Breeding method of early-matured aromatic type late rice

Abstract

The application provides a breeding method of early-matured aromatic type late rice, and the method comprises the following steps: providing a first primer group for an Ef-cd gene and a second primer group for an fgr gene; screening a first rice strain with homozygous Ef-cd gene and homozygous fgr gene by using the first primer group and the second primer group; crossing the first rice strain with a second rice strain to obtain F1 generation rice, and self-crossing the F1 generation rice to obtain F2 generation rice; screening a large number of F2 generation single plants with homozygous Ef-cd gene and homozygous fgr gene by using the first primer group and the second primer group; selecting a fertile single plant rice with target traits in F3 generation rice for one-generation or multi-generation self-crossing selection; and selecting a fertile single plant rice with target traits in each generation self-crossing selection for next-generation self-crossing selection; and obtaining a fertile rice strain with target traits with genetic stability. The method of the application effectively ensures the accuracy of the breeding direction and improves the cultivation efficiency of new products.

Description

Technical Field

[0001] This application relates to the field of agricultural biotechnology, and in particular to a breeding method for an early-maturing, aromatic late-season rice. Background Technology

[0002] Rice is an important food crop in my country and the world. Due to the vast territory, complex terrain, and significant climate differences, as well as the complex farming methods, there are many types of rice. Rice includes early-season rice, mid-season rice, and late-season rice, all of which are planted on a large scale, with numerous varieties and large quantities.

[0003] Late-season rice, due to its larger diurnal temperature range during the grain-filling period compared to mid-season and early-season rice, generally has better quality. Furthermore, the demand for early-maturing rice varieties is increasingly strong, especially for extra-early-maturing late-season rice. Therefore, aroma is a crucial aspect of high-quality late-season rice, and its characteristics should be given greater consideration in the breeding of extra-early-maturing late-season rice. Currently, there are as many as 200 substances related to aroma, but the one directly related to rice aroma is mainly 2-acetyl-1-pyrrolin (2-AP). Further research has revealed that mutations in the Badh2 gene on rice chromosome 8 are the fundamental cause of aroma formation, and this mutation is recessive. Currently, many deletion or mutation types have been reported, but the most widely used are mainly the 7bp deletion of exon 2 and the 8bp deletion and 3bp mutation of exon 7. Among them, the exon 7 mutation type is the main type of fragrant rice in my country. In 1979, Huang Fasong introduced the fragrant rice resource 066 (exon 7 mutation type) from the International Rice Research Institute and systematically bred 80-65 and 80-66 from it. Subsequently, it was introduced to 22 provinces across the country, which opened the prelude to the breeding of fragrant rice in China.

[0004] Therefore, how to provide a breeding method for a mature and fragrant late-season rice is the focus of research. Summary of the Invention

[0005] This application provides a breeding method for early-maturing, aromatic late-season rice, which effectively ensures the accuracy of the breeding direction, improves the breeding efficiency of new varieties, and promotes the successful breeding of early-maturing, high-quality, aromatic late-season rice varieties.

[0006] In a first aspect, embodiments of this application provide a breeding method for early-maturing aromatic late-season rice, comprising:

[0007] Provides a first primer set targeting the Ef-cd gene and a second primer set targeting the fgr gene.

[0008] The first rice line with homozygous Ef-cd gene and homozygous fgr gene was screened using the first primer set and the second primer set;

[0009] The first rice line was crossed with the second rice line to obtain F1 generation rice, wherein the second rice line has the characteristics of stress resistance, environmental adaptability and high yield.

[0010] The F1 generation rice was self-pollinated to obtain the F2 generation rice;

[0011] Select fertile single rice plants with the target trait from the F2 generation rice for one or more generations of inbred line selection; and select fertile single rice plants with the target trait for the next generation of inbred lines in each generation of inbred line selection; to obtain genetically stable fertile rice lines with the target trait; the target trait includes rice seeds with fragrance, leaves with fragrance, stress resistance, environmental adaptability, and high yield characteristics.

[0012] In some embodiments, the first primer set is as shown in SEQ ID NO.1-3.

[0013] In some embodiments, the second primer set is shown in SEQ ID NO.4-6.

[0014] In some embodiments, the second rice variety is Yuehe material.

[0015] In some embodiments, the F2 generation has homozygous Ef-cd gene and homozygous fgr gene.

[0016] In some embodiments, the selection of fertile single rice plants with the target trait for each generation of inbred lines includes: F2 generation rice is raised indoors, and then more than 1,000 Ef-cd and fgr gene homozygous seedlings are selected using the "20-to-1" method and single plant testing. These seedlings are then transplanted, and dominant single plants are harvested in combination based on plant type and agronomic traits to ensure the accuracy of the breeding direction; F3 planting populations are more than 300 plants, and dominant single plants are harvested in combination after maturity to reduce labor costs; F4-F6, single plants are selected based on agronomic traits, grain type, resistance, and other phenotypes; F7, more than 100 plants are planted to select lines with good rice quality and cooked taste.

[0017] The technical solutions provided in this application have the following advantages compared with the prior art:

[0018] The breeding method provided in this application constructs a breeding strategy for extra-early maturing, high-quality, aromatic late-season rice. By identifying the Ef-cd and fgr genes in the lines, safe, efficient, and accurate KASP marker primers were designed. Based on conventional methods, a breeding strategy for early-maturing, high-quality, aromatic late-season rice varieties was constructed, effectively ensuring the accuracy of the breeding direction, improving the breeding efficiency of new varieties, and promoting the successful breeding of early-maturing, high-quality, aromatic late-season rice varieties.

[0019] In addition, a second rice line with good resistance, strong adaptability, and high yield was introduced into the first rice line containing homozygous Ef-cd and fgr genes. The two lines were then hybridized, and the F1 generation was planted, self-pollinated, and the seeds were harvested. The F2 generation achieved homozygosity of the target genes, ensuring the focus of the breeding and providing more possibilities for the next stage of screening. Ultimately, a genetically stable fertile rice line with the target traits was obtained, including rice quality and good cooking taste. Attached Figure Description

[0020] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The diagram shows the detection results of the fgr gene and Ef-cd gene in 49 rice materials from embodiments of this application;

[0023] Figure 2 This is a schematic diagram illustrating the breeding method of early-maturing aromatic late-season rice according to an embodiment of this application. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0025] The concept of this application includes: selecting two major genes, Ef-cd and fgr, that are closely related to rice maturity and aroma without affecting yield; and successfully designing KASP marker primers based on gene sequence characteristics: the primers for fgr gene detection are FGR-F, FGR-Rt, and FGR-Rc, and the primers for Ef-cd gene detection are 122500-1Rt, 122500-1Rc, and 122500-1F. Simultaneously, combining conventional breeding methods for high-quality rice with aroma and early maturity traits, a breeding strategy suitable for early-maturing, high-quality, and aromatic late-season rice was constructed.

[0026] In a first aspect, embodiments of this application provide a breeding method for early-maturing aromatic late-season rice, comprising:

[0027] Provides a first primer set targeting the Ef-cd gene and a second primer set targeting the fgr gene.

[0028] The first rice line with homozygous Ef-cd gene and homozygous fgr gene was screened using the first primer set and the second primer set;

[0029] The first rice line was crossed with the second rice line to obtain F1 generation rice, wherein the second rice line has the characteristics of stress resistance, environmental adaptability and high yield.

[0030] The F1 generation rice was self-pollinated to obtain the F2 generation rice;

[0031] Select fertile single rice plants with the target trait from the F2 generation rice for one or more generations of inbred line selection; and select fertile single rice plants with the target trait for the next generation of inbred lines in each generation of inbred line selection; to obtain genetically stable fertile rice lines with the target trait; the target trait includes rice seeds with fragrance, leaves with fragrance, stress resistance, environmental adaptability, and high yield characteristics.

[0032] According to the embodiments of this application, the first rice line with homozygous Ef-cd gene and homozygous fgr gene is screened using the first primer set and the second primer set. This method utilizes the developed gene detection markers, fully leverages the speed advantage of fluorescent labeling detection, avoids the application of agarose gel and polyacrylamide gel in previous detection methods, saves manpower and time costs, reduces human error, and significantly improves the safety and efficiency of detection. In addition, the breeding strategy for early-maturing, high-quality, and aromatic late-season rice focuses on the detection of major traits from the early stage, avoids the loss of target genes, and leaves more possibilities for later selection, increasing the probability of breeding target new varieties.

[0033] According to the embodiments of this application, the Ef-cd gene and its derivatives in rice can significantly advance the heading period of rice by 7-20 days, without affecting yield, and even showing varying degrees of yield increase in multiple regions. Rice materials containing Ef-cd exhibit significantly enhanced expression of genes related to nitrogen metabolism, chlorophyll metabolism, and photosynthesis; Ef-cd significantly improves the absorption capacity of nitrate and ammonium nitrogen in rice, as well as the photosynthetic efficiency of leaves. The Ef-cd gene combines early maturity and high yield, demonstrating significant characteristics of efficient resource utilization.

[0034] According to the embodiments of this application, the fgr locus is related to the aroma production of rice. Aromatic aroma is recessive, while lack of aroma is dominant. Using RFLP markers, fgr was located approximately 5 cM away from marker RG28 on rice chromosome 8.

[0035] According to the embodiments of this application, the Ef-cd gene and fgr gene in the strain are identified.

[0036] Safe, efficient, and accurate KASP marker primers were designed to screen out the first rice line with homozygous Ef-cd and fgr genes, effectively ensuring the accuracy of the breeding direction and improving the breeding efficiency of new varieties. This has important guiding significance for the breeding of early-maturing, high-quality, and aromatic late-season rice varieties. Furthermore, the introduction of a second rice line with good resistance, strong adaptability, and high yield further ensured the accuracy of the breeding direction, improved the breeding efficiency of new varieties, and promoted the successful breeding of early-maturing, high-quality, and aromatic late-season rice varieties.

[0037] In the embodiments of this application, the target trait includes at least one of the following: flowering habit, ideal plant and leaf morphology, high yield, disease and pest resistance, and lodging resistance. Genetically stable fertile rice lines with the target trait can be propagated by seed.

[0038] In some embodiments, the first primer set is as shown in SEQ ID NO.1-3.

[0039] In some embodiments, the second primer set is shown in SEQ ID NO.4-6.

[0040] In some embodiments, the second rice variety is Yuehe material.

[0041] In some embodiments, the second rice line has Wx b Gene. According to the embodiments of this application, the wx gene in rice is a key gene controlling the amylose content of rice. Its functional variation directly leads to changes in amylose content and affects rice quality, and has important value in rice quality breeding.

[0042] In some embodiments, the F2 generation has homozygous Ef-cd gene and homozygous fgr gene.

[0043] In some embodiments, the selection of fertile single rice plants with the target trait for each generation of inbred lines includes: F2 generation rice is raised indoors, and then more than 1,000 Ef-cd and fgr gene homozygous seedlings are selected using the "20-to-1" method and single plant testing. These seedlings are then transplanted, and dominant single plants are harvested in combination based on plant type and agronomic traits to ensure the accuracy of the breeding direction; F3 planting populations are more than 300 plants, and dominant single plants are harvested in combination after maturity to reduce labor costs; F4-F6, single plants are selected based on agronomic traits, grain type, resistance, and other phenotypes; F7, more than 100 plants are planted to select lines with good rice quality and cooked taste.

[0044] The method of the present invention will now be described in detail with reference to embodiments, comparative examples and experimental data.

[0045] Example 1

[0046] 1.1 Provide rice materials

[0047] The rice varieties derived from the Hunan Provincial Rice Research Institute are: ZX-1, ZX-2, ZX-3, ZX-4, ZX-5, ZX-6, ZX-7, ZX-8, ZX-9, ZX-10, ZX-11, ZX-12, ZX-13, ZX-14, ZX-15, ZX-16, TW-1, TW-2, TW-3, TW-4, TW-5, TW-6, TW-7, TW-8, TW-9, TW-10, T... W-11, TW-12, TW-13, TW-14, TW-15, TW-16, TW-17, TW-18, TW-19, TW-20, TW-21, TW-22, TW-23, TW-24, TW-25, TW-26, TW-27, TW-28, TW-29, TW-30, Yuzhenxiang, Nongxiang 42, Fengliangyouxiang No. 1; among them, TW and ZX are materials for comparing extra-early maturing late rice and ratooning rice varieties.

[0048] 1.2 Test Methods

[0049] 1.2 DNA Extraction

[0050] 1) Place a leaf approximately 1 cm long and wide into a deep-well plate (1.2 ml for 96 wells).

[0051] 2) Add 100 μL of 0.3 M sodium hydroxide and grind the sample at 50 Hz for 2 minutes using a Shanghai Jingxin tissue homogenizer (until the sample is completely ground).

[0052] 3) After grinding, centrifuge at 3000 rpm for 1 min, then bath in boiling water for 2 min.

[0053] 4) Add 200 μL of 0.2 M Tris-HCl (pH 6.8-7.0), mix well, and then boil in a water bath for 2 min again.

[0054] 5) After the water bath, centrifuge at 3000 rpm for 1 min, take the supernatant, dilute it 20 times, and freeze it at -20℃ as a template for subsequent PCR amplification.

[0055] 1.3 PCR amplification

[0056] Design primers and PCR amplification primer sequences:

[0057]

[0058]

[0059] The PCR amplification reaction system is as follows (PARMS mix, Wuhan Jingtai).

[0060] Element volume Final concentration 2×PARMS master mix 5μL 1× Allele X primer (10μM) 0.15μL 150nM Allele Y primer (10μM) 0.15μL 150nM Common primer (10μM) 0.4μL 400nM DNA template 10-100ng 10-100nM ddH2O Add to 10 μL

[0061] The PCR amplification reaction conditions are as follows (ABI Gene Amp9700 dual-head 384 PCR instrument).

[0062]

[0063]

[0064] 1.4 Genotyping

[0065] After PCR, the fluorescence signal was read using a TECANinfinite M1000 microplate reader. Then, the converted fluorescence signal was analyzed using the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) to obtain a clear and intuitive genotyping diagram. Based on the different colors, the genotype results were output.

[0066] 1.5 Validation of Aroma and Early Maturation Phenotype

[0067] Aroma phenotype identification: The aroma of leaves and seeds was identified using the KOH method and chewing method, respectively.

[0068] Early maturity phenotype identification: Early maturity is determined by adjusting the heading date of each material, that is, the time taken from rice sowing to 10% heading.

[0069] 1.6 Results and Analysis

[0070] In the fgr gene detection (Table 1 below), materials ZX-1, ZX-3, and ZX-5 showed green fluorescence, indicating the absence of the fgr gene; both grains and leaves showed no aroma. Materials ZX-2, ZX-6, and ZX-7 showed red fluorescence, indicating heterozygotes of the fgr gene; their leaves showed no aroma, but some grains showed aroma – aroma is a recessive gene, and heterozygous plants should show no aroma, but the grains produced by these plants showed phenotypic segregation, with some showing aroma. Materials ZX-9, ZX-10, and TW-6 showed blue fluorescence, indicating homozygotes of the fgr gene; both grains and leaves showed aroma. Thus, the phenotypic identification results of the 49 materials are consistent with the genotypes confirmed in this invention.

[0071] Forty-nine experimental materials were planted at the Gaoqiao Base of Hunan Rice Research Institute in Changsha County, Changsha City, Hunan Province. They were sown on June 10 and transplanted on July 2. The transplanting size was 20cm×20cm. The sowing time was recorded to determine the early maturity characteristics of the materials. The results of Ef-cd gene detection and sowing-to-start dates for the experimental materials are shown in the table below. Materials ZX-1, ZX-11, and ZX-12 showed green fluorescence, indicating the absence of the Ef-cd gene, with a sowing-to-start date of 81-90 days. Materials ZX-5, ZX-9, and ZX-14 showed red fluorescence, indicating heterozygotes for the Ef-cd gene, with a sowing-to-start date of 74-75 days. Materials ZX-6, ZX-7, and ZX-8 showed blue fluorescence, indicating homozygotes for the Ef-cd gene, with a sowing-to-start date of 72-74 days. Materials ZX-2, ZX-3, and ZX-4 showed no signal, indicating the presence of other alleles, with a sowing-to-start date of 72-84 days. In summary, the sowing-to-start date of materials containing the Ef-cd gene was significantly shorter than that of materials without this gene, and other genes regulating rice maturity were also present.

[0072] Table 1. Results of gene detection and phenotypic identification of 49 rice materials.

[0073]

[0074]

[0075]

[0076] + indicates homozygous gene, - indicates no gene, - / + indicates heterozygous gene, and unknown indicates no signal.

[0077] The fluorescence signal of the PCR product was read by a TECANinfinite M1000 microplate reader, and the results were obtained as follows: Figure 1 The results shown, along with phenotypic analysis, demonstrate the high efficiency and reliability of the gene detection. The left figure displays the detection results for the fgr gene in 49 rice materials, and the right figure displays the detection results for the Ef-cd gene in 49 rice materials. The horizontal and vertical axes represent fluorescence signal intensity, respectively.

[0078] Example 2: Breeding of Extra-early Mature, High-Quality, Fragrant Late Rice

[0079] "Jade Needle Fragrance" (containing Ef-cd, fgr, Wx) bUsing donors (such as Ef-cd and fgr genes), the "Yuehe material" with good resistance, strong adaptability, and high yield was introduced for hybridization. F1 plants were planted, self-pollinated, and seeds were harvested. For F2, indoor seedling cultivation was used, followed by screening using a "20-to-1" method and single-plant testing to select over 1000 homozygous seedlings with Ef-cd and fgr genes. These were then transplanted, and dominant individual plants were mixed and harvested based on plant type and agronomic traits, ensuring the accuracy of the breeding direction and providing greater possibilities for subsequent gene recombination. The F3 planting population was over 300 plants, and dominant individual plants were continued to be mixed and harvested after maturity to reduce labor costs. For F4-F6, individual plants were selected based on agronomic traits, grain type, resistance, and other phenotypes, with a scale of approximately 50 plants per plant. For F7, over 100 plants were planted, with a focus on rice quality and cooked taste to determine the final strain. F8 was mainly used for seed propagation and named "Daoxiaxiang No. 4" for participation in trials. A detailed flowchart is shown below. Figure 2 As shown.

[0080] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0081] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A breeding method of early-matured aromatic type late rice, characterized by, The application comprises the following steps: providing a first primer set for the Ef-cd gene and a second primer set for the fgr gene, wherein the first primer set is shown as SEQ ID NO. 1-3; the second primer set is shown as SEQ ID NO. 4-6; screening a first rice line having homozygous Ef-cd gene and homozygous fgr gene using the first primer set and the second primer set, the first rice line being "Yu-zhenxiang" containing Ef-cd gene, fgr gene and Wx b gene. crossing the first rice strain with a second rice strain to obtain F1 generation rice, wherein the second rice strain is "Yuehe material" with stress resistance, environmental adaptability, and high yield characteristics; self-crossing the F1 generation rice to obtain F2 generation rice, and the F2 generation rice is screened for more than 1000 Ef-cd and fgr gene homozygous seedlings; selecting the F2 generation rice with target traits for one or more generations of self-crossing selection, and each generation of self-crossing selection is selected for the next generation of self-crossing selection; a hereditarily stable rice strain with target traits is obtained; the target traits include rice seeds with fragrance, leaf blades with fragrance, stress resistance, environmental adaptability, and high yield characteristics.

2. The breeding method according to claim 1, characterized by, Each generation of self-crossing selection is selected for the next generation of self-crossing selection, which comprises the following steps: indoor seedling of the F2 generation rice, then "20 mix 1" and single plant detection, screening for more than 1000 Ef-cd and fgr gene homozygous seedlings, then transplanting, and mixing the superior single plants according to the plant type and agronomic traits to ensure the accuracy of the breeding direction; the F3 planting population is more than 300, and the mature plants are mixed to reduce the labor cost; F4-F6, single plant selection according to the agronomic traits, grain type, and resistance phenotype; F7, planting more than 100 plants to select a strain with rice quality and rice cooking taste.

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