A method for creating a new high-quality disease-resistant and compact japonica rice material

Abstract

The present invention relates to a method for creating a new material of high-quality disease-resistant and compact japonica rice, that is, selecting high-quality fragrant japonica rice as the recipient parent, and using a compact tillering gene to produce a new material of high-quality disease-resistant and compact japonica rice. TAC4 <supgt; T < / supgt;作为供体亲本A、含有广谱抗稻瘟病基因 Pigm The line was used as donor parent B, and the KASP marker was used for marker-assisted selection. Through backcrossing, selfing and polyhybridization, combined with molecular marker-assisted selection, the compact tillering gene was obtained. TAC4 <supgt; T < / supgt;、稻瘟病广谱抗性基因 Pigm and aroma genes fgr The aggregation in breeding materials has achieved the improvement of the rice blast resistance and plant lodging resistance of the parent materials on the basis of ensuring the excellent appearance quality and aroma characteristics of rice, and created a new japonica rice material with high-quality disease-resistant and compact plant type.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and particularly relates to a method for creating a new material of high-quality, disease-resistant and compact-type japonica rice. Background Art

[0002] With the improvement of people's living standards and the pursuit of healthy diets, the market and consumers have an increasingly strong demand for high-quality and disease-resistant varieties. At the same time, during production, the phenomenon of lodging is common while applying excessive heavy fertilizers to pursue high yields, which seriously restricts the high and stable yields and quality improvement of rice. Plant type is one of the important traits affecting population yield. Compact plant type varieties often have better lodging resistance and stable yield potential (Ding et al., Compact plant type rice has higher lodging and Nresistance under machine transplanting, Journal of Integrative Agriculture, 2021, 20:65 - 77). In recent years, a number of rice varieties such as "Nanjing 9108" and "Nanjing 46" with low amylose content and excellent eating quality have been bred, but at the same time, they show problems such as poor appearance quality and weak blast resistance.

[0003] As an important eating quality trait of rice, fragrance is mainly controlled by the Badh2 / fgr gene on chromosome 8; among the cloned blast resistance genes, Pigm (Deng et al., Epigenetic regulation ofantagonistic receptors confers rice blast resistance with yield balance, Science, 2017, 355:962 - 965) shows stable broad-spectrum resistance in both indica and japonica rice genetic backgrounds.

[0004] The tiller angle is the angle between the lateral tiller and the main stem, which is the core element affecting plant type. Rice varieties with an ideal tiller angle show high light energy utilization efficiency, fertilizer tolerance and lodging resistance, as well as higher yield and harvest index; at the same time, reasonable high-density planting is beneficial to increasing the population yield per unit area. TAC4 encodes a plant-specific and highly conserved nuclear protein, and the loss of its function will lead to a significant increase in the tiller angle.

[0005] At present, breeding compact plant type rice varieties with good appearance transparency, strong fragrance and blast resistance has always been a technical problem to be solved in this field. Summary of the Invention

[0006] To solve some problems existing in the above-mentioned prior art, the present invention provides a method for creating a new material of high-quality, disease-resistant and compact-type japonica rice.

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] The present invention provides a method for creating a new material of high-quality disease-resistant compact japonica rice, comprising the following steps: (1) Using a fragrant and high-yield japonica rice line (i.e., the line carrying the fgr fragrant gene) as the recipient parent, and using a breeding material containing the compact tillering gene TAC4 T as the donor parent A, crossing the recipient parent with the donor parent A to obtain hybrid F1 generation seeds, designated as F1-A;

[0009] Using a breeding material containing the broad-spectrum blast resistance gene Pigm as the donor parent B, crossing it with the recipient parent to obtain hybrid F1 generation seeds, designated as F1-B;

[0010] (2) Planting the F1-A and F1-B seeds respectively, backcrossing the F1-A seeds at the heading stage with the recipient parent to obtain the BC1F1 population of the first backcross generation, designated as BC1F1-A; backcrossing the F1-B seeds at the heading stage with the recipient parent to obtain the BC1F1 population of the first backcross generation, designated as BC1F1-B;

[0011] (3) Planting the BC1F1-A and BC1F1-B seeds respectively, using high-throughput KASP molecular markers during the tillering stage of rice seedlings, and using the donor parent A and the donor parent B as controls respectively, conducting molecular marker comparison and detection of the TAC4 T and Pigm genes, and screening out the heterozygous single plants of the target gene TAC4 T and the Pigm heterozygous single plants respectively;

[0012] Using the TAC4 T heterozygous single plants with good agronomic traits as the female parent and the Pigm heterozygous single plants with good agronomic traits as the male parent to cross at the heading stage to obtain the target F1 seeds;

[0013] (4) Planting the target F1 seeds to obtain the selfed first-generation population F2 seeds;

[0014] (5) Planting the F2 population seeds, conducting molecular marker detection at the seedling stage, and conducting rice quality screening at the maturity stage, screening out the lines that simultaneously carry TAC4 T , Pigm and fgr genes (homozygous and / or heterozygous), have good grain appearance transparency (chalkiness degree ≤ 2%, chalky grain rate < 10%) and good agronomic traits, and harvesting the seeds F3 of all lines;

[0015] (6) After planting the F3 lines, self-crossing, repeating the above step (5), screening out the lines that simultaneously carry TAC4 T , Pigm and fgr genes homozygous, have good grain appearance transparency and good agronomic traits, and harvesting the seeds F4 of all lines;

[0016] (7) Plant the F4 lines, preferably the lines with good transparency of grain appearance, excellent and stable agronomic traits (no trait segregation occurs in self-crossed offspring), and harvest the seeds F5 of all lines.

[0017] (8) Plant the F5 lines, combine the identification of panicle blast resistance at the booting stage and the screening of rice quality at the maturity stage, and preferably select the lines with good transparency of grain appearance, strong panicle blast resistance (the disease incidence level of panicle blast ≤ 3) and excellent agronomic traits, and finally create new japonica rice materials with high quality, panicle blast resistance and compact plant type.

[0018] Preferably, the above recipient parents include at least one of Yangjing 805, Suken 118, and the lines carrying the fgr fragrance gene in Jiahexiang 1.

[0019] Preferably, the above donor parent A includes at least one of rice lines such as Jinjing 616 and Ningxiangjing 9 containing the compact tillering gene TAC4 T

[0020] Preferably, the above donor parent B includes Yangjing 7311 containing the broad-spectrum blast resistance gene Pigm.

[0021] In some embodiments of the present invention, in step (3), the target genes TAC4 T The heterozygous single plants of TAC4 and the heterozygous single plants of Pigm refer to: respectively extract the genomic DNA of all lines, and use the preferred KASP molecular marker PM-TAC4 of the target gene TAC4 T to detect the TAC4 T gene; use the KASP molecular marker PM-Pigm reported by Qing Dongjin et al. to detect the Pigm gene (Qing Dongjin et al., Development of molecular markers for the blast resistance gene Pigm based on PARMS technology, Southwest China Journal of Agricultural Sciences, 2018, 31: 1617-1621); screen out the lines containing TAC4 T and Pigm. T

[0022] Preferably, in the above step (5), at the tillering stage of the F2 rice seedlings, using the recipient parent, donor parent A, and recipient parent B as controls, respectively conduct molecular marker comparison detections of TAC4 T , Pigm and fgr genes, and screen out the lines containing TAC4 T ​​, strains of Pigm and fgr genes. Among these strains, select the strains with good transparency of grain appearance for continuous self-crossing, and gradually compare and screen the strains of each generation to obtain new japonica rice materials with high quality, disease resistance and compact plant type. The specific steps are as follows: extract the genomic DNA of all strains respectively, and detect the TAC4T gene by using the preferred KASP molecular marker PM-TAC4T of the target gene TAC4T; detect the Pigm gene by using the KASP molecular marker PM-Pigm reported by Qing Dongjin et al. (Qing Dongjin et al., Southwest China Journal of Agricultural Sciences 2018(31)1617 - 1621); detect the fgr gene by using the preferred KASP molecular marker PM-fgrE2 of the target gene fgr; screen out the strains containing TAC4T, Pigm and fgr.

[0023] Preferably, in the above step (8), it also includes self-crossing the screened F5 generation strains for 1 generation, and combining the identification of blast resistance at the booting stage to obtain new japonica rice materials with strong panicle blast resistance and compact plant type. The inoculation identification of blast resistance at the booting stage adopts the artificial inoculation method of the intermediate test of rice varieties in Jiangsu Province (Qi Zhongqiang et al., Evaluation of the blast resistance of new rice varieties (lines) and main cultivated varieties in Jiangsu Province from 2016 to 2020, Jiangsu Agricultural Sciences, 2022, 50(01), 91 - 96). 5 - 7 days before the rice booting and heading stage, use a syringe to suck 1 mL of the mixed solution of mycelial fragments and thin-walled conidia of the blast physiological race, the conidia suspension of the blast fungus, with a concentration of about 2×105 spores mL-1. Inject it into the rice booting bract from the side until it overflows. Select cloudy days or evenings (15:00 - 18:00 in the afternoon of the same day) with an air temperature of 25 - 28°C and a relative humidity of 90%, inoculate 10 panicles, and make corresponding marks. Different physiological races cannot be repeatedly inoculated on each panicle. The blast identification standard: 0 level: disease-free; 1 level: incidence rate ≤ 5%; 3 level: incidence rate is 5.1% - 10.0%; 5 level: incidence rate is 10.1% - 25.0%; 7 level: incidence rate is 25.1 - 50%; 9 level: incidence rate is 50.1 - 100%. In this application, the term "strong panicle blast resistance" refers to the rice strains with a panicle blast disease incidence level ≤ 3 according to the above method of identification.

[0024] In this application, the term "good transparency of grain appearance" refers to rice grains with chalkiness degree ≤ 2% and chalky grain rate < 10%. The term "good agronomic traits" refers to the strain that simultaneously meets the following property requirements: plant height is between 85 - 100 cm, growth period ≤ 153.0 days, panicle type is upright, and grain weight ≥ 26 grams.

[0025] In some embodiments of the present invention, the primer group of the gene PM-TAC4 T includes: the reverse primer PM-TAC4 with the nucleotide sequence shown in SEQ ID NO.1 T-Rc, the reverse primer PM-TAC4 with the nucleotide sequence shown in SEQ ID NO.2 T -Ra, the forward primer PM-TAC4 with the nucleotide sequence shown in SEQ ID NO.3 T -F.

[0026] In some embodiments of the present invention, the primer set for the gene PM-Pigm includes: the forward primer PM-Pigm-Ft with the nucleotide sequence shown in SEQ ID NO.4, the forward primer PM-Pigm-Fc with the nucleotide sequence shown in SEQ ID NO.5, and the reverse primer PM-Pigm-R with the nucleotide sequence shown in SEQ ID NO.6.

[0027] In some embodiments of the present invention, the primer set for the gene PM-fgrE2 includes: the reverse primer PM-fgrE2-Rp1 with the nucleotide sequence shown in SEQ ID NO.7, the reverse primer PM-fgrE2-Rn1 with the nucleotide sequence shown in SEQ ID NO.8, and the forward primer PM-fgrE2-F with the nucleotide sequence shown in SEQ ID NO.9.

[0028] In the embodiments of this application, the receptor parent Yangjing 805 is a high-quality japonica variety, whose rice quality reaches the second-class high-quality rice standard of the national standard "High-quality Paddy Rice". The grains have good appearance transparency and have a fragrance (carrying the fragrance gene fgr), and the eating quality is relatively good; the donor parent A Jinjing 616 is a japonica variety carrying the excellent haplotype TAC4 with compact tillering T ; Yangjing 7311 (GY31-Pigm) is a breeding intermediate material created by the applicant of the present invention and carrying the broad-spectrum blast resistance gene Pigm.

[0029] The present invention uses molecular marker-assisted selection (MAS) to aggregate the broad-spectrum blast resistance gene Pigm, the fragrance gene fgr, and the compact tillering gene TAC4 T together to breed a new japonica rice material with good appearance transparency, strong fragrance, and blast resistance, meeting the needs of rice production. Compared with the prior art, the method for creating the new high-quality disease-resistant compact japonica rice material provided in this application has the following beneficial effects: while retaining the fragrance gene fgr, it efficiently aggregates the compact tillering gene TAC4 T and the broad-spectrum blast resistance gene Pigm, thereby creating a new high-quality disease-resistant compact japonica rice material, and realizing further improvement of the lodging resistance and blast resistance of the parental materials. The KASP molecular marker PM-TAC4 developed and designed by the present invention TTogether with PM-fgrE2, it can achieve rapid and accurate identification of target genes in rice germplasm resources or breeding populations, and can simultaneously complete high-throughput detection of large-scale samples, significantly shortening the breeding cycle and improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Results of genome-wide association analysis of tiller angle and TAC4 haplotype analysis for 255 japonica rice materials;

[0031] Among them, A: Genome-wide association analysis of tiller angle for 255 japonica rice materials mapped the tiller angle regulatory gene TAC4; B: Positions and variation types of significant SNPs on the TAC4 genome; C: TAC4 haplotype analysis for 255 japonica rice materials; D: Linkage KASP marker genotyping map in Example 1 of the present invention. Green dots are distribution points of the TAC4T allele genotype with small tiller angle, blue dots are distribution points of the TAC4C allele genotype with large tiller angle, red dots are distribution points of heterozygous genotypes, and gray dots are distribution points of negative controls.

[0032] Figure 2 For the linkage KASP marker PM-TAC4 in Example 2 T Genotyping map;

[0033] Among them, green dots represent distribution points of the compact tiller allele genotype, blue dots represent distribution points of the less compact allele genotype, red dots represent distribution points of heterozygous genotypes, and gray dots are distribution points of negative controls.

[0034] Figure 3 For the linkage KASP marker PM-Pigm genotyping map in Example 2;

[0035] Among them, green dots represent distribution points of the susceptible allele genotype, blue dots represent distribution points of the resistant allele genotype, red dots represent distribution points of heterozygous genotypes, and gray dots are distribution points of negative controls.

[0036] Figure 4 For the linkage KASP marker PM-fgrE2 genotyping map in Example 2;

[0037] Among them, green dots represent distribution points of the non-aromatic allele genotype, blue dots represent distribution points of the aromatic allele genotype, red dots represent distribution points of heterozygous genotypes, and gray dots are distribution points of negative controls.

[0038] Figure 5 Schematic diagram of the breeding process for obtaining new japonica rice materials with high quality, disease resistance and compact plant type through marker-assisted selection.

[0039] Figure 6Photographs of new japonica rice materials with high quality, disease resistance and compact plant type obtained by molecular marker-assisted selection in the examples; among them, A: Appearance quality identification of new japonica rice materials with high quality, disease resistance and compact plant type; B: Comparison of plant types between new japonica rice materials with high quality, disease resistance and compact plant type (B-2) and the control (B-1, Yangjing 805); C: Identification of rice blast resistance of the control (Yangjing 805); D: Identification of rice blast resistance of new japonica rice materials with high quality, disease resistance and compact plant type. Detailed implementation manners

[0040] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified.

[0041] The abbreviations and term definitions involved in the present invention are as follows:

[0042] KASP: Competitive allele-specific PCR.

[0043] SNP: Single nucleotide polymorphism.

[0044] Example 1 Mining of rare base variation type TAC4 that regulates compact tillering T of

[0045] In this example, 255 japonica rice varieties selected from the middle and lower reaches of the Yangtze River in China were collected in the early stage, and 30-fold deep resequencing was carried out on them. The tiller angle (flowering stage) data of the above japonica rice varieties were investigated in 2021 and 2022. To mine tiller angle regulatory genes / alleles with breeding value to guide the improvement of the compact plant type of japonica rice. Using the Nipponbare genome (IRGSP-1.0, https: / / rapdb.dna.affrc.go.jp) as the reference genome, sequence alignment was performed through the BWA (http: / / bio-bwa.sourceforge.net) analysis software. The quality control parameters for SNP information extraction were set as follows: the mapping quality value of each locus was greater than 20, the variant quality value was greater than 50, and each base had at least 2 or more reads data support, and the MAF value > 0.05. The SNP extraction software was GATKV4.1.4.1. The obtained material genotypes and corresponding tiller angle data were respectively imported into the Tassle (5.0) software. Using the mixed linear model method, there was a significant SNP locus at position 14,683,773 on chromosome 2 of rice, located at the 1793rd nucleotide of the CDS of the tiller angle regulatory TAC4 gene ( Figure 1)。The effective nucleotide difference of this SNP is C / T, resulting in the mutation of the 598th amino acid of TAC4 from threonine (ACT) to isoleucine (ATT). In addition, haplotype analysis found that most varieties carried TAC4 with a larger tiller angle C haplotype, and a few varieties carrying TAC4 T haplotype had smaller tiller angles, and this locus was designed as the linked KASP marker (SNP locus) of TAC4 T .

[0046] According to the sequences 300 bp upstream and downstream of the above SNP locus, KASP marker primers were designed using primer premier5.0 software. It includes a reverse primer Rc with a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer Ra with a nucleotide sequence as shown in SEQ ID NO.2, and a forward primer F with a nucleotide sequence as shown in SEQ ID NO.3. The specific sequences are shown in Table 1.

[0047] Table 1 Sequence information of the markers used for marker-assisted selection

[0048]

[0049]

[0050] In Table 1, the underlined part of the Rc primer sequence is the FAM universal fluorescent tag sequence, and the underlined part of the Ra primer sequence is the HEX universal fluorescent tag sequence.

[0051] The above KASP marker can be used to detect the TAC4 locus in rice materials in a high-throughput manner: if only the FAM fluorescence signal corresponding to Rc is detected in the PCR product, the base at the detection locus is C, and the test material contains the TAC4 C larger tiller angle allele. If only the HEX fluorescence signal corresponding to Ra is detected, the base at the detection locus is T, and the test material contains the TAC4 T smaller tiller angle allele; if both FAM and HEX fluorescence signals are detected, the test material is a heterozygous genotype. The tiller angle of rice containing allele T is significantly smaller than that of rice containing allele C.

[0052] Example 2 Creation of new materials for high-quality, disease-resistant and compact japonica rice

[0053] The source of the rice varieties involved in this example:

[0054] (1) The recipient parent, "Yangjing 805 (Approval number: Jiangsu Approval Rice 201307; Variety right: CNA20150052.6)", is a high-quality fragrant japonica rice variety jointly selected by the Agricultural Science Research Institute of the Lower Yangtze River Region in Jiangsu and Jiangsu Jintudi Seed Industry Co., Ltd., and can be purchased from Jiangsu Jintudi Seed Industry Co., Ltd.

[0055] (2) The donor parent of the compact tillering gene TAC4 T The donor parent of "Jinjing 616" is a conventional japonica rice variety selected by the Tianjin Rice Research Institute and can be purchased from the Tianjin Rice Research Institute; the donor parent of the broad-spectrum blast resistance gene Pigm, "Yangjing 7311", is an intermediate breeding material selected by the Agricultural Science Research Institute of the Lower Yangtze River Region in Jiangsu, using Yangjing 4227 (Approval number: Jiangsu Approval Rice 200912; Variety right: CNA20090109.7) as the recurrent female parent and the donor material Gumei 4 containing the broad-spectrum blast resistance gene Pigm as the male parent, through continuous backcrossing for 3 generations and self-crossing for 4 generations, and has applied for variety right protection (Application number: 20211001521).

[0056] Aggregate the compact tillering gene TAC4 T and the broad-spectrum blast resistance gene Pigm to create a new high-quality disease-resistant compact plant type japonica rice material. The creation steps are as follows;

[0057] (1) Using the donor parent Jinjing 616 carrying the compact tillering gene TAC4 T and the donor parent Yangjing 7311 of the broad-spectrum blast resistance gene Pigm, respectively cross with the recurrent parent Yangjing 805 (high-quality fragrant japonica rice line) to obtain 50 F1 seeds each, and represent them as F1-TAC4 T and F1-Pigm respectively;

[0058] (2) Plant the F1-TAC4 T and F1-Pigm seeds respectively, and backcross with Yangjing 805 at the heading stage of rice to obtain 100 backcross generation BC1F1 seeds, and represent them as BC1F1-TAC4 T and BC1F1-Pigm respectively;

[0059] (3) Plant the BC1F1-TAC4 T and BC1F1-Pigm seeds respectively, and use Jinjing 616 and Yangjing 7311 as controls at the seedling stage of rice, and use PCR to identify the compact tillering gene TAC4 T(Primers are shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3) and high-throughput KASP markers for the broad-spectrum blast resistance gene Pigm in rice (primers are shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6) were used for PCR molecular detection (primer information is shown in Table 1), and heterozygous single plants of TAC4 T and heterozygous single plants of Pigm were respectively screened out. At the heading stage, TAC4 T heterozygous single plants with good agronomic traits were used as female parents, and Pigm heterozygous single plants with good agronomic traits (that is, strains that simultaneously meet the following criteria: plant height 85-100 cm, growth period ≤ 153.0 days, erect panicle type, and grain weight ≥ 26 grams, and the following judgment criteria are the same) were used as male parents for hybridization to obtain 50 F1 seeds;

[0060] The above-mentioned compact tillering gene TAC4 T The PCR detection method is as follows:

[0061] To detect the compact tillering gene TAC4 T : When synthesizing the KASP molecular marker primers for the TAC4 gene, a fluorescent signal tag of carboxyfluorescein (FAM) was added to the 5' end of the reverse primer PM-TAC4 T -Rc; a fluorescent signal tag of 5-hexachlorofluorescein phosphoramidite (HEX) was added to the 5' end of the reverse primer PM-TAC4 T -Ra.

[0062] 1) Take the leaves of a single plant and obtain genomic DNA by the CTAB extraction method;

[0063] 2) PCR amplification system (10 μL): 2.5 μL of DNA at 20 ng / μL, 5.0 μL of 2×KASP Master mix, 0.4 μL of PM-TAC4 T -F primer (10 μM), 0.15 μL of PM-TAC4 T -Rc primer (10 μM), 0.15 μL of PM-TAC4 T -Ra primer (10 μM), 1.8 μL of ddH2O.

[0064] The system was placed in a 384-well PCR instrument for reaction. The PCR amplification program: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing at 58 °C for 60 s, 10 cycles (decreasing 0.8 °C per cycle); denaturation at 94 °C for 20 s, renaturation at 57 °C for 60 s, 32 cycles. After the cycle ended, it was placed in a 384-well fluorescence quantitative PCR instrument for typing detection, and the detection results are as Figure 2 shown.

[0065] The PCR detection method for the broad-spectrum blast resistance gene Pigm is as follows:

[0066] 1) Take single-plant leaves and obtain genomic DNA by the CTAB extraction method;

[0067] 2) PCR amplification system (10 μL): 2.5 μL of 20 ng / μL DNA, 5.0 μL of 2×KASP Master mix, 0.4 μL of PM-Pigm-R primer (10 μM), 0.15 μL of PM-Pigm-Ft primer (10 μM), 0.15 μL of PM-Pigm-Fc primer (10 μM), 1.8 μL of ddH2O.

[0068] Place the system in a 384-well PCR instrument for reaction. The PCR amplification program: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 58°C for 60 s, for 10 cycles (decreasing 0.8°C per cycle); denaturation at 94°C for 20 s, renaturation at 57°C for 60 s, for 32 cycles. After the cycles are completed, place it in a 384-well fluorescence quantitative PCR instrument for genotyping detection, and the detection results are as Figure 3 shown.

[0069] (4) Plant F1 seeds to obtain 3000 F2 seeds of the self-crossed first-generation population;

[0070] (5) Plant the F2 population seeds. At the seedling stage, using the parental Jinjing 616, Yangjing 7311, and Yangjing 805 as controls, use the KASP detection markers of the compact tillering gene TAC4 T , the broad-spectrum blast resistance gene Pigm, and the fragrance gene fgr (primer information is shown in Table 1) to compare the molecular markers of individual plants in the F2 population, and screen out 437 individual plants (homozygous or / and heterozygous) carrying TAC4 T , Pigm, and fgr; combined with the screening of agronomic traits at maturity and the observation of rice quality in the later stage, 162 lines carrying TAC4 T , Pigm, and fgr genes (homozygous and / or heterozygous), with good grain appearance transparency (chalkiness degree ≤ 2%, chalky grain rate < 10%) and good agronomic traits are preferably selected;

[0071] Detect the fragrance gene fgr: When synthesizing the KASP molecular marker primers of the fgr gene, add a fluorescent signal tag of carboxyfluorescein (FAM) to the 5' end of the reverse primer PM-fgrE2-Rp1; add a fluorescent signal tag of 5-hexachlorofluorescein phosphoramidite (HEX) to the 5' end of the reverse primer PM-fgrE2-Rn1.

[0072] The above molecular marker method for detecting the fragrance gene fgr is as follows:

[0073] 1) Take single-plant leaves and obtain genomic DNA by CTAB extraction method;

[0074] 2) Perform PCR amplification using the above primer set, and the reaction system is 10 μL.

[0075] The reaction system is as follows: 2.5 μL of DNA at 20 ng / μL, 5.0 μL of 2×KASP Master mix, 0.4 μL of PM-fgrE2-F primer (10 μM), 0.15 μL of PM-fgrE2-Rp1 primer (10 μM), 0.15 μL of PM-fgrE2-Rn1 primer (10 μM), 1.8 μL of ddH2O. Place the system in a 384-well PCR instrument for reaction.

[0076] PCR amplification program: Pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing at 58 °C for 60 s, cycle 10 times (decrease 0.8 °C per cycle); denaturation at 94 °C for 20 s, renaturation at 57 °C for 60 s, cycle 32 times.

[0077] 3) After the cycle ends, place it in a 384-well fluorescence quantitative PCR instrument for genotyping detection, as Figure 4 shown.

[0078] In Figure 2 - 4 , the X-axis is the FAM fluorescence signal coordinate axis, and the Y-axis is the HEX fluorescence signal coordinate axis. Figure 2 The green dots in represent homozygous single plants carrying the compact tillering allele, the blue dots represent homozygous single plants not carrying the compact tillering allele, the red dots represent heterozygous genotype single plants, and the gray dots are the distribution points for negative controls; Figure 3 The green dots in represent homozygous single plants carrying the susceptible allele, the blue dots represent homozygous single plants carrying the resistant allele, the red dots represent heterozygous genotype single plants, and the gray dots are the distribution points for negative controls; Figure 4 The green dots in represent homozygous single plants carrying the non-aromatic allele, the blue dots represent homozygous single plants carrying the aromatic allele, the red dots represent heterozygous genotype single plants, and the gray dots are the distribution points for negative controls.

[0079] (6) Plant the 162 single plants obtained in step (5) into separate families (F3 generation), and screen out the lines that are all homozygous for the TAC4 T , Pigm and fgr genes according to step (5); at the maturity stage, select 43 lines with good grain appearance transparency and excellent agronomic traits from the above-mentioned homozygous lines.

[0080] (7) Plant the 43 individual plants separated into families (F4 generation) obtained in step (6), and preferably select 17 lines with good transparency of grain appearance, good and stable agronomic traits (no trait segregation occurs in the offspring) at the mature stage.

[0081] (8) Plant the 17 individual plants separated into families (F5 generation) obtained in step (7), and conduct panicle blast resistance identification on each line at the booting stage. Combine the panicle blast resistance identification at the booting stage and the rice quality screening at the mature stage, and preferably select 5 lines with good transparency of grain appearance, strong panicle blast resistance (panicle blast disease incidence level ≤ 3), good and stable agronomic traits (Table 2). Finally, create new japonica rice materials with high quality, disease resistance, and compact plant type (all self-named by the applicant).

[0082] Table 2 Genotypes and partial phenotypes of new japonica rice materials with high quality, disease resistance, and compact plant type

[0083]

[0084] The screening process of the above examples is as Figure 5 shown. Select high-quality fragrant japonica rice as the recipient parent, and develop and design KASP markers for identifying the compact tillering gene TAC4 T and the fragrance gene fgr for marker-assisted selection. Through backcrossing, self-crossing, and pyramiding hybridization, and combined with molecular marker-assisted selection, the pyramiding of the compact tillering gene TAC4 T , the broad-spectrum blast resistance gene Pigm, and the fragrance gene fgr in the breeding materials was achieved. The improvement of the blast resistance and plant type lodging resistance of the parental materials was realized, and the physical photos of the new japonica rice materials with high quality, disease resistance, and compact plant type are as Figure 6 shown.

[0085] The above examples are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above examples without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for creating a new material of high-quality disease-resistant compact japonica rice, characterized in that, The specific steps are as follows: 1) Using rice carrying the fgr gene as the recipient parent and rice carrying the gene TAC4 T as the donor parent A to obtain F1-A generation seeds; Using rice carrying the gene Pigm as donor parent B, cross it with the recipient parent to obtain F1-B generation seeds; The rice carrying the gene TAC4 T refers to the rice in which the KASP molecular marker PM-TAC is detected with nucleotide sequences as shown in SEQ ID NO.1 - SEQ ID NO.3 4 T to detect the TAC 4 gene, and the rice containing the allele T; 2) Plant F1-A and F1-B seeds respectively. Backcross the F1-A seeds at the heading stage with the recipient parent to obtain BC1F1-A; backcross the F1-B seeds at the heading stage with the recipient parent to obtain BC1F1-B; 3) Sow the BC1F1-A and BC1F1-B seeds separately. At the seedling stage, using the donor parent A as a control, screen for individual plants carrying the gene TAC4 T . Using the donor parent B as a control, select individual plants carrying the gene Pigm ; Then, using the single plant carrying the gene TAC4 T and having good agronomic traits as the female parent, cross it with the single plant carrying the gene Pigm and having good agronomic traits as the male parent to obtain the target F1 seeds; 4) Plant the target F1 seeds to obtain the selfed first-generation population F2 seeds; 5) Sow the seeds of the F2 population, and screen for seeds that carry the genes TAC4 T , Pigm and fgr at the seedling stage. Then, screen for lines with a chalkiness degree ≤ 2%, a chalky grain rate < 10%, and good agronomic traits from the seeds obtained through screening. These are the F3 lines; 6) After planting the F3 lines, self-cross and repeat the above step 5) to obtain the F4 lines; 7) Plant the F4 lines and screen for lines with a chalkiness degree ≤ 2%, a chalky grain rate < 10%, good and stable agronomic traits to obtain the F5 lines; 8) Plant the F5 lines and screen for lines with a chalkiness degree ≤ 2%, a chalky grain rate < 10%, a rice blast disease incidence level ≤ 3, and good agronomic traits, and thus obtain the new high-quality disease-resistant compact-type japonica rice material; The good agronomic traits refer to a rice strain with a plant height of 85 - 100 cm, a growth period ≤ 153.0 days, an upright panicle type, and a grain weight ≥ 26 grams.

2. The method for creating a new high-quality disease-resistant and compact japonica rice material according to claim 1, characterized in that, The recipient parent includes at least one of Yangjing 805, Suken 118, and Jiahexiang 1.

3. The method for creating a new high-quality disease-resistant and compact japonica rice material according to claim 1, characterized in that, The donor parent A includes at least one of Jinjing 616 and Ningxiangjing 9.

4. The method for creating a new high-quality disease-resistant and compact japonica rice material according to claim 1, characterized in that, The donor parent B is Yangjing 7311.

5. The method for creating a new material of high-quality disease-resistant compact japonica rice according to claim 1, characterized in that, The single plant screened for carrying the gene TAC4 T refers to using the KASP molecular marker PM - TAC4 T to detect TAC4 T the gene. The single plant screened for carrying the gene Pigm refers to using the KASP molecular marker PM - Pigm to detect Pigm the gene.

6. The method for creating a new high-quality disease-resistant and compact japonica rice material according to claim 1, characterized in that, Select seeds that carry the genes TAC4 T , Pigm and fgr . The seeds refer to using the KASP molecular marker PM- TAC4 T to detect TAC4 T gene, using the KASP molecular marker PM- Pigm to detect Pigm gene, and using the KASP molecular marker PM-fgrE2 to detect the fgr gene.

7. The method for creating a new high-quality disease-resistant and compact japonica rice material according to claim 5, characterized in that, The primer set of the KASP molecular marker PM- TAC4 T has a nucleotide sequence as shown in SEQ ID NO. 1-3; The primer set of the KASP molecular marker PM- Pigm has a nucleotide sequence as shown in SEQ ID NO. 4-6.

8. The method for creating a new high-quality disease-resistant and compact-type japonica rice material according to claim 6, characterized in that, The primer set of the KASP molecular marker PM- TAC4 T has a nucleotide sequence as shown in SEQ ID NO.1-3; The nucleotide sequences of the primer sets of the KASP molecular marker PM- Pigm are shown in SEQ ID NO.4 - 6; the nucleotide sequences of the primer sets of the KASP molecular marker PM-fgrE2 are shown in SEQ ID NO.7 - 9.

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