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A method for genome haplotype assembly using pear pollen single cells

A single-cell, haplotype technology, applied in genomics, biochemical equipment and methods, proteomics, etc., can solve the problem of high heterozygosity, difficulty in haplotype genome assembly, and limited read length and short bases of next-generation sequencing Bias and other issues

Active Publication Date: 2022-07-19
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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  • A method for genome haplotype assembly using pear pollen single cells
  • A method for genome haplotype assembly using pear pollen single cells
  • A method for genome haplotype assembly using pear pollen single cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of pear pollen protoplasts

[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 it slightly to make the pollen fully contact with the medium, place it on a shaker at 28°C, 130-210 rpm, and cultivate in the dark for 40-60 minutes. Observation of pollen hydration under a microscope ( figure 1 ). 2. Add cell lysate to the medium at a ratio of 3:1 (cell lysate: pollen medium), then pipette and mix slowly, shake at 30°C for 40-100 rpm, and incubate in the dark for 5 ~20 minutes, observed under a microscope after the end of the culture, to determine the preparation of protoplasts. 3. Set the parameters of the flame needle pu...

Embodiment 2

[0052] Example 2 Amplification and purification of pear pollen single cells

[0053] According to Sygni TruePrime TM Instructions for use of the single-cell whole genome amplification kit Steps to add neutralization solution, reaction buffer solution and amplification enzyme in sequence, react at 30°C for 8 hours, and inactivate for 10 minutes at 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 expansion.

[0056] 2. Vortex or use a pipette 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 bind the DNA to the beads.

[0058] 4. Transfer all the mixed liquid into a 1.5mL EP tube and use a magnetic separation...

Embodiment 3

[0064] Example 3 Amplification and purification of pear pollen single cells

[0065] Using the plant genomic DNA extraction kit (TianGen, Beijing, China), the pear leaf DNA was extracted according to the procedure of the kit. According to the reference genome sequence, 11 marker primers were designed (Table 1) and tested by leaf DNA ( image 3 ). After the test was completed, the integrity of each single-cell purified product was verified by PCR through 11 marker primers. PCR program: 94°C for 10 minutes, 94°C for 30 seconds, 61°C annealing for 30 seconds, 72°C for 1 minute (35 cycles), and 72°C for 10 minutes.

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

[0067]

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Abstract

The invention discloses a method for using pear pollen single cells to assemble genome haplotypes, utilizes the characteristics of in vitro pollen growth combined with enzymatic removal of cell walls to prepare single pollen protoplasts without cell walls, and uses animal single cell amplification technology to assemble haplotypes. Whole-genome amplification and sequencing of individual pollen cells. Using the haplotype pollen genome information obtained by sequencing, the 38,304 artificial chromosomes were numbered with a '12-bit binary code' to establish the connection between each artificial chromosome and the two parents of 'Dangshansuli'. Finally, combined with the localization of most BACs in the anchored reference genome, the independent haplotype assembly of the 34 haplotype chromosomes of Dangshansu pear was carried out. This method is helpful for the follow-up research on fruit tree plant genomes, provides new genetic resources for follow-up molecular breeding, and provides new genetic resources for the implementation of 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 perform haploid typing, and in particular relates to the preparation of pollen protoplasts from the single pollen cells and the utilization of animal single cells The amplification kit was used to amplify the whole genome. Finally, 38,304 artificial chromosomes (BACs) in the pear genome were haplotyped through the sequencing results of 12 single pollen cells. The combination of this method is suitable for the assembly of plant haplotype genomes as well as for the assembly of animal haplotype genomes, and our method can combine artificial chromosomes (BAC), fosmid, 10X genome and single-molecule sequencing sequences of equal length Read-length sequencing technology makes haplotype genome assembly more perfect. Background technique [0002] As an economic cr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6881C12Q1/6869G16B20/30G16B30/10G16B30/20
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