A method for rapid identification of homozygous diploid Populus xiaohei

Abstract

The present invention discloses a method for rapidly identifying homozygous diploids of Populus dasyphylla. Based on the information of whole genome resequencing of Populus dasyphylla, the present invention screens out specific primers that can identify specific SNP sites of Populus dasyphylla, and identifies homozygous diploids obtained from Populus dasyphylla by combining SNP molecular marker technology with PCR technology, which has the characteristics of high efficiency, accuracy and high repeatability. Compared with traditional identification methods, the present invention can obtain results in a very short time, greatly reducing working time and workload, and the experimental results are more authentic and reliable. The present invention is the first to establish an efficient homozygous diploid identification method for Populus dasyphylla, which can provide a valuable reference for haploid breeding of other poplars.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and mainly relates to a method for quickly identifying homozygous diploid Populus dasyphylla. Background Art

[0002] Poplars are highly adaptable, widely distributed, and have the advantages of rapid growth and easy reproduction, making them the main afforestation species in the world today. In addition, poplars are also the first woody plant in the world to complete genome sequencing (Tuskan et al., Science, 2006, 313 (5793): 1596-0601.), which provides great convenience for molecular biology research on poplars. At present, they have become a model plant for molecular biology research on woody plants. Most natural wild species of poplars are diploid (Zhao Fengbin, et al. Journal of Northeast Forestry University, 2006, 44 (06): 23-27.), that is, they contain two sets of chromosomes, one from the father and one from the mother. Since poplars are dioecious, the offspring all come from inter-individual hybridization, resulting in a high degree of heterozygosity, which brings many difficulties to the improvement and genetic research of poplars. Haploid poplars have only one set of chromosomes, which plays an irreplaceable role in the fields of genome sequencing, functional identification and construction of genetic maps. Since the early 1970s, forestry workers in my country have been studying the induction of haploids in poplars using anther culture, and have obtained haploid materials in multiple poplar species. Populus somoni×P. nigra is a hybrid of Populus simonii and Populus nigra. Due to its strong stress resistance and fast growth, it has become a widely cultivated poplar species in Northeast my country. It has been used for many studies on haploid breeding of poplars (Tree Breeding Group of Northeast Forestry University, Journal of Northeast Forestry University, 1976, 2: 9-18; Zhu Xiangyu et al., Forestry Science, 1980, 06(3): 190-196); Yang et al., Journal of Forestr Research, 29: 321-330).

[0003] Poplar haploid materials can be detected by flow cytometry, but there has been no efficient method for the identification of homozygous diploids, which has seriously affected the haploid breeding research of poplars. With the development of molecular biology, ploidy identification methods based on DNA molecules have been applied in forest trees, such as the use of SSR molecular marker technology for the identification of homozygous diploids, but the SSR molecular marker technology process is complicated and has a certain error rate (Deutsch et al., Physiologia Plantarum, 2001, 120: 613-622; Yang et al., Journal of Forestr Research, 29: 321-330). The present invention has developed a method based on the combination of SNP molecular markers and PCR technology, which can quickly identify homozygous diploids of Populus dasyphylla, and the accuracy rate is 0.60%. This method is a technical breakthrough in the field of haploid breeding research of forest trees, and will provide theoretical reference and technical support for its haploid breeding research. Summary of the invention

[0004] The present invention provides a method for rapidly identifying homozygous diploid Populus xiaohei, the specific contents of which are as follows:

[0005] First, based on the information of multiple whole genome resequencing of Populus xiaohei, six SNP sites were selected in the Populus xiaohei genome and specific primers were designed for the sequences. PCR amplification was performed using the haploid DNA obtained by anther induction of Populus xiaohei as a template. After the PCR reaction was completed, 5 μL of PCR product was taken for 1% agarose gel electrophoresis detection. The results showed that all templates had specific bands at 250 bp. The remaining PCR products were subjected to Sanger sequencing. By comparing the sequencing results with the specific primer sequences, the presence of SNP sites was heterozygous, and the absence of SNP sites was homozygous diploid. Finally, two pairs of specific primers, Chr01 and Chr06, were screened out, which can quickly identify whether the diploid callus induced by the anther of Populus xiaohei or the regenerated diploid seedlings are homozygous. We identified homozygous diploid materials for genome resequencing, and the k-mer analysis results showed that all homozygous diploids detected had only one main peak, indicating that the genome was in a homozygous state. The above proves that the combination of SNP molecular markers and PCR technology can accurately detect whether the genome is homozygous.

[0006] The PCR amplification system of the present invention comprises: a total system of 50 μL, including 5 μL 06×Ex Taq Buffer, 4 μL dNTP, 2 μL DNA template, 2 μL upstream and downstream primers, 0.5 μL Ex Taq, 32.5 μL ddH 2O. The PCR amplification program was: 95℃ pre-deformation for 3min, 95℃ denaturation for 30sec, 59℃ annealing for 30sec, 72℃ extension for 15sec, 35 cycles, 72℃ post-extension for 7min, and 16℃ insulation. The haploid callus induced from the anthers of Populus nigra was H1, the homozygous diploid formed by the doubling of H1 chromosomes was DH1, and the callus with heterozygous genome was D1. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Attached Figure 1 Gel electrophoresis test results of PCR reaction of 6 SNP locus-specific primers on the mother plant of Populus dasyphylla and H1 M.DL5000 marker; 1. Mother plant of Populus dasyphylla; 2. H1

[0008] Attached Figure 2 Sanger sequencing results of PCR reaction of Populus xiaohei mother plant and H1 with 6 pairs of specific primers

[0009] (A) Sanger sequencing results of the amplified products of Chr01-F / R; 1. Populus dasyphylla; 2. H1; (B) Sanger sequencing results of the amplified products of Chr02-F / R; 1. Populus dasyphylla; 2. H1; (C) Sanger sequencing results of the amplified products of Chr03-F / R; 1. Populus dasyphylla; 2. H1; (D) Sanger sequencing results of the amplified products of Chr01-F / R; 1. Populus dasyphylla; 2. H1; (E) Sanger sequencing results of the amplified products of Chr05-F / R; 1. Populus dasyphylla; 2. H1; (F) Sanger sequencing results of the amplified products of Chr06-F / R; 1. Populus dasyphylla; 2. H1

[0010] Attached Figure 3 Gel electrophoresis results, Sanger sequencing and k-mer analysis results of the amplified products using specific primers Chr01 and Chr06

[0011] (A) Gel electrophoresis results of the products amplified by Chr01 and Chr06 primers; M.DL5000 marker; 1. Populus dasyphylla; 2. H1; 3. DH1; 4. D1; (B) Sanger sequencing of the products amplified by Chr01 and Chr06 primers; 1. Populus dasyphylla; 2. H1; 3. DH1; 4. D1; (C) k-mer analysis results of Populus dasyphylla, H1, DH1 and D1; 1. Populus dasyphylla; 2. H1; 3. DH1; 4. D1 DETAILED DESCRIPTION

[0012] Example 1 Design and screening of primer sets for identifying haploid or homozygous diploid Populus xiaohei

[0013] 1. Plant Materials

[0014] Populus xiaohei is a heterozygous diploid. In this study, a healthy male Populus xiaohei was selected as the mother plant. Flower branches were taken for hydroponics in April, and anther-induced plants were obtained by anther in vitro culture technology. Flow cytometry detection, molecular marker identification, and resequencing analysis determined that H1 was haploid, DH1 was a homozygous diploid formed by the natural doubling of H1 chromosomes, and D1 was a heterozygous.

[0015] 2. Primer design and screening

[0016] (1) Primer design

[0017] Based on the information of whole genome resequencing of multiple Populus dasyphylla, HaplotypeCaller in GATK was used to detect variations (SNP / INDEL), and accurate SNP and INDEL detection was achieved through local recombination of haplotypes. VariantFiltration was used to filter the detection results, FS>30.0, QD<2.0, and 35 bases as a window. If there were more than 3, it was filtered to obtain the final vcf file. According to the final vcf file obtained, SNP sites were found, and samtools was used to manually check and further confirm. Six SNP sites were selected in the Populus dasyphylla genome and specific primers were designed for the sequence, with a product length of 250bp.

[0018] (2) Primer screening

[0019] The genomic DNA of the mother plant of Populus xiaohei and the anther-induced haploid H1 was extracted by the CTAB method and used as a template for PCR amplification with the synthesized 6 pairs of primers (Table 1). The PCR kit used Ex Taq enzyme (Dalian Bao Biotechnology). The total reaction system was 50 μL, including 5 μL 06×Ex Taq Buffer, 4 μL dNTP, 2 μL DNA template, 2 μL upstream and downstream primers, 0.5 μL Ex Taq, 32.5 μL ddH 2 O. The PCR amplification program was: 95℃ pre-deformation for 3min, 95℃ denaturation for 30sec, 59℃ annealing for 30sec, 72℃ extension for 15sec, 35 cycles, 72℃ post-extension for 7min, and 16℃ incubation. After the PCR reaction was completed, 5μL of PCR product was taken for 1% agarose gel electrophoresis. The results showed that all templates had a specific band ( Figure 1 ). The remaining PCR products were then subjected to Sanger sequencing, and the sequencing results were compared with the specific sequences (Table 2) using BioEdit software, and the primers for amplifying the SNP site sequence were selected from the 06 pairs of primers ( Figure 2 ), and finally screened out two pairs of specific primers Chr01 and Chr06, which can be used to identify whether the ploidy of plants induced by anthers of Populus nigra is homozygous.

[0020] Table 1 Information of six SNP locus-specific primers

[0021]

[0022]

[0023] Table 2 Sequence information of sequence-specific alignment containing SNP sites

[0024]

[0025] 3. Resequencing assisted verification of the accuracy of detecting genome homozygosity using SNP molecular markers

[0026] The genomic DNA of the mother plant of P. xiaohei, H1, DH1, and D1 plants was used as a template, and PCR amplification was performed with two pairs of primers, Chr01 and Chr06, respectively. The results were tested by 1% agarose gel electrophoresis, and the PCR products were sequenced by Sanger sequencing. Then, the genomic DNA of the mother plant of P. xiaohei, H1, DH1, and D1 callus was resequenced and k-mer analyzed. The results showed that there was a main peak and a heterozygous peak in the mother plant of P. xiaohei and the D1 plant, which were judged as heterozygous, while there was only one main peak in the haploid H1 and the homozygous diploid DH1 plant, that is, the genome was homozygous ( Figure 3 ). The above results show that the two pairs of primers screened can quickly and specifically identify haploid plants induced by anthers of P. xiaohei.

Claims

1. Application of primers in identifying whether plants induced by anthers of Populus nigra are haploid or homozygous diploid. It is characterized in that The primer is Chr01, and its nucleotide sequence is 5'-GCGAGTATGATGATAACGAGGAGCGATAG-3' and 5'-CGACGAATAGACCGCACATCCGTAA-3'.

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