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Genomic single-fold type assembly method using pear pollen single cell

A single-cell and genome technology, applied in biochemical equipment and methods, special data processing applications, microbial measurement/inspection, etc., can solve the difficulty of haplotype genome assembly and the limited read length and base bias of next-generation sequencing , high heterozygosity, etc.

Active Publication Date: 2018-10-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high degree of heterozygosity and the existence of a large number of repetitive sequences, the genome assembly of many fruit tree species is still in the draft stage, and it is difficult to achieve a true whole genome assembly and chromosome anchoring, mainly due to the limitation of next-generation sequencing. Read length and base bias
Therefore, in order to technically solve the genome sequencing problem, it was established based on second-generation sequencing technology, such as BAC, fosmid, and other long-read sequencing technologies; single-molecule sequencing technology called third-generation sequencing technology and some assisted genome assembly Hi-seq and other sequencing technologies have been continuously applied, and the continuous update of bioinformatics technology has also accelerated the improvement of the assembly quality of fruit tree genomes, but the assembly of haplotype genomes is still a difficult problem

Method used

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  • Genomic single-fold type assembly method using pear pollen single cell
  • Genomic single-fold type assembly method using pear pollen single cell
  • Genomic single-fold type assembly method using pear pollen single cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The preparation of embodiment 1 pear pollen protoplast

[0049] Pollen medium formula: 1.60mM boric acid, 1.40mM magnesium sulfate, 0.2mM calcium nitrate, 292mM sucrose, 25mM MES (Methanesulfonic acid hydrate), pH 6.0-6.5.

[0050] Cell lysate formula: 35-40% (g / 100ml) sorbitol, 0.4% (w / v, g / 100ml) isolated enzyme R-10, 1.0% (w / v, g / 100ml) cellulase.

[0051] Experimental steps: 1. Add a small amount of pear pollen to the medium, shake slightly to make the pollen fully contact with the medium, place on a shaker at 28°C, 130-210 rpm, and incubate in the dark for 40-60 minutes. The pollen hydration was observed under a microscope ( figure 1 ). 2. Add cell lysate to the culture medium according to the ratio of 3:1 (cell lysate: pollen medium), then pipette the liquid and mix slowly, shake at 30°C at 40-100 rpm, and incubate in the dark for 5 After ~20 minutes, observe under a microscope after the culture is over to confirm the preparation of protoplasts. 3. Set the par...

Embodiment 2

[0052] Embodiment 2 Expansion and purification of pear pollen single cell

[0053] According to Sygni TruePrime TM Instructions for the use of the Single Cell Whole Genome Amplification Kit Steps Add neutralization solution, reaction buffer solution and amplification enzyme in sequence, react at 30°C for 8 hours, and inactivate for 10 minutes after 65 minutes.

[0054] In order to efficiently purify single-cell amplification products, Aliquot AMPure XP beads (Bechman, California, America) were used for separation and purification in this experiment. Proceed as follows:

[0055] 1. Add 100-150 microliters of Ampure XP beads to the reaction solution after each single cell amplification.

[0056] 2. Vortex or use a pipette gun to repeatedly beat and mix at least ten times to fully mix the solution.

[0057] 3. Place at room temperature for at least 10 minutes to fully combine the DNA with the beads.

[0058] 4. Transfer all the mixed liquid into a 1.5mLEP tube and use a magne...

Embodiment 3

[0064] Embodiment 3 Expansion and purification of pear pollen single cell

[0065] Using the Plant Genomic DNA Extraction Kit (TianGen, Beijing, China), the DNA of pear leaves was extracted according to the procedure of the kit. 11 marker primers (Table 1) were designed according to the reference genome sequence, and tested by leaf DNA ( image 3 ). After the test was completed, 11 marker primers were used to verify the integrity of each single-cell purification product by PCR. PCR program: 94°C for 10 minutes, 94°C for 30 seconds, 61°C for 30 seconds, 72°C for 1 minute (35 cycles), 72°C for 10 minutes.

[0066] Table 1 is the marker primer designed according to the species genome information in the present invention

[0067]

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Abstract

The invention discloses a genomic single-fold type assembly method using a pear pollen single cell, which comprises the following steps of: utilizing the characteristic of in vitro pollen growth, combined with an enzymatic method, to remove the cell wall to prepare a single cell wall-free pollen protoplast, carrying out full genome amplification and sequencing on the single-fold type pollen cell by using animal single-cell amplification technology, By using the genome information of the single-fold pollen obtained by sequencing, establishing the relationship between each artificial chromosomeand both of the two parents of a 'Dangshan pear' by using a method of numbering 38, 304 chromosomes using '12-bit binary code'. Finally, carrying out separate single-fold type assembly on the 34 single-fold type chromosomes of the Dangshan pear, in combination with the localization of the majority of BAC in the anchor reference genome. The method can help the research of the plant genome of the follow-up fruit trees, provide new gene resources for subsequent molecular breeding, and provide new genetic resources for implementing green agriculture.

Description

technical field [0001] The invention belongs to the field of plant genomes, and relates to the whole genome amplification of pear pollen single cells, that is, using the whole genome information of a single pollen cell to carry out haploid typing, in particular to preparing pollen protoplasts from pollen single cells and using animal single cells The amplification kit was used to amplify the whole genome, and finally, the 38,304 artificial chromosomes (BAC, artificial chromosome) in the pear genome were haplotyped based on the sequencing results of 12 pollen single cells. The combination of this method is suitable for the assembly of plant haplotype genomes, and is also suitable for the assembly of animal haplotype genomes. At the same time, our method can combine artificial chromosomes (BAC), fosmid, 10X genome and single-molecule sequencing sequences of equal length Long-read sequencing technology makes haplotype genome assembly more perfect. Background technique [0002]...

Claims

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Application Information

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IPC IPC(8): C12Q1/6881C12Q1/6869G06F19/22
CPCG16B30/00C12Q1/6869C12Q1/6881C12Q2531/113
Inventor 张绍铃施冬青吴俊唐海宝吴巨友谷超殷豪钱铭
Owner NANJING AGRICULTURAL UNIVERSITY