Development and application of new dwarf gene locus in rice

Abstract

The application discloses development and application of a rice dwarf gene site and belongs to the field of genome sequences and plant biotechnologies. The application comprises detecting a genotype of a to-be-tested rice, and identifying or assisting in identifying the dwarf according to the genotype of the to-be-tested rice gene site. The OsPH9_M13 site is an InDel site in a rice genome, the nucleotide type of which is GC or deletion (-), which is the 101-102th nucleotide of SEQ ID No. 1 in the sequence listing. The application uses a rice dwarf difference material to prepare a positioning population, mines a gene site related to the dwarf, and the site can be used for commercial rice dwarf molecular breeding. The molecular marker assisted selection has a higher efficiency, can be used in early molecular marker assisted selection, reduces breeding cost, and speeds up the rice dwarf breeding process.

Description

Technical Field

[0001] This invention relates to the fields of genome sequencing and plant biotechnology, specifically to the development and application of rice dwarf genes. Background Technology

[0002] Rice (Oryza sativa L.) is the most important food crop globally and one of the highest-yielding grain varieties in my country. Dwarf rice breeding triggered the first global Green Revolution, significantly improving plant height, lodging resistance, density tolerance, and fertilizer tolerance, leading to a leap in rice yield per unit area and increasing rice production by 20%–30%. However, due to the limited number of commonly used dwarfing genes, primarily the sd1 gene, the singular use of dwarf genes and their narrow genetic background may result in genetic vulnerability. Therefore, discovering new dwarfing genes is extremely important for rice dwarfing breeding (Chen Wenjuan, Liu Yanan, Sun Yali, Li Wanchang, Li Jingyuan. Research progress in cloning rice dwarf genes [J]. Henan Agricultural Sciences, 2017, 46(03):1-7.). This study discovered a new rice dwarfing gene locus through population genetic linkage analysis, providing a theoretical basis for in-depth research on the mechanism of rice dwarfing and technical support for the breeding of dwarf rice varieties. Summary of the Invention

[0003] The technical problem to be solved by this invention is to improve the breeding efficiency of dwarf rice varieties by developing new rice dwarf gene loci and their SNP molecular markers. To address the technical problems mentioned in the background section, the following technical solution is adopted:

[0004] The main steps in developing rice dwarf gene loci are as follows:

[0005] This study investigates plant height and plant type identification in different rice materials for the preparation of gene mapping populations, localization analysis of dwarf gene loci, and development and application of molecular markers for gene loci. The gene locus is OsPH9_M13. OsPH9_M13 is an InDel locus in the rice genome, with nucleotides either GC or deleted (- / -), and is located at nucleotides 101-102 of SEQ ID No. 1 in the sequence listing.

[0006] As a preferred example, when the genotype of the OsPH9_M13 locus is - / -, the rice is dwarf or a candidate for dwarf, wherein the - / - genotype indicates that the nucleotide type of the OsPH9_M13 locus in the rice genome is a deletion homozygous type.

[0007] As a preferred example, the PCR primers for OsPH9_M13 are a primer set consisting of single-stranded DNA whose nucleotide sequence is the single-stranded DNA at positions 22-41 of SEQ ID No. 2 in the sequence listing, single-stranded DNA whose nucleotide sequence is the single-stranded DNA at positions 22-41 of SEQ ID No. 3 in the sequence listing, and single-stranded DNA whose nucleotide sequence is the single-stranded DNA at position 4 of SEQ ID No. 4 in the sequence listing.

[0008] A product developed using the KASP molecular marker of the rice dwarf gene locus OsPH9_M13, the product comprising the following substances.

[0009] I. The substance used to detect the polymorphism or genotype of the OsPH9_M13 site in the rice genome contains PCR primers for amplifying rice genomic DNA fragments including the OsPH9_M13 site.

[0010] II. The substance used to detect the polymorphism or genotype of the OsPH9_M13 site in the rice genome is a PCR reagent containing the PCR primers.

[0011] III. A kit containing the PCR primers described in I or the PCR reagents described in II.

[0012] The beneficial effects of this invention are as follows: This invention develops a new rice dwarf gene locus OsPH9_M13 and develops corresponding KASP molecular markers for rapid genotyping and identification, which can be applied to rice molecular breeding; at the same time, the phenotypic selection efficiency of the above locus is high, and the dwarf gene in rice can be detected quickly and accurately; moreover, the detection process is simple, efficient and safe, without aerosol pollution or the use of toxic substances such as ethidium bromide, and can be used for molecular marker-assisted selection in the early stage of breeding, thereby improving breeding efficiency and accelerating the breeding process. Attached Figure Description

[0013] Figure 1 Phenotypic diagrams of different plant types: a) tall plant type, b) short plant type;

[0014] Figure 2 Linkage map of genome-wide marker QTLs in the target population;

[0015] Figure 3 Primer amplification diagrams with different typing effects: a, b, and c are primers with poor typing, and d is a primer with good typing.

[0016] Figure 4 Alignment of sequencing results for site OsPH9_M13 with reference sequence;

[0017] Figure 5 Genotyping diagram of the segregating population. Detailed Implementation

[0018] To facilitate a clear understanding of the technical means, creative features, objectives, and effects of this invention, the invention will be further described below in conjunction with specific embodiments.

[0019] Example 1

[0020] Plant height identification and gene mapping population preparation of different rice materials

[0021] To obtain rice materials with different plant heights, seven rice varieties were first planted in the field, with three replicates for each variety and eight rows planted in each plot. Conventional cultivation and management were implemented at the field experimental base. Plant height was then measured for the seven rice varieties using the following method: after maturity, ten plants of each variety were measured. To minimize measurement error, the plant height of each plant was measured three times, and the average value was taken as the plant height. The plant type was defined as follows: below 110 cm was considered short, 110-120 cm was considered medium-tall, and above 120 cm was considered tall. The plant height measurement results for the seven test materials are shown in Table 1. The tallest variety was Jiayou Zhongke 6, and the shortest was CZ315. Figure 1 .

[0022] Table 1. Plant height data of 7 tested rice varieties

[0023] Material Name Plant height / cm Plant type Lianjing No. 11 101.4 Medium and high B Two Advantages 105.8 Medium and high CZ315 87.6 short pole Jia Yifu Rice 969 113.8 Medium and high Dragon Yang 11 113.4 Medium and high Huizhou Two Excellent Silky Seedlings 107.6 Medium and high Jiayou Zhongke No. 6 121.4 High pole

[0024] To prepare a gene mapping population, CZ315 was used as the male parent and Jiayou Zhongke 6 as the female parent for hybridization, resulting in a hybrid population containing 417 individual plants. After continuous self-pollination to the F2 generation, the F2 generation mapping population was obtained. Planting and hybridization were carried out at a field experimental base using conventional cultivation and management methods.

[0025] Example 2

[0026] Linked gene locus localization and molecular marker development and optimization

[0027] To obtain the gene loci linked to dwarf rice, field planting and plant height measurement were conducted on the aforementioned F2 generation mapping population. Field planting was carried out at a field experimental base, and plant height measurement was performed using the method described in Example 1. Then, DNA extraction and genotyping were performed on 417 individual plants using 385 pairs of rice background genetic markers developed in our laboratory. The methods for DNA extraction and genotyping are as follows:

[0028] 1) DNA extraction: Genomic DNA was extracted from rice leaves using the conventional CTAB method;

[0029] 2) The KASP reaction test was performed using the Douglas Scientific ArrayTape platform.

[0030] The 1.6 μL PCR ArrayTape platform reaction system includes: 0.8 μL of genomic DNA (50 ng / μL) and 0.03 μL of primer mixture (preferred primer mixture ratio: forward primers Primer X and Primer Y 100 pmol·L⁻¹). -1 12 μL each of the following: reverse primer Primer R 100 pmol·L⁻¹ 30 μL, ddH₂O 46 μL (other reasonable primer mixture ratios can also achieve the same detection purpose), LGC 2×KASP Mix (Std Rox) 0.8 μL. According to the ArrayTape platform instrument operation manual, compile the sample table, run the program, and read the data.

[0031] The 2×KASP Mix consists of fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B, as well as high-fidelity Taq enzyme, dNTPs, and Mg. 2+ The amplification program consists of the following components: Fluorescent probe A has the nucleotide sequence 5'-GAAGGTCGGAGTCAACGGATT-3', with a VIC fluorescent group attached to its 5' end; Fluorescent probe B has the nucleotide sequence 5'-GAAGGTGACCAAGTTCATGCT-3', with a FAM fluorescent group attached to its 5' end; Quencher probe A has the nucleotide sequence 5'-AATCCGTTGACTCCGACCTTC-3', with a BHQ quencher group attached to its 3' end; Quencher probe B has the nucleotide sequence 5'-AGCATGAACTTGGTCACCTTC-3', with a BHQ quencher group attached to its 3' end. The amplification program is as follows: 95℃ pre-denaturation for 10 min, 1 cycle; 95℃ denaturation for 20 s, annealing at 55-62℃ (preferably 55℃) for 60 s, 40 cycles.

[0032] The above reaction system is the preferred reaction system for Douglas Scientific's ArrayTape platform. Other reasonable reaction systems can also achieve the same detection purpose.

[0033] Note: The above are recommended detection methods. Other detection methods that can achieve the same detection purpose can also be applied to the molecular marker-assisted breeding process of the above markers.

[0034] Phenotypic measurements of plant height showed that among 417 F2 individual plants, 53 were tall, 297 were medium-tall, and 67 were short. The Kolmogorov-Smirnov test confirmed that the phenotypic data conformed to a normal distribution. Gene mapping of plant height phenotypic values ​​and genotypes was performed using Windows QTL cartographer. A genome-wide scan was conducted using composite interval mapping with a window size of 10 cM, and Model 6 (the standard model) was selected for both forward and reverse regression calculations. At a p-value of 0.05, the LOD threshold for the locus controlling sweetness was determined to be 8.31 after 1000 permutation tests. Figure 2 Three gene loci were identified: OsPH4_M19, OsPH5_M08, and OsPH9_M13. These loci showed high genetic linkage to rice dwarf genes and can be used for rice dwarf gene detection. Phenotypic contribution analysis of these three loci revealed that OsPH9_M13 had the highest phenotypic contribution and is considered the major locus controlling rice sweetness.

[0035] To better utilize the OsPH9_M13 gene locus for dwarf rice breeding, the molecular markers at this locus were optimized. The method is as follows: flanking sequences of the OsPH9_M13 gene locus were downloaded from the NCBI database (Reference Genome IRGSP-1.0), primers were designed using Primer 5.0 software, and detection was performed using the Douglas platform. Figure 3 As can be seen, while the primers in Figure a exhibit product typing, their amplification efficiency is uneven across different samples; the primers in Figure b lack typing ability; although the primers in Figure c also exhibit typing, their insufficient specificity leads to erroneous typing results, mostly concentrated at heterozygous sites; Figure d shows primers with better typing, consistent amplification efficiency across different samples, strong primer specificity, and accurate typing results, making them suitable for subsequent gene detection. The SNP marker primers used to detect the rice dwarf gene locus OsPH9_M13 are shown in sequences 2 to 4.

[0036] To confirm the specificity of the nucleic acid composition, the PCR amplification products were cloned and sequenced. The cloning and sequencing were commissioned to Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were compared with the rice reference genome (Reference genome IRGSP-1.0). Figure 4 The comparison results showed that the amplification product of the two primer-probe combinations was indeed a fragment of the rice site OsPH9_M13, which was in line with expectations.

[0037] Example 3

[0038] Application of SNP markers linked to the rice dwarfing gene locus OsPH9_M13 in marker-assisted selection of dwarf rice plants

[0039] To test the practicality of the OsPH9_M13 locus of this invention, an F1 population was obtained by crossing the dwarf material CZ315 with the medium-tall material Longyang 11. The F1 population was then naturally self-pollinated to produce a segregating F2 population of 136 plants. SNP marker detection and plant height measurement were performed on the segregating population (Table 2). The methods for marker detection and plant height measurement are as described in Example 1. Figure 5 Consistency analysis was performed on plant height phenotypic and genotypic data. A total of 34 plants with the genotype - / - at the OsPH9_M13 locus were identified, of which 29 plants were determined to be dwarf, resulting in a dwarf plant selection efficiency of 85.29%. These results indicate that using the OsPH9_M13 locus for dwarf plant selection is highly efficient and yields stable results, demonstrating the practical applicability of the OsPH9_M13 gene locus in screening dwarf rice plants.

[0040] Table 2. Plant height phenotypic data and genotypic information of the population

[0041]

[0042]

[0043] Note: '*' indicates no detection signal.

[0044] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and various changes and modifications can be made without departing from the spirit and scope of the invention, all of which fall within the scope of the claims. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A primer set for identifying or assisting in the identification of KASP molecular markers for the dwarf trait in rice, characterized in that, The KASP molecular marker is the InDel site OsPH9_M13 in the rice genome corresponding to positions 101-102 of SEQ ID No. 1, and its nucleotide type is GC or deletion. The primer set used to detect the KASP molecular marker consists of the following three primers: forward primer 1 is the sequence shown in positions 22-41 of SEQ ID No. 2, forward primer 2 is the sequence shown in positions 22-41 of SEQ ID No. 3, and reverse primer is the sequence shown in SEQ ID No.

4.

2. A kit for detecting the dwarf trait in rice, comprising the primer set of the KASP molecular markers for identifying or assisting in the identification of the dwarf trait in rice as described in claim 1.

3. The application of the primer set according to claim 1 or the kit according to claim 2 in the identification of dwarf traits in rice or in molecular breeding of dwarf rice, characterized in that, When the OsPH9_M13 locus genotype is a deletion homozygous type, the rice is either dwarf or a candidate for dwarf.

Images