CN117904344A - SNP molecular marker combination for constructing large-diameter material populus americana DNA fingerprint and application thereof - Google Patents
SNP molecular marker combination for constructing large-diameter material populus americana DNA fingerprint and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker combination for constructing a large-diameter-material populus tomentosa DNA fingerprint, and application and a method thereof, belonging to the field of biological breeding of populus tomentosa. The invention discloses an SNP molecular marker combination for constructing a large-diameter material populus americana DNA fingerprint, which consists of 8 SNP molecular markers, wherein the nucleotide sequences of the SNP molecular markers respectively correspond to SEQ ID NO.1-8. According to the invention, 24 large-diameter populus americana DNA are used as templates, the screened 20 pairs of SNP primers are adopted for PCR amplification and then subjected to high-throughput sequencing comparison, 8 SNP molecular markers are finally determined as SNP molecular marker combinations for constructing the large-diameter populus americana DNA fingerprint, and finally the DNA fingerprint of the large-diameter populus americana consisting of 8 SNP loci is obtained. The single nucleotide marking site for detection has the advantages of simplicity, rapidness and low cost, and can be applied to production.
Description
Technical Field
The invention relates to a SNP molecular marker combination for constructing a large-diameter-material populus americana DNA fingerprint and application thereof, belonging to the field of biological breeding of populus americana.
Background
Poplar is an important fast-growing industrial wood tree species and greening afforestation tree species in China. Since the beginning of the seventies of the 20 th century, american black poplar such as I-69, I-72, I-63, I-214 and the like and its hybrid clone were introduced, poplar resources have been the subject of forest resources in Jiangsu province through the development of half an excessive century. The populus americana and the hybrid thereof have the advantages of fast growth, high yield, short rotation period, wide adaptability and the like, and are not only the preferred tree species for cultivating large-diameter high-quality industrial resource materials, but also pioneer tree species for urban and rural greening forestation. The populus americana is an important industry of economy in the North China, and plays an extremely important role in relieving the contradiction between wood supply and demand in China, maintaining ecological balance, realizing agricultural synergy, increasing income of farmers and the like.
The populus americana is a populus plant of populus of salicaceae, totally comprises more than 100 species of populus asiatica, is widely distributed worldwide, is more common in a warm zone or a warm zone region of 30-60 degrees N, is a short-term rotation of forestation tree species, and is beneficial to solving the problems of ecological environment management and wood shortage. The new and superior species of populus jaborandi promoted in the development of Chinese forestry at present mainly originate from artificial hybridization breeding, have the characteristics of early fast growth, good materials, strong resistance and the like, and create great ecological benefit, economic benefit and social benefit. Therefore, cross breeding is still an important means for breeding fine varieties of populus jaborandi at present or even in future. The populus americana is native to the downstream region of the Missiubi river in North America, is one of important fast-growing industrial tree species and greening afforestation tree species in the south plain region of China introduction, and is a common parent for artificially hybridizing and breeding new varieties. At present, the development process of the populus americana also has a plurality of problems which need to be solved urgently, such as single variety, a plurality of low-yield forest stands, low improved seed conversion rate, serious flocculation and the like, seriously influences the production and development of the populus americana in the middle and downstream plain areas of the Yangtze river, and needs to select and select new species of the southern populus americana which are suitable for local fast growth, high quality, high yield and no flocculation for updating. Therefore, the development of a high-efficiency and accurate large-diameter material American black poplar identification technical means has great significance for the breeding of large-diameter material American black poplars in China.
Genetic markers based on inter-individual nucleotide sequence variation are direct reflections of DNA-level genetic polymorphisms. The single nucleotide polymorphism (Single nucleotide polymorphisms, SNP) refers to the polymorphism of a DNA sequence at the genome level caused by the variation of single nucleotide, and the SNP molecular marker has the characteristics of large quantity, wide distribution, high genetic stability, co-dominance and the like, and is an important technical means in the current molecular genetics research. DNA fingerprints constructed by SNP can reflect the individual differences of organisms essentially, have high individual specificity and stable environment, and can accurately and rapidly identify new varieties and existing varieties. Therefore, aiming at the populus americana with large diameter materials on the market, the construction of the DNA fingerprint based on the SNP molecular marker has a very positive effect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an SNP molecular marker combination for constructing a large diameter material American black poplar DNA fingerprint and application thereof, which are used for rapidly and accurately identifying the clone genotype of the poplar large diameter material.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
The SNP molecular marker combination for constructing the large-diameter material populus americana DNA fingerprint consists of 8 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers respectively correspond to SEQ ID NO.1-8.
Each SNP molecular marker corresponds to one SNP site:
The first SNP molecular marker corresponds to SNP site SNP2: 5_5078125;
the second SNP molecular marker corresponds to SNP site SNP3:5_5468930;
the third SNP molecular marker corresponds to SNP site SNP5:8_43714;
the fourth SNP molecular marker corresponds to SNP site SNP7: 9_853188;
the fifth SNP molecular marker corresponds to SNP site SNP8:9_8784110;
the sixth SNP molecular marker corresponds to SNP locus SNP10:11_986759;
The seventh SNP molecular marker corresponds to SNP locus SNP11: 11_1822946;
the eighth SNP molecular marker corresponds to SNP site SNP14:13_276618.
The construction method for constructing the SNP molecular marker combination of the large diameter material populus americana DNA fingerprint comprises the following steps:
1) Initially screening 20 SNP loci which are obviously associated with growth traits as candidate loci;
2) Designing 20 pairs of SNP primers according to the positions of the candidate SNP loci;
3) Taking 24 large-diameter material populus americana DNA as a template, and carrying out PCR amplification on the screened 20 pairs of SNP primers to obtain a PCR amplification product;
4) Purifying the PCR amplified product, and then carrying out high-throughput sequencing to determine the genotype of the SNP locus;
5) According to the genotyping result of the SNP locus, 8 SNP molecular markers are finally determined as SNP molecular marker combinations for constructing the large-diameter material populus americana DNA fingerprint.
A PCR amplification primer pair combination of a SNP molecular marker combination, comprising:
SNP2-F：5’-GGCCACAAAATGAATTTCAGAAG-3’，
SNP2-R：5’-GCTATCACAGTTGCAAATTGTCA-3’；
SNP3-F：5’-GTTTATTGATTGGGTGTTGTTTG-3’，
SNP3-R：5’-GATGAAATGCGCTAAAAATCC-3’；
SNP5-F：5’-AGTCTACACGAGCCCTTCATTT-3’，
SNP5-R：5’-ATGGAAGCAAGATGGCTAGACT-3’；
SNP7-F：5’-GTCCCTGGATTTCAATCTCA-3’，
SNP7-R：5’-TACGAATCCCATATGTACCTCC-3’；
SNP8-F：5’-TGATATTATCAGCTGTGTTGGGG-3’，
SNP8-R：5’-TCCTGGTTCTGAAGTTAGCTTCT-3’；
SNP10-F：5’-ACTGCTTTAACCAGCTTGTATCCT-3’，
SNP10-R：5’-CTCAACCCAAATCCACACAATA-3’；
SNP11-F：5’-TTAGGCTGAGAGAACCTCGTG-3’，
SNP11-R：5’-TTCCTCGTATACCGCTCTCTA-3’；
SNP14-F：5’-GGAATTCAAGAACCTGCAATCA-3’，
SNP14-R：5’-TTCAATTGCTAGAACTCTGGGTC-3’。
The SNP molecular marker combination for constructing the DNA fingerprint of the populus tremuloides is applied to constructing the DNA fingerprint of the populus tremuloides.
The PCR amplification primer pair combination for amplifying the SNP molecular marker combination is applied to the construction of the large-diameter populus americana DNA fingerprint.
The application of the SNP molecular marker combination for constructing the DNA fingerprint of the large diameter material populus tremuloides in preparing the kit for screening the large diameter material populus tremuloides.
The application of a PCR amplification primer pair combination for amplifying SNP molecular marker combinations in the preparation of a kit for screening large-diameter populus americana.
The invention has the beneficial effects that:
According to the invention, 20 SNP loci which are obviously associated with growth traits are screened out as candidate loci; designing 20 pairs of SNP primers according to the positions of the candidate SNP loci; taking 24 large-diameter material populus americana DNA as a template, and carrying out PCR amplification on the screened 20 pairs of SNP primers to obtain a PCR amplification product; purifying the PCR amplified product, and then carrying out high-throughput sequencing to determine the genotype of the SNP locus; according to the genotyping result of the SNP loci, 8 SNP molecular markers are finally determined as SNP molecular marker combinations for constructing the DNA fingerprint of the large-diameter populus tremuloides, and the DNA fingerprint of the large-diameter populus tremuloides consisting of 8 SNP loci is finally obtained. The single nucleotide marking site for detection has the advantages of simplicity, rapidness and low cost, and can be applied to production.
Drawings
FIG. 1 is a genotyping map of 20 SNP loci.
Detailed Description
The present invention will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art.
The big diameter material American black poplar refers to a log with a small head peeling diameter of at least 24cm and a length of more than 2.5 m.
Example 1
1. The 20 SNP sites with significant association to growth traits in the previous GWAS study (Xu et al:A computational framework for mapping the timing of vegetative phase change.New Phytol 2016.) were initially screened and used as candidate sites, the detailed information of which is as follows:
TABLE 1 information about candidate SNP loci
2. Based on the position of the candidate SNP site, 20 pairs of SNP primers were designed using Primer 5.0 software. The design method is as follows: firstly, determining the position of a sequence where an SNP locus is located, and then respectively designing a forward primer and a reverse primer at the upstream and downstream of the SNP locus, wherein the parameters of primer design are that the primer length is 18-23bp; the Tm is 59-61 ℃, so that the Tm values of the upstream and downstream primers are ensured to be consistent as far as possible, and the Tm value is generally not more than 2 ℃; GC content is 40-60% (45-55% most preferred); the size of the PCR product is 350-400bp. The primer sequences are shown below:
TABLE 2 primer sequence information for 20 SNPs
3. The 24 large diameter material America black Yang Moxing lines (named MY1-24 respectively) on the farm of siyang county siyang in Suzhou-Suqian, were selected, 3-5 complete leaves without plant diseases and insect pests were selected for each clone for DNA extraction, and placing in a dry ice box, storing and transporting to a Nanjing forestry university laboratory, and transferring to an ultralow temperature refrigerator at-80 ℃ for later use. Extraction of 24 american black Yang Moxing-line sample DNA was performed according to the instructions of TIANGEN (DP 360) kit. After extraction, degradation and contamination of DNA was detected on a 1.0% agarose gel. The DNA purity was checked using a spectrophotometer (Implen, WESTLAKE VILLAGE, CA, USA). Placing the extracted DNA sample in an ultralow temperature refrigerator at-20 ℃ for preservation.
4. Taking 24 large-diameter material populus americana DNA as a template, and carrying out PCR amplification by adopting 20 screened SNP primers to obtain a PCR amplification product; after the reaction, 3. Mu.L of the PCR product was added to 4. Mu. LLoading buffer, and then 3. Mu.L of ddH 2 O was added thereto, and the PCR amplification product was detected by electrophoresis on an 8% polyacrylamide gel and then silver staining. The target band was detected without other bands and primer dimer. The 10. Mu.L LA-taq enzyme PCR reaction system was as follows: 5 μL of Premix Taq (LA Taq version2.0plus dye); 0.2. Mu. Mol/L SNP primer; 40ng of DNA template; the remaining ddH 2 O was filled in. The PCR reaction procedure was: pre-denaturation at 94℃for 3min;35 cycles at 94℃for 30s, optimum Tm for 30s,72℃for 30s; extending at 72deg.C for 1min, and storing at 4deg.C.
5. After purification of the PCR product, double-ended 250X high throughput sequencing was performed using Illumina Miseq. Obtaining raw data (raw reads) in FASTQ format first filters the quality using FASTP software to obtain clean data (CLEAN READS) from the low quality read from the raw data. And counting the sequencing data of all samples, and obtaining 166897 strips CLEAN READS by quality control, wherein the GC content is basically kept between 40.35 and 46.82 percent, the percentage of Q20 bases of each sample is more than 89.86 percent, and the content of Q30 bases is not less than 86.8 percent, which indicates that the quality of the data of each sample is qualified. Clean data were then aligned to the reference sequence using bwa-mem2 (v2.2.1). Candidate SNPs were genotyped using SAMtools (v 1.9). SNP sites of BAM files were checked manually using IGVs (INTEGRATIVE GE-nomics viewer, v2.11.0). Genotyping of 24 populus americana is shown in figure 1.
6. And finally, determining a set of 8 SNP molecular markers by utilizing a Perl program, wherein the set of 8 SNP molecular markers can distinguish the large-diameter populus tremuloides clone and is used for constructing SNP molecular marker combinations of DNA finger-print of the large-diameter populus tremuloides. The molecular marker combinations are shown below:
The first SNP molecular marker has a nucleotide sequence shown as SEQ ID NO.1, wherein Y is C or T;
the second SNP molecular marker has a nucleotide sequence shown as SEQ ID NO.2, wherein R is A or G;
The nucleotide sequence of the third SNP molecular marker is shown as SEQ ID NO.3, wherein R is A or G;
a fourth SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.4, wherein R is A or G;
A fifth SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.5, wherein R is A or G;
a sixth SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.6, wherein K is T or G;
a seventh SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.7, wherein W is T or A;
And the eighth SNP molecular marker has a nucleotide sequence shown in SEQ ID NO.8, wherein Y is C or T.
Each SNP molecular marker corresponds to one SNP site, the first SNP molecular marker corresponds to SNP2 (5_5078125), the second SNP molecular marker corresponds to SNP3 (5_5468930), the third SNP molecular marker corresponds to SNP5 (8_43714), the fourth SNP molecular marker corresponds to SNP7 (9_853188), the fifth SNP molecular marker corresponds to SNP8 (9_8784110), the sixth SNP molecular marker corresponds to SNP10 (11_986759), the seventh SNP molecular marker corresponds to SNP11 (11_184946), and the eighth SNP molecular marker corresponds to SNP14 (13_276618).
Example 2
And detecting the typing consistency of 8 SNP loci in table 2 in 24 large diameter material populus americana according to SNP molecular markers (SNP information in a sequencing database), and constructing a fingerprint of the large diameter material populus americana, wherein the result is shown in table 3. The SNP molecular markers form the molecular identity card of each variety according to the sequence. For example, the SNP molecular identity card of the populus americana MY1 clone (populus americana T20) is CARRAKWT.
TABLE 3 DNA finger print of 24 large diameter American black poplar clones
Wherein R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C, and W is A or T.
The American black poplar varieties corresponding to MY1-24 are respectively:
MY1 is populus americana NLT2-20; MY2 is populus nigra NL-T2; MY3 is populus jaborandi NL-T120; MY4 is populus nigra NL-T1; MY5 is populus jaborandi NL-S392; MY6 is populus jaborandi NL-S3907; MY7 is populus jaborandi NL-S3814; MY8 is a national fine variety NL-S3804 of populus americana; MY9 is populus jaborandi NL-S3801; MY10 is populus jaborandi NL-S372; MY11 is populus nigra NL-S3702; MY12 is populus nigra NL-S3700; MY13 is populus nigra NL-S3261; MY14 is populus jaborandi NL-S3244; MY15 is populus jaborandi NL-S3240; MY16 is populus nigra NL-S3239; MY17 is populus jaborandi NL-S3236; MY18 is populus nigra NL-S3229; MY19 is populus jaborandi NL-S3201; MY20 is populus nigra NL-S3183; MY21 is populus jaborandi NL-S3121; MY22 is populus jaborandi NL-S3109; MY23 is populus jaborandi NL-S3503; MY24 is Populus americana NL-S3016.
Example 3
A kit for screening large diameter material populus americana, which comprises the following molecular marker combination:
The nucleotide sequence of the first SNP molecular marker is shown as SEQ ID NO. 1; the second SNP molecular marker has a nucleotide sequence shown as SEQ ID NO.2, wherein R is A or G; the nucleotide sequence of the third SNP molecular marker is shown as SEQ ID NO.3, wherein R is A or G; a fourth SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.4, wherein R is A or G; a fifth SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.5, wherein R is A or G; a sixth SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.6, wherein K is T or G; a seventh SNP molecular marker, the nucleotide sequence of which is shown as SEQ ID NO.7, wherein W is T or A; and the eighth SNP molecular marker has a nucleotide sequence shown in SEQ ID NO.8, wherein Y is C or T.
Each SNP molecular marker corresponds to one SNP site, the first SNP molecular marker corresponds to SNP2 (5_5078125), the second SNP molecular marker corresponds to SNP3 (5_5468930), the third SNP molecular marker corresponds to SNP5 (8_43714), the fourth SNP molecular marker corresponds to SNP7 (9_853188), the fifth SNP molecular marker corresponds to SNP8 (9_8784110), the sixth SNP molecular marker corresponds to SNP10 (11_986759), the seventh SNP molecular marker corresponds to SNP11 (11_184946), and the eighth SNP molecular marker corresponds to SNP14 (13_276618).
Each SNP site corresponds to a corresponding SNP primer shown in table 2.
Therefore, different large-diameter varieties of the populus tomentosa can be simply and accurately identified in different environments and different growing seasons, the growing and developing conditions of the populus tomentosa can be predicted, the genetic improvement process of the populus tomentosa is accelerated, and the breeding period is shortened.
Claims (8)
1. The SNP molecular marker combination for constructing the large-diameter material populus americana DNA fingerprint consists of 8 SNP molecular markers, and the nucleotide sequences of the SNP molecular markers respectively correspond to SEQ ID NO.1-8.
2. The SNP molecular marker combination for constructing a large diameter populus americana DNA fingerprint according to claim 1, wherein each SNP molecular marker corresponds to one SNP site:
The first SNP molecular marker corresponds to SNP site SNP2: 5_5078125;
the second SNP molecular marker corresponds to SNP site SNP3:5_5468930;
the third SNP molecular marker corresponds to SNP site SNP5:8_43714;
the fourth SNP molecular marker corresponds to SNP site SNP7: 9_853188;
the fifth SNP molecular marker corresponds to SNP site SNP8:9_8784110;
the sixth SNP molecular marker corresponds to SNP locus SNP10:11_986759;
The seventh SNP molecular marker corresponds to SNP locus SNP11: 11_1822946;
the eighth SNP molecular marker corresponds to SNP site SNP14:13_276618.
3. The method for constructing the SNP molecular marker combination for constructing the large diameter material populus americana DNA fingerprint spectrum, which is disclosed in claim 1, comprises the following steps:
1) Initially screening 20 SNP loci which are obviously associated with growth traits as candidate loci;
2) Designing 20 pairs of SNP primers according to the positions of the candidate SNP loci;
3) Taking 24 large-diameter material populus americana DNA as a template, and carrying out PCR amplification on the screened 20 pairs of SNP primers to obtain a PCR amplification product;
4) Purifying the PCR amplified product, and then carrying out high-throughput sequencing to determine the genotype of the SNP locus;
5) According to the genotyping result of the SNP locus, 8 SNP molecular markers are finally determined as SNP molecular marker combinations for constructing the large-diameter material populus americana DNA fingerprint.
4. The PCR amplification primer pair combination of the SNP molecular marker combination of claim 1, comprising:
SNP2-F：5’-GGCCACAAAATGAATTTCAGAAG-3’，
SNP2-R：5’-GCTATCACAGTTGCAAATTGTCA-3’；
SNP3-F：5’-GTTTATTGATTGGGTGTTGTTTG-3’，
SNP3-R：5’-GATGAAATGCGCTAAAAATCC-3’；
SNP5-F：5’-AGTCTACACGAGCCCTTCATTT-3’，
SNP5-R：5’-ATGGAAGCAAGATGGCTAGACT-3’；
SNP7-F：5’-GTCCCTGGATTTCAATCTCA-3’，
SNP7-R：5’-TACGAATCCCATATGTACCTCC-3’；
SNP8-F：5’-TGATATTATCAGCTGTGTTGGGG-3’，
SNP8-R：5’-TCCTGGTTCTGAAGTTAGCTTCT-3’；
SNP10-F：5’-ACTGCTTTAACCAGCTTGTATCCT-3’，
SNP10-R：5’-CTCAACCCAAATCCACACAATA-3’；
SNP11-F：5’-TTAGGCTGAGAGAACCTCGTG-3’，
SNP11-R：5’-TTCCTCGTATACCGCTCTCTA-3’；
SNP14-F：5’-GGAATTCAAGAACCTGCAATCA-3’，
SNP14-R：5’-TTCAATTGCTAGAACTCTGGGTC-3’。
5. the use of the SNP molecular marker combination for constructing the large diameter material populus tomentosa DNA fingerprint of claim 1 in constructing the large diameter material populus tomentosa DNA fingerprint.
6. The use of the PCR amplification primer pair combination for amplifying SNP molecular marker combinations as set forth in claim 4 for constructing large diameter material populus americana DNA fingerprint.
7. The use of the SNP molecular marker combination for constructing large diameter populus tomentosa DNA fingerprint of claim 1 in the preparation of a kit for screening large diameter populus tomentosa.
8. The use of the PCR amplification primer pair combination for amplifying SNP molecular marker combination as set forth in claim 4 for preparing a kit for screening large diameter populus americana.
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