阅读说明：本技术 基于转录组测序分析确定特定染色体上物种特异性基因的方法 (Method for determining species-specific genes on specific chromosomes based on transcriptome sequencing analysis ) 是由 王金英 党江波 温国 郭启高 梁国鲁 何桥 杨垚 于 2019-09-24 设计创作，主要内容包括：本发明公开了一种基于转录组测序分析确定特定染色体上物种特异性基因的方法。所述方法包括：(1)取外源染色体来源材料、异源单体附加系材料和未附加外源染色体的寄主材料进行转录组测序；(2)根据所述转录组测序结果设计PCR引物；(3)使用所述PCR引物对所述外源染色体来源材料、所述异源单体附加系材料和所述未附加外源染色体的寄主材料进行PCR验证；(4)对所述PCR验证的结果进行统计并确定染色体上物种特异基因。本发明以异源单体附加系为材料、利用转录组分析和PCR验证,不进行染色体分离和测序,相比之下过程简易,成本较低。(The invention discloses a method for determining species-specific genes on a specific chromosome based on transcriptome sequencing analysis. The method comprises the following steps: (1) taking exogenous chromosome source material, heterologous monomer additional system material and host material without exogenous chromosome to perform transcriptome sequencing; (2) designing a PCR primer according to the sequencing result of the transcriptome; (3) performing PCR validation of the exogenous chromosome source material, the heteromonomer addition line material, and the host material to which no exogenous chromosome is added, using the PCR primers; (4) and counting the PCR verification result and determining the species specific gene on the chromosome. The invention uses the heterologous monomer addition system as material, uses transcriptome analysis and PCR verification, does not carry out chromosome separation and sequencing, and has simple process and lower cost compared with the prior art.)

1. A method for determining species-specific genes on a specific chromosome based on transcriptome sequencing analysis, the method comprising:

(1) taking exogenous chromosome source material, heterologous monomer additional system material and host material without exogenous chromosome to perform transcriptome sequencing;

(2) designing a PCR primer according to the sequencing result of the transcriptome;

(3) performing PCR validation of the exogenous chromosome source material, the heteromonomer addition line material, and the host material to which no exogenous chromosome is added, using the PCR primers;

(4) and counting the PCR verification result and determining the species specific gene on the chromosome.

2. The method of claim 1, wherein taking exogenous chromosome source material, heterologous monomer addition line material, and host material without the exogenous chromosomes attached for transcriptome sequencing comprises:

respectively extracting total RNA from an exogenous chromosome source material, a heterologous monomer additional system material and a host material without an exogenous chromosome;

respectively carrying out reverse transcription on the extracted total RNA, and then respectively carrying out sequencing;

removing reads containing adapter, ploy-N and low quality to obtain clean reads;

the clean reads were aligned to the approved transcript to obtain the count number, and the FPKM value was calculated.

3. The method of claim 1, wherein designing PCR primers based on the transcriptome sequencing results comprises:

selecting a sequence with FPKM value of 0 in host material without exogenous chromosome and 0 in exogenous chromosome source material and heterologous monomer addition system material as an alternative sequence;

and (3) designing a primer for the alternative sequence.

4. The method of claim 1, wherein said performing PCR validation of said exogenous chromosome source material, said heterologous monomeric addition line material, and said host material without the addition of an exogenous chromosome using said PCR primers comprises:

extracting total genomic DNA from the exogenous chromosome source material, the heterologous monomer addition line material, and the host material to which the exogenous chromosome is not added;

using the total DNA of the genome of the 3 materials as a template, and respectively amplifying by different primers;

the amplification products of the 3 materials are subjected to polyacrylamide gel electrophoresis, silver staining, photographing and observation respectively.

5. The method of claim 4, wherein the primer, when amplified,

the amplification system is as follows: 3.4. mu.L of 10 XBuffer, 0.4. mu.L of 2.5mmol/L dNTP, 0.1. mu.L of 5U/. mu.L rTaq, 1. mu.L of 25-50 ng/. mu.L DNA template, 0.5. mu.L of each of 10. mu.mol/L forward and reverse primers, ddH₂O14.1. mu.L, wherein the 10 XBuffer contains Mg²⁺15mmol/L；

Reaction procedure: pre-denaturation at 94 ℃ for 2.5 min; 30-35 cycles; each circulation: denaturation at 94 ℃ for 30s, annealing at 53-60 ℃ for 30s, and extension at 72 ℃ for 30 s; extending for 10min at 72 ℃, and storing for later use at 4 ℃.

6. The method according to claim 1, wherein the counting the results of the PCR verification and determining specific genes comprises:

and counting electrophoresis results, namely selecting primers which can amplify the same bands in the total DNA of the exogenous chromosome source material genome and the total DNA of the heterologous monomer addition system material genome and can not amplify corresponding bands in the total DNA of the host material genome without the exogenous chromosome as specific primers, wherein the sequences of the specific primers are species specific gene sequences on the chromosome.

Technical Field

The invention relates to the technical field of biology, in particular to a method for determining species-specific genes on a specific chromosome based on transcriptome sequencing analysis.

Background

The location of a gene on a chromosome affects the expression of the gene and its regulation, which is called the location effect of the gene. Therefore, chromosomal localization of genes is important for the study of chromosomal function and expression patterns.

During distant hybridization, the introduction of an exogenous chromosome leads to the introduction of a large number of exogenous genes. Introduction of foreign genes often results in new phenotypes of the host material, which can lead to the production of new species or the acquisition of new breeding material. The analysis of these foreign genes is very important for species evolution and the study of new germplasm. The first step in the analysis of these foreign genes requires the excavation of these foreign genes.

The method for mining genes on specific chromosomes mainly comprises the following steps: (1) separating and sequencing chromosomes to obtain gene sequence information; (2) constructing a linkage map by using the EST markers, and obtaining chromosome specific molecular markers through the chromosome linkage map so as to obtain chromosome specific genes; (3) known genes are mapped to chromosomes by fluorescence in situ hybridization.

With the improvement of sequencing technology and the development of omics technology, the cost of sequence analysis on the whole genome and transcriptome is greatly reduced, and the analysis on the difference sequences among different materials is easy to realize.

A heteromonomeric addition line is material that contains a single chromosome of one species and a single chromosome of another species, and in which specific sequences on the foreign chromosome are expressed to varying degrees during growth and development, but not in material to which the foreign chromosome is not added. These expressed specific sequences can be obtained by screening by transcriptome analysis using the heterologous monomer addition system as a material and a material without added exogenous chromosomes as a control.

Disclosure of Invention

To address the problems of the prior art, embodiments of the present invention provide a method for determining species-specific genes on specific chromosomes based on transcriptome sequencing analysis. The technical scheme is as follows:

in a first aspect, a method for determining a species-specific gene on a specific chromosome based on transcriptome sequencing analysis, the method comprising:

(1) taking exogenous chromosome source material, heterologous monomer additional system material and host material without exogenous chromosome to perform transcriptome sequencing;

(2) designing a PCR primer according to the sequencing result of the transcriptome;

(3) performing PCR validation of the exogenous chromosome source material, the heteromonomer addition line material, and the host material to which no exogenous chromosome is added, using the PCR primers;

(4) and counting the PCR verification result and determining specific genes according to species on the chromosome.

Further, taking exogenous chromosome source material, heterologous monomer addition line material and host material without exogenous chromosome to perform transcriptome sequencing, wherein the transcriptome sequencing comprises the following steps:

respectively extracting total RNA from an exogenous chromosome source material, a heterologous monomer additional system material and a host material without an exogenous chromosome;

respectively carrying out reverse transcription on the extracted total RNA, and then respectively carrying out sequencing;

removing reads containing adapter, ploy-N and low quality to obtain clean reads;

the clean reads were aligned to the approved transliteration to obtain the count, and the FPKM (fragments per knowledge mile) value was calculated.

Further, the designing of the PCR primer according to the transcriptome sequencing result comprises:

selecting a sequence with FPKM value of 0 in host material without exogenous chromosome and 0 in exogenous chromosome source material and heterologous monomer addition system material as an alternative sequence;

and (3) designing primers for the alternative sequences by using Primer Premier5.0. software, and selecting primers with the scores of more than 90 for carrying out PCR verification.

Further, said performing PCR verification of said exogenous chromosome source material, said heterologous monomer addition line material, and said host material to which no exogenous chromosome is added using said PCR primers comprises:

extracting the exogenous chromosome source material, the heterologous monomer addition system material and the host material genome total DNA without the exogenous chromosome by adopting an improved CTAB method;

using the total DNA of the genome of the 3 materials as a template, and respectively amplifying by different primers;

the amplification products of the 3 materials are subjected to polyacrylamide gel electrophoresis, silver staining, photographing and observation respectively.

Further, when the primer is used for amplification,

the amplification system is as follows: 3.4 μ L of 10 XBuffer (containing Mg)²⁺15mmol/L, Takara Co., Ltd.), dNTP 0.4. mu.L (2.5mmol/L, Takara Co., Ltd.), rTaq 0.1. mu.L (5U/. mu.L, Takara Co., Ltd.), 1. mu.L of DNA template (25-50 ng/. mu.L), forward and reverse primers (10. mu. mol/L) each 0.5. mu.L, ddH₂O14.1. mu.L, 20. mu.L of PCR amplification system;

reaction procedure: pre-denaturation at 94 ℃ for 2.5 min; 30-35 cycles; each circulation: denaturation at 94 ℃ for 30s, annealing at 53-60 ℃ (depending on the primer) for 30s, and extension at 72 ℃ for 30 s; extending for 10min at 72 ℃, and storing for later use at 4 ℃.

Further, the counting of the results of the PCR verification and the determination of the specific gene include:

and counting electrophoresis results, namely selecting primers which can amplify the same bands in the total DNA of the exogenous chromosome source material genome and the total DNA of the heterologous monomer addition system material genome and can not amplify corresponding bands in the total DNA of the host material genome without the exogenous chromosome as specific primers, wherein the sequences of the specific primers are species specific gene sequences on the chromosome.

The technical scheme provided by the embodiment of the invention has the following beneficial effects: the invention uses the heterologous monomer addition system as material, uses transcriptome analysis and PCR verification, does not carry out chromosome separation and sequencing, and has simple process and lower cost compared with the prior art. The invention is based on transcriptome sequencing and analysis, uses differentially expressed genes as alternatives, and obtains sequences which are species-specific sequences, thereby being beneficial to the research of specific genes. Because the transcriptome data is more, and the non-reference analysis is adopted, the specific chromosomes of the species without research base and reference genome can be analyzed, and the number of the obtained specific genes is also more.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail with reference to examples.