CN112501270A - Identification method of POUCTUS COCONUS CHLORILORIDE genome and germplasm - Google Patents
Identification method of POUCTUS COCONUS CHLORILORIDE genome and germplasm Download PDFInfo
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Abstract
The invention relates to a POUCTASSIUM COCONUS chloroplast genome and a germplasm identification method for POUCTASSIUM COCONUS kindred species by using the POUCTASSIUM COCONUS chloroplast genome. The method utilizes chloroplast genomes to select the Jinguang bamboo joint coconuts, pocket coconuts and tassel bamboo joint coconuts to extract DNA of the golden-guang bamboo joint coconuts, the pocket coconuts and the tassel bamboo joint coconuts, and utilizes a specific primer and a PCR amplification method designed by the pocket coco chloroplast genomes to carry out PCR amplification on the golden-guang bamboo joint coconuts and the pocket cocoanut bamboo joint coconuts.
Description
Technical Field
The invention relates to a POUCTASSIUM COCONUS chloroplast genome and a germplasm identification method for POUCTASSIUM COCONUS kindred species by using the POUCTASSIUM COCONUS chloroplast genome.
Background
Pocket coconut (chamaedora elegans), also known as dwarf coconut and pocket palm, is a bamboo-joint coconut of the family palmaceae, is an indoor health-care flower, can purify benzene, trichloroethylene and formaldehyde in the air, has a certain sterilization function, and is beneficial to increasing the concentration of indoor negative ions. Since pockmarkets are good at improving indoor air quality, also known as "high efficiency air purifiers" in biology, a reasonable plan for protecting, preserving and developing the palmaceae wildlife resources should be made, enhancing innovation, protection and utilization of the pockmarkets germplasm resources.
Chloroplast genomes contain rich genetic information, are well known for conservation of structure, size and gene types, are widely applied to species identification and phylogenetic research, and have important contribution to deciphering phylogenetic relationships among plant groups with close relativity. The method for reconstructing species evolution and systematic classification positions among families and genera based on the mode of combining multiple genes or sequence fragments provided by the chloroplast whole genome sequence gradually becomes a new research trend and obtains better results.
The method is used for determining the chloroplast genome sequence of the pocket coconut, accurately annotating all genes of the chloroplast through bioinformatics software, and further exploring the chloroplast genome structure, gene components, codon usage, repetitive sequences and SSR sites of the pocket coconut so as to reveal the diversity and evolutionary characteristics of the pocket coconut cPDNA and provide important genetic data for germplasm resource protection, fine variety breeding, molecular marker development, and evolutionary and origin relations among various genera in the Palmae.
Disclosure of Invention
The invention aims to provide a pocket-size coconut chloroplast genome, which is used for filling the blank that a gene database in the prior art has no pocket-size coconut chloroplast genome sequence, establishing a germplasm identification method for closely related species of pocket-size coconut by utilizing a chloroplast genome, and providing powerful theoretical and technical support for theoretical research and germplasm identification.
In order to achieve the purpose, the invention provides a method for identifying a genome and germplasm of a chloroplast of a pockmark coconut, which comprises the following steps:
(1) extraction of plant DNA
Fully grinding fresh plant leaves by using liquid nitrogen, and extracting DNA of the pocket coconut and the closely related species thereof by using an improved CTAB method;
(2) chloroplast genome sequencing
After the sample genome DNA is qualified, fragmenting the DNA by using ultrasonic waves, then carrying out fragment purification, end repairing, adding A at the 3' end, connecting a sequencing joint on the fragmented DNA, then carrying out fragment size selection by using agarose gel electrophoresis, carrying out PCR amplification to form a sequencing library, carrying out library quality inspection on the built library, and sequencing the library qualified by quality inspection by using an Illumina Novaseq platform;
(3) quality control of sequencing data
Filtering the original data by using fastp v0.20.0 software, and obtaining high-quality sequencing data after filtering;
(4) assembly of genomic sequencing data
Aligning an NCBI chloroplast genome database by using a bowtie2 v2.2.4very-sensitive-local mode, using the aligned sequencing sequence as a chloroplast genome sequencing sequence of a sample, assembling a chloroplast genome by using a SPAdes v3.10.1 software in an assembly core module, and assembling a non-dependent reference genome by using 55, 87 and 121 for kmer respectively;
(5) chloroplast genome map
Using OGDRAW to make a chloroplast genome map;
(6) germplasm identification of pockmarkets and closely related species thereof
PCR amplification was performed using the extracted DNA as a template, using designed Primer1 and Primer2 as amplification primers.
Preferably, the modified CTAB method in step (1) is to grind plant leaves into powder in liquid nitrogen, take 0.1g of the powder to transfer into a 2ml microcentrifuge tube, add 630 μ L of CTAB buffer solution 1.5 times preheated to 65 ℃, add 70 μ L of absolute ethanol, then heat in a 65 ℃ water bath for 30min, shake 4-5 times in the middle, take out the microcentrifuge tube to cool, then add 700 μ L of chloroform: isoamyl alcohol 24: 1, uniformly mixing, centrifuging at 10000rpm for 10min, taking supernatant into a new microcentrifuge tube, adding isopropanol with the volume of 2/3 of that of the supernatant, uniformly mixing, settling, standing, centrifuging at 4000rpm for 3min, taking supernatant, or directly picking out filamentous DNA (deoxyribonucleic acid) for washing, adding 500 mu L76% ethanol for washing for 2 times, placing the mixture on a clean bench for drying, adding 100 mu L TE (potassium titanate) buffer solution or ddH (ddH), drying, and adding20 dissolving DNA, adding 20 μ g/L ribonuclease, digesting at 37 deg.C for 30min, adding equal volume of chloroform and isoamyl alcohol 24: 1, mixing, centrifuging at 1000rpm for 10min, adding 2 times volume of anhydrous ethanol, mixing, precipitating, adding 500 μ L ethanol, washing for 2 times, drying in ultra-clean bench, adding 50 μ L ethanol, and air dryingddH2O dissolves the DNA.
Preferably, the sequencing read length of the chloroplasts in step (2) is PE 150.
Preferably, the filtering criteria of step (3) is to cut off sequencing adapters and primer sequences in Reads, filter out Reads with an average quality value of less than Q5, and filter out N Reads with a number of more than 5.
Preferably, the assembling of genome sequencing data of step (4) comprises the following steps:
(1) assembling the cpDNA sequence by SPAdes software to obtain the SEED sequence of the chloroplast genome;
(2) kmer iterative extended seed, if the result of step (2) is one contig, the result is determined as pseudo genome sequence, and step (6) is directly carried out;
(3) connecting the contig sequences obtained in the step (2) by using SSPACE v2.0 software to obtain scafffolds;
(4) performing GAP supplement on the scaffolds sequence obtained in the step (3) by using Gapfiller v2.1.1 software;
(5) if GAP still exists after the operation is finished, designing a primer, carrying out PCR sequencing, and then assembling until a complete pseudo genome sequence is obtained;
(6) aligning the sequencing sequence to pseudo genome for genome correction;
(7) and (3) according to the structure of the chloroplast, carrying out coordinate rearrangement on the corrected pseudo genome to obtain a complete chloroplast circular genome sequence.
Preferably, the PCR primer of step (6) is designed by chloroplast based on group sequence, and the product is 1046 bp:
primerl：5’-TCCGCCTGAGGAAGCAGGGGCA-3’；
primer2：5’-ACGGGACTGGCTTTAGAAACC-3’。
preferably, the PCR reaction amplification program conditions of step (6) are: pre-denaturation at 94 ℃ for 4 minutes, denaturation at 94 ℃ for 50 seconds, annealing at 54 ℃ for 37 seconds, annealing at 72 ℃ for 90 seconds, and denaturation, annealing and extension for 30 cycles, and finally fully extending at 72 ℃ for 6 minutes, wherein the whole PCR reaction process is amplified on an Eppendorf Mastercycler nexus PCR instrument.
The invention has the beneficial effects that: at present, researches on evolution, identification and the like of the pocket coconut are still few, the invention discloses the chloroplast genome of the pocket coconut for the first time, and the chloroplast genome of the pocket coconut is applied to identification of the pocket coconut and kindred plants thereof, so that the invention can provide help for reasonable plan of protecting, storing and developing wild resources of palmaceae and strengthening innovation, protection and utilization of germplasm resources of the pocket coconut.
Drawings
FIG. 1 is a chloroplast genome map;
FIG. 2 is a diagram of a PCR-based assay of a pockmarked coconut by agarose gel electrophoresis;
FIG. 3 Main flow chart of library construction.
Detailed Description
The present invention will be further described with reference to examples.
Example 1
Chloroplast genome total DNA extraction
The extraction of the total DNA of the plant is a precondition for obtaining a whole genome sequence, and the quality of the total DNA directly influences whether a target gene segment can be successfully amplified. In the experiment, the DNA is extracted from pocket coconuts, golden-luster bamboo coconuts, bamboo coconuts and tassel-vein bamboo coconuts. Taking different paulownia samples stored in a refrigerator at minus 80 ℃, extracting DNA by adopting an improved CTAB method, grinding plant leaves into powder in liquid nitrogen, taking 0.1g of powder, transferring the powder into a 2ml microcentrifuge tube, adding 630. mu.L of 1.5 times CTAB buffer solution preheated to 65 ℃, adding 70. mu.L of absolute ethyl alcohol, heating in 65 deg.C water bath for 30min, shaking for 4-5 times, taking out the microcentrifuge tube, cooling, then adding 700 μ L chloroform and isoamyl alcohol 24: 1, mixing well, centrifuging at 10000rpm for 10min, taking supernatant to a new microfuge tube, adding isopropanol 2/3 of the volume of the supernatant, mixing, precipitating, standing, centrifuging at 4000rpm for 3min, collecting supernatant, or directly picking out filamentous DNA for washing, adding 500. mu.L 76% ethanol for washing for 2 times, placing on an ultra-clean workbench for drying, and adding 100. mu.L TE buffer solution or ddH.2Dissolving DNA with O, adding 20 μ g/L ribonuclease, digesting at 37 deg.C for 30min, adding equal volume of chloroform and isoamylol (24: 1), and mixingHomogenizing at 1000rpm for 10min, adding 2 times volume of anhydrous ethanol, mixing, precipitating, adding 500 μ L ethanol, washing for 2 times, blow drying on ultra-clean bench, adding 50 μ L ddH2O dissolves the DNA.
Example 2
Chloroplast genome sequencing
Extracting qualified genome DNA, fragmenting the qualified genome DNA by using a mechanical breaking method (ultrasonic wave), purifying, repairing the tail end, adding A to the 3' end, connecting a sequencing joint, selecting the size of the fragment by using agarose gel electrophoresis, performing PCR amplification on the fragment with proper size to form a sequencing library, wherein the specific flow of the construction of the sequencing library is shown in figure 3, chloroplast genome sequencing is performed on an Illumina Hiseq 2000 platform (Illumina, San Diego, CA), and the sequencing strategy is PE 150.
Example 3
Quality control of sequencing data
Filtering the original data by using fastp (v0.20.0, https:// github. com/OpenGene/fastp) software to obtain high-quality sequencing data (Reads), cutting sequencing joints and primer sequences in the Reads, filtering Reads with the average mass value of less than Q5, and filtering Reads with the number of N being more than 5.
Example 4
Assembly of genomic sequencing data
To reduce the complexity of subsequent sequence assembly, the NCBI chloroplast genome database was aligned using the bowtie2(v2.2.4) very-sensitive-local pattern, and the aligned sequenced sequences were taken as chloroplast genome sequencing sequences (cpDNA sequences) of the samples. The assembly core module adopts SPAdes v3.10.1 software to assemble chloroplast genome, and kmer respectively uses 55, 87 and 121 to assemble independent reference genome. However, in general, due to the characteristics of second-generation sequencing, repeated genome sequences, specific genome structures, and the like, the complete circular genome sequence cannot be directly obtained by one-time splicing, and other strategies are adopted to obtain the complete circular genome sequence. The assembly process is divided into the following 7 steps:
step 1: assembling the cpDNA sequence by SPAdes software to obtain the SEED sequence of the chloroplast genome;
step 2: kmer iterative extended seed, if the result of Step2 is one contig, the result is determined as pseudo genome sequence, and Step6 is directly carried out;
step 3: connecting contig sequences obtained by Step2 by using SSPACE v2.0 software to obtain scafffolds;
step 4: GAP supplementation is carried out on the scaffolds sequence obtained from Step3 by using Gapfiller v2.1.1 software;
step 5: if GAP still exists after the operation is finished, designing a primer, carrying out PCR sequencing, and then assembling until a complete pseudo genome sequence is obtained;
step 6: aligning the sequencing sequence to pseudo genome for genome correction;
step 7: and (3) according to the structure of the chloroplast, carrying out coordinate rearrangement on the corrected pseudo genome to obtain a complete chloroplast circular genome sequence.
Finally, the published chloroplast genome (NC-045907.1) is used as a reference, and the complete chloroplast genome sequence is assembled by using the Geneius Prime to obtain the complete sequence of the pockmarked chloroplast genome (particularly, see a sequence file).
Example 5
Chloroplast genome mapping using OGDRAW (https:// chlorobox. mpimp-golm. mpg.de/0gdraw. html) a chloroplast genome mapping was made, and the results are shown in fig. 1, in which the forward-encoded gene is located outside the circle and the reverse-encoded gene is located inside the circle. The inner gray circles represent the GC content.
Example 6
Germplasm identification of pockmarkets and closely related species thereof
Based on the above-mentioned sequence of chloroplast genome of pockmarked coconut, the germplasm identification method of pockmarked coconut and its kindred species includes the following steps: the plant DNA was extracted, and amplification was performed using the extracted DNA as a template using designed Primer1 and Primer2 as amplification primers.
The PCR primers were designed based on the group sequences using chloroplasts and synthesized by Biotechnology engineering (Shanghai) GmbH, the product was 1046 bp:
primer1：5’-TCCGCCTGAGGAAGCAGGGGCA-3’
primer2：5’-ACGGGACTGGCTTTAGAAACC-3’
and (3) PCR reaction system: the specific reaction is as follows:
the PCR amplification program conditions are as follows: pre-denaturation at 94 ℃ for 4 minutes, denaturation at 94 ℃ for 50 seconds, annealing at 54 ℃ for 37 seconds, annealing at 72 ℃ for 90 seconds, and fully extending at 72 ℃ for 6 minutes after 30 cycles of three steps of denaturation, annealing and extension. The whole PCR reaction process is amplified on an Eppendorf Mastercycler nexus PCR instrument.
Example 7
Gel electrophoresis analysis of PCR products
The amplification result is detected by agarose gel electrophoresis, and the result is shown in fig. 2, wherein the Marker is a DL2000 DNA Marker produced by baobaozhen biotechnology (Dalian) limited, and the sizes of the fragments are sequentially from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp. 1-3 parts of golden-luster bamboo joint coconut, 4-6 parts of bamboo joint coconut, 8-10 parts of pocket coconut and 13-15 parts of tassel-ludwigia bamboo joint coconut, 7, 11 and 12 parts of blank lanes, wherein the size of a specific amplification product of the pocket coconut is about 1000bp through gel electrophoresis, the size of a fragment of the bamboo joint coconut is 750bp, and the size of a fragment of the tassel-ludwigia bamboo joint coconut is 1500bp, so that the pocket coconut and related plants thereof can be identified through the size of the gel electrophoresis fragment after PCR specific amplification.
Claims (7)
1. The identification method of the genome and germplasm of the chloroplast of the pockmarkets is characterized by comprising the following steps: the method for identifying the genome and the germplasm comprises the following steps:
(1) extraction of plant DNA
Fully grinding fresh plant leaves by using liquid nitrogen, and extracting DNA of the pocket coconut and the closely related species thereof by using an improved CTAB method;
(2) chloroplast genome sequencing
After the sample genome DNA is qualified, fragmenting the DNA by using ultrasonic waves, then carrying out fragment purification, end repairing, adding A at the 3' end, connecting a sequencing joint on the fragmented DNA, then carrying out fragment size selection by using agarose gel electrophoresis, carrying out PCR amplification to form a sequencing library, carrying out library quality inspection on the built library, and sequencing the library qualified by quality inspection by using an Illumina Novaseq platform;
(3) quality control of sequencing data
Filtering the original data by using fastp v0.20.0 software, and obtaining high-quality sequencing data after filtering;
(4) assembly of genomic sequencing data
Aligning an NCBI chloroplast genome database by using a bowtie2 v2.2.4very-sensitive-local mode, using the aligned sequencing sequence as a chloroplast genome sequencing sequence of a sample, assembling a chloroplast genome by using a SPAdes v3.10.1 software in an assembly core module, and assembling an independent reference genome by using 55, 87 and 121 for kmers respectively;
(5) chloroplast genome map
Using OGDRAW to make a chloroplast genome map;
(6) germplasm identification of pockmarkets and closely related species thereof
PCR amplification was performed using the extracted DNA as a template, using designed Primer1 and Primer2 as amplification primers.
2. The identification of the genome and germplasm of claim 1, wherein: the improved CTAB method in the step (1) is that plant leaves are ground into powder in liquid nitrogen, 0.1g of the powder is taken and transferred into a 2mL microcentrifuge tube, 630 muL of CTAB buffer solution which is 1.5 times of that preheated to 65 ℃ is added, 70 muL of absolute ethyl alcohol is added, then the mixture is heated in a water bath kettle at 65 ℃ for 30min, the middle of the mixture needs to be shaken for 4-5 times, the microcentrifuge tube is taken out for cooling, and then 700 muL of chloroform is added: isoamyl alcohol 24: 1, uniformly mixing, centrifuging at 10000rpm for 10min, taking supernatant into a new microcentrifuge tube, adding isopropanol with the volume of 2/3 of that of the supernatant, uniformly mixing, precipitating and standing, centrifuging at 4000rpm for 3min, taking supernatant, or directly picking out filamentous DNA (deoxyribonucleic acid) for washing, adding 500 mu L76% ethanol for washing for 2 times, placing on a superclean bench for drying, addingInto 100. mu.L of TF buffer or ddH2O dissolving DNA, adding 20. mu.g/L ribonuclease, digesting at 37 ℃ for 30min, adding equal volume of chloroform: isoamyl alcohol 24: 1, uniformly mixing, centrifuging at 1000rpm for 10min, adding 2 times of volume of absolute ethyl alcohol, uniformly mixing, precipitating, adding 500 mu L of ethyl alcohol, washing for 2 times, drying in an ultra-clean bench, adding 50 mu L of ddH20 dissolves the DNA.
3. The identification of the genome and germplasm of claim 1, wherein: the sequencing read length of the chloroplast in the step (2) is PF 150.
4. The identification of the genome and germplasm of claim 1, wherein: the filtering standard of the step (3) is as follows: the sequencing adapters and primer sequences in Reads were truncated, and Reads with average quality values less than Q5 were filtered out, and N Reads with numbers greater than 5 were filtered out.
5. The identification of the genome and germplasm of claim 1, wherein: the assembling of the genome sequencing data of the step (4) comprises the following steps:
(1) assembling the cpDNA sequence by SPAdes software to obtain the SEED sequence of the chloroplast genome;
(2) a kmer iterative extended seed, if the result of the step (2) is one contig, the result is determined as a pseudo genome sequence, and the step (6) is directly carried out;
(3) connecting the contig sequences obtained in the step (2) by using SSPACE v2.0 software to obtain scafffolds;
(4) performing GAP supplement on the scaffolds sequence obtained in the step (3) by using Gapfiller v2.1.1 software;
(5) if 6AP still exists after the operation is finished, designing a primer, carrying out PCR sequencing, and then assembling until a complete pseudo genome sequence is obtained;
(6) aligning the sequencing sequence to pseudo genome for genome correction;
(7) and (3) according to the structure of the chloroplast, carrying out coordinate rearrangement on the corrected pseudo genome to obtain a complete chloroplast circular genome sequence.
6. The identification of the genome and germplasm of claim 1, wherein: the PCR primer in the step (6) is designed by utilizing chloroplast to design the PCR primer based on a group sequence, and the product is 1046 bp:
primer1：5’-TCCGCCTGAGGAAGCAGGGGCA-3’；
primer2：5’-ACGGGACTGGCTTTAGAAACC-3’。
7. the identification of the genome and germplasm of claim 1, wherein: the PCR amplification program conditions of the step (6) are as follows: pre-denaturation at 94 ℃ for 4 minutes, denaturation at 94 ℃ for 50 seconds, annealing at 54 ℃ for 37 seconds, annealing at 72 ℃ for 90 seconds, and denaturation, annealing and extension for 30 cycles, and finally fully extending at 72 ℃ for 6 minutes, wherein the whole PCR reaction process is amplified on an Eppendorf Mastercycler nexus PCR instrument.
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