CN119876248B

CN119876248B - Cloning and application of major gene QT12 with rice quality and yield resistant to field natural high temperature

Info

Publication number: CN119876248B
Application number: CN202510316742.3A
Authority: CN
Inventors: 李一博; 李威; 胡超凡; 常鑫源; 张建; 杨柯
Original assignee: Huazhong Agricultural University
Current assignee: Hubei Hongshan Laboratory; Huazhong Agricultural University
Priority date: 2025-03-18
Filing date: 2025-03-18
Publication date: 2025-07-04
Anticipated expiration: 2045-03-18
Also published as: CN119876248A

Abstract

The present invention relates to the application of QT12 gene in rice to synergistically improve rice quality and yield under natural high temperature environment in the field. QT12 gene was discovered by map-based cloning. High expression alleles under high temperature destroyed the homeostasis of endosperm storage substances, and negatively regulated the heat resistance of rice quality. Large-scale field high temperature experiments showed that non-functional or weakly functional QT12 not only gave rice grains low chalkiness, high storage protein, low amylose and other quality traits stronger heat resistance, but also significantly improved the fruit setting rate and plot yield. The G/A variation of QT12 in indica rice and the 6bp insertion and deletion variation between indica and japonica explain the genetic basis of the difference in heat resistance within indica rice and between indica and japonica, so that the A variation in indica rice has a lower expression and higher heat resistance relative to G, or the deletion of 6bp in indica rice has a lower expression and higher heat resistance relative to the insertion of 6bp in japonica rice, which is of great significance for breaking the breeding bottleneck of crop resistance and growth, yield and quality contradictions.

Description

Cloning and application of major gene QT12 with rice quality and yield resistant to field natural high temperature

Technical Field

The invention belongs to the technical field of molecular breeding, and particularly relates to cloning and application of a major gene QT12 with rice quality and yield resistant to natural high temperature of a field.

Background

Global warming severely threatens global agricultural production and constitutes a significant threat to global food security. The average yield of main crops can be reduced by 3.1-7.4% every 1 ℃ of the global average air temperature, and the global average air temperature is a serious threat to global grain safety. In addition, global food safety is not only related to yield but also closely related to quality, and rice determines human nutrition, market value and income of farmers, especially in southeast Asia and Africa regions where high temperature is frequent and high dependence on grains. The high temperatures can deteriorate the appearance, milling, cooking and eating of the cereal and the nutritional quality of the cereal. These problems highlight the need for sustainable agriculture that improves the high temperature resistance of crops to improve rice quality and yield at high temperatures.

The rice is more important to the appearance and taste quality from the previous single pursuit of high yield to the present pursuit of high yield, the rice quality comprises appearance, nutrition, cooking, taste, grinding, sensory characteristics and the like, is an important index for measuring the economic value and the edible value of the rice, and the quality directly determines the market price of the rice and the purchase wish of consumers, thereby greatly influencing the income of peasants. Chalky is the most visual property of appearance quality of rice, is also the most direct and sensitive index for deteriorating rice quality caused by high temperature, directly influences appearance quality of rice, cooking taste quality, nutrition quality and whole polished rice rate/yield, and is a great problem for determining market value of rice in a global rice production area and human grain consumption. Meanwhile, as the constitution factors of the quality characteristics are complex and are greatly influenced by other factors, the pace of improving the rice quality is greatly limited.

Innovations in global high quality rice present a great challenge to frequent high temperatures, manifested by low proportions and poor market competitiveness in global high quality rice, due to the low heat resistance of most modern high quality rice varieties to rice quality. If the rice is not sufficiently attractive in price, the peasant's enthusiasm is suppressed and the rice production is lowered. In order to improve the heat resistance of crops, it is important to identify germplasm resources that are stable against heat in a variety of natural high temperature environments. Traditional methods for identifying heat resistant phenotypes and potential QTL or genes are usually focused on identifying yield traits such as survival rate in seedling stage or fruiting rate in reproductive stage in controllable environments such as greenhouses, and failure to accurately simulate natural high temperature in field environments often results in failure to successfully identify true heat resistant germplasm. Therefore, germplasm and QTL with true heat resistance suitable for breeding purposes are still few. Therefore, it is necessary to identify true thermotolerant germplasm in the field at high temperatures, clone QTLs for true thermotolerance, and elucidate potential mechanisms to overcome the challenges of developing naturally high Wen Zuowu breeding.

Disclosure of Invention

The invention separates and clones the rice quality variety Chenghui 448 and OM1723 from the high-quality rice variety Chenghui 448 and OM1723 under the environment of natural large Tian Gaowen for many years to a main effect QTL gene QTL 12 for improving the rice quality and yield under the environment of large Tian Gaowen, and provides a new gene resource for synergistically improving the rice yield and quality breeding under the global warming background.

The invention aims to provide application of QT12 gene or coded protein thereof in rice in improving rice quality (chalky appearance quality, storage substance content, taste quality and the like) and yield in a multi-year-old Tian Gaowen environment.

The invention discovers that the gene has great effect on chalkiness rate from rice quality analysis of F ₂ genetic population of rice Chenghui 448 and OM1723 under large Tian Gaowen. The method of F ₃ genetic large populations and map-based cloning was used to pinpoint QT12 into a 14-kb chromosomal segment containing 1 ORF. Further genotype and expression level analysis determined it as a reliable candidate gene. Only 1 full-length cDNA is provided, the full sequence of the gene in rice Chenghui 448 is shown as SEQ ID NO.1, the full sequence of the gene in rice OM1723 is shown as SEQ ID NO.2, the full sequence of the gene in japonica rice ZH11 is shown as SEQ ID NO.3, and the full-length cDNA comprises a promoter, a5 'UTR, a CDS, an intron and a 3' UTR, wherein the CDS sequence is shown as SEQ ID NO.4, and the coded protein is shown as SEQ ID NO. 5.

The protection scope of the invention comprises:

The application of QT12 gene or coded protein thereof in regulating and controlling the quality (chalky appearance quality, storage material content and the like) and yield of rice at natural high temperature is characterized in that the sequences of the QT12 gene in Chengfu 448, OM1723 and ZH11 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, the CDS sequence is shown as SEQ ID NO.4, and the protein sequence is shown as SEQ ID NO. 5.

The application is specifically as follows:

The expression, function or activity of QT12 gene or coded protein thereof in rice is knocked out, inhibited or reduced to improve the heat resistance of the rice at high temperature in a field, namely, the stable balance of the content of storage substances such as rice storage protein, amylose and the like, the quality characters such as low chalkiness (good appearance quality), higher rice taste value and the like are better maintained at high temperature, and simultaneously, the setting rate, the solid grain number increase, the yield increase and the like are improved.

The expression quantity of QT12 gene in rice or the expression quantity or activity of coded protein thereof is improved to reduce the heat resistance of the rice under the condition of large Tian Gaowen, namely, the quality of rice storage protein is reduced, amylose is increased, chalkiness are increased (poor appearance quality), the taste value of the rice is reduced and the like at high temperature are poor, and simultaneously, the setting rate, the solid grain number and the yield are reduced and the like.

The substance for inhibiting the expression quantity or the function of the QT12 protein in the rice is a CRISPR/Cas9 vector with a knocked-out target sequence being a polynucleotide shown as SEQ ID NO.6, and the rice with improved quality and heat resistance under Tian Gaowen after the CRISPR/Cas9 system is edited has the following polynucleotides:

①AATGGCGATGGCCCTGCTCAGAGG

②AATGGCGATGGCCCTGCCTAGAGG

AATGGCGATGGCCCTCCAGAGG

③AATGGCGATGGCCCTGCCGCAGAGG

AATGGCGATGGCCCTGCTCAGAGG

Any molecular marker designed by using two main allelic variants G/A including QT12 comprises CAPS, KASP and the like, and molecular marker assisted breeding or gene editing technology is utilized to introduce/replace a high-expression QT12 allele QT12 ^A (high-quality variety Chenghui 448) into/from a high-expression QT12 allele QT12 ^G (high-quality main cultivated variety Huazhan) of rice so as to improve the heat resistance of the rice in a high field, namely, the stable balance of the storage substance content of rice storage protein, amylose and the like, the quality properties such as low chalkiness (good appearance quality), higher rice taste value and the like are better maintained at a high temperature, and simultaneously, the fruiting rate, the solid grain number and the yield are increased. The low-expression QT12 allele comprising Chenghui 448 and the like is a genome fragment shown in SEQ ID NO. 1. The QT12 clone Huazhan ^A generated by molecular marker assisted selection contains the allele of the genomic fragment shown in SEQ ID No. 1.

Any molecular marker designed by utilizing two main 6-bp InDel variations between indica and japonica rice QT12 comprises CAPS, KASP, inDel and the like, and molecular marker assisted breeding or gene editing technology is utilized to introduce/replace low-expression QT12 ^I alleles in indica rice into japonica rice (QT 12 ^J) to improve the heat resistance of rice under the condition that the japonica rice is large Tian Gaowen, namely, the stable balance of the content of storage substances such as rice storage proteins and amylose is better maintained at high temperature, the quality characters such as low chalkiness (good appearance quality), higher rice taste value and the like are better maintained, and simultaneously, the fruiting rate, the solid grain number and the yield are increased.

The introduction/replacement of the high expression QT12 allele QT12 ^G (high quality variety OM 1723) into/for the low expression QT12 allele QT12 ^A (high quality variety chenhui 448) in rice or the introduction of a substance that increases the expression level of QT 12-encoding protein in rice into rice is carried out by using any molecular markers designed to include CAPS, KASP and the like for molecular marker assisted breeding or gene editing techniques to decrease the heat resistance of rice at a high temperature, that is, decrease the quality of rice storage protein, increase amylose, increase chalk (poor appearance quality), decrease of rice taste value and the like, while decreasing the seed setting rate, solid grain number, and the like at a high Tian Gaowen. The recombinant QT12 allele comprises genome fragments shown as SEQ ID NO.2 and SEQ ID NO.3, wherein the substance is a nucleic acid molecule containing a coding QT12 protein or an expression frame, a recombinant vector and a recombinant microorganism thereof, and the nucleic acid molecule containing the coding QT12 protein is shown as SEQ ID NO. 4. The QT 12-derived fragment of the introduction line NIL ^OM generated by molecular marker assisted selection contains a genomic fragment of the allele shown in SEQ ID No. 2.

Any molecular marker designed by utilizing two main 6-bp InDel variations of indica type and japonica type QT12 comprises CAPS, KASP, inDel and the like, and molecular marker assisted breeding or gene editing technology is utilized to introduce a high-expression QT12 allele QT12 ^J in japonica type rice (ZH 11) into indica type rice (Sanhuang accounts for-2; QT12 ^I) to reduce the heat resistance of rice under the condition of large Tian Gaowen, namely, the quality deterioration such as reduction of rice storage protein, increase of amylose, increase of chalkiness (poor appearance quality), reduction of rice taste value and the like at high temperature, and simultaneously, the fruiting rate, the number of solid grains and the yield are reduced. The QT12 allele in japonica rice ZH11 is a genome fragment shown in SEQ ID NO. 3.

Specifically, the present invention provides any one of the following applications:

a) The application of the rice QT12 protein, a nucleic acid molecule for encoding the protein, or an expression cassette, a recombinant vector, a transgenic cell line or a recombinant bacterium containing the nucleic acid molecule in regulating heat resistance of rice quality and yield or preparing a product for regulating heat resistance of rice quality and yield;

B) Application of agent for reducing QT12 protein functional activity in cultivating quality and yield resistant rice germplasm or preparing cultivating quality and yield resistant rice germplasm products;

the amino acid sequence of the QT12 protein is shown as SEQ ID NO. 5.

Further, rice quality includes chalky appearance quality including chalky rate and chalky whiteness, storage substance content including storage protein and amylose, and taste quality, and a nucleotide sequence encoding the QT12 protein is shown in SEQ ID No. 4.

The invention provides a method for enhancing rice quality and yield at high temperature and/or cultivating quality and yield resistant rice germplasm, which improves rice quality and yield heat resistance by knocking out, inhibiting or reducing expression, function or activity of QT12 genes or coded proteins thereof in rice, namely the obtained rice better maintains stable balance of rice storage proteins and amylose content at high temperature, has low chalkiness and higher rice taste value, and simultaneously has high fruiting rate, high solid grain number and increased yield, wherein the protein sequence coded by the QT12 genes is shown as SEQ ID NO. 5.

Further, the substance for inhibiting the expression quantity or the function of the QT12 protein in the rice is a CRISPR/Cas9 vector, and a target sequence aimed by the CRISPR/Cas9 vector is a polynucleotide site shown as SEQ ID NO. 6.

The invention provides an SNP molecular marker which can distinguish rice quality and yield heat resistance between indica rice and indica rice, wherein the SNP molecular marker is positioned at the upstream-1455 position (corresponding to 3718373 th base on chromosome 12 of rice, the reference genome version is MSU version 7.0) of a QT12 gene promoter, A/G polymorphism exists, particularly the A/G mutation exists at the 2372 th position of a sequence shown as SEQ ID NO.3, wherein when the SNP locus is A, the rice is identified as indica rice, and the rice shows lower quality heat damage index, namely higher quality heat resistance and lower QT12 expression level.

The invention provides an indel molecular marker which can distinguish indica rice and is related to rice quality and yield heat resistance, wherein the indel molecular marker is specifically the deletion of 6bp in the 2828 th-2833 th (corresponding to the 3718829-3718834 th base on chromosome 12 of rice and the reference genome version is MSU version 7.0) position of a sequence shown in SEQ ID NO.3, and is identified as indica rice when the result is-6 bp.

The invention provides a molecular marker combination related to the QT12 expression level, rice quality and yield heat resistance, which is characterized in that the SNP and the indel are defined as above, rice germplasm is further divided into three haplotypes according to the SNP and the indel result, hap1: G/+6 bp, hap3: G/-6bp and Hap7: A/-6bp, wherein the haplotype Hap7 has the lowest QT12 expression level, hap3 is centered, the expression level of QT12 in Hap1 is highest, and the lower the QT12 expression level represents higher quality heat resistance.

The invention provides application of any one of the molecular markers or the combination thereof in identification and auxiliary identification of indica rice and/or cultivation quality and yield resistant rice germplasm, wherein rice with SNP result of A and/or indel result of-6 bp is identified as indica rice, rice with indel result of +6bp is identified as japonica rice, and rice with A, -6bp and A/-6bp is selected according to SNP and/or indel result to carry out synergistic cultivation of quality and yield resistant rice germplasm.

The invention provides a method for identifying and assisting in identifying indica rice, which utilizes any one of the molecular markers or the combination thereof to detect rice, identifies the rice with SNP result of A and/or indel result of-6 bp as indica rice, and identifies the rice with indel result of +6bp as japonica rice.

The invention provides a method for cultivating quality and yield resistant rice germplasm, which utilizes any one of the molecular markers or the combination thereof to detect rice, and selects A, -6bp and A/-6bp rice for the synergistic cultivation of quality and yield resistant rice germplasm according to SNP and/or indel results.

The invention clones a main effect gene for negative regulation of rice quality and yield heat resistance at high temperature in rice, provides new gene resources for high-quality and high-yield breeding of cereal crops such as rice and the like, and provides genetic basis for heat resistance breeding and research in other crops due to the highly conserved characteristic.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of the present invention.

FIG. 2 shows the initial positioning of QT12 in the present invention. a, the appearance quality character chalkiness of rice in the grouting period of Chenghui 448 and OM1723 at six different field natural temperatures. The seeding time is adjusted according to the heading time of the two parents, so that the heading time and the grouting time are kept consistent. b, particle widths of the recovery 448 and the OM1723 are formed at different field temperatures. c, identifying the high temperature resistant QTL gene QTL 12 of the chalky appearance quality of RICE by using the RICE 6K chip technology. The region within the ellipse is the QT12 site. d, determination of candidate QT12 sites using co-segregation criteria, i.e. two homozygous genotypes of QT12, can significantly distinguish between two extreme chalky phenotypes, wherein a (yellow), B (gray) and H (brown) represent high-value homozygous, low-value homozygous and heterozygous genotypes of QT12 sites, respectively.

FIG. 3 is a diagram showing the determination of QT12 candidate genes according to the present invention. a, carrying out fine localization and cloning on QT12 by using 57 recombinant single strains screened by 4310F ₃ large-population chalk phenotype and genotype. Phenotyping was performed by detecting the offspring of each recombinant individual to infer QT12 genotype. b, comparative sequencing of QT12 in the parents. c, the expression level of two QT12 alleles in the endosperm of 5-DAF (5 days after flowering) of two NILs at different temperatures. d, quality heat damage index of both haplotypes of Hap ^CH（QT12^A) and Hap ^OM（QT12^G) in rice microkernel germplasm, and expression level of QT12 in 5-DAF endosperm. The rice chalkiness at high temperature minus the rice chalkiness at normal temperature is defined as the rice quality heat damage index, the lower the quality heat damage index is, the stronger the quality heat resistance is.

FIG. 4 shows the analysis of the expression level and the heat resistance of different QT12 genotypes in the micro-core germplasm according to the present invention. a, the expression level of QT12 in the endosperm of 5-DAF of heat-resistant (R) and sensitive (S) varieties identified from field high temperature trials over the years. b, distribution of G/A variation of QT12 in 533 parts of microkernel germplasm. QT12 ^A and QT12 ^G are resistant haplotypes and sensitive haplotypes, respectively. c, quality heat damage index of QT12 ^A and QT12 ^G in indica and QT12 expression level in 5-DAF endosperm.

FIG. 5 shows a haplotype analysis and an indica differentiation analysis of QT12 in the present invention. analysis of QT12 haplotype in a,4726 rice core germplasm. The green letter indicates a functional G/A SNP variation on the QT12 promoter between Hui 448 and OM 1723. QT12 ^G of OM1723 and QT12 ^A haplotype of chenghui 448 are marked with circles, respectively. The ratio after corresponding haplotypes is the number of indica rice and japonica rice varieties respectively. b, QT12 naturally varies between typical indica trifoliate-2 (SHZ)/OM 1723 (OM)/chenghui 448 (CH)/hua-ZH (HZ) and japonica ZH 11. The variance in the box is the representative variance G/A of QT12 in this study. c, distribution of 6-bp InDel on QT12 in indica, japonica, aus and Intermediate. +6bp and-6 bp represent the presence or absence of a 6-bp variation, respectively. d, transient double luciferase reporter gene experiments of QT12 promoters in indica rice and japonica rice at different temperatures. e, expression level of QT12 in three haplotypes determined by 6-bp InDel and functional variation G/A.

FIG. 6 shows the quality phenotypes of the QT12 transgenic complementary families according to the present invention at different field temperatures. a, the QT12 transgenic complementary family had chalky appearance quality phenotype at different field temperatures in the arms of 2022. b, chalky appearance quality phenotype of QT12 transgenic complementary families at different field temperatures in the arms of 2023. c, content of each storage substance of the QT12 transgenic complementary family at different field temperatures. d, ratio of content of each stored substance of QT12 transgenic complementary families at different field temperatures.

FIG. 7 shows the quality phenotype of QT12 transgenic knockout families according to the present invention at different field temperatures. a, the QT12 transgenic knockout line had a chalky appearance quality phenotype at different field temperatures in the 2022 martial arts. b, the QT12 transgenic knockout line had a chalky appearance quality phenotype at different field temperatures in the arms of 2023. c, content of each stored substance of the QT12 transgenic knockout family at different field temperatures. d, ratio of content of each stored substance of QT12 transgenic knockout families at different field temperatures.

FIG. 8 is a rice quality phenotype of the QT12 ^J allele of japonica rice variety ZH11 complemented to the complement family of indica rice variety SHZ (QT 12 ^I) at natural elevated temperatures and normal temperatures. a, the QT12 ^J complements the chalky appearance quality phenotype of the rice of the family at different field temperatures. b, content of each stored substance of the rice of the QT12 ^J complementary families under different field temperatures. c, the ratio of rice storage protein to amylose content of the QT12 ^J complementary families at different field temperatures.

FIG. 9 is a graph showing the quality phenotype of two NIL materials of QT12 of the present invention at different field temperatures. The chalky appearance quality phenotype of two NIL materials of QT12 at different field temperatures in the arms of 2022. b, content of each stored substance of two NIL materials of QT12 under different field temperatures. c, ratio of contents of stored substances of two NIL materials of QT12 at different field temperatures.

FIG. 10 is a subcellular structural view of endosperm at different field temperatures for two NIL materials of QT12 of the present invention. a, scanning electron microscope observation of two NIL mature endosperm under natural high temperature and normal temperature conditions. b, transmission electron microscopy was used to observe subcellular structures of NIL ^CH and NIL ^OM 10-DAF endosperm cells. PBI and PBII represent proteosomes I and II, respectively, and SG represents starch grains. Scale bar, 2 μm. Statistical field of view area for size and area of protein bodies and starch grains were 875 μm ².c,d,NIL^CH and the number and area of starch grains (c) and protein bodies (d) per 875 μm ² of the NIL ^OM 10-DAF endosperm abdomen. e, the ratio of the number and area of protein bodies to starch grains. f, the maintenance degree of the ratio of protein bodies to starch grains. The degree of maintenance refers to the ratio of the amount or area of protein bodies to starch granules at high temperature compared to the ratio at normal temperature.

FIG. 11 shows plant types and quality traits of the Czochralski introduced line of QT12 under natural high temperature and normal temperature conditions. a, naturally and at normal temperature, the rice is in the plant type of the introduced line and has chalky appearance quality character phenotype. b, naturally and at normal temperature, the water accounts for the content of each storage substance in the introducing system. And c, naturally increasing the ratio of the contents of various storage substances in the introduction system at high temperature and normal temperature.

FIG. 12 shows the yield and taste quality phenotypes of QT12 different genetic material under the present invention at large Tian Gaowen. The NIL (a) of QT12, the complementary transgenic line (b), the knockout line (c) and the QT12 ^J complementary line (d) are set at different field temperatures, the yield per plant and the rice taste value.

FIG. 13 shows the effect of QT12 of the present invention on other rice yield traits at Tian Gaowen. Seed setting rate of two QT12 haplotypes in 533 parts of rice micro-core germplasm and indica rice subpopulations. b, other yield traits and chalky phenotype of both NIL at large Tian Gaowen. c-e, thousand seed weight, number of single plants and number of glume flowers of QT12 complementary family (c), CRISPR family (d) and QT12 ^J complementary family (e) under natural high temperature of the field.

FIG. 14 shows the phenotype of various yield traits of QT12 different genetic materials according to the present invention at normal temperatures in the Wuhan field. The complementary transgenic family (a) of QT12, the knockout family (b) and the complementary family (c) of QT12 ^J have the seed setting rate, the solid grain number of single plant, the Yinghua number of single plant and thousand grain weight at normal temperature of a field.

FIG. 15 is a large-scale field high temperature test of NIL and the introgression line of QT12 in two elite varieties. The highest air temperature of flowering and grouting stages of the Wuhan, hangzhou and Changsha paddy rice in 2000 to 2024. b-h, QT12 NILs (b-e) and Huazhan importation lines (f-h) cell yield (5 square meters), seed setting rate, number of solid plants per plant, yield per plant, chalkiness and chalkiness under natural high temperature conditions of Wuhan, hangzhou and Changsha in 2024. The genetic material of each city was based on extensive field trials using a randomized block design with 3-4 cell replicates per family, each cell having an area of about 5 square meters and 12 x 15 = 180 individuals.

FIG. 16 is a large-scale field high temperature test of QT12 different transgenic genetic material according to the present invention. a-g, QT12 complementation line (b-d) and CRISPR knockout line (e-g) cell yield (5 square meters), seed setting rate, number of solid plants per plant, yield per plant, chalk, and chalkiness under natural high temperature conditions of wuhan, hangzhou and changsha in 2024.

FIG. 17 shows grain width of QT12 different genetic materials according to the present invention in a high temperature environment of the Wuhan in 2024.

FIG. 18 shows plant height and growth period of QT12 different genetic material according to the present invention at normal temperature of Wuhan in 2024. The QT12 near isogenic line (g, h), the buddha-introducing line (i, j), the transgenic complementing family (k) and the transgenic CRISPR knockout family (l) are of plant height and heading date.

FIG. 19 shows the mRNA and protein expression levels of QT12 in wild-type Chenghui 448 and QT12 complementary families at different temperatures according to the present invention.

Detailed Description

The following examples are only for more clearly illustrating the technical aspects of the present application, and thus are merely examples, which should not be construed as limiting the scope of the present application. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.

The parents used in the invention are rice Chenghui 448 and OM1723, which belong to rice micro-core germplasm resources, and the grain widths are consistent.

Example 1 Natural high temperature treatment in Rice field during the filling period

In order to make each rice genetic material receive the natural high temperature and normal temperature treatment in the grouting period, batch planting is carried out on each genetic material in the sowing period, the respective planting time is adjusted according to the heading period of each material, and the sowing is generally carried out once every half month, so that the heading and grouting early stage is ensured to be in the period Tian Gaowen of great Wuhan as much as possible. For materials with longer heading times (> 95 days), such as genetic materials with background of chenghui 448 or OM1723, it is common to sow in late middle of 4 months, and for materials with shorter heading times (< 80 days), it is common to sow in around mid 5 months. Different sowing dates can lead genetic materials in different growth periods to be unified in the middle and late 7 months or the beginning of 8 months to begin heading and grouting, and in the period, the Wuhan is often subjected to extremely high-temperature weather exceeding 35 ℃, and the record has been continued for more than 60 years. For normal temperature treatment experiments, all materials were sown in the middle of 6 months or at the end of the month, so that the short heading period materials generally began heading and grouting in the middle and upper 9 months, while the long growth period materials began heading and grouting in the middle and upper 9 months. During this period, the average air temperature of the martial arts is relatively low and normal (about 30 ℃). And (3) measuring dynamic air temperature data automatically recorded once every 5 minutes during 6-10 months of agricultural university in China and 2-5 months of the field of the south numerous bases in the Lingshui city by using a USB temperature and humidity recorder (USB-TH). The early grouting period is the most sensitive period of rice endosperm, so that the daily average temperature of each material in the early grouting period of 6-12 days after flowering is counted, and the time is 9 am to 7 pm. When the rice chalkiness of OM1723, NIL ^OM, ZH11, etc. sensitive materials are significantly increased, it is indicated that these materials are subjected to high temperature treatment during grouting. Conversely, when all sensitive wild-type materials still maintained a low grain chalkiness, it was demonstrated that they were grouted at normal temperatures.

Example 2 quality and yield characterization at Natural temperature in Rice fields

The harvested mature seeds are thoroughly baked or sun dried and stored at room temperature for at least three months before investigating the appearance quality traits such as chalkiness. Rice chalkiness, including abdominal, heart and back, are generally detected by visual inspection. Mature dried seeds were threshed and dehulled into brown rice, 100 whole grains were randomly selected and the percentage of chalky grains counted was expressed as the Grain Chalky Rate (GCR). Chalky rice was laid flat and visually examined, and the percentage of the projected area of the chalky portion to the projected area of the whole rice was expressed as the chalky area (GCA) of the rice. Chalkiness (GCD) is the product of the grain chalkiness rate and chalkiness area. All chalky properties are expressed as percentages. Chalkiness in rice can degrade the appearance, milling, cooking and eating quality of rice and the yield of whole polished rice, so that the increase of chalkiness is the most direct indicator of rice quality, and the high temperature is very liable to cause sensitive changes in chalkiness. Thus, having lower rice chalkiness at high temperatures indicates that rice quality has greater resistance to high temperatures.

The total protein content of the brown rice grains and the amylose content of the fine rice grains were measured using an XDS near infrared fast content analyzer (FOSS) and near infrared reflectance spectroscopy. The milled rice is milled to flour and the total starch content is measured using conventional chemistry. The amylopectin content is equal to the total starch content minus the amylose content. The total storage material content is approximately equal to the total starch content plus the total protein content. The ratio between different stored substances can be better understood about the balance and unbalance of the content of the stored substances at different temperatures. The taste value of cooked rice was measured by the STA1B apparatus (zobamboos japan). Briefly, 30 g of whole polished rice was weighed, washed and soaked in water in a ratio (indica rice 1:1.4, japonica rice 1:1.35) for 30 minutes, then boiled for 40 minutes, kept warm for 10 minutes, and cooled in a cooler for 20 minutes. 7 g of rice is taken to test the taste value, and the method is repeated for 6 times. The score ranges from 0 to 100, representing a composite value of taste quality. Yuzhenxiang (YZX) is a famous quality grain in China and a variety with high grain taste value, and has very similar excellent quality to the famous KDML105 variety consumed globally. We set the cereal taste value of YZX to 90 as a calibration standard, measure the taste value simultaneously with the experimental samples, and finally calibrate and normalize the taste value of each sample. The higher the value, the higher the taste quality.

For investigation of different yield traits, threshing after seed collection of an artificial single plant, manually counting the number of solid grains, the number of empty grains and the yield on the single plant level, and further calculating other traits based on the three traits. Yinghua number=solid number+empty number, setting rate=solid number/Yinghua number, thousand seed weight=individual yield/solid number×1000. The Wuhan, hangzhou and Changsha are three major cities of the rice producing areas in the Yangtze river of China, and extremely high temperatures occur frequently, especially in 2024 summer. Cell yield and other yield, quality trait data for all genetic material in each city were based on extensive field trials using a randomized block design, repeating 3-4 cells per family, with each block of land having an area of about 5 square meters and about 12 x 15 = 180 individual plants.

Example 3 discovery of chalky Heat resistance Gene QT12 for appearance quality traits of Rice

Quick positioning and cloning are carried out on QT12 by adopting RapMap method, and the flow is shown in figure 1, and is specifically as follows:

1) QT12 gene primary localization:

Using natural temperature treatment for many years, a high Wen Pinzhi heat resistant parent Chenghui 448 (CH) and a high temperature sensitive parent OM1723 (OM) were identified, and the effect of grain width on chalky quality was excluded due to the consistent grain width of the two parents (FIGS. 2a, b). Hybridizing rice Chenghui 448 with OM1723 to obtain F ₁, and selfing to generate F ₂ genetic isolate. The chalky phenotype of rice of the population under the natural environment Tian Gaowen is examined, an extremely high-low pool is constructed according to a BSA method, 10 full seeds are selected from each single plant in the extremely high-low pool, and the high-low pool is mixed for germination. After two weeks, equal amount of leaves of each plant were ground with liquid nitrogen, and sent to China seed group (Wuhan) for RICE6K SNP chip detection (FIG. 2 c);

The "Variation ID" of the InDel polymorphism Variation was found using RICEVARMAP database (http:// ricevarmap. Ncpgr. Cn /), and then the InDel marker was designed by the "DESIGN PRIMER by Variation ID" function. The PCR fragment with 3-8bp deletion of InDel difference is preferentially selected in design, and the PCR fragment has about 100-200bp. Template DNA was amplified to recover 448 and OM1723 using all primers/markers designed and detected by 4% PAGE gel electrophoresis. Primers L12W1, L12W2, L12W3, L12W4 and L12W5 with polymorphisms between parents are selected, and genotyping is performed on each individual strain of the population by using the primers.

Based on the analysis of the genotypes of the L12W1 and L12W2 markers with the individual phenotypes (FIG. 2 d), the segment homozygous genotypes separated the two extreme chalky phenotypes at high temperature and the chalky difference reached very significant levels, indicating that the locus belongs to single-factor Mendelian inheritance and that further fine localization and map cloning is possible.

2) Fine localization of QT12 gene:

To further narrow the localization interval of QT12, F ₂ individuals with heterozygous initial localization segments were developed into a large population of 4310 strains of F ₃, from which recombinant individuals were selected.

Screening was first performed with Indel markers L12W1 and L12W2 (table 1), from 4310 individuals, 57 recombinant individuals were screened together, and these individuals were subjected to a progeny test to confirm their previous generation phenotypes, the line of trait segregation indicated that the previous generation phenotype was heterozygous, the progeny trait was not segregating and that the higher value trait indicated that the previous generation phenotype was homozygous from OM1723, and the lower value indicated that the previous generation phenotype was homozygous from chenghui 448. These 57 recombinant individuals were then analyzed using the 5 InDel markers developed (L12W 1, L12W7, L12W8, L12W9, L12W10 and L12W 2). Thus, QT12 is ultimately located between L12W7 and L12W8, which interval corresponds to a physical range of about 14-kb on the genomic sequence of Nipponbare. The segment contains only 1 candidate gene, and a reliable candidate gene, called QT12 gene, is determined by genotyping and expression level analysis (fig. 3 a).

TABLE 1 primers for map-based cloning and gene function verification of the invention

In genome of rice adult Hui 448 and OM1723, the complete sequences of the genes are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, the genes comprise a promoter, a 5 'UTR, a CDS, an intron and a 3' UTR, the CDS sequence is shown as SEQ ID NO.4, and the coded protein is shown as SEQ ID NO. 5.

Example 4 QT12 candidate Gene analysis

Sequencing of QT12 target segments from chenghua 448 and OM1723 revealed 9 polymorphic variants between the 3-kb promoter and the two varieties within the CDS, both occurring on the promoter upstream of the translation initiation site, these variants including substitution, insertion and deletion three mutation types (fig. 3 b). qRT-PCR in the endosperm of 5-DAF in the natural temperature environment of the field showed no significant difference in QT12 gene expression between the two NILs. However, under natural high temperature conditions, QT12 expression in NIL ^OM was significantly increased, while the QT12 expression level in NIL ^CH remained essentially unchanged (fig. 3 c), indicating that high temperature was able to induce QT12 expression in NIL ^OM, i.e. the QT12 haplotype in sensitive parent OM1723 was dominant genotype.

Analysis of the QT12 haplotypes found that, in 533 parts of the microkernel germplasm, hap ^CH had a lower quality heat damage index than Hap ^OM (the quality heat damage index is the difference between the chalkiness of 533 germplasm materials at high temperature and normal temperature, i.e. the degree of chalkiness increase of the seeds at high temperature, the lower the quality heat damage index is, the better the heat resistance), indicating that the Hap ^CH haplotype variety has higher quality heat resistance (fig. 3 d). Analysis of 5-DAF endosperm expression data from 119 kernel germplasm found that Hap ^CH had lower QT12 expression levels (fig. 3 d), consistent with the low expression pattern in NIL ^CH. These results demonstrate that resistant haplotypes may have lower QT12 expression at high temperature, resulting in higher quality heat resistance.

Example 5 analysis of QT12 Natural variation in parents

The resistant (R) and sensitive (S) varieties identified in the martial arts at natural high temperature for many years were subjected to RNA-Seq analysis by sampling endosperm at different field temperatures, and the expression level of QT12 in the resistant varieties at high temperature was found to be significantly lower than that of the sensitive varieties, but not significantly different at normal temperature (FIG. 4 a). Further analysis revealed that the SNP variation of one G/A on the Hui 448 QT12 promoter was almost exclusively distributed in indica (FIG. 4 b), and that within indica varieties, varieties containing QT12 ^A had lower quality heat damage index and lower QT12 expression level (FIG. 4 c), indicating that the QT12 ^A within indica had higher heat resistance, explaining the genetic basis of the heat resistance difference within indica. The above results demonstrate that the G/A variation on the QT12 promoter present only in indica rice may result in lower expression of QT12 at high temperature, thereby enhancing the heat resistance and explaining the cause of the difference in heat resistance in indica rice.

Example 6 QT12 haplotype analysis in 533 core germplasm resources

QT12 was divided into 7 haplotypes using 7 representative variations on QT12, where chenghui 448 belongs to Hap7 and OM1723 belongs to Hap3 (fig. 5 a). Analysis of the distribution of each haplotype in the indica subpopulation found that QT12 had significant indica differentiation (fig. 5 a). In addition, the 5' UTR region of QT12 has a 6-bp InDel (CGCCGC) which can substantially distinguish indica (-6 bp) from japonica (+6 bp) (FIGS. 5b, c). To further compare QT12 promoter activity between indica and japonica, we found that the QT12 promoter activity of japonica was significantly higher than that of indica at 25 ℃ and 37 ℃ using a dual-luciferase reporter gene experiment. And at high temperature, the activity of the non-glutinous rice promoter QT12 ^J is obviously enhanced, but the activity of the indica rice promoter QT12 ^I is not obviously changed (figure 5 d). The present invention previously identified that the functional variation G/A of QT12 was present only in indica (FIG. 4 b), so three major haplotypes Hap1, hap3 and Hap7 could be combined from this functional variation G/A present only in indica and the 6-bp InDel variation between indica and japonica (FIG. 5a, b). These three haplotypes can clearly differentiate the expression level of QT12 in the endosperm of 119 core germplasm and show gradient change, i.e. QT12 ^A haplotypes in indica rice have the lowest QT12 expression level, QT12 ^G expression level in indica rice is centered, and QT12 ^G expression level in japonica rice is highest (fig. 5 e), explaining the heat-resistant difference in indica rice and the genetic basis of indica rice being more heat-resistant than japonica rice.

Example 7 application of Rice QT12 Gene in regulating Heat resistance of Rice quality (chalky appearance quality, storage substance content, taste quality, etc)

1) Reduced grain quality after the OM1723 sensitive genotype QT12 ^G complements the resistant parent back to 448

The QT12 gene of OM1723 was amplified by designing a PCR specific primer QT12-flag (Table 1) with restriction endonuclease KpnI and EcoRI linkers, and the amplified sequence contained the sequence shown in SEQ ID NO.2, i.e., TATGACATGATTACGAATTC 'end of SEQ ID NO.2 was added and GGTACCCGGGGATCCTCTAG was added at its 3' end. The recombinant vector pCAMBIA 1301-QT 12 ^OM1723 was obtained by ligation to pCAMBIA 1301 using the Gibson ligation method (Gibson et al 2009, nat. Methods 6:343-345).

The correctly cloned plasmid is led into Chenghui 448 by agrobacterium-mediated indica genetic transformation system by adopting a transgenic method, and the transgenic rice plantlet is obtained through induction, subculture, infection, co-culture, screening of calli, differentiation, rooting and seedling hardening transplanting with hygromycin resistance. Transgenic complementary positive individuals are detected by QT12-F/1301-flag-R (table 1), agarose gel electrophoresis is adopted for detection, and the positive individuals are obtained after the presence of the bands.

The heat resistance of transgenic positive families compared with wild families to recover 448 rice quality was examined. At natural normal temperatures in the martial arts, both transgenic complementary positive families QT12-Com and wild-type chenghui 448 exhibited lower chalkiness, whereas QT12-Com families chalkiness were significantly increased compared to Cheng Hui 448 under natural high temperature conditions (fig. 6a, b), representing poorer grain quality. Also the storage protein content of QT12-Com at high temperature is significantly reduced compared to normal temperature conditions, the amylose content is increased, resulting in a significantly reduced ratio of storage protein to amylose or starch content leading to an imbalance of storage material and thus to the formation of grain chalkiness (fig. 6c, d). In contrast, the product return 448 maintained a relative balance of stored material content and exhibited a low chalky phenotype (fig. 6). These results demonstrate that QT12 alleles from susceptible parents can significantly reduce rice quality thermotolerance by disrupting the balance of stored material content in endosperm tissue.

2) Synergistic improvement of seed quality after CRISPR knockout of sensitive parent OM1723 (QT 12 ^G) (knockout target is sequence shown in SEQ ID NO. 6)

Amplifying by using a forward primer of QT12-U3 and a reverse primer of U3 as templates, amplifying by using a U3 plasmid as templates and a reverse primer of QT12-U3 and a forward primer of U3, respectively recovering the two amplified products, mixing the two amplified products in equal amounts, amplifying by using the mixture as templates and using the U3 primer, recovering the products, and introducing the recovered products into pCXUN-CAS9 plasmid (cut by KpnI firstly) by using a Gibson ligation method (refer to CN 201610639854.3), thereby obtaining the pCXUN-CAS9-QT12-U3 knockout vector.

The obtained correctly cloned pCXUN-CAS9-QT12-U3 knockout vector is introduced into OM1723 through agrobacterium-mediated indica rice genetic transformation system, and the transgenic rice plantlet is obtained through induction, subculture, infection, co-culture, screening of calli with hygromycin resistance, differentiation, rooting and seedling transplanting. Transgenic individuals obtained were detected by QT12-CR (Table 1) and positive individuals that were knocked out were confirmed by sequencing using QT12-U3-SEQ (Table 1).

Examine the rice quality of CRISPR transgenic positive plants at Tian Gaowen a greater than wild type OM1723 rice. Transgenic knockout line QT12-CR and sensitive wild-type OM1723 both exhibited lower chalkiness at natural normal temperatures in the martial arts, whereas QT12-CR line chalkiness remained lower under natural high temperature conditions, showing better grain quality (fig. 7a, b). Compared with normal temperature, the QT12-CR family has little change of total protein and amylose/starch content at high temperature, better maintains the balance of endosperm tissue storage substances, thus maintaining lower grain chalkiness and better quality (figures 7c and d), which shows that mutation or low expression of QT12 can endow rice with high temperature resistance by better balancing the content of storage substances, thereby obtaining better rice quality at high temperature.

3) The heat-resistant differentiation of the QT12 indica rice verifies that the ZH11 japonica rice genotype QT12 ^J complements to indica rice variety Sanhuang-2 (SHZ; QT12 ^I) and then reduces the quality of seeds:

The QT12 gene of japonica rice ZH11 is amplified by designing a PCR specific primer QT12-flag (table 1) with restriction endonuclease KpnI and EcoRI joints, and the amplified sequence comprises a sequence shown in SEQ ID NO.3, namely TATGACATGATTACGAATTC is added at the 5 'end of the SEQ ID NO.3, and GGTACCCGGGGATCCTCTAG is added at the 3' end of the SEQ ID NO. 3. The recombinant vector pCAMBIA 1301-QT 12 ^ZH11 was obtained by ligation to pCAMBIA 1301 using the Gibson ligation method (Gibson et al 2009, nat. Methods 6:343-345).

The obtained plasmid with correct clone is led into indica rice SHZ through agrobacterium-mediated indica rice genetic transformation system by adopting a transgenic method, and the transgenic rice plantlet is obtained through induction, subculture, infection, co-culture, screening of callus with hygromycin resistance, differentiation, rooting and seedling hardening transplanting. Transgenic complementary positive individuals are detected by QT12-F/1301-flag-R (table 1), agarose gel electrophoresis is adopted for detection, and the positive individuals are obtained after the presence of the bands.

The heat resistance of transgenic positive families compared with the rice quality of wild family SHZ was examined. Because QT12 shows obvious indica-japonica differentiation, in order to study the influence of the QT12 indica-japonica differentiation on the differentiation of the heat-resistant difference of the indica-japonica, genetic materials QT12 ^J -Com, the genotypes of which are complementary to those of the indica-japonica rice QT12 ^J, in the SHZ of the indica-japonica rice are planted in different field temperature environments. At normal temperature, both the complementary positive line and the wild-type SHZ were of low chalky phenotype (fig. 10 a), whereas in the large Tian Gaowen environment, the complementary positive line had a significantly reduced protein content, a significantly increased amylose content, resulting in a significantly reduced protein to amylose ratio, a disturbed balance of stored materials, resulting in increased chalkiness and poor quality (fig. 8). Whereas wild-type SHZ is able to maintain a certain balance of storage protein to amylose ratios and therefore appears to be of better quality (fig. 8). These results indicate that the QT12 ^J genotype of japonica rice can reduce the quality and heat resistance of indica rice, while QT12 indica differentiation may resolve the genetic basis of indica rice that is more resistant to high temperatures than japonica rice.

4) Near Isogenic Line (NIL) constructed to restore 448 background verifies the effect of QT12 two alleles on quality thermotolerance

And (3) taking Chenghui 448 as a donor parent, taking OM1723 as a recurrent parent, continuously backcrossing for more than 4 times (each backcrossing is supplemented with MAS, selecting a single plant with the heterozygous L12W7 and L12W8 markers and the plant type consistent with that of Chenghui 448 as a male parent), and then separating by selfing to screen pure NIL ^CH and NIL ^OM.

NIL ^OM and NIL ^CH show similar plant types, but NIL ^CH shows lower rice chalkiness, lower amylose and higher protein content at high temperatures (fig. 9). NIL ^CH maintains relatively lower rice chalkiness by maintaining a balance of protein and amylose/starch ratios at high temperatures compared to ambient temperatures. However, NIL ^OM shows a lower protein to amylose/starch ratio, and the balance is disrupted, thus leading to chalky formation.

5) Near Isogenic Line (NIL) cytology electron microscopy (SEM) observation to verify the effect of QT12 two allelic forms on endosperm storage substance development at high temperature

To assess the effect of QT12 on grain stock at high temperature, we observed the starch structure of the stock in both NIL maturing endosperm by Scanning Electron Microscopy (SEM). The chalky endosperm of NIL ^OM at high temperature is filled with loosely packed spherical storage starch particles with very large voids, whereas the non-chalky grain of NIL ^CH at the same high temperature consists of a dense and regularly packed polyhedral crystal storage particle structure. Whereas the chalky endosperm structures of NIL ^CH and NIL ^OM at normal temperature are consistent with NIL ^CH at high temperature, exhibiting a dense crystal structure (fig. 10 a). To further analyze the subcellular causes of QT12 at high temperature leading to structural changes in the storage material of the kernel endosperm, we compared the ultrastructural structure of 10-DAF endosperm cells in NIL ^CH and NIL ^OM at different temperature conditions using Transmission Electron Microscopy (TEM). The number and area of starch granules for both NIL's did not change significantly at high temperature compared to normal temperature, while the number and area of protein bodies I and II in both NIL's was reduced, but the reduction of both protein bodies in the heat resistant family NIL ^CH was much lower than that of the sensitive family NIL ^OM, resulting in a higher maintenance of the ratio of protein bodies to starch granule area and number of resistant NIL ^CH, better maintaining the steady state balance of the two stored substances (fig. 10 b-f). These subcellular evidences further support the conclusion that the balance and imbalance between storage protein and starch content at high temperatures leads to high temperature resistance and sensitive phenotypes, respectively.

6) Construction of Huazhan introduced line to verify the breeding potential of QT12 resistance genotype in improving the heat resistance of main cultivated high quality varieties

In order to evaluate the breeding potential of the QT12 gene in improving rice quality, we select the most common restorer line widely used in Chinese high-yield hybrid rice breeding at present, namely the excellent variety Huazhan (Huazhan) (the QT12 allele is genotype QT12 ^G with high-temperature sensitivity of rice quality) as an improvement object. The method comprises the steps of taking Chenghui 448 as a donor parent, taking Huazhan as a recurrent parent, continuously backcrossing for more than 4 times (each backcrossing is supplemented with MAS, selecting a single plant with the same plant type as Huazhan as a male parent), then carrying out selfing separation, and screening Huazhan ^A and Huazhan ^G from the separated plants (figure 11). Huazhan ^A（QT12^A) has a lower chalky and amylose content and a higher protein content than Huazhan ^G（QT12^G) under natural high temperature conditions, showing a better rice appearance and taste quality (fig. 11). The Huazhan ^A/G heterozygous and Huazhan ^G homozygous lines did not differ significantly in various quality traits (fig. 11), indicating QT12 ^G is a dominant allele. Taken together, these results indicate that QT12 has great potential for improving the quality of high-quality varieties of rice at high temperatures.

Example 8 extensive field trials over many years verify the use of the rice QT12 gene for synergistically improving rice quality (chalky appearance, stored matter content, taste quality, etc.) and yield and heat resistance

The seed setting rate of QT12 ^A haplotype was significantly higher than QT12 ^G in 533 parts of core germplasm and indica subpopulation germplasm (fig. 13 a). The genetic material of QT12 was examined for yield traits and taste quality at different temperatures. Wherein the rice taste value represents an evaluation of the taste of the cooked rice. The rice taste value of the high-quality rice variety Yuzhixi (YZX) with high taste value of China is set to 90, and the taste value of each experimental sample is obtained by conversion and standardization by taking the rice taste value as a calibration standard.

The chalkiness, fruiting rate and individual yield of the two NIL's were not significantly different at normal temperature of martial in 2023 in summer, whereas at high temperature, the setting rate and individual yield of NIL ^CH were significantly higher than (+18.1%) NIL ^OM, but the chalkiness rate of the seeds was significantly lower than NIL ^OM. In addition, the cooked rice taste value of NIL ^CH was always higher than NIL ^OM under both temperature conditions, and the taste value of NIL ^OM was significantly reduced at high temperature (fig. 12a,13 b). The QT12 complementation line QT12-Com has significantly reduced seed setting rate, individual yield and rice taste values compared to wild type chenghui 448 (high resistance), significantly increased chalkiness and an individual yield reduced by 40.7% (fig. 12b,13 c). In the CRISPR family QT12-CR, the setting rate, grain number and thousand grain weight were significantly increased, resulting in a significant 1.7-fold increase in individual grain yield, while the rice chalkiness rate was significantly reduced and the taste value of cooked rice was also significantly increased (fig. 12c,13 d). Meanwhile, the positive family of the complementary material QT12 ^J -Com with the genotype complementary to that of indica rice SHZ is remarkably reduced in fruiting rate and yield at high temperature, and the taste value of rice is further reduced (figures 12d and 13 e). However, at normal temperatures, the yield traits of the various QT12 transgenic families did not change significantly (fig. 14).

To further evaluate QT12 effects on rice quality and yield, we performed large-scale field trials in 2024 summer on three major cities (martial arts, hangzhou and changsha) of three provinces of various genetic materials of QT12 in the Yangtze river basin of china (3 or4 cells were planted per genetic material family, each cell having an area of about 5 square meters and 12×15=180 plants). Yangtze river basin is the main paddy rice producing area in China, and the extremely high temperature breaks records continuously in the last 20 years, especially in 2024 summer (FIG. 15 a). Through examination of various yield traits, we found that the seed setting rate of NIL ^CH was increased by 50.6%, 55.8% and 14.6% respectively, and the individual yield was increased by 62.4%, 74.2% and 34.9% respectively, compared with NIL ^OM under the condition of large Tian Gaowen (fig. 15 b-d). Meanwhile, large-scale field experiments show that compared with NIL ^OM at high temperature, the cell yield (4×5m ² or3×5m ²) of NIL ^CH is respectively improved by 67.4 percent, 68.1% and 40.6% (fig. 15 c). Further investigation of the rice quality phenotype found that the chalkiness and chalkiness of NIL ^CH were significantly lower than NIL ^OM (fig. 15d, e). In addition, we further evaluated the breeding potential of QT12 gene in improving the heat tolerance of the yield of chinese elite variety huaman (Huazhan ^G). We introduced QT12 ^A from chenghui 448 into warfarin by backcrossing, creating an introduced line Huazhan ^A. Large-scale field experiments in martial arts, hangzhou and Changsha showed that Huazhan ^A significantly increased grain yield by 13.6%, 32.5% and 10.9% respectively, individual yield by 46.9%, 80.8% and 28.0% respectively, and cell yield by 49.1%, 77.9% and 31.2% respectively, compared to Huazhan ^G (fig. 15f, g). At the same time, we found that Huazhan ^A also had a much lower grain chalkiness and chalkiness than Huazhan ^G (fig. 15g, h). Extensive field trials at these multiple sites further demonstrate the impact of QT12 ^A on grain yield and quality at high temperatures.

In addition, we performed large-scale field trials of two major transgenic materials of QT12 in three major cities in 2024 in summer at the same high temperature treatment and performed phenotypic investigation. Compared to wild-type adult-back 448 (high resistance), the seed setting rates of QT 12-complementing families at high temperature were significantly reduced by 30.23%, 31.5% and 23.0%, respectively, and the individual yield was reduced by 52.6%, 50.0% and 44.1%, respectively (fig. 16 a-c). At the same time, the cell yield of QT 12-complementary families at high temperature was significantly reduced by 49.7%, 52.1% and 40.3%, respectively, compared to chenghui 448 (high resistance) (fig. 15 b). Further investigation of the grain quality showed a significant increase in both the grain chalkiness and chalkiness in the complementary families (fig. 16c, d). In contrast, CRISPR knockout line set-up rates were significantly improved by 37.6%, 43.5% and 24.6% over sensitive line OM1723, resulting in a significant improvement in individual yield of 88.2%, 67.9% and 60.5%, respectively (fig. 16e, f). Meanwhile, in three main cities of Wuhan, hangzhou and Changsha, the cell yield of the CRISPR family is respectively improved by 92.5%, 64.1% and 54.7% (figure 16 e), and the CRISPR family is very high-temperature resistant. Further examination of rice quality revealed that CRISPR families exhibited lower grain chalkiness and chalkiness, which were shown to be better rice quality (fig. 16f, g). Notably, other agronomic traits (grain width, plant height, and growth period, etc.) were not significantly altered for all QT 12-related genetic materials (fig. 17, 18). These results show that QT12, which is expressed low or nonfunctional, can simultaneously improve heat resistance of rice quality and yield in a natural field high temperature environment, has significance for synergistically improving rice quality and yield at high temperature, and is beneficial to breaking breeding bottlenecks of contradiction between crop stress and growth, yield and quality.

Example 9 Effect of high temperature on QT12 mRNA and the expression level of the encoded protein

In FIG. 3c, the high temperature was able to significantly up-regulate the mRNA expression level of QT12 in NIL ^OM, in order to further verify the effect of high temperature on the expression of QT12 mRNA and protein levels, we performed immunoblotting experiments of QT12-Com transgenic lines with qPCR and QT12-Flag fusion proteins, and found that high temperature could significantly induce the expression level of QT12 ^G mRNA and protein in QT12-Com without significantly changing the mRNA level of heat resistant wild type QT12 ^A (FIG. 19). These results further demonstrate that high temperature negatively regulates rice grain heat tolerance by inducing QT12 protein levels in QT12 ^G sensitive haplotypes.

These results demonstrate that the QT12 gene is a negative regulator for controlling rice quality and yield heat resistance, and demonstrate that this gene can be used to improve rice varieties by genetic transformation, molecular marker assisted breeding, and the like, including but not limited to improving rice quality and yield heat resistance using QT 12.

The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.

Claims

1. The application is any one of the following:

the amino acid sequence of the QT12 protein is shown as SEQ ID NO. 5;

the rice quality includes chalky appearance quality of rice, storage material content, and taste quality.

2. The use according to claim 1, wherein the chalky appearance quality comprises chalky rate and chalky degree, the storage substance comprises storage protein and amylose, and the nucleotide sequence encoding the QT12 protein is shown in SEQ ID No. 4.

3. A method for enhancing rice quality and yield at high temperature and/or cultivating quality and yield resistant rice germplasm is characterized in that expression, function or activity of QT12 genes or coded proteins thereof in rice is knocked out, inhibited or reduced to improve heat resistance of rice quality and yield, namely the obtained rice better maintains stable balance of rice storage protein and amylose content, low chalkiness and higher rice taste value at high temperature, and simultaneously, maturing rate, solid grain number increase and yield increase, and protein sequences coded by the QT12 genes are shown as SEQ ID NO. 5.

4. The method of claim 3, wherein the substance that inhibits QT12 protein expression or function in rice is a CRISPR/Cas9 vector, and the target sequence for which the CRISPR/Cas9 vector is directed is the polynucleotide site shown in SEQ ID No. 6.

5. An SNP molecular marker capable of distinguishing the quality and yield heat resistance of indica rice from that of nonglutinous rice, which is characterized in that the SNP molecular marker is shown as SEQ ID NO.3, has A/G variation at 2372 th position, wherein when the SNP locus is A, the rice is identified as indica rice, and lower QT12 expression level and lower quality heat damage index at high temperature, namely higher quality heat resistance are shown.

6. An indel molecular marker related to heat resistance and capable of distinguishing quality and yield of indica rice, which is characterized in that the indel molecular marker is shown as SEQ ID NO.3, a deletion of CGCCGC bp exists or does not exist at 2828-2833, and the indel molecular marker is identified as indica rice when the result is-6 bp.

7. A molecular marker combination related to QT12 expression level and rice quality and yield heat resistance, characterized in that the molecular marker combination comprises the SNP molecular marker of claim 5 and the indel molecular marker of claim 6, and further the rice germplasm is divided into three haplotypes according to the SNP and indel results, hap1: G/+6 bp, hap3: G/-6bp, hap7: a/-6bp, wherein the haplotype Hap7 has the lowest QT12 expression level, hap3 is centered, the expression level of QT12 in Hap1 is the highest, and the lower QT12 expression level represents higher quality and yield heat resistance.

8. Use of the molecular markers according to any one of claims 5-7 or a combination thereof for the identification, assisted identification of indica rice and/or rice cultivars of quality and yield resistant rice, characterized in that rice with a SNP result of a and/or indel result of-6 bp is identified as indica rice, rice with an indel result of +6bp is identified as japonica rice, and rice with a, -6bp, a/-6bp is selected for co-cultivation of rice germplasm of quality and yield resistant rice according to the SNP and/or indel result.

9. A method for identifying and assisting in identifying indica rice, which is characterized in that the molecular marker or the combination thereof in any one of claims 5-7 is utilized to detect rice, the rice with SNP result of A and/or indel result of-6 bp is identified as indica rice, and the rice with indel result of +6bp is identified as japonica rice.

10. A method for cultivating quality and yield-tolerant rice germplasm, characterized in that the molecular markers or the combination thereof according to any one of claims 5-7 are used for detecting rice, and rice with the length of A, -6bp and A/-6bp is selected according to SNP and/or indel results for synergistic cultivation of quality and yield-tolerant rice germplasm.