PCR primers and identification method for identifying female and male sex of five poplar varieties and sex genotypes of poplar variety

Abstract

This invention discloses PCR primers and identification methods for distinguishing the sex of poplar trees from five major groups and the sex genotype of the white poplar group. This invention is based on the sex-determining gene in poplar trees. FERR Genes and FERR-R Specific primers were designed for each gene, and a PCR primer set for detecting the sex of poplar trees from five different groups was finally obtained through screening. PCR detection results of poplar materials with known sex using this primer set were completely consistent with the actual poplar sex phenotype, demonstrating that the PCR primer set provided by this invention can accurately identify the sex of poplar trees from the five different groups at the molecular level, making poplar sex identification more universal. Furthermore, the specific amplification provided by this invention... FERR The primer pairs for the gene can accurately identify the sex chromosome composition of poplar species. This invention has significant application prospects in the identification of male and female sexes in the five major poplar species, molecular breeding of poplars, and the control of female tree fluff.

Description

Technical Field

[0001] This invention relates to methods for identifying the sex and sex genotype of poplar trees, and particularly to PCR primers and identification methods for identifying the sex and sex genotype of poplar trees from five major groups and the white poplar group, belonging to the field of identifying the sex and sex genotype of poplar trees and the white poplar group. Background Technology

[0002] Poplar sex chromosomes are still in the evolutionary stage, and morphologically, sex chromosomes and autosomes cannot be directly distinguished. Furthermore, there are no significant morphological differences between homologous chromosomes (such as X and Y), making karyotype analysis difficult. Therefore, the most accurate and intuitive method for determining the sex of poplar trees is currently through the developmental phenotype of their reproductive organs. However, poplar trees have a long juvenile period, making phenotypic sex determination impossible before commercial use. Simultaneously, due to the sexual dimorphism of poplar trees (female trees produce catkins in spring, while male trees shed pollen), sex selection during the juvenile stage before planting is essential and crucial for better realizing the ecological and economic value of poplar trees and for selecting and breeding them.

[0003] Because of the spatiotemporal specificity of sex-determining genes, which are expressed only in floral organs during the flowering stage at maturity, analyzing and comparing the genomic sequences of sex-determining genes is expected to achieve specific markers of sex-determining genes at the DNA level, thereby achieving the purpose of identifying the sex and sex composition of poplar trees.

[0004] Existing marker-based methods for poplar sex identification can only achieve early sex identification for some of the five major poplar groups. To date, there is a lack of PCR primers and identification methods that can achieve early sex identification for both male and female poplars of the five major groups, as well as for the sex genotype of poplar species. Summary of the Invention

[0005] One of the objectives of this invention is to provide a PCR primer set for identifying the sex of poplar trees from five major groups.

[0006] The second objective of this invention is to apply the PCR primer set described above for identifying the sex of poplar trees from the five major groups to identify the sex of poplar trees from the five major groups. The third objective of this invention is to provide PCR primer pairs for identifying the sex genotype of poplar species.

[0007] The fourth objective of this invention is to apply the PCR primer pair for sex genotypes of poplar species to identify sex genotypes of poplar species.

[0008] The above-mentioned objectives of the present invention are mainly achieved through the following technical solutions:

[0009] One aspect of the present invention is to provide a PCR primer set for identifying the sex of poplar trees from five major groups, wherein the PCR primer set is selected from any one of the following two sets of PCR primer sets:

[0010] PCR primer set (I): consists of PCR primer pairs 1, 2, 3 and 4 as shown below: PCR primer pair 1: consists of the left primer with nucleotide sequence shown in SEQ ID No. 11 and the right primer with nucleotide sequence shown in SEQ ID No. 12; PCR primer pair 2: consists of the left primer with nucleotide sequence shown in SEQ ID No. 45 and the right primer with nucleotide sequence shown in SEQ ID No. 46; PCR primer pair 3: consists of the left primer with nucleotide sequence shown in SEQ ID No. 93 and the right primer with nucleotide sequence shown in SEQ ID No. 94; PCR primer pair 4: consists of the left primer with nucleotide sequence shown in SEQ ID No. 95 and the right primer with nucleotide sequence shown in SEQ ID No. 96.

[0011] PCR primer set (II): Composed of PCR primer pairs 2, 3, 4 and 5 as shown below: PCR primer pair 2: Composed of the left primer with nucleotide sequence SEQ ID No. 45 and the right primer with nucleotide sequence SEQ ID No. 46; PCR primer pair 3: Composed of the left primer with nucleotide sequence SEQ ID No. 93 and the right primer with nucleotide sequence SEQ ID No. 94; PCR primer pair 4: Composed of the left primer with nucleotide sequence SEQ ID No. 95 and the right primer with nucleotide sequence SEQ ID No. 96; PCR primer pair 5: Composed of the left primer with nucleotide sequence SEQ ID No. 17 and the right primer with nucleotide sequence SEQ ID No. 18.

[0012] Another aspect of the present invention is to apply the two sets of PCR primers to identify the sex of poplar trees from five different schools, including: (1) extracting DNA from the poplar sample to be tested; (2) using the extracted DNA from the poplar sample as a template, establishing a PCR amplification system using each pair of PCR primers from either PCR primer set (I) or PCR primer set (II) as detection primers; if the amplification results of all amplification systems only amplify the 250bp product, then the poplar sample to be tested is female; if If no bands are amplified in any of the amplification systems, the poplar sample to be tested is male; if, in addition to the 250bp product, any one or more bands of 400bp, 500bp, or 700bp are amplified in all the amplification systems, the poplar sample to be tested is male; if any one or more bands of 400bp, 500bp, or 700bp are amplified in all the amplification systems, the poplar sample to be tested is male.

[0013] In a preferred embodiment of the present invention, the PCR amplification system is preferably: 10 μL Master Mix, 0.5 μL PCR left primer, 0.5 μL PCR right primer, 2 μL DNA template, and 7 μL dd H2O.

[0014] In a preferred embodiment of the present invention, the PCR amplification procedure is preferably as follows:

[0015] Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 sec, annealing at 60℃ for 30 sec, extension at 72℃ for 30 sec, 34 cycles; 72℃ for 5 min.

[0016] The five major schools of poplar trees mentioned in this invention are the white poplar, black poplar, green poplar, desert poplar, and large-leaved poplar; among them, the white poplar includes: the white poplar clonal strain GM107 (… Populus alba × Populus davidiana Populus tomentosa clonal strain GM15 ( Populus adenopoda × Populus alba var . pyramidlis Populus tomentosa ( ) Populus adenopoda Maxim.), Silver Poplar ( Populus alba L.), Aspen ( Populus davidiana Dode); the black poplar species mentioned include: Giant Poplar (… Populus euramericana ), Sino-Dutch No. 1 ( Populus euramericana ), I-69 Populus deltoides 'I-69'), I-69 ( Populusdeltoides 'I-69', I-214 Populus canadensis 'I-214'); the poplar species mentioned include: small-leaved poplar ( Populus simonii ), Beijing Yang ( P. nigra × P. cathayana ), hairy poplar ( Populus trichocarpa The poplar species mentioned include Populus euphratica 1 ( Populus euphratica Populus euphratica 2 ( Populus euphratica Populus euphratica 3 ( Populus euphratica Populus euphratica 14 ( Populus euphratica The aforementioned Populus macrophylla species includes: Populus macrophylla 1 ( Populus lasiocarpa Populus macrocarpa 2 ( Populus lasiocarpa Populus macrocarpa 3 ( Populus lasiocarpa ).

[0017] Another aspect of the present invention provides a PCR detection kit for detecting the sex of poplar trees from five major groups, comprising: a thermostable DNA polymerase, dNTPs, PCR primers, and double-distilled water; wherein the PCR primers are selected from any one of the PCR primer groups (I) or (II) mentioned above.

[0018] Another aspect of the present invention provides a PCR primer pair for identifying the sex chromosome composition of Populus tomentosa species, wherein the PCR primer pair 1 consists of a left primer with the nucleotide sequence shown in SEQ ID No. 11 and a right primer with the nucleotide sequence shown in SEQ ID No. 12.

[0019] Another aspect of the present invention is to apply the PCR primer pair to identify the sex chromosome composition of poplar species, including: (1) using the DNA of female or male poplar species to be identified as templates, establishing a fluorescence quantitative PCR system with PCR primer pair 1 to amplify the corresponding Ct values; (2) using poplar species with known sex genotypes as standards to draw a standard curve and a sex genotype reference table; (3) matching the Ct values ​​of the poplar species samples to be identified with the drawn sex genotype reference table to determine the sex chromosome composition of the poplar species to be identified.

[0020] Another aspect of the present invention provides a PCR detection kit for detecting the sex chromosome composition of Yangpai tree species, comprising: a thermostable DNA polymerase, dNTPs, PCR primers, and double-distilled water; wherein the PCR primers are a PCR primer pair 1 consisting of a left primer with the nucleotide sequence shown in SEQ ID No. 17 and a right primer with the nucleotide sequence shown in SEQ ID No. 18.

[0021] This invention utilizes the NCBI database and 73 poplar genomes already released by the National Center for Biotechnology Information to identify sex-determining genes for each tree species through sequence alignment. FERR and FERR-R In different poplar species, sex-determining genes are present. FERR The sequence is highly conserved. FERR-R It exhibits multiple sequence structures, representing different groups of poplars. FERR-R A summary of various sequence structures revealed that, among the currently released poplar genomes, only the white poplar species possess... FERR-R -Class I structure; Black poplar species only have FERR-R -Class II structure; Poplar species have FERR-R -II and FERR-R -Class III structure; Populus euphratica species have FERR-R -Class III structure.

[0022] This invention is based on the sex-determining gene of poplar trees. FERR Genes and FERR-R Specific primers were designed for each gene, and finally, primers capable of specific amplification were screened. FERR Two pairs of specific primers for the gene and three types of specific primers. FERR-R Three pairs of gene-specific primers were used to obtain a PCR primer set for detecting the sex of poplar trees from five major groups. PCR detection results using this primer set on 21 poplar materials with known sexes were completely consistent with the actual poplar sex phenotypes, demonstrating that the PCR primer set provided by this invention can accurately identify the sex of poplar trees from five major groups at the molecular level, making poplar sex identification more universal. Furthermore, the specific amplification provided by this invention... FERR The primer pairs for the gene can accurately identify the sex chromosome composition of poplar species. This invention has significant application prospects in poplar sex identification, molecular breeding, and the control of female tree fluff. Attached Figure Description

[0023] Figure 1 for FERR Genes and FERR-R Gene sequence diagram; Note: White arrows represent promoters; red arrows represent... FERR Gene sequences and partial sequences; pink areas indicate FERR-R Longest copy FERR The middle position of the fourth exon; the black arrow indicates the position of the primer pair.

[0024] Figure 2 for FERR gel image of gene-specific primer screening process; M represents marker; numbers 1-12 correspond to poplar sample numbers in Table 2; the blue boxes highlight the selected specific amplifications. FERRGene-specific primers.

[0025] Figure 3 for FERR-R-Ⅰ Gel image of the specific primer screening process; M represents marker; numbers 1-12 correspond to the poplar sample numbers in Table 2; the blue boxes highlight the specific amplifications selected. FERR-R-Ⅰ Gene-specific primers.

[0026] Figure 4 for FERR-R-Ⅱ , FERR-R-Ⅲ gel image of gene-specific primer screening process; M represents marker; numbers 1-12 correspond to poplar sample numbers in Table 2; the blue boxes highlight the selected specific amplifications. FERR-R-Ⅱ , FERR-R-Ⅲ Gene-specific primers.

[0027] Figure 5 A quantitative real-time PCR reaction procedure for identifying the sex chromosome composition of poplar trees.

[0028] Figure 6 This is a PCR gel image of primer pairs A, C, D, and E for 21 poplar tree samples.

[0029] Figure 7 This is a standard curve showing the relationship between gene copy number and Ct value for the standard. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0031] Experimental Example 1: Design and Screening of Primers for Sex Identification in Five Major Poplar Varieties

[0032] The expression of sex in poplar trees is determined by sex-determining genes. FERR and FERR-R Regulation, FERR The gene belongs to the A-class RR gene family and is involved in the transmission of cytokinin signals; FERR-R Genes are FERR Partially repetitive sequences in genes often consist of multiple FERR-R The combination occurs, and after transcription, a double-stranded long non-coding RNA is formed. Then, through modification and splicing, a functional mature siRNA is formed. FERR-R siRNA target FERR Methylation modification of the gene inhibits FERRGene expression. This experiment utilized the NCBI database and 73 poplar genomes released by the National Center for Biotechnology Information to identify sex-determining genes for each tree species through sequence alignment. FERR and FERR-R In different poplar species, sex-determining genes are present. FERR The sequence is highly conserved. FERR-R It exhibits multiple sequence structures. We studied different groups of poplar... FERR-R The various sequence structures of poplar trees were summarized and generalized. FERR-R There are three types of sequence structures, named as follows: FERR-R -Ⅰ、 FERR-R -Ⅱ、 FERR-R -Ⅲ( Figure 1 Of the poplar genomes that have been released so far, only the white poplar species possess... FERR-R -Class I structure; Black poplar species only have FERR-R -Class II structure; Poplar species have FERR-R -II and FERR-R -Class III structure; Populus euphratica species have FERR-R -Class III structure. Due to the spatiotemporal specificity of sex-determining genes, they are only expressed in floral organs during the flowering stage at maturity. Therefore, analyzing and comparing the genomic sequences of sex-determining genes aims to achieve specific markers of these genes at the DNA level, thereby enabling the identification of male and female sex and sex chromosome composition in poplar trees.

[0033] 1 FERR Gene-specific primer design and screening

[0034] FERR Gene-specific primer design approach: selection FERR-R Specific primers were designed for the sequence other than the repeat fragment, specifically the position between the fourth exon and the terminator. Figure 1 The area shown in the red box in the middle region. A total of 15 primer pairs were designed (Table 1), and specific primers were screened using adult poplar trees of known sex from Table 2 as standards.

[0035] Table 1 FERR Design of gene-specific marker primers

[0036]

[0037] Table 2. Adult poplar trees with known sex.

[0038]

[0039] according to Figure 2 The screening results Figure 2The blue boxes in the middle highlight the selected specific primers. A total of two pairs of specific primers were selected, namely primer pairs 6 and 9 in Table 1.

[0040] 2 FERR-R Gene-specific primer design and screening

[0041] FERR-R Gene-specific primer design approach: based on three FERR-R Specific primers were designed based on the structural characteristics of the genes, and selected... Figure 1 Specific primers were designed for regions 2, 3, 4, 5, 6, and 7 in the study, resulting in a total of 33 primer pairs (Table 3). Adult poplar trees of known sex in Table 2 were used as standards for screening specific primers.

[0042] Table 3 FERR-R Design of gene-specific marker primers

[0043]

[0044] Figure 3 This is a gel image showing the screening process for specific primers for the FERR-R-Ⅰ gene. The specific primers selected are circled in blue (primer pair 8 in Table 3).

[0045] Figure 4 This is a gel image showing the screening process for specific primers for the FERR-R-II and FERR-R-III genes. The specific primers selected are circled in blue (primer pair 32 and primer pair 33 in Table 3). According to... Figure 3 and Figure 4 The screening results identified three pairs of specific primers: primer pair 8 corresponds to the FERR-R-Ⅰ gene, primer pair 32 corresponds to the FERR-R-Ⅱ gene, and primer pair 33 corresponds to both the FERR-R-Ⅱ and FERR-R-Ⅲ genes. Due to the diverse species of poplar, the sequence conservation of primer pair 33 at the FERR-R-Ⅱ and FERR-R-Ⅲ gene positions could not be guaranteed. Therefore, primer pair 32 supplemented primer pair 33, further improving the accuracy of specific primer pairs for poplar sex identification.

[0046] In summary, this experiment screened out 5 pairs of specific primers, named A, B, C, D, and E. Among them, primer pair A and primer pair B have functional redundancy and can both be used as... FERRGene-specific primers; primer pairs C, D, and E correspond to three types of poplar trees, respectively. FERR-R Gene structure, serving as a specific primer for each structure.

[0047] Example 2: Verification Experiment of Four Pairs of Specific Primers for Sex Identification in Poplar Trees from Five Major Categories

[0048] There are two sex determination systems in poplar trees: the XY sex determination system and the ZW sex determination system. Genome-wide association analysis revealed that in both systems, there is a consistent hierarchical relationship between sex chromosomes and sex-determining genes; the X and W sex chromosomes contain these sex-determining genes. FERR The Y chromosome contains genes that determine sex. FERR and FERR-R Neither of the two sex-determining genes is present on the Z sex chromosome. Therefore, the identification of the sex of poplar trees is transformed into the identification of sex-determining genes by focusing on the relationship between sex-determining genes and sex chromosomes. FERR and FERR-R The identification is based on sex-determining genes. FERR and FERR-R The sex of poplar trees is determined by their presence or absence.

[0049] Due to the diverse species of poplar, the sequence conservation of primer pair E at the FERR-R-II and FERR-R-III positions cannot be guaranteed. Therefore, primer pair C complements primer pair D, further improving the accuracy of specific primer pairs for poplar sex identification. Because poplars exhibit high heterozygosity, natural or artificial hybridization events are common among different poplar species; therefore, testing a specific poplar sample... FERR-R During specific identification, the ability to amplify any one of the criteria proves the presence of [a specific marker / symbol]. FERR-R The presence of the gene. Although the nuclear genome information of the Populus macrophylla is currently unavailable, the sex-determining gene of the Populus macrophylla has been verified by collecting samples of adult Populus macrophylla trees (Table 4) and using primer pairs A, C, D, and E for sex determination. FERR It is highly conservative compared to other schools of poplar. FERR-R Its sequence structure is similar to that of Populus tomentosa, and it also has... FERR-R -Class II structure and FERR-R -Class III structure.

[0050] 1. Experimental Methods

[0051] Poplars with known sex and known sex chromosomes are shown in Table 4. The samples were dried with silica gel and stored at room temperature for genomic DNA extraction.

[0052] Table 4. Adult poplar trees with known sex.

[0053]

[0054] Table 5. Nucleotide sequences of the four primer pairs and lengths of the amplification products.

[0055]

[0056] Using poplar genomic DNA as a template, PCR experiments were conducted with four pairs of specific primers. The sex of the sample trees was determined by analyzing the presence or absence of bands in the four pairs of primers. All possible experimental results are summarized in Table 6.

[0057] Table 6. Reference Table for Male and Female Poplar Trees

[0058]

[0059] The PCR reaction solution components and reaction procedures for all four primer pairs were identical. The PCR reaction solution components are shown in Table 7, and the reaction procedures are as follows: Figure 5 .

[0060] Table 7 PCR Reaction Solution Preparation Table

[0061]

[0062] 2. Experimental Results

[0063] The four specific primer pairs described in Table 5 were used to perform PCR detection on 21 poplar materials of known sex in Table 4. The amplification results of the PCR detection of the four specific primer pairs on the 21 poplar materials of known sex in Table 4 are shown in the figure. Figure 6 Referring to the known sex conclusions of poplar trees detected in Table 6, the PCR detection results of 21 poplar materials with known sex in Table 4 using 4 specific primer pairs are completely consistent with the actual poplar sex phenotypes, proving that these 4 primer pairs can achieve accurate identification of the sex of the five major groups of poplar trees at the molecular level.

[0064] Example 3: Screening of fluorescent quantitative primers for sex genotyping in the Poplar School and verification experiment for sex genotyping in the Poplar School.

[0065] Based on the results of sex identification of Populus tomentosa species in Experiments 1 and 2, this experiment further uses primer pair A to identify the sex genotype of Populus tomentosa species.

[0066] The experimental approach is as follows: Genes are determined by sex. FERR Starting with the copy number in the poplar genome, and utilizing different sex genotypes and sex-determining genes... FERR The relationship between copy numbers can be quantified using quantitative fluorescence techniques.FERR The copy number is used to determine the different sex genotypes of poplar species. When identifying the sex of poplar trees, the sex-determining genes... FERR The products of specific primers A and B are both approximately 250 bp in length and can be used as primers for quantitative real-time PCR. Primer pair A was selected based on conserved sequences from Populus tomentosa species, and primer pair B was selected based on conserved sequences from Populus euphratica species. Therefore, primer pair A was chosen as the sex-determining gene. FERR Primers for quantitative fluorescence experiments.

[0067] Ten 2-year-old hybrid offspring of *Populus deltoides* and *Populus tomentosa* were randomly selected. Leaf samples were collected using liquid nitrogen flash freezing for DNA extraction, followed by sex genotyping. First, primer pairs A, C, D, and E (Table 5) were used to identify the sex of these 10 offspring. Then, quantitative real-time PCR was performed on both female and male offspring using primer A, and Ct values ​​were obtained. To ensure data reliability and avoid random errors in each quantitative real-time PCR experiment for sex genotyping, a standard curve was constructed using a tree species with known sex genotypes as a standard (Table 8). Figure 7 The standard curves for gene copy number versus Ct value and the sex genotype reference table were constructed (Table 9). The Ct values ​​of the samples to be identified were matched with the sex chromosome composition reference table to obtain the sex chromosome composition of the samples.

[0068] Table 8 Poplar Standards for Plotting Standard Curves

[0069]

[0070] Table 9. Reference Table for Sex Genotypes

[0071]

[0072] Table 10 Sex Genotypes of Populus spp. and Populus tomentosa Offspring

[0073]

[0074] The experimental results show that primer pair A meets the requirements for sex genotyping of Populus alba and can accurately identify the sex genotyping of Populus alba.

Claims

1. A PCR primer set for identifying the sex of poplar trees from five major groups, characterized in that, The PCR primer set consists of PCR primer pairs 1, 2, 3, and 4 as shown below: PCR primer pair 1: consists of a left primer with the nucleotide sequence shown in SEQ ID No. 11 and a right primer with the nucleotide sequence shown in SEQ ID No. 12; PCR primer pair 2: consists of a left primer with the nucleotide sequence shown in SEQ ID No. 45 and a right primer with the nucleotide sequence shown in SEQ ID No. 46; PCR primer pair 3: consists of a left primer with the nucleotide sequence shown in SEQ ID No. 93 and a right primer with the nucleotide sequence shown in SEQ ID No. 94; PCR primer pair 4: consists of a left primer with the nucleotide sequence shown in SEQ ID No. 95 and a right primer with the nucleotide sequence shown in SEQ ID No.

96.

2. The application of the PCR primer set described in claim 1 in identifying the sex of five major types of poplar trees; the five major types of poplar trees are white poplar, black poplar, green poplar, Euphratica poplar and large-leaved poplar.

3. The application according to claim 2, characterized in that, include: (1) Extract DNA from the poplar samples to be identified; (2) Using the extracted poplar sample DNA as a template, a PCR amplification system is established using each pair of PCR primers in the PCR primer set described in claim 1 as detection primers; if the amplification results of all amplification systems only amplify the 250bp product, then the poplar sample to be identified is female. If no bands are amplified in all amplification systems, the poplar sample to be identified is male. If, in addition to the 250bp product, all amplification systems produce one or more bands of 400bp, 500bp, or 700bp, then the poplar sample to be identified is male.

4. The application according to claim 3, characterized in that, The PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 esc, 60℃ annealing for 30 sec, 72℃ extension for 30 sec, 34 cycles; 72℃ for 5 min.

5. The application according to claim 2, characterized in that, The poplar species mentioned include: Populus tomentosa clonal lines GM107, GM15, Populus elongata, Populus simonii, or Populus spp.; the poplar species mentioned include: Populus japonica, Populus nigra 1, I-69, or I-214; the poplar species mentioned include: Populus microphylla, Populus pekinensis, or Populus pubescens.

6. PCR detection kits for identifying the sex of poplar trees from five major groups, including: Thermostable DNA polymerase, dNTPs, PCR primers, double-distilled water; characterized in that the PCR primers are the PCR primer set described in claim 1.

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