers are as follows:

[0009] 5'-GAAGGTGACCAAGTTCATGCTAATTACATCCAGGTCCTCGAGCA-3',

[0010] The primers for qHRY4-10-T are as follows:

[0011] 5'-GAAGGTCGGAGTCAACGGATTAATTACATCCAGGTCCTCGAGCT-3',

[0012] The primer for qHRY4-10-C was as follows: 5′-CAACTTATTCACAGATAGGCCCTCC-3′.

[0013] The present invention also provides application of the detection method of the molecular marker of rice head milled rice rate in identifying the rice head milled rice rate.

[0014] Specifically, the detection method is gene sequencing or molecular amplification.

[0015] The present invention provides a method for identifying rice polished rice rate, which adopts the detection method of rice polished rice rate molecular marker to obtain the result of the molecular marker to judge the rice polished rice rate, wherein the polished rice rate of the germplasm carrying the A nucleotide allele variation at the site is extremely significantly higher than that of the germplasm carrying the T nucleotide allele variation.

[0016] Specifically, the primers are used for detection.

[0017] A more specific method is as follows: extracting genomic DNA of the rice to be tested, using the genomic DNA as a template, and using the primers to perform PCR amplification to obtain a PCR product;

[0018] Specifically, in the PCR amplification system, the concentrations of qHRY4-10-A and qHRY4-10-T are 30-45 μmol / μL, the concentration of qHRY4-10-C is 80-100 μmol / μL, and the three primers are mixed at a volume ratio of 1:1:1 to form KASP Primer Mix; the total reaction volume of the reaction system is 10.14 μL, and the reaction system includes: DNA 5 μL, 2x KASP Master Mix 5 μL, KASP Primer Mix 0.14 μL;

[0019] The amplification program was as follows: (1) pre-denaturation at 94°C for 15 min; (2) denaturation at 94°C for 20 s, extension at 61°C for 60 s, ramping at a rate of 0.6°C / cycle, for 10 cycles; (3) denaturation at 94°C for 20 s, extension at 55°C for 60 s, for 26 cycles.

[0020] Furthermore, the detection step includes: identifying the whole polished rice rate of the rice to be tested according to the nucleotide sequence of the PCR product: if the typing of the PCR product is type A, the rice to be tested is or is a candidate for a rice variety with a higher whole polished rice rate; if the typing of the PCR product is type T, the rice to be tested is or is a candidate for a rice variety with a lower whole polished rice rate.

[0021] The method for identifying the whole polished rice rate of rice disclosed by the invention is verified to be accurate and reliable and has application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Results of genome-wide association study (GWAS) of 164 japonica rice accessions. Figure A and Figure B respectively show the Manhattan plot and QQ plot of GWAS results, and qHRY4-10 in the present invention is marked with an arrow in the figure.

[0023] Figure 2 :Comparison of head rice rate between two allelic variant germplasms of qHRY4-10.

[0024] Figure 3 :Detection results of specific molecular markers of qHRY4-10 (A) and validation of the effectiveness of qHRY4-10 in identification of whole polished rice rate (B). DETAILED DESCRIPTION

[0025] Example 1: Identification of rice head rice rate-related locus qHRY4-10 and its specific molecular marker

[0026] I. Identification of the locus qHRY4-10 associated with rice head rice rate

[0027] 1. Test materials and identification of polished rice rate

[0028] A total of 164 representative japonica rice germplasms from different rice-growing areas in my country were used as research materials. These 164 japonica rice varieties were planted at the experimental base of the Rice Research Institute of Anhui Academy of Agricultural Sciences in 2018. After the seeds were harvested, the whole polished rice rate of rice was determined according to NY / T2334-2013.

[0029] 2. Genome-wide association analysis of rice head rice rate

[0030] A genome-wide association study (GWAS) was performed using 1,580,393 SNP data from the whole genome of 164 japonica rice varieties and data on the head rice rate. The genome-wide association study was performed using the software package Tassel (v5) (reference: Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES. (2007) TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 23: 2633-2635.), combined with the kinship matrix of the population and the population structure Q matrix, using a mixed linear model (MLM) for calculation. P < 10 -4 As the significance threshold, a locus qHRY4-10 ( Figure 1 ).

[0031] 3. Allelic variation analysis of qHRY4-10 loci

[0032] In order to test the correlation between qHRY4-10 and head milled rice rate, this study compared the head milled rice rate of germplasms carrying different alleles at this locus. It was found that the head milled rice rate of germplasms carrying the A nucleotide allele was significantly higher than that of germplasms carrying the T nucleotide allele ( Figure 2 , P<0.01).

[0033] 2. Development of qHRY4-10 specific molecular markers

[0034] 1. Design of KASP primers

[0035] According to the nucleotide difference of qHRY4-10 at the physical position 2480324bp, the sequence of 100bp above and below 2480324bp was obtained from the Nipponbare reference genome (IRGSP-1.0) (URL: https: / / plants.ensembl.org / Oryza_sativa / Info / Index), and the specific KASP markers for qHRY4-10 were designed using Primer3Plus (URL: https: / / www.primer3plus.com / ), including the high head milled rice rate allele specific primer qHRY4-10(A), the low head milled rice rate allele specific primer qHRY4-10(T) and the common primer qHRY4-10(Common).

[0036] The molecular marker qHRY4-10 (A) primer sequence:

[0037] 5'-GAAGGTGACCAAGTTCATGCTAATTACATCCAGGTCCTCGAGCA-3',

[0038] qHRY4-10(T) primer sequence:

[0039] 5'-GAAGGTCGGAGTCAACGGATTAATTACATCCAGGTCCTCGAGCT-3',

[0040] qHRY4-10 (Common) primer sequence: 5′-CAACTTATTCACAGATAGGCCCTCC-3′.

[0041] Embodiment 2, the identification method of rice head rice rate

[0042] 1. Method for identifying the polished rice rate of the rice to be tested

[0043] The details are as follows: extracting genomic DNA of rice to be tested, using the genomic DNA as a template and molecular markers to perform PCR amplification to obtain PCR products;

[0044] In the above experiment, the DNA extraction method steps are as follows: (1) Place a small amount of fresh rice leaves in a 2 mL centrifuge tube filled with steel balls, immerse in liquid nitrogen for 10 minutes, and then quickly place in a sampler to grind into powder; (2) Add 600 μL CTAB buffer and place in a 65°C water bath for 30 minutes; (3) Add an equal volume of chloroform:isoamyl alcohol (24:1) solution and shake vigorously to mix thoroughly; (4) Centrifuge at 12,000 rpm for 10 minutes, and draw 400 μL of the supernatant into a new 1.5 mL centrifuge tube; (5) Add 400 μL of pre-cooled isopropanol and place in a -20°C refrigerator for at least 20 minutes to precipitate DNA; (6) Centrifuge at 12,000 rpm for 10 minutes and discard the supernatant; (7) Air dry at room temperature and add 400 μL of double distilled sterile water (ddHO). 2 O) Dissolve DNA.

[0045] Molecular marker primers were prepared as follows: (1) the primers were purified by ULTRPAGE; (2) the primer powder was dissolved, the concentration of qHRY4-10 (A) and qHRY4-10 (T) was 36 μmol / μL, and the concentration of qHRY4-10 (Common) was 90 μmol / μL; (3) the three primers were then mixed in a volume ratio of 1:1:1 to form KASP primer Mix.

[0046] The KASP genotyping experiment was as follows: a 96-well plate was used for reaction and loading, with a total reaction volume of 10.14 μL per well, and the reaction system included: 5 μL DNA, 5 μL 2x KASP Master Mix, and 0.14 μL KASP primer mix. After completion, the 96-well plate was sealed with a membrane that was centrifuged and fluorescently viewed, and PCR amplification was performed after confirming that there were no air holes. Amplification procedure: (1) 94°C pre-denaturation for 15 min; (2) 94°C denaturation for 20 s, 61°C extension for 60 s, ramping at a rate of 0.6°C / cycle, 10 cycles; (3) 94°C denaturation for 20 s, 55°C extension for 60 s, 26 cycles, and (4) data reading and analysis.

[0047] 2. Identify the polished rice rate of the rice to be tested based on the nucleotide sequence of the PCR product:

[0048] If the PCR product is type A, the rice to be tested is or is a candidate for a rice variety with a higher polished rice rate;

[0049] If the typing of the PCR product is T-type, the rice to be tested is or is a candidate rice variety with a lower polished rice rate.

[0050] Example 3: Application of qHRY4-10 specific molecular marker in identification of head rice rate of rice varieties

[0051] 1. Test Materials

[0052] The tested materials were Wufeng 1, Aisizhan, Fuxin, Shuangchao 25, Fanfengruanzhan, Shuangkang 42, Sansizhan 1, 9598, Fengliuzhan, Sanerai, Kaihong 5, Honghui 237, Honghui 255, 14W-157, Fuhui 838 and Chuanguanghui 909.

[0053] 2. Determination of the polished rice rate of rice according to NY / T 2334-2013

[0054] The polished rice rate of the test material was identified according to the method in step 1 of Example 1.

[0055] The results are shown in Table 1. It can be seen from the table that among the 16 rice varieties, the polished rice rates of Wufeng 1, Aisizhan, Fuxin, Shuangchao 25, Fanfengruanzhan, Shuangkang 42, Sansizhan 1, 9598, Fengliuzhan, Sanerai and Kaihong 5 were all above 65%, and they were all rice varieties with relatively high polished rice rates; the polished rice rates of Honghui 237, Honghui 255, 14W-157, Fuhui 838 and Chuanguanghui 909 were all below 31%, and they were all rice varieties with relatively low polished rice rates.

[0056] Table 1. Genotype and head rice rate identification results of the tested rice varieties

[0057]

[0058]

[0059] 3. Rice genotype identification

[0060] The genomic DNA of the test material in step 1 is extracted, and the obtained genomic DNA is used as a template to perform typing using the specific molecular marker of qHRY4-10. The specific experimental process is the same as step 1 of Example 2.

[0061] The allelic variation at the physical position 2480324 bp of chromosome 4 was detected using the qHRY4-10 specific molecular marker. The detection results are shown in Table 1 and Figure 3 As shown, the PCR product typing of Wufeng 1, Aisizhan, Fuxin, Shuangchao 25, Fanfengruanzhan, Shuangkang 42, Sansizhan 1, 9598, Fengliuzhan, Sanerai and Kaihong 5 were all A-type, and according to the identification method of rice polished rice rate in Example 2, Wufeng 1, Aisizhan, Fuxin, Shuangchao 25, Fanfengruanzhan, Shuangkang 42, Sansizhan 1, 9598, Fengliuzhan, Sanerai and Kaihong 5 were all rice varieties with higher polished rice rates; the PCR product typing of Honghui 237, Honghui 255, 14W-157, Fuhui 838 and Chuanguanghui 909 were all T-type, and according to the identification method of rice polished rice rate in Example 2, Honghui 237, Honghui 255, 14W-157, Fuhui 838 and Chuanguanghui 909 were all rice varieties with lower polished rice rates.

[0062] It can be seen that the identification method of the rice head rice rate of the present invention is completely consistent with the head rice rate identification result in step 2, and the head rice rate of the rice variety with allele variation A is 66.5% on average, which is significantly higher than the head rice rate of the rice variety with allele variation T (mean value is 25.9%) ( Figure 3 , P<0.05). This indicates that the method for identifying the polished rice rate of the rice of the present invention is accurate and reliable.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A molecular marker for rice head rice rate, characterized in that: It is a locus located at 2480324bp on chromosome 4 that is significantly associated with the head rice rate. qHRY4-10 , specifically the nucleotide difference at the physical position 2480324bp, and the whole polished rice rate of the germplasm carrying the A nucleotide allele at this site was significantly higher than that of the germplasm carrying the T nucleotide allele.

2. A primer for detecting the molecular marker as claimed in claim 1, preferably, a KASP primer.

3. The primer according to claim 2, characterized in that The invention comprises a high whole rice polishing rate allele variation specific primer for detecting the A nucleotide allele variation site. qHRY4-10 -A. Specific primers for detecting allele variation of low head milled rice carrying T nucleotide allele variation qHRY4-10 -T, optionally also includes a universal primer qHRY4-10 -C.

4. The primer according to claim 3, characterized in that The molecular marker qHRY4-10 -A primer is as follows: 5'-GAAGGTGACCAAGTTCATGCTAATTACATCCAGGTCCTCGAGCA-3', qHRY4-10 -T primer is as follows: 5'-GAAGGTCGGAGTCAACGGATTAATTACATCCAGGTCCTCGAGCT-3', qHRY4-10 The -C primer was as follows: 5′-CAACTTATTCACAGATAGGCCCTCC-3′.

5. Use of the method for detecting molecular markers for rice head rice rate as claimed in claim 1 in identifying rice head rice rate.

6. The use according to claim 1, characterized in that The detection method is gene sequencing and molecular amplification.

7. A method for identifying the polished rice rate of rice, characterized in that: The method for detecting the molecular marker of rice head rice rate as described in claim 1 is used to obtain the result of the molecular marker to determine the rice head rice rate, wherein the head rice rate of the germplasm carrying the A nucleotide allele variation at this site is extremely significantly higher than that of the germplasm carrying the T nucleotide allele variation.

8. The method according to claim 7, characterized in that The detection is performed using the primers described in any one of claims 2 to 4.

9. The method according to claim 8, characterized in that The specific method is as follows: extracting genomic DNA of rice to be tested, using the genomic DNA as a template, and using the primers to perform PCR amplification to obtain a PCR product; Specifically, in the PCR amplification system, qHRY4-10 -A and qHRY4-10 The concentration of -T is 30-45 μmol / μL, the concentration of qHRY4-10-C is 80-100 μmol / μL, and the three primers are mixed at a volume ratio of 1:1:1 to form KASP Primer Mix; the total reaction volume of the reaction system is 10.14 μL, and the reaction system includes: DNA 5 μL, 2x KASP Master Mix 5 μL, KASP Primer Mix 0.14 μL; The amplification program was as follows: (1) pre-denaturation at 94°C for 15 min; (2) denaturation at 94°C for 20 s, extension at 61°C for 60 s, ramping at a rate of 0.6°C / cycle, for 10 cycles; (3) denaturation at 94°C for 20 s, extension at 55°C for 60 s, for 26 cycles.

10. The method according to claim 8 or 9, characterized in that The detection step includes: identifying the whole polished rice rate of the rice to be tested according to the nucleotide sequence of the PCR product: if the typing of the PCR product is type A, the rice to be tested is or is a candidate for a rice variety with a higher whole polished rice rate; if the typing of the PCR product is type T, the rice to be tested is or is a candidate for a rice variety with a lower whole polished rice rate.

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