CN109295185B

CN109295185B - Method for determining genome size of unicellular eukaryotic algae

Info

Publication number: CN109295185B
Application number: CN201811032498.4A
Authority: CN
Inventors: 张成武; 黄罗冬; 高保燕
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-09-05
Filing date: 2018-09-05
Publication date: 2022-03-22
Anticipated expiration: 2038-09-05
Also published as: CN109295185A

Abstract

The invention discloses a method for determining the size of a genome of unicellular eukaryotic algae. The invention optimizes the extraction and staining method of cell nucleus based on the characteristics of unicellular eukaryotic algae, obtains more accurate flow cell analysis result, and combines with high-throughput second-generation illumina sequencing technology to comprehensively evaluate the genome size of algae cells and determine the characteristics of the genome. The technical method optimizes the traditional plant flow cytometry, utilizes methanol to remove the interference of algae cytochrome and cell wall, greatly improves the dyeing effect of PI (polyimide), improves the accuracy of flow cytometry, simultaneously combines the prior advanced high-throughput sequencing technology, comprehensively analyzes the structural characteristics of the genome of the species to be detected, and can provide a theoretical basis for the genetic structure research of the subsequent species and the establishment of the assembly strategy of whole genome sequencing.

Description

Method for determining genome size of unicellular eukaryotic algae

Technical Field

The invention relates to a method for determining the size of a genome of unicellular eukaryotic algae, which analyzes the size of the genome of the algae by combining flow cytometry analysis and a second-generation sequencing technology, in particular to microalgae species.

Background

In order to determine the complexity of an unknown species, it is first necessary to know the characteristics of the genome from the genome level and to quickly obtain the genome size of the unknown species. The cell nucleus is usually fluorescence-stained with Propidium Iodide (PI) by an external standard method or an internal standard method using Flow Cytometry (FCM), and then measured by a flow cytometer. FCM is one of the most commonly used methods for estimating the size of a species genome at present, has the obvious advantages of simple operation, short sample preparation flow and the like compared with earlier means such as pulsed field gel electrophoresis and the like, and has better application in animals, plants and part of fungi. However, different species, especially different types of species, require optimization of the FCM assay methods, such as cell wall disruption, nuclear extraction and optimization of staining conditions, to improve the accuracy of FCM technology.

Microalgae (unicellular eukaryotic algae) are a phylogenetically diverse, small individual, usually unicellular or colony, photosynthetic aquatic lower organism. Currently, there is less research on the genomic evaluation of unicellular eukaryotic microalgal species, which, because of their smaller cells and generally thicker cell walls, in addition to the large amount of pigments and secondary metabolites present in the cells, reduce the fluorescence intensity of PI. In addition, due to the large evolutionary scale among different algae species, the genome size of species in the same family may be very different, so that the accuracy of the genome size determination by the flow cytometry method is seriously influenced by the selection of internal reference species, and a method which is further definitely suitable for the genome determination of the unicellular eukaryotic algae is needed.

Disclosure of Invention

In order to solve the technical difficulty of accurate determination of the genome size of the unicellular eukaryotic microalgae at present, the invention aims to provide a method suitable for determining the genome size of the unicellular eukaryotic microalgae.

At present, the low-depth sequencing data of the genome small fragment library based on the second-generation sequencing technology can effectively evaluate the genome size, GC content, heterozygosity, content of a repetitive sequence and other related information through K-mer analysis, and is also an effective method for comprehensively understanding the genome characteristics of a certain species. Therefore, the flow cytometry analysis method is optimized, and the size of the genome of the alga species to be detected is accurately and comprehensively evaluated by combining with high-throughput sequencing data, so that the method has important theoretical significance on subsequent systematic and genomics research of alga resources. Accurate genome size estimation can provide help for the subsequent genetic and evolutionary analysis and the establishment of the whole genome sequencing assembly strategy.

The purpose of the invention is realized by the following technical scheme:

a method for determining genome size of unicellular eukaryotic algae comprises the following steps:

(1) preparing materials: centrifuging a unicellular eukaryotic algae sterile culture to remove a supernatant, rinsing with sterile water for 2-3 times, and removing culture medium components to obtain algal cells;

(2) internal reference selection: predicting the fluorescence intensity and genome size of a sample after debugging an instrument, selecting Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) as a main internal reference and Nannochloropsis oculata (Nannochloropsis oculata) and/or Nannochloropsis salina (Microchlorophpsis salina) as a second internal reference according to the predicted genome size, and performing mutual correction according to the genome size of the internal references;

(3) cell wall disruption and pigment removal: the algae to be tested and the reference algae are 10 times⁵Adding 1-2 mL of methanol into each algae cell; adding methanol into algae cells of algae to be detected and reference algae respectively, magnetically stirring at normal temperature to destroy cell walls and chloroplast structures to remove interference of chlorophyll, centrifuging for 1-2 h each time to remove supernatant, and repeatedly extracting for 3-5 times until the cells become white; then according to each 10⁵Adding 0.1-0.2 mL of lysine buffer LB01 into each algae cell for resuspending the cells, and performing Lysis treatment on ice to obtain a cell nucleus suspension;

(4) algae nucleus staining: adding 50 mu g of PI, 50 mu g of RNase and 5 mu L of beta-mercaptoethanol into 1mL of cell nucleus suspension, uniformly mixing, dyeing in a dark place, analyzing the cell nucleus suspension by using a flow cytometer to determine the fluorescence values of the algae to be detected and the internal reference algae, and calculating the genome size of the algae to be detected.

In order to better achieve the aim of the invention, the method further comprises the following steps:

(5) and (3) verifying the genome of the algae to be detected by high-throughput sequencing, and comprehensively evaluating the size and complexity of the genome of the algae to be detected.

Preferably, the algal cell density in the unicellular eukaryotic algal sterile culture in the step (1) is 1 × 10³～1×10⁵Per mL; more preferably 1X 10⁵one/mL.

Preferably, the speed of the centrifugation in the step (1) is 3000-5000 rpm; more preferably 3000 rpm.

Preferably, the algae to be tested in step (3) is an alga of euglenophyceae, more preferably euglenophyceae (Eustigmatos cf. polyphem) or widmannheimia stellatoides (Vischeria stellata).

Preferably, the speed of the centrifugation in the step (3) is 3000-5000 rpm; more preferably 3000 rpm.

The formula of the lysine buffer LB01 in the step (3) is as follows: 363.4mg Tris, 148.9mg Na in 200mL₂EDTA, 34.8mg of speramine tetrahydrochloride (spermine tetrahydrate), 1.193g of KCl, 233.8mg of NaCl and 200. mu.L of 0.1% (v/v) Triton X-100.

Preferably, the time of the cracking treatment in the step (3) is 0.5-1 h; more preferably 0.5 h.

Preferably, the dyeing in the step (4) is carried out at 4 ℃ in the dark for 30-60 min.

Preferably, the flow cytometer analysis conditions described in step (4) are as follows: excitation wavelength of 488nm, flow rate of 20 μ L/min, and collecting at least 10 per time⁴Repeating the cells for more than 3 times, wherein the coefficient of variation is less than 5%, and performing image and data processing analysis by using Flowjo X V10 software to obtain PI fluorescence intensity (PI-A) of the algae to be detected and the reference algae; the genome size of a unicellular eukaryotic algae can be given by the following calculation: the size of the genome of the alga to be detected is equal to the fluorescence intensity of the alga to be detected/(fluorescence intensity of the reference alga x the size of the genome of the reference alga).

Preferably, the high throughput sequencing step of step (5) is as follows:

(A) preparing materials: centrifuging a unicellular eukaryotic algae sterile culture solution to remove a supernatant, collecting 100-200 mg of algae mud, and extracting a sample DNA;

(B) DNA sequencing: constructing a 170-350 bp small fragment DNA library, sequencing double-end PE150bp by using an Illumina or BGI 500 platform, wherein the sequencing data volume is 5-10G, the sequencing depth is not less than 30 x, and the sequencing is completed by Shenzhen Huada gene science and technology service Limited;

(C) and (3) data analysis: detecting the base quality by using FastQC software, and detecting whether the base is polluted or not and the level of a repeated sequence by GC separation and GC peak diagram; then removing the joint sequence and filtering low-quality reads by using NGSQCToolkit software, and further correcting the error base in the reads by using Bless software; after obtaining high-quality clean reads, the size, the content of repeated sequences, the heterozygosity rate and the like of the genome are evaluated on the basis of Kmer-17 by using GCE (genome diagnostics) software.

(D) And (3) carrying out primary assembly on a target genome by utilizing SOAPdenovo software, then extracting GC content and sequencing depth by utilizing Samtools software, calculating a GC content distribution map of algae cells, and analyzing whether the genome is polluted by other exogenous species.

Preferably, the speed of the centrifugation in the step (A) is 3000-5000 rpm; more preferably 3000 rpm.

Preferably, the small fragment DNA library in step (B) is a 270bp small fragment DNA library;

preferably, the amount of sequencing data described in step (B) is 10G.

Preferably, the sequencing depth in step (B) is 30X to 100X.

According to the nature of the unicellular algae, the method starts from two methods of flow cytometry analysis and high-throughput sequencing analysis, and comprehensively and accurately evaluates the size of the genome.

Compared with the prior art, the invention has the following advantages and effects:

the invention optimizes the extraction and staining method of cell nucleus based on the characteristics of unicellular eukaryotic algae, obtains more accurate flow cell analysis result, and comprehensively evaluates the genome size of algae cells by combining with high-throughput second-generation illumina sequencing technology, thereby defining the characteristics of the genome. The technical method optimizes the traditional plant flow cytometry, utilizes methanol to remove the interference of algae cytochrome and cell wall, greatly improves the dyeing effect of PI, provides the accuracy of flow cytometry, simultaneously combines the prior advanced high-throughput sequencing technology, comprehensively analyzes the structural characteristics of the genome of the species to be detected, and can provide a theoretical basis for the genetic structure research of the subsequent species and the establishment of the assembly strategy of whole genome sequencing.

Drawings

FIG. 1 is a fluorescent peak plot of 5 unicellular eukaryotic algae.

FIG. 2 is a comparison of peak patterns and fluorescence values of 5 kinds of algae.

FIG. 3 shows the result of agarose gel electrophoresis of DNA samples; wherein, 1, 2: euglena powensis; 3,4: widmanspipe stellatus; m1: lambda-Hind III digest (Takara); m2: d2000 (Tiangen).

FIG. 4 is a base mass distribution diagram of FastQC analysis.

FIG. 5 is a Kmer-17 histogram of Euglena borealis

FIG. 6 is a Kmer-17 histogram of Weissella stellata.

FIG. 7 is a graph of the GC content of Euglena powellia and sequencing depth of scafffolds.

FIG. 8 is a graph of GC content of Weissella stellata versus sequencing depth of scaffolds.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

The euglena poboldii (eustigmaos cf. polyphem) H4302 was purchased from czech bragg charles university. Widmanassia stellata (Vischia stellata) SAG 33.83, Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) SAG 54.72, Nannochloropsis oculata (Nannochloropsis oculata) SAG 38.85, and Nannochloropsis salina (Microchloropsis salina) SAG 40.85 were purchased from the algae species Collection of the university of Gettin, Germany.

Example 1

Two strains of unicellular eukaryotic microalgae such as Nonconsome Boehringer euglena (E.cf. polyphem) and Weishi star are used as the genome unknown strainsFor example, the alga (v. stellata) is the first internal reference, chlamydomonas reinhardtii (c. reinhardtii), nannochloropsis oculata (n. oculata) and nannochloropsis salina (m. salina), respectively, and 3 known species are genomically aligned with each other. 5 microalgae cultures (about 50mL, 1X 10)⁵each/mL) are respectively centrifuged, 50mL of methanol is added, magnetic stirring is carried out, 1-2 h is carried out each time, supernatant is removed by centrifugation at 3000rpm, and extraction is repeated for 3-5 times until cells become white; centrifuging to remove supernatant, adding 5mL lysine buffer LB01 for resuspension, treating and cracking cell nucleus for 0.5h to obtain cell nucleus suspension, and standing on ice for use; taking 1mL of cell nucleus suspension, adding 50 mu L of PI (1mg/mL), 50 mu L of RNase (1mg/mL) and 5 mu L of beta-mercaptoethanol, uniformly mixing, and dyeing for 0.5h at 4 ℃ in a dark place; collecting 300 μ L of the stained Cell suspension, adding into special flow-type round bottom test tube (FACS), analyzing in BD FACSCAnto II flow cytometer with excitation wavelength of 488nm and flow rate of 20 μ L/min, collecting no less than 10 μ L each time⁴Each cell was repeated 3 times with a coefficient of variation of < 5%, and images and data were analyzed by processing using Flowjo X V10 software to obtain fluorescence peak maps, as shown in fig. 1 and 2. Therefore, the fluorescence values of eustigmatophycus boldii and widmanspium stellatus are 140109 and 91119 respectively, as shown in table 1, the PI fluorescence intensities (table 1) of the sample to be detected and the sample with the known genome size are obtained, and the PI fluorescence intensities are calculated according to the calculation formula of the genome size: the size of the genome of the alga to be detected is equal to the fluorescence intensity of the alga to be detected/(fluorescence intensity of the reference alga x the size of the genome of the reference alga). Finally, the estimated genome of Weissella stellata is about 90-110 Mb, and the estimated genome of Eustigmaea powelliana is about 180-220 Mb.

TABLE 1 flow cytometric estimation of genome size

To confirm the results, the flow cytometry obtained a preliminary estimate, and to extract the whole Genomic DNA of Eustigmatophyceae and Weissella stellata to be tested using Takara Plant DNA Extraction Kit (MiniBEST Plant Genomic DNA Extraction Kit), 2 tubes of DNA were extracted, DNA concentration and purity measurements were determined using a Qubit Fluorometer, see Table 2, and the integrity of the samples was checked by agarose gel electrophoresis, FIG. 3.

TABLE 2DNA extraction test results

The detection results show that the 4 tubes of DNA have good quality, the concentration is more than 30 ng/mu L, the total amount of DNA of each alga is more than 10 mu g, gel electrophoresis images show that the algae are free from pollution such as protein, RNA/salt ions and the like, samples are not degraded, the main peak is more than 20Kb, and the integrity is high, thereby meeting the requirements of library construction.

A270 bp small fragment DNA library is constructed, the Illumina HiSeq 4000 platform is utilized to carry out double-end PE150bp sequencing, the sequencing data volume is 10G, and the sequencing is completed by Shenzhen Hua Dagen science and technology service GmbH, as shown in Table 3. The sequencing quality is subjected to FastQC quality control, low-quality sequences (figure 4) are removed, and further Bless software is used for correcting wrong bases in reads to obtain high-quality clear reads.

TABLE 3 sequencing raw data statistics

After obtaining clean reads, using GCE software, Kmer-17 frequency distribution maps of two species were obtained, as shown in fig. 5 and 6. There are two distinct peaks in euglena pophonii, the main depth Peak is about 27, and according to the formula Genome Size K-mer num/Peak depth, the Genome Size of the species can be estimated to be about 267 Mb. The second peak at depth is at 61, indicating the presence of a large number of repeats in the genome. The Kmer-17 histogram of Weissella stellata has three distinct peaks with depth peaks of 14, 58 and 117 (FIG. 6), a main depth Peak of 58, a Genome Size of about 103Mb according to the formula Genome Size K-mer num/Peak depth, and two other peaks indicating the presence of a certain proportion of heterozygous and repeated sequences in the Genome, respectively, as shown in Table 4.

TABLE 417-Kmer analysis statistics

Initial assembly was performed using clean reads obtained by sequencing, as shown in Table 5, with total lengths of scaffolds of Eustigmatophycus poidomestis and Weissella stellata of 220Mb and 114Mb, respectively, and N50 of 6328bp and 19101bp, respectively. Meanwhile, scaffolds with the length of more than 2Kbp are selected, the relation between the coverage of the scaffolds and the GC content is calculated, and a GC content distribution diagram of two algae is calculated, as shown in FIG. 7, the GC depth of the eustigmatophycus boviensis is mainly about 30, and the corresponding GC content is about 50%. In contrast, the widmanspium stellatus, as shown in fig. 8, the GC distribution shows a two-part regional distribution, the gravity centers of the two parts are respectively about 20 and about 100 in depth, the central region about 20 in depth is the GC distribution region of the hybrid scaffold, and at the same time, the GC contents corresponding to the two depths are also at 50%, which is basically consistent with the GC contents of the genome assembly calculated by us, which indicates that the genome is not polluted by other exogenous species.

TABLE 5 preliminary Assembly results of two Euglena

	Euglena powensis (Fr.) Pilat	Chordaria stellata
			Genome scaffolds size(bp)	230490304	119222094
Genome contig size(bp)	223863295	116367984
			Scaffolds number	276658	96294
Contigs number	400415	153276
			GC Rate(％)	53.4％	53.5％
Scaffold N50	6328	19101
			Contig N50	2024	4378
Longest length(bp)	519339	211848
			Total length>＝1kb	184992717	104796597
Total length>＝2kb	167673725	100696179
			Total length>＝3kb	153087041	97238189

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for determining genome size of unicellular eukaryotic algae is characterized by comprising the following steps:

(1) preparing materials of algae to be tested: centrifuging the algae sterile culture to be detected to remove supernatant, rinsing with sterile water for 2-3 times, and removing culture medium components to obtain algae cells; the cell density of the algae in the algae sterile culture to be detected is 1 multiplied by 10³～1×10⁵Per mL; the centrifugation speed is 3000-5000 rpm;

(2) internal reference selection: predicting the fluorescence intensity and genome size of the sample after debugging the instrument, and selecting Chlamydomonas reinhardtii (C.reinhardtii) according to the predicted genome sizeChlamydomonas reinhardtii) Nannochloropsis oculata (Nannochloropsis oculata) as the main internal controlNannochlropsis oculata) And/or Nannochloropsis salina (or salt thereof)Microchloropsis salina) The second internal reference is obtained, and mutual correction is carried out according to the size of the internal reference genome;

(3) cell wall disruption and pigment removal: the algae to be tested and the reference algae are 10 times⁵Adding 1-2 mL of methanol into each algae cell; adding methanol into algae cells of algae to be detected and reference algae respectively, magnetically stirring at normal temperature to destroy cell walls and chloroplast structures to remove interference of chlorophyll, centrifuging for 1-2 h each time to remove supernatant, and repeatedly extracting for 3-5 times until the cells become white; then according to each 10⁵Adding 0.1-0.2 mL of lysine buffer LB01 into each algae cell to resuspend the cellsPerforming cracking treatment on ice to obtain a cell nucleus suspension;

the algae to be detected is Euglena pohuanii (A. pohuanensis)Eustigmatoscf. polyphem) Or Weissella stellata (Weissella stellata) (III)Vischeria stellata）；

The centrifugation speed is 3000-5000 rpm;

the formula of the lysine buffer LB01 is as follows: 363.4mg Tris, 148.9mg Na in 200mL₂EDTA, 34.8mg spermine tetrahydrate, 1.193g KCl, 233.8mg NaCl and 200 muL 0.1% v/v Triton X-100;

(4) algae nucleus staining: adding 50 mug PI, 50 mug RNase and 5 mug beta-mercaptoethanol according to 1mL of the cell nucleus suspension, uniformly mixing, dyeing in a dark place, taking the cell nucleus suspension, analyzing and determining fluorescence values of the algae to be detected and the internal reference algae by using a flow cytometer, and calculating the genome size of the algae to be detected.

2. The method for determining the genome size of a unicellular eukaryotic algae according to claim 1, wherein the method comprises: further comprising:

3. The method for determining the genome size of a unicellular eukaryotic algae according to claim 1 or 2, wherein the method comprises the following steps:

the time of the cracking treatment in the step (3) is 0.5-1 h;

and (4) dyeing at 4 ℃ in a dark place for 30-60 min.

4. The method for determining the genome size of a unicellular eukaryotic algae according to claim 1 or 2, wherein the method comprises the following steps:

the flow cytometer analysis conditions described in step (4) are as follows: excitation wavelength 488nm, flow rate 20 mu L/min, at least 10 collections each time⁴Repeating the above steps for more than 3 times, wherein the coefficient of variation is less than5%, performing image and data processing analysis by using Flowjo X V10 software to obtain PI fluorescence intensity of the algae to be detected and the internal reference algae; the genome size of the algae to be detected is given by the following calculation formula: the size of the genome of the alga to be detected = fluorescence intensity of the alga to be detected/(fluorescence intensity of the internal reference alga × size of the genome of the internal reference alga).

5. The method for determining genome size of a unicellular eukaryotic algae according to claim 2, wherein the method comprises:

the high-throughput sequencing step (5) comprises the following steps:

(A) preparing materials of algae to be tested: centrifuging to remove supernatant from the algae sterile culture solution to be detected, collecting 100-200 mg of algae mud, and extracting sample DNA;

(B) DNA sequencing: constructing a small fragment DNA library of 170-350 bp, and sequencing double-end PE150bp by using an Illumina or BGI 500 platform, wherein the sequencing data volume is 5-10G, and the sequencing depth is not less than 30 x;

(C) and (3) data analysis: detecting the base quality by using FastQC software, and detecting whether the base is polluted or not and the level of a repeated sequence by GC separation and GC peak diagram; then removing the joint sequence and filtering low-quality reads by using NGSQCToolkit software, and further correcting the error base in the reads by using Bless software; after high-quality clean reads are obtained, using GCE software to evaluate the size, the content of repeated sequences and the heterozygosity of the genome based on Kmer-17;

6. The method for determining genome size of a unicellular eukaryotic algae according to claim 5, wherein the method comprises:

the centrifugation speed in the step (A) is 3000-5000 rpm;

the small fragment DNA library in the step (B) is a 270bp small fragment DNA library;

the sequencing data volume in the step (B) is 10G;

the sequencing depth described in step (B) is 30X to 100X.