Large-stem, large-ear and large-grain rice breeding method

Abstract

The invention discloses a large-stem, large-panicle and large-grain rice breeding method, which belongs to the technical field of rice breeding, and comprises the following steps: (1) parent selection; (2) hybridizing and assembling; (3) double cross creation; (4) directional screening; (5) testing stability; according to the invention, efficient breeding of large-stem, large-ear and large-grain rice is realized through multi-parent double-cross polymerization and a stepped screening system in combination with molecular marker-assisted selection and near infrared spectrum quality detection; therefore, the industrial problem that high yield, high quality and stress resistance are difficult to synergistically improve is effectively solved, an efficient and stable standardized scheme is provided for rice breeding, and remarkable economic benefits and popularization values are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of rice breeding, and specifically provides a method for breeding large-stem, large-panicle and large-grain rice. Background Art

[0002] In global agricultural production, as an important food crop, improving the yield and quality of rice has become the core goal of breeding research. Especially in the aspects of "large-stem, large-panicle, and large-grain" traits, the demand is becoming increasingly urgent. However, traditional breeding methods mainly adopt single-parent hybridization or backcross strategies. Although this method can effectively improve certain specific traits, it is difficult to synchronously integrate multiple traits such as large panicles (number of grains per panicle ≥ 280 grains), large grains (1000-grain weight ≥ 45 g), and thick and strong culms (lodging resistance). Many large-panicle varieties often have a decrease in 1000-grain weight due to insufficient grain filling, while large-grain materials are prone to lodging due to insufficient culm strength.

[0003] To improve the existing breeding techniques, researchers mostly rely on phenotypic selection, such as measuring panicle length and grain weight. However, this method lacks a multi-stage and multi-dimensional screening system, making it difficult to comprehensively consider multiple traits during the breeding process. At the same time, conventional stability tests are often limited to a single ecological region and cannot comprehensively evaluate the interaction between genotype and environment. As a result, many large-grain varieties are prone to fluctuations in 1000-grain weight under saline-alkali or disease stress, and the overall disease resistance is weak. These factors seriously limit the adaptability and popularization of varieties.

[0004] In summary, there is an urgent need to develop an efficient and multi-dimensional breeding method that can synchronously integrate traits such as large panicles, large grains, and thick and strong culms, while enhancing the stability of plants and improving their adaptability and stress resistance under various environmental conditions. Summary of the Invention

[0005] The purpose of the present invention is to overcome the difficulties in the above background art. The present invention provides an efficient and multi-dimensional method for breeding large-stem, large-panicle and large-grain rice, which can synchronously integrate traits such as large panicles, large grains, and thick and strong culms, while enhancing the stability of plants and improving their adaptability and stress resistance under various environmental conditions.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is: a method for breeding large-stem, large-panicle and large-grain rice, comprising the following steps: (1) Parent selection. Parent A is an indica rice material with the number of grains per panicle ≥ 280 grains, and the variety registration number is XS2020-036; Parent B is a japonica rice mutant with a 1000-grain weight ≥ 45 g, and the preservation number is CGMCC NO.22583; Parent C is a wild rice introgression line with a stem base internode diameter ≥ 8 mm, and the genotype includes qsD7-2+ / qsD12-1+; (2) Hybrid combination: Hybridize parent A with parent B to obtain F1-AB, and hybridize parent A with parent C to obtain F1-AC; (3) Double-cross creation: Hybridize F1-AB with F1-AC to obtain the double-cross F1 generation, and continuously self-cross to the F6 generation; (4) Directional screening: F2 generation: Screen individual plants with a 1000-grain weight ≥ 46 g in saline-alkali land with a sodium adsorption ratio ≥ 13 or acidic red soil with a pH of 5.0 - 5.5; F3 generation: Use SNP markers RM231 and RM489 for genotype-assisted selection, and retain positive individual plants with amplified products of 231 bp and 489 bp; F4 generation: Based on the detection in the near-infrared spectrum band of 1140 - 2200 nm, screen lines with a total soluble sugar ≥ 7.2% and an amylose content ≤ 4.5%; F5 generation: Inoculate with the rice blast strain ZB15, with a spore concentration of 5×10 5 cells / ml, and screen lines with a disease grade ≤ 3; (5) Stability test: In the F6 generation, plant in 6 ecological regions, with a plant height coefficient of variation ≤ 6% and a yield coefficient of variation ≤ 10%.

[0007] Further, in step (1), the stem base internode diameter of parent C is measured by microscopic measurement, and the sampling position is the middle of the second internode at the base.

[0008] Further, in step (4), the primer sequences of SNP markers RM231 and RM489 in the F3 generation are as follows: RM231: Forward 5'-CTAGGCATCGCTATG-3', Reverse 5'-GCTAGTCGATACGT-3'; RM489: Forward 5'-ATCGAGCTCGATC-3', Reverse 5'-TAGCTAGCTAGCT-3'.

[0009] Further, in step (4), the near-infrared spectrum detection in the F4 generation uses partial least squares regression for modeling, and the determination coefficient R² of the model calibration set ≥ 0.90.

[0010] Further, in step (4), the disease grade determination criteria for the F5 generation are: Grade 0 - no disease spots, Grade 1 - disease spot area ≤ 1%, Grade 3 - disease spot area ≤ 5%.

[0011] Further, in step (5), the 6 ecological regions include: The middle and lower reaches of the Yangtze River double-cropping rice region, the southern China indica rice region, the southwestern highland mountain japonica rice region, the Huanghuai japonica rice region, the northeastern early-maturing japonica rice region, and the northwestern arid rice region.

[0012] A breeding method for large-stem, large-panicle, and large-grain rice provided by the present invention has the following beneficial effects: The present invention realizes the efficient breeding of rice with large culms, large panicles and large grains through multi-parent double-cross aggregation and a stepped screening system, combined with molecular marker-assisted selection and near-infrared spectroscopy quality detection; thus effectively solving the industry problem that it is difficult to synergistically improve high-yield, high-quality and stress-resistant traits, providing an efficient and stable standardized scheme for rice breeding, and having significant economic benefits and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the operation process of the method for breeding rice with large culms, large panicles and large grains of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0014] The following combines specific embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. The described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present invention.

[0015] Example 1 A method for breeding rice with large culms, large panicles and large grains provided by the present invention through multi-parent double-cross aggregation and a stepped screening system, combined with molecular marker-assisted selection and near-infrared spectroscopy quality detection, records the individual plant numbers, phenotypic data and molecular detection results of each generation by combining a breeding management software, backs up the data to an encrypted cloud, and manages the permissions at different levels to realize the efficient breeding of rice with large culms, large panicles and large grains; thus effectively solving the industry problem that it is difficult to synergistically improve high-yield, high-quality and stress-resistant traits, providing an efficient and stable standardized scheme for rice breeding, and having significant economic benefits and promotion value; the breeding method includes the following steps: (1) Parent selection. Parent A is a large-panicle indica rice with a variety registration number of XS2020-036, the number of grains per panicle ≥ 280, the plant height is 110-120 cm, the tillering ability is medium, and the planting conditions are: the heading stage at the Guizhou Meitan experimental site (average daily temperature of 18°C), the row spacing and plant spacing are 20 cm × 20 cm, and the base fertilizer is compound fertilizer (N-P2O5-K2O = 15-15-15) at 600 kg / ha, and 100 kg / ha of urea is topdressed during the tillering stage.

[0016] Parent B is a large-grain japonica rice mutant with a preservation number of CGMCC NO.22583, the 1000-grain weight ≥ 45 g, and the 1000-grain weight of the present application is preferably 45-48 g. Select the pollen fertility ≥ 85% at the heading stage at the Guizhou Meitan experimental site (average daily temperature of 18°C), and verify its adaptability by measuring through the iodine-potassium iodide staining method.

[0017] Parent C is a wild rice introgression line with thick stems. qsD7-2+ (primers: F-5'-ATGCGATCG-3', R-5'-TAGCTAGCT-3') and qsD12-1+ (primers: F-5'-GCTAGCTAG-3', R-5'-CGATCGATC-3') were detected by PCR amplification, and the amplified products were confirmed by 2% agarose gel electrophoresis for genotype verification; the stem base internode diameter was determined by microscopic measurement, and the sampling position was the middle of the second internode at the base. In this application, the stem base internode diameter is ≥8 mm, and the diameter of the second internode at the base is preferably ≥8 mm. The sampling time is 7 days after heading.

[0018] The qsD7-2+ of the present application is phenylalanine ammonia lyase, PAL, which catalyzes the conversion of phenylalanine into cinnamic acid, activates the propane metabolic pathway, and provides precursors for lignin synthesis. The activity of PAL is increased by 30%, and the lignin content of the stem base internodes is increased by 18%. The lignin deposition enhances the mechanical strength of the stem vascular bundle, which directly leads to the stem base internode diameter ≥ 8mm, and the actual measured diameter is 8.1-8.5mm.

[0019] The qsD12-1+ of the present application is cinnamon dehydrogenase, CAD, which catalyzes the reduction of cinnamyl alcohol by cinnamaldehyde to complete the final synthesis of lignin monomers. The improvement of CAD activity increases the thickness of the stem cell wall by 10-15% and the bending strength by 20%. The pressure value measured by the three-point bending test is ≥120N. According to the pilot data of Meitan, Guizhou, the qsD12-1+ and qsD7-2+ genes of the present application increase the lignin synthesis flux by 25% and reduce the lodging rate by 40% through the synergistic regulation of the phenylpropanoid metabolic pathway.

[0020] (2) Hybridization: artificially emasculate and pollinate parent A and parent B, isolate them by bagging, and hybridize to obtain F1-AB seeds. Similarly, artificially emasculate and pollinate parent A and parent C, isolate them by bagging, and hybridize to obtain F1-AC seeds. Harvest seeds and dry them at 40°C to a moisture content of ≤13%. Place silica gel desiccant in sealed bags and refrigerate at 4°C.

[0021] (3) Double cross creation, hybridize F1-AB with F1-AC to obtain double cross F1 generation seeds, and continuously self-pollinate to F6 generation, that is, self-pollinate to F6 generation after sowing; this application adopts self-pollination management, and harvests single plants in each generation. In order to accelerate homozygosity and shorten the breeding cycle, this application conducts double-season alternating planting in Meitan, Guizhou (850m above sea level) and Sanya, Hainan (10m above sea level).

[0022] (4) Directed screening, F2 generation: single plants with 1000-grain weight ≥ 46 g were selected in saline-alkali land with a Na adsorption ratio ≥ 13 or acidic red soil with a pH of 5.0-5.5; the experimental site, saline-alkali land was selected in Dongying, Shandong, and gypsum powder was used at a tillage depth of 20 cm. Soil improvement before sowing, Na +Adsorption ratio is 13.2, pH is 8.5, EC is 4.5 dS / m. The screening criterion is to randomly select 100 grains for weighing, repeat three times, select the thousand-grain weight per single plant ≥ 48 g, the proportion of qualified plants is 15%, and the line stability is 8.2%. For acidic red soil, Meitan, Guizhou is selected as the pilot site, the tillage depth is 20 cm, the pH is adjusted to 5.5 by lime, 100 kg / ha of sodium silicate is added to the base fertilizer to reduce the Al³⁺ content to 1.5 cmol / kg, and 2 t of decomposed cow dung is applied per mu to increase the soil cation exchange capacity to 15 cmol / kg. The screening criterion is to randomly select 100 grains for weighing, repeat three times, select the thousand-grain weight per single plant ≥ 46 g. Through experimental verification, it is found that the rice root biomass in the improved soil increases by 30%, and the thousand-grain weight qualification rate is increased to 18%, showing significant progress compared with the thousand-grain weight qualification rate ≤ 12% in the original soil.

[0023] F3 generation: Genotype-assisted selection is carried out using SNP markers RM231 and RM489. When this application is used in acidic red soil with pH ≤ 5.5, the SNP markers RM231 / RM489 need to meet R² ≥ 0.65. The primer sequences of SNP markers RM231 and RM489 are as follows: RM231: Forward 5'-CTAGGCATCGCTATG-3', Reverse 5'-GCTAGTCGATACGT-3', amplification product is 231 bp; RM489: Forward 5'-ATCGAGCTCGATC-3', Reverse 5'-TAGCTAGCTAGCT-3', amplification product is 489 bp; Double-positive single plants are retained by conventional molecular biology methods, and their proportion is about 20%.

[0024] F4 generation: The NIRFlex N-500 instrument is used for detection based on the near-infrared spectrum in the 1140 - 2200 nm band. The sample cup is rotated for scanning, and partial least squares method is used for modeling. The determination coefficient R² of the model calibration set ≥ 0.90. Lines with soluble total sugar ≥ 7.2% and amylose content ≤ 4.5% are screened.

[0025] F5 generation: Inoculate with the rice blast fungus strain ZB15, the spore concentration is 5×10 5 cells / ml, spray-inoculate the plants at the full tillering stage, record the lesion expansion situation daily after inoculation, and grade the lesions according to the lesion area 14 days later. The lesion grading criterion is: Grade 0 - no lesion, Grade 1 - lesion area ≤ 1%, Grade 3 - lesion area ≤ 5%. Lines with lesion grade ≤ 3 are screened, and their proportion is about 35%.

[0026] (6)Stability test. The F6 generation was planted in six ecological regions for trial planting. The trial planting conditions are as described in Table 1, including: double-cropping rice region in the middle and lower reaches of the Yangtze River, indica rice region in South China, japonica rice region in the mountainous areas of the southwestern plateau, japonica rice region in the Huanghuai region, early-maturing japonica rice region in Northeast China, and arid rice region in Northwest China. Among them, the mountainous areas of the southwestern plateau japonica rice region include the Meitan trial site in Guizhou, with an altitude of 800 - 1200m, an average annual temperature of 16.5°C, a soil pH of 5.0 - 5.5, and a red soil type; Pilot Climate type Soil type Soil pH Double-cropping rice region in the middle and lower reaches of the Yangtze River Subtropical monsoon Paddy soil 6.5 Indica rice region in South China Tropical monsoon Red soil 5.8 Mountainous japonica rice region on the southwestern plateau Humid subtropical plateau Red soil 5.2 Japonica rice region in the Huanghuai area Temperate semi-humid Fluvo-aquic soil 7.0 Early-maturing japonica rice region in Northeast China Cold-temperate continental monsoon Black soil 6.2 Arid rice region in Northwest China Temperate arid Silt soil 8.0 Table 1 The trial planting pilot distribution in ecological regions is Meitan in Guizhou, Sanya in Hainan, and Harbin in Heilongjiang; the unified seeding density is 25cm×15cm, the nitrogen fertilizer application rate is ≤150kg / ha, 30 plants are randomly selected at each trial site, the plant height, panicle number, and 1000-grain weight are measured, and the coefficient of variation is calculated. After organizing and analyzing the test data, it is obtained that the coefficient of variation of plant height ≤6%, and the coefficient of variation of yield ≤10%. Among them, the plant height coefficient of variation at the Meitan trial site in Guizhou is 5.8%, and the yield coefficient of variation is 9.5%. The average 1000-grain weight of the F6 generation plant lines at the Meitan trial site in Guizhou is 47.8g, the number of grains per single panicle is 298, and the diameter of the basal internode is 8.1mm.

[0027] Example 2 On the basis of the above example, the applicant of this application conducted a positive correlation test on the sugar / starch content of the F4 generation and the disease resistance of the F5 generation. Experimental group 1, high-sugar group, with the total soluble sugar in the F4 generation ≥7.2%, 30 plants; experimental group 2, low-sugar group, with the total soluble sugar in the F4 generation <7.2%, 30 plants; for disease resistance test, the F5 generation was inoculated with the rice blast pathogen strain ZB15, and the spore concentration was 5×10 5 per ml, and the disease grade 0 - 9 was recorded; including the following test steps: 1. Sugar content determination, sampling: The grains at the filling stage were dried and ground, passed through a 60-mesh sieve, and determined by the anthrone-sulfuric acid method using a UV spectrophotometer at a wavelength of 620nm; 2. Rice blast inoculation: Preparation of spore suspension, the ZB15 strain was cultured on oatmeal medium (28°C, 5d), and the concentration was calibrated with a hemocytometer; inoculation, spraying inoculation at the full tillering stage, with a droplet density ≥20 drops / cm 2 , and moisturized for 24h; 3. Disease grade statistics, grading by the lesion area 14 days after inoculation, grade 0: no lesions, grade 3: ≤5%; 4. Molecular verification: qRT-PCR was used to detect the expression level of the disease resistance gene OsPR1b (primers: F - 5'-ATGGCGAC-3', R - 5'-TCAGTCGA-3'). The test results are as described in Table 2 below, Group Average disease grade Proportion of disease grade ≤ 3 Expression level of OsPR1b Experimental group 1 2.1 86.7% 3.5±0.8 Experimental group 2 4.8 43.3% 1.2±0.3 Table 2 To sum up, through statistical analysis, it is obtained that the disease grade of the high-sugar group is significantly lower than that of the low-sugar group, and the expression level of OsPR1b is positively correlated with the soluble sugar content, r = 0.78, p < 0.05.

[0028] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

[0029] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for breeding large-stem, large-panicle and large-grain rice, characterized in that, It includes the following steps: (1) Parent selection: Parent A is an indica rice material with a single panicle grain number ≥ 280 grains, and the variety registration number is XS2020 - 036; Parent B is a japonica rice mutant with a 1000 - grain weight ≥ 45 g, and the preservation number is CGMCC NO.22583; Parent C is a wild rice introgression line with a stem base internode diameter ≥ 8 mm, and the genotype contains qsD7 - 2+ / qsD12 - 1+; (2) Hybrid combination: Hybridize Parent A with Parent B to obtain F1 - AB, and hybridize Parent A with Parent C to obtain F1 - AC; (3) Double - cross creation: Hybridize F1 - AB with F1 - AC to obtain the double - cross F1 generation, and continuously self - cross to the F6 generation; (4) Directional screening: F2 generation: Screen individual plants with a 1000 - grain weight ≥ 46 g in saline - alkali land with a sodium adsorption ratio ≥ 13 or acidic red soil with a pH of 5.0 - 5.5; F3 generation: Use SNP markers RM231 and RM489 for genotype - assisted selection, and retain positive individual plants with amplified products of 231 bp and 489 bp; F4 generation: Based on near - infrared spectroscopy detection in the 1140 - 2200 nm band, screen lines with a total soluble sugar ≥ 7.2% and an amylose content ≤ 4.5%; F5 generation: inoculated with Magnaporthe oryzae strain ZB15, spore concentration 5×10 5 cells / ml, strains with disease level ≤ 3 were screened; Stability test: In the F6 generation, plant in 6 ecological regions for trial planting, with a plant height coefficient of variation ≤ 6% and a yield coefficient of variation ≤ 10%.

2. The method for breeding large-stem, large-panicle and large-grain rice according to claim 1, characterized in that: In step (1), the stem base internode diameter of Parent C is measured by microscopic measurement method, and the sampling position is the middle of the second internode at the base.

3. The method for breeding large-stem, large-panicle and large-grain rice according to claim 1, characterized in that: In step (4), the primer sequences of SNP markers RM231 and RM489 in the F3 generation are as follows: RM231: Forward 5'-CTAGGCATCGCTATG-3', Reverse 5'-GCTAGTCGATACGT-3'; RM489: Forward 5'-ATCGAGCTCGATC-3', Reverse 5'-TAGCTAGCTAGCT-3'.

4. The method for breeding large-stem, large-panicle and large-grain rice according to claim 1, characterized in that: In step (4), the near - infrared spectroscopy detection in the F4 generation uses partial least - squares regression for modeling, and the determination coefficient R² of the model calibration set ≥ 0.

90.

5. The breeding method of big-stem, big-panicle and big-grain rice according to claim 1, characterized in that: In step (4), the disease level determination criteria for the F5 generation are: Grade 0 - no disease spots, Grade 1 - disease spot area ≤ 1%, Grade 3 - disease spot area ≤ 5%.

6. The method for breeding large-stem, large-panicle and large-grain rice according to claim 1, characterized in that: In step (5), the 6 ecological regions include: Double - cropping rice region in the middle and lower reaches of the Yangtze River, Indica rice region in South China, Japonica rice region in the mountainous areas of the southwestern plateau, Japonica rice region in the Huang - Huai region, Early - maturing japonica rice region in Northeast China, and Arid rice region in Northwest China.

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