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CN116590462A - Development and application of novel gene locus of rice dwarf - Google Patents

Development and application of novel gene locus of rice dwarf Download PDF

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Publication number
CN116590462A
CN116590462A CN202310766275.5A CN202310766275A CN116590462A CN 116590462 A CN116590462 A CN 116590462A CN 202310766275 A CN202310766275 A CN 202310766275A CN 116590462 A CN116590462 A CN 116590462A
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rice
locus
osph9
dwarf
gene
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辛威
王敬国
刘化龙
杨洛淼
贾琰
王新鹏
邹德堂
马天泽
申审
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Northeast Agricultural University
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    • C12Q2600/156Polymorphic or mutational markers

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Abstract

The invention discloses development and application of rice dwarf gene loci, and belongs to the technical fields of genome sequences and plant biology. The method comprises the steps of detecting the genotype of rice to be detected, and identifying or assisting in identifying short bars according to the genotype of the rice gene locus to be detected; the OsPH9_M13 locus is an InDel locus in a rice genome, the nucleotide variety of the locus is GC or deletion (-) and is 101-102 nucleotides of SEQ ID No.1 in a sequence table. The invention utilizes the rice dwarf differential material to prepare a positioning group, and excavates gene loci related to the dwarf, and the loci can be used for commercialized rice dwarf molecular breeding. The molecular marker assisted selection efficiency is higher, the molecular marker assisted selection can be carried out in the early stage of breeding, the breeding cost is reduced, and the rice dwarf breeding process is accelerated.

Description

Development and application of novel gene locus of rice dwarf
Technical Field
The invention relates to the field of genome sequences and plant biotechnology, in particular to development and application of rice dwarf genes.
Background
Rice (Oryza sativa L.) is the most important food crop worldwide, and is one of the most productive food varieties in China. The dwarf rice breeding initiates the first green revolution worldwide, the plant height is reduced, the lodging resistance, the confidentiality and the fertilizer resistance of plants are greatly improved, the single yield level of rice is leaved, and the rice yield is improved by 20% -30%. However, the single and genetic background of dwarf gene utilization is narrow because of the few dwarf genes which are commonly used at present, mainly sd1 genes, which can cause genetic vulnerability. Therefore, the mining of novel dwarf genes is extremely important for rice dwarf breeding (Chen Wenjuan, liu Yanan, sun Yali, li Mochang, li Jingyuan. Development of cloning of dwarf genes for rice [ J ]. Henan agricultural science, 2017,46 (03): 1-7.). The research digs a new rice dwarf gene locus through group genetic linkage analysis, thereby providing a theoretical basis for deeply researching a rice dwarf mechanism and providing a technical support for breeding rice dwarf varieties.
Disclosure of Invention
The invention aims to solve the technical problems that the breeding efficiency of rice dwarf varieties is improved by developing new rice dwarf gene loci and SNP molecular markers thereof, and the technical problems mentioned in the background art are solved by adopting the following technical scheme:
the development of the rice dwarf gene locus mainly comprises the following steps:
the method is used for detecting the plant height and identifying the plant type of different rice materials, and is used for preparing gene positioning groups, positioning and analyzing the gene loci of short stems, and developing and applying molecular markers of the gene loci. The gene locus is OsPH9_M13 locus. The gene locus OsPH9_M13 is an InDel locus in a rice genome, and the nucleotide is GC or deletion (-/-) and is 101-102 nucleotides of SEQ ID No.1 in a sequence table.
As a preferred example, when the OsPH9_M13 locus genotype is-/-wherein the-/-genotype indicates that the nucleotide species of the OsPH9_M13 locus in the rice genome is homozygous for deletion, rice is short or is a candidate for short.
As a preferred example, the PCR primer of OsPH9_M13 is a primer set composed of a single-stranded DNA whose nucleotide sequence is the 22 th to 41 th positions of SEQ ID No.2 in the sequence table, a single-stranded DNA whose nucleotide sequence is the 22 nd to 41 th positions of SEQ ID No.3 in the sequence table, and a single-stranded DNA whose nucleotide sequence is the SEQ ID No.4 in the sequence table.
A product developed by KASP molecular marker of rice dwarf gene locus OsPH9_M13, which comprises the following substances,
I. the substance for detecting polymorphism or genotype of the OsPH9_M13 locus in the rice genome comprises a PCR primer for amplifying a rice genome DNA fragment comprising the OsPH9_M13 locus;
II. The substance for detecting the polymorphism or genotype of the OsPH9_M13 locus in the rice genome is a PCR reagent containing the PCR primer;
III, a kit containing the PCR primer described in I or the PCR reagent described in II.
The beneficial effects of the invention are as follows: the invention develops a novel rice dwarf gene locus OsPH9_M13, develops a corresponding KASP molecular marker for rapid genotyping identification, and is applied to rice molecular breeding; meanwhile, the phenotype selection efficiency of the locus is high, and the dwarf gene of rice can be rapidly and accurately detected; the detection process is simple, efficient and safe, no aerosol pollution and no toxic substances such as ethidium bromide are used, molecular marker auxiliary selection can be carried out in the early stage of breeding, the breeding efficiency is improved, and the breeding process is accelerated.
Drawings
FIG. 1 shows phenotype diagrams of materials of different plant types, wherein a) is a high-stalk plant type variety, and b) is a low-stalk plant type variety;
FIG. 2. Map of linkage of whole genome markers QTLs of a mapped population;
FIG. 3 shows amplification diagrams of primers with different typing effects, wherein a, b and c are primers with poor typing, and d is a primer with good typing;
FIG. 4 shows an alignment of the sequencing result of the OsPH9_M13 site with a reference sequence;
FIG. 5 genotyping map of isolated populations.
Detailed Description
The invention will be further described with reference to the following embodiments in order to make the technical means, the creation features, the achievement of the objects and the effects of the invention easy to understand.
Example 1
Plant height identification of different rice materials and preparation of gene positioning population
In order to obtain rice materials with different plant heights, 7 rice varieties of materials are planted in the field, each variety is planted in 3 cells repeatedly, 8 rows of rice are planted in each cell, conventional cultivation and management are carried out, and the planting work is carried out on a field test base. 7 parts of rice variety materials are subjected to plant height measurement, and the detection method comprises the following steps: after the plants are mature, 10 plants are taken for each variety to be measured, the measurement is repeated for 3 times for each plant height in order to reduce the measurement error, and the average value is taken as the plant height of the plant. The judging threshold of the plant type is that the plant type is lower than 110 cm and is short, 110-120 cm is medium and high, and the plant type is higher than 120 cm and is high. The plant height measurement results of 7 parts of the test materials are shown in Table 1, wherein the highest variety of the plant height is Jiayou Zhongke No. 6, and the lowest variety of the plant height is CZ315, which is shown in FIG. 1.
TABLE 1.7 plant height data for test rice varieties
Material name Plant height/cm Plant type
Lingjing No. 11 101.4 Middle and high altitude
B two-best 105.8 Middle and high altitude
CZ315 87.6 Short rod
Jiayi Fu rice 969 113.8 Middle and high altitude
Long Yang 11 113.4 Middle and high altitude
Hui's two excellent silk seedling 107.6 Middle and high altitude
Jiayou Zhongke No. 6 121.4 High pole
In order to prepare a gene localization population, hybridization is carried out by taking CZ315 as a male parent and Jiayou Zhongke No. 6 as a female parent, so as to obtain a hybridization population containing 417 single plants. And F2 generation positioning groups are obtained through continuous selfing to F2 generation. Planting and hybridization work is carried out in a field test base, and conventional cultivation and management are carried out.
Example 2
Linkage gene locus positioning and molecular marker development and optimization
To obtain the gene locus linked to the rice dwarf, the F2 generation positioning population is subjected to field planting and plant height measurement, the field planting work is carried out on a field test base, and the plant height measurement method is described in example 1. Then, DNA extraction and genotype detection were performed on 417 individuals with a laboratory self-developed 385 against rice background genetic markers. The DNA extraction and genotype detection methods were as follows:
1) DNA extraction: extracting genome DNA from rice leaves by adopting a conventional CTAB method;
2) Kasp reaction test selection Douglas Scientific company ArrayTape platform detection
1.6 mu L PCR ArrayTape platform detection reaction system comprising: contains genome DNA 50 ng/. Mu.L 0.8. Mu.L, primer mixture 0.03. Mu.L (preferably Primer mixture ratio: forward Primer X, primer Y100 pmol.L) -1 The same detection objective can be achieved by using other reasonable Primer Mix ratios, 12. Mu.L each, reverse Primer R100 pmol.L-130. Mu.L, ddH2O 46. Mu.L, LGC 2 XKASP Mix (Std Rox) 0.8. Mu.L, and according to the ArrayTape platform instrument manual, a sample table was written, a program was run, and data was read.
Wherein the 2 xKASP Mix is prepared from fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B, high-fidelity Taq enzyme, dNTP, mg 2+ And the nucleotide sequence of the fluorescent probe A is as follows: 5'-GAAGGTCGGAGTCAACGGATT-3',5' end is linked to a VIC fluorophore; the nucleotide sequence of fluorescent probe B is: 5'-GAAGGTGACCAAGTTCATGCT-3', the 5' end of which is connected with a FAM fluorescent group; the nucleotide sequence of quenching probe a is: 5'-AATCCGTTGACTCCGACCTTC-3', the 3' end of which is connected with a quenching group BHQ; the nucleotide sequence of quenching probe B is: 5'-AGCATGAACTTGGTCACCTTC-3' it has a quenching group BHQ attached to its 3' end. Amplification procedure: pre-denaturation at 95 ℃ for 10min,1 cycle; denaturation at 95℃for 20s, annealing at 55-62℃for 60s (preferably 55 ℃) and setting 40 cycles.
The reaction system is a preferable reaction system of an ArrayTape platform of Douglas Scientific company, and other reasonable reaction systems can achieve the same detection purpose.
Note that: the above is a recommended detection method, and other detection methods capable of achieving the same detection purpose can be applied to the molecular marker assisted breeding process of the above markers.
The strain height phenotype measurement shows that 53 strains are tall rods, 297 strains are medium and tall rods, 67 strains are short rods, and the phenotype data accords with normal distribution through the Kolmogorov-Smirnov test. And (5) carrying out gene localization on the phenotype value and genotype of the plant height by using Windows QTL cartographer. The whole genome scan was performed using the composite interval mapping method with the window size set to 10cM and Model 6 (The standard Model) was selected for forward and reverse regression. At the level of p=0.05, the LOD value threshold for the control sweetness trait locus was determined to be 8.31 by 1000 displacement tests (persistence Test) (fig. 2). 3 gene loci, namely OsPH4_M19, osPH5_M08 and OsPH9_M13, are screened out, which shows that the loci have high genetic linkage with rice dwarf genes and can be used for rice dwarf gene detection. The three sites were analyzed for their phenotypical contribution rate, and OsPH9_M13 was the most effective site for controlling sweetness of rice.
In order to better apply the gene locus OsPH9_M13 to seed-selection of short-stalk rice, the locus molecular marker is optimized. The method comprises the following steps: flanking sequences of the OsPH9_M13 gene locus were downloaded from NCBI database (Reference genome IRGSP-1.0), and primers were designed using Primer5.0 software and tested using the Douglas platform. As can be seen from FIG. 3, the primers in the a-chart have non-uniform amplification efficiency among different samples although the primers have product typing; b, the primer of the diagram has no parting capability; the primers in the c diagram are also typed, but the typing result is wrong due to insufficient specificity of the primers, and the primers are concentrated at heterozygous sites; and d, the primer with better typing is high in primer specificity and correct in typing result, and the primer can be used for subsequent gene detection, and the amplification efficiency among different samples is consistent. The SNP marker primer for detecting the rice dwarf gene locus OsPH9_M13 is shown as a sequence 2 to a sequence 4.
To confirm the specificity of the nucleic acid composition, the amplified products of the PCR were subjected to clone sequencing, which was performed by the company Shanghai, inc. of the biological engineering of attorney docket. Comparing the sequencing result with the rice reference genome (Reference genome IRGSP-1.0) (FIG. 4), the comparison result shows that the amplified product of the two primer probe combinations is really a fragment of the rice locus OsPH9_M13, which accords with the expectation.
Example 3
Application of SNP marker of gene locus OsPH9_M13 linked with rice dwarf in molecular marker-assisted selection of dwarf rice plants
To examine the utility of the osph9_m13 locus of the present invention, the short stalk material CZ315 was hybridized with the medium and tall stalk material Long Yang 11 to obtain F1 populations, and F2 isolate 136 strains were generated by natural selfing of F1. SNP marker detection and strain height measurement (Table 2) were performed on the isolated population, and the marker detection and strain height measurement were performed as described in example 1 (FIG. 5). And carrying out consistency analysis on the plant height phenotype data and genotype data, wherein 34 plants with OsPH9_M13 locus genotype-/-are total, wherein the plant height measurement result is that 29 plants are short plants, and the screening efficiency of the short plants is 85.29%. The result shows that the efficiency of the short stalk single plant screening by using the locus OsPH9_M13 is high and the result is stable, and simultaneously the locus OsPH9_M13 has higher practicability in the rice short stalk plant screening.
TABLE 2 plant height phenotype data and genotype information for populations
Note that: the method comprises the steps of carrying out a first treatment on the surface of the ' indicates no detection signal.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be appreciated by persons skilled in the art that the present invention is not limited to the embodiments described above, but is capable of numerous variations and modifications without departing from the spirit and scope of the invention as hereinafter claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
Sequence 1
AAGATCTTCCTCGAGAACGTCATCCGCGACGCCGTCACCTACACCGAG
CACGCCCGCCGCAAGACCGTCACCGCCATGGACGTCGTCTACGCCCTC
AAGCGCCAGGGCCGCACCCTCTACGGCTTCGGCGGCTAGGAGCCTTTT
GCATTGTGTTTGTCTTGTTAGGAATCTGCTTGTTTCGGATGTAGCCCTGT
ATCCTG
Sequence 2
5’-GAAGGTCGGAGTCAACGGATTTCGTCTACGCCCTCAAGCGC-3’
Sequence 3
5’-GAAGGTGACCAAGTTCATGCTTCGTCTACGCCCTCAAGCCA-3’
Sequence 4
5’-GGCTCCTAGCCGCCGAAG-3’

Claims (3)

1. The development of the rice dwarf gene locus is characterized by comprising the following main steps: the plant height identification of different rice materials is used for preparing gene positioning groups, positioning and analyzing dwarf gene loci, and developing and applying molecular markers of the gene loci;
the gene locus OsPH9_M13 is an InDel locus in a rice genome, and the nucleotide is GC or deletion (-/-) and is 101-102 nucleotides of SEQ ID No.1 in a sequence table.
2. The development of a KASP molecular marker at the rice dwarf gene locus osph9_m13 according to claim 1, wherein when the osph9_m13 locus genotype is-/-the rice is dwarf or is candidate as dwarf, wherein the-/-genotype indicates that the nucleotide species of the osph9_m13 locus in the rice genome is homozygous for the deletion; wherein KASP molecular marker of OsPH9_M13 is a primer group composed of single-stranded DNA of which the nucleotide sequence is the 22 th-41 th site of SEQ ID No.2 in the sequence table, single-stranded DNA of which the nucleotide sequence is the 22 nd-41 th site of SEQ ID No.3 in the sequence table and single-stranded DNA of which the nucleotide sequence is the SEQ ID No.4 in the sequence table.
3. The product developed by molecular markers of the rice dwarf gene locus OsPH9_M13 according to claim 2, wherein the product comprises,
I. the substance for detecting polymorphism or genotype of the OsPH9_M13 locus in the rice genome comprises a PCR primer for amplifying a rice genome DNA fragment comprising the OsPH9_M13 locus;
II. The substance for detecting the polymorphism or genotype of the OsPH9_M13 locus in the rice genome is a PCR reagent containing the PCR primer;
III, a kit containing the PCR primer described in I or the PCR reagent described in II.
CN202310766275.5A 2023-06-27 2023-06-27 Development and application of novel gene locus of rice dwarf Pending CN116590462A (en)

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