阅读说明：本技术 植物多基因控制性状的单基因分离群体构建方法 (Single gene segregation population construction method for plant polygene control character ) 是由 杨行海 张宗琼 夏秀忠 农保选 李丹婷 邓国富 曾宇 吴艳艳 许娟 高菊 梁海福 于 2019-11-20 设计创作，主要内容包括：本发明涉及生物技术领域，特别涉及植物多基因控制性状的单基因分离群体构建方法，本发明所公开的一种水稻多基因控制性状的单基因分离群体构建方法，该方法针对开关基因控制的相关性状，通过对后代基因的定向筛选，能够快速、高效、准确地进行复杂性状中单个基因的效应及精细定位研究，方法还通过申请人采用F<Sub>2</Sub>代与显性亲本不断回交的方法，解决了基因测序样本量过大，表型表达不能快速锁定目标基因基因型的问题，能更快速的找到目的样本减少回交代数，提高筛选的精确性和效率。(The invention relates to the field of biotechnology, in particular to a method for constructing a single-gene segregation population of plant polygene control traits, and discloses a method for constructing a single-gene segregation population of rice polygene control traits 2 The method for continuous backcross of generations and dominant parents solves the problems that the gene sequencing sample amount is too large, and the phenotype expression cannot quickly lock the target gene genotype, can more quickly find a target sample to reduce the number of backcross generations, and improves the accuracy and efficiency of screening.)

1. The method for constructing the single-gene segregation population of plant polygene control traits is characterized by comprising the following steps of:

(1) selecting parents with obvious differential characters to obtain F₁Generation, F₁Selfing to obtain F₂Generation, construction of F₂Generation group;

(2) sequencing the parents and comparing the parents with a reference genome sequence; after the comparison result is normal, designing a primer according to the InDel locus;

(3) gene localization: for F in step (1)₂Performing gene localization on a population, and associating a plurality of related genes for controlling a certain trait, wherein the trait comprises a switch gene; selecting a certain non-switch gene as a target gene for subsequent analysis;

(4) InDel marker screening: designing two pairs of polymorphic InDel markers according to the multiple related genes or gene candidate regions in the step (3);

(5) construction of single-gene segregating population: analyzing F by using the polymorphic InDel marker screened in the step (4)₂Group or F₂Screening out plants of which the target genes are heterozygous fragments and other non-target genes are dominant homozygous fragments according to the genotypes of the backcross progeny of the population; and developing the plant into a population, namely constructing a single-gene segregation population.

2. The method of constructing a monogenic segregating population for a polygenic control trait of a plant of claim 1, wherein the plant is rice.

3. The method for constructing a monogenic segregating population for a polygenic control trait of a plant of claim 1, wherein the parents of step (1) are Orchium Oriental and Huanghuazhan.

4. The method for constructing a monogenic segregating population for a polygenic control trait of a plant of claim 1, wherein said trait of step (3) is a species of anthocyanin.

5. The method of claim 1, wherein the genes involved in step (3) are selected from the group consisting of: switch gene Kala4, related gene Rd, BP2, BP3, BP3.1, BP3.2, OsCHI, BP6, BP9 and BP 12; the target gene is BP 3.1.

6. The method of claim 1, wherein said analysis F of step (5) is performed₂Group or F₂The genotype method of the colony backcross progeny comprises the following steps: InDel labeling to analyze F₂Genotype of each individual plant in the population, finding out the filial plant with heterozygous target gene and dominant other genes, if in F₂If no related plants are found in the population, F is determined₂Backcrossing the generations with dominant homozygous parents,backcross 1 generation F₃Generation, backcross 2 generation is marked as F₄Generation, then harvest (F)₂、F₃、F₄) Planting seeds, and continuously identifying the genotype by adopting a molecular marker method until a plant which is heterozygous for a target gene and is dominant and homozygous for other linked genes is screened.

7. The method for constructing a monogenic segregating population for a polygenic control trait of a plant of claim 1, wherein the method for fine mapping the target gene further comprises: and positioning the target gene by using the constructed single-gene segregation population through a substitution mapping method.

8. The method for constructing the single-gene segregating population of the plant polygene control traits as claimed in claim 1, wherein the primer sequence screened by the InDel marker in the step (4) is shown in SEQ ID No. 1-40.

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of biology, in particular to a method for constructing a single-gene segregation population of plant polygene control traits.

[ background of the invention ]

Increasing grain yields presents a significant challenge in situations where global climate is warming and farm land is diminishing. The complex character of the plant has important significance in adapting to the environment and artificially selecting. Complex traits are commonly controlled by multiple genes, a single gene may have relatively little phenotypic effect and be susceptible to environmental influences. Therefore, it has been difficult to analyze a control gene for a complex trait.

The construction of a suitable mapping population is the key to realizing accurate gene positioning. In order to analyze the genetic effect of each gene in the complex traits of rice, researchers adopt backcross and selfing to obtain an introduced fragment population with only one or few donor parent allele differences, including Near Isogenic Lines (NILs), single segment replacement lines (SSSLs), chromosome segment replacement lines (CSSLs), and the like. The groups can eliminate the interference of genetic background, and the gene positioning is accurate and sensitive. However, as for the character with the switch gene, the interaction between other genes can be shown in the phenotype only when the switch gene exists, and the research in the prior art finds that the character with the switch gene is influenced by the switch gene, and besides, the close linkage effect also exists between different genes to generate the influence of the mutual correlation between related characters, so that the phenotype is not obvious; therefore, the gene control of the existing switch gene character is much more complex than other gene control mechanisms, and is greatly influenced by the linkage regulation among genes; if the target individual is determined according to the phenotype of the offspring according to the prior art, so that the genotype is determined inaccurately, in order to further strengthen the positioning research of the gene, a certain gene in the character is positioned according to the control characteristics of the switch gene, and the construction of the single-gene segregation population provides more accurate and precise genetic materials for the subsequent gene research, and can promote the development of a gene positioning system.

[ summary of the invention ]

In view of the above, it is necessary to target the trait of the presence of the switch gene and provide a precise mapping system for precisely mapping the trait of the presence of the switch gene.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for constructing a single-gene segregating population of a plant with a polygenic control trait, the method comprising the steps of:

(1) selecting parents with obvious differential characters to hybridize to obtain an F1 generation, selfing an F1 generation to obtain an F2 generation, and constructing an F2 generation population;

(2) sequencing the parents and comparing the parents with a reference genome sequence; after the comparison result is normal, designing a primer according to the InDel locus;

(3) gene localization: performing gene mapping on the F2 population in the step (1) to associate a plurality of related genes for controlling a certain trait, wherein the trait comprises a switch gene; selecting a certain non-switch gene as a target gene for subsequent analysis;

(4) InDel marker screening: designing two pairs of polymorphic InDel markers according to the multiple related genes or gene candidate regions in the step (3);

(5) construction of single-gene segregating population: analyzing the genotype of each individual plant in the F2 population or the F2 population and the progeny population backcrossed with the dominant parent by using the polymorphic InDel marker screened in the step (4), and screening out plants of which the target genes are heterozygous segments and other non-target genes are dominant homozygous segments; and developing the plant into a population, namely constructing a single-gene segregation population.

Further, the plant is rice.

Further, the parents in the step (1) are Orchium and Huazhan.

Further, the character in the step (3) is seed anthocyanin.

Further, the related genes in the step (3) are respectively: switch gene Kala4, related gene Rd, BP2, BP3, BP3.1, BP3.2, OsCHI, BP6, BP9 and BP 12; the target gene is BP 3.1.

Further, the analysis F of step (5)₂Group or F₂The genotype method of the colony backcross progeny comprises the following steps: InDel labeling to analyze F₂Genotype of each individual plant in the population, finding out the filial plant with heterozygous target gene and dominant other genes, if in F₂If no related plants are found in the population, F is determined₂Backcrossing the generation with dominant homozygous parent, and marking the backcross 1 generation as F₃Generation, backcross 2 generation is marked as F₄Generation, then harvest (F)₂、F₃、F₄) Planting seeds, and continuously identifying the genotype by adopting a molecular marker method until a plant which is heterozygous for a target gene and is dominant and homozygous for other linked genes is screened.

Further, the fine localization method of the target gene further comprises: and positioning the target gene by using the constructed single-gene segregation population through a substitution mapping method.

Further, the primer sequence screened by the InDel marker in the step (4) is shown in SEQ ID NO. 1-40.

The invention has the following beneficial effects:

the invention discloses a method for constructing a single-gene segregation population of rice with polygenic control traits, which aims at controlling traits by switch genesThe method can effectively remove other noises, and can quickly, efficiently and accurately research the effect and fine positioning of a single gene in complex characters; mainly aims at the characters controlled by the switch gene, directionally screens plants with target genes as heterozygous segments and other non-target genes as dominant homozygous segments to construct a single-gene segregation population, and provides a more accurate genetic material for subsequent genotype research; because the phenotype of the trait controlled by the switch gene is possibly not uniform with the genotype, if the phenotype is only used for screening and judging the genotype of the offspring, fine positioning is difficult to carry out, and if the phenotype is not prominent in the research process, the workload of researchers can be increased due to the existence of a large number of samples, so that when the research sample is constructed, the applicant adopts F₂The method for continuous backcross of generations and dominant parents can more quickly find a target sample to reduce the number of backcross generations, solve the problems that the gene sequencing sample amount is too large, and the phenotype expression cannot quickly lock the target genotype, and improve the accuracy and efficiency of screening.

[ description of the drawings ]

FIG. 1 is an electrophoretogram of molecular markers closely linked to 10 genes according to an embodiment of the present invention; in the figure, 1 is the homozygous recessive parent huanghuazhan; 2 is homozygous dominant parent east blue black rice; 3-31 are different progeny; a-j correspond to Rd, BP2, BP3.2, BP3.1, BP3, oSCHI, Kala4, BP6, BP9 and BP12 respectively.

FIG. 2 is a graph showing the expression amount of the candidate gene LOC _ Os03g32470 in Huanghuazhan and Toyland black rice in an embodiment of the present invention.

[ detailed description ] embodiments

All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.

Any feature disclosed in this specification (including any accompanying claims, abstract) is merely an example of a generic series of equivalent or similar features, unless explicitly described as such.