Rice milled rice rate site qhry4-10 and molecular marker and application thereof

Abstract

The application belongs to the field of agricultural biotechnology engineering, and particularly relates to a milled rice rate locus of rice qHRY4-10 and a molecular marker and application thereof. The molecular marker is a milled rice rate locus significantly related to a 2480324bp on chromosome 4 qHRY4-10 , and the milled rice rate of a kind of germplasm carrying A nucleotide allelic variation is extremely significantly higher than that of a kind of germplasm carrying T nucleotide allelic variation. The milled rice rate identification method of the application is accurate and reliable.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural biotechnology engineering, and particularly relates to a rice milled rice rate site qHRY4-10 and a molecular marker and application thereof. BACKGROUND

[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 main food. On the basis of current stable and increased rice production, further improving rice quality has become the main direction of rice breeding and industry development. The milled rice rate is used to measure the processing quality and edible quality of rice, and is an important indicator of rice commodity. High milled rice rate rice has high milling rate, less broken rice, uniform rice, high edible part utilization rate, high commodity economic value, and large economic benefit of farmers, so improving the milled rice rate is of great significance to the economic value and edible quality of rice.

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

[0004] Through research, the present application finds that there is a site qHRY4-10 significantly related to milled rice rate at 2480324bp on chromosome 4 of rice, and the milled rice rate of germplasm carrying A nucleotide allelic variation is significantly higher than that of germplasm carrying T nucleotide allelic variation, thereby completing the present application.

[0005] The present application provides a rice milled rice rate molecular marker, which is a site qHRY4-10 significantly related to milled rice rate at 2480324bp on chromosome 4, specifically a nucleotide difference at physical position 2480324bp, and the milled rice rate of germplasm carrying A nucleotide allelic variation at the site is significantly higher than that of germplasm carrying T nucleotide allelic variation.

[0006] The present application provides primers for detecting the molecular marker, preferably KASP primers.

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

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

[0009] 5'-GAAGGTGACCAAGTTCATGCTAATTACATCCAGGTCCTCGAGCA-3',

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

[0011] 5'-GAAGGTCGGAGTCAACGGATTAATTACATCCAGGTCCTCGAGCT-3',

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

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

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

[0015] The present invention provides a method for identifying rice head rice rate, which adopts the detection method of the rice head rice rate molecular marker to obtain the result of the molecular marker to judge the rice head rice rate, wherein the head 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 from the rice to be tested, using the genomic DNA as a template and employing 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: 5 μL DNA, 5 μL 2x KASP Master Mix, and 0.14 μL KASP primer mix;

[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 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 head rice rate of the rice to be tested based on 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 head 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 head rice rate.

[0021] The method for identifying the polished rice rate of the rice disclosed by the present 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. Figures A and B show the Manhattan plot and QQ plot of the GWAS results, respectively. 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 the specific molecular marker qHRY4-10 (A) and validation of the effectiveness of qHRY4-10 in identifying the head rice rate (B). DETAILED DESCRIPTION

[0025] Example 1: Identification of the Rice Head Rice Rate-Related Locus qHRY4-10 and Its Specific Molecular Markers

[0026] Identification of the locus qHRY4-10 associated with rice head rice yield

[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 head rice rate of the 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 and head rice rate data from the whole genome of 164 japonica rice varieties. The genome-wide association analysis was performed using the Tassel (v5) software package (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.). The kinship matrix and population structure Q matrix of the population were combined to calculate the mixed linear model (MLM). P < 10 -4 As the significance threshold, a locus qHRY4-10 ( Figure 1 ).

[0031] 3. Allelic Variation Analysis of qHRY4-10 Loci

[0032] 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] II. Development of qHRY4-10-specific molecular markers

[0034] 1. Design of KASP primers

[0035] Based on the nucleotide difference at the physical position 2480324bp of qHRY4-10, the sequence of 100bp above and below 2480324bp was obtained from the Nipponbare reference genome (IRGSP-1.0) (https: / / plants.ensembl.org / Oryza_sativa / Info / Index). Specific KASP markers for qHRY4-10 were designed using Primer3Plus (https: / / www.primer3plus.com / ). These markers include primers specific for the high head rice rate allele, qHRY4-10(A); primers specific for the low head rice rate allele, qHRY4-10(T); and a universal 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] Example 2: Method for Identifying Rice Polished 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 and grind into powder; (2) Add 600 μL of CTAB buffer and incubate 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 remove 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 (ddH2O) to dissolve the DNA.

[0045] Molecular marker primers were prepared as follows: (1) the primers were purified by ULTRPAGE; (2) the primer powders were dissolved, the concentrations of qHRY4-10(A) and qHRY4-10(T) were 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 performed as follows: The reaction was performed using a 96-well plate. The total reaction volume per well was 10.14 μL. 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 then fluorescently examined. After confirming that there were no air holes, PCR amplification was performed. The amplification procedure was as follows: (1) 94°C pre-denaturation for 15 min; (2) 94°C denaturation for 20 s, 61°C extension for 60 s, and ramp rate of 0.6°C / cycle for 10 cycles; (3) 94°C denaturation for 20 s, 55°C extension for 60 s, and 26 cycles; (4) data reading and analysis.

[0047] 2. Identify the head 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 rice variety with a higher head 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 low head 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 head 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. As can be seen from the table, among the 16 rice varieties, Wufeng 1, Aisizhan, Fuxin, Shuangchao 25, Fanfengruanzhan, Shuangkang 42, Sansizhan 1, 9598, Fengliuzhan, Sanerai, and Kaihong 5 all had head rice rates above 65%, indicating that they were all rice varieties with relatively high head rice rates. Meanwhile, Honghui 237, Honghui 255, 14W-157, Fuhui 838, and Chuanguanghui 909 all had head rice rates below 31%, indicating that they were all rice varieties with relatively low head 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 was extracted, and the obtained genomic DNA was used as a template to perform typing using the specific molecular marker qHRY4-10. The specific experimental process was the same as step 1 of Example 2.

[0061] The allelic variation at the physical position 2480324bp on 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 head 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 head rice rate; 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 head rice rate in Example 2, Honghui 237, Honghui 255, 14W-157, Fuhui 838 and Chuanguanghui 909 were all rice varieties with lower head rice rate.

[0062] It can be seen from this that the identification method of 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 rice varieties with allelic variation A is 66.5% on average, which is significantly higher than the head rice rate of rice varieties with allelic 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 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 within the scope of protection of the present invention.

Claims

1. A method for detecting a molecular marker of rice head rice rate and its application in identifying rice head rice rate, wherein the molecular marker of rice head rice rate is qHRY4-10 , which is located at 2480324bp on chromosome 4 of rice, and the base is A or T; and the whole polished rice rate of germplasm carrying A nucleotide allele variation at this site is extremely significantly higher than that of germplasm carrying T nucleotide allele variation; the reference genome is Nipponbare IRGSP-1.

0.

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

3. A method for identifying the polished rice rate of rice, characterized in that: A method for detecting molecular markers of rice head rice rate is used to obtain the results of the molecular markers to determine the rice head rice rate; The molecular marker for rice head rice rate is qHRY4-10 , which is located at 2480324bp on chromosome 4 of rice, and the base is A or T; and the whole polished rice rate of germplasm carrying A nucleotide allele variation at this site is extremely significantly higher than that of germplasm carrying T nucleotide allele variation; the reference genome is Nipponbare IRGSP-1.

0.

4. The method according to claim 3, wherein It is detected using KASP primers.

5. The method according to claim 4, wherein The KASP primers include primers specific for detecting the high head rice rate allele variation site carrying the A nucleotide allele variation site. qHRY4-10 -A. Specific primers for detecting allele variants of low head rice rate carrying T nucleotide allele variants qHRY4-10 -T, universal primer qHRY4-10 -C; in, qHRY4-10 -A primer is as follows: 5'-GAAGGTGACCAAGTTCATGCTAATTACATCCAGGTCCTCGAGCA-3', qHRY4-10 -T primers are as follows: 5'-GAAGGTCGGAGTCAACGGATTAATTACATCCAGGTCCTCGAGCT-3', qHRY4-10 The -C primer was as follows: 5′-CAACTTATTCACAGATAGGCCCTCC-3′.

6. The method according to claim 5, wherein The specific method is as follows: extracting genomic DNA from the rice to be tested, using the genomic DNA as a template and using the KASP primers to perform PCR amplification to obtain a PCR product; In the PCR amplification system, qHRY4-10 -A and qHRY4-10 The concentration of qHRY-T was 30-45 μmol / μL, the concentration of qHRY4-10-C was 80-100 μmol / μL, and the three primers were mixed at a volume ratio of 1:1:1 to form KASP Primer Mix. The total reaction volume was 10.14 μL, and the reaction system included: 5 μL DNA, 5 μL 2x KASP Master Mix, and 0.14 μL KASP Primer Mix. 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 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.

7. The method according to claim 6, wherein The detection step includes: identifying the head-finished 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 head-finished 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 head-finished rice rate.

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