CN108715901B - SNP marker related to millet plant height character and detection primer and application thereof - Google Patents
SNP marker related to millet plant height character and detection primer and application thereof Download PDFInfo
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Abstract
The application discloses an SNP marker related to the plant height trait of millet and a detection primer and application thereof. The SNP marker is positioned in bin markers from 18023413bp to 21734440bp of a fourth chromosome, and is closely linked with the height of a millet plant. The SNP marker related to the millet plant height character can be used for molecular marker assisted breeding of the millet plant height character, the millet plant height can be predicted through SNP marker detection, scientific basis is provided for early identification or screening breeding of the millet plant height character, and important theoretical and practical guiding significance is achieved for accelerating genetic breeding or improvement process of millet varieties. The primer pair for detecting the SNP markers can be used for typing the plant height of the millet and detecting the difference of SNP expression.
Description
Technical Field
The application relates to the technical field of millet breeding, in particular to an SNP marker related to the plant height character of millet and a detection primer and application thereof.
Background
China is the country with the largest cultivation area and the highest yield in the native place of millet and the world, and the yield accounts for about 80 percent of the total amount of the world. Meanwhile, China is also the country with the largest quantity of millet genetic resources and the most abundant diversity.
The plant height, the ear length and the like are important factors influencing the millet yield, and the research on genes related to the yield traits such as the plant height of the millet and the like and the position function thereof has important significance for guiding the genetic breeding of the millet.
Single Nucleotide Polymorphism (SNP) is a DNA sequence polymorphism caused by variation of a single nucleotide base at the genome level, and is large in number, widely distributed, and good in genetic stability in the genome. With the development of gene sequencing technology and the reduction of cost, the same gene or gene fragment of different individuals is directly sequenced and compared with the sequence, so that whether the base has variation or not can be determined. Therefore, SNP detection is beneficial to genotyping and is suitable for rapidly and massively screening the relation between unknown or known SNP and certain genetic traits.
At present, QTL positioning and SNP marker research related to the quantitative traits of millet mainly focuses on the development and utilization of SSR markers, and the like, but the development and application research of SNP molecular markers for detecting polymorphism by utilizing degenerate genome sequencing of a population inbred line has not been reported. Therefore, the development of the quantitative character SNP molecular marker of the millet is developed, and an auxiliary breeding system is established, so that the method has important significance for improving the yield of the millet and saving the breeding cost.
Disclosure of Invention
The application aims to provide a novel SNP marker related to the millet plant height character, and a detection primer and application thereof.
In order to achieve the purpose, the following technical scheme is adopted in the application:
one aspect of the application discloses an SNP marker related to the height trait of a millet plant, wherein the SNP marker is positioned in bin markers from 18023413bp to 21734440bp of a fourth chromosome, and is tightly linked with the height of the millet plant.
It should be noted that, through research, a sequence closely linked with the height of the millet plant is discovered, and the SNP marker in the sequence directly affects the height property of the millet plant, namely the bin marker from 18023413bp to 21734440bp of the fourth chromosome.
Further research shows that the bases of the bin markers from 18023413bp to 21734440bp of the fourth chromosome, especially SNP markers, are C or T and are closely linked with the height of the millet plant.
In one implementation mode of the application, one SNP marker, namely PH-04-413 site is specifically disclosed, the PH-04-413 site is located at 18023413bp position of the fourth chromosome sequence, the PH-04-413 site is located in the sequence shown in Seq ID No.1, and the PH-04-413 site is 163 th base from the 5' end in the sequence shown in Seq ID No. 1.
It should be noted that bin markers from 18023413bp to 21734440bp of the fourth chromosome are closely linked with the height of the millet plant, wherein a plurality of SNP markers may be contained, the PH-04-413 site is only the SNP marker related to the height trait of the millet plant confirmed in one implementation mode of the application, and other SNP markers in the bin markers are not excluded under the guidance of the application.
Preferably, the SNP marker related to the millet plant height trait in the application utilizes a composite interval mapping method for the SNP marker, the total significance level is 5%, the LOD value detected by QTL is 3.2792, and the phenotypic variation interpretation rate is 2.76%.
The other side of the application discloses a primer pair for detecting the SNP marker of the application, wherein an upstream primer of the primer pair is a sequence shown by Seq ID No.2, and a downstream primer of the primer pair is a sequence shown by Seq ID No. 3;
Seq ID No.2：5’-AAACGAGCAACGGGTAGGC-3’
Seq ID No.3：5’-GGAAAGACAGCAGGAACAGAG-3’。
the other side of the application discloses a kit for detecting the SNP marker of the application, and the kit comprises the primer pair of the application.
Preferably, the kit of the present application further comprises reagents for PCR amplification.
The application also discloses a method for detecting the SNP marker, which comprises the steps of carrying out PCR amplification on the genomic DNA of the millet to be detected by adopting the primer pair or the kit, and obtaining the base condition of the SNP marker according to the sequencing result of the PCR amplification product.
The application also discloses a method for predicting the plant height of the millet, which comprises the steps of carrying out PCR amplification on the genomic DNA of the millet to be predicted by adopting the primer pair or the kit, obtaining the base condition of the SNP marker of the application according to the sequencing result of the PCR amplification product, and predicting the plant height of the millet according to the base condition of the SNP marker.
The application also discloses application of the SNP marker in predicting the height of the millet plants, early identifying the height characters of the millet plants or assisting screening and breeding of the millet molecules.
The application also discloses application of the primer pair or the kit in predicting the height of the millet plants, identifying the height characters of the millet plants at early stage or screening and breeding assisted by millet molecules.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the SNP marker related to the millet plant height character can be used for molecular marker assisted breeding of the millet plant height character, the millet plant height can be predicted through SNP marker detection, scientific basis is provided for early identification or screening breeding of the millet plant height character, and important theoretical and practical guiding significance is achieved for accelerating genetic breeding or improvement process of millet varieties. The primer pair for detecting the SNP markers can be used for typing the plant height of the millet and detecting the difference of SNP expression.
Drawings
FIG. 1 is an electrophoretogram of parental genomic DNA of millet in the present example, wherein the first lane is lane M, which represents DL2000marker, the second lane, which is the lane marked with 1, represents genomic DNA of paternal "Ji Zhang Gu No." and the third lane, which is the lane marked with 2, represents genomic DNA of A2;
FIG. 2 is an electrophoresis diagram of PCR products of the parental genomic DNA of foxtail millet in the present example, wherein the first lane is the lane marked with A16-1, which shows the PCR product with the genomic DNA of the female parent "A2" as the template and 16_1 as the primer, the second lane is the lane marked with 316-1, which shows the PCR product with the genomic DNA of the male parent "Ji Zhang Gu No. one" as the template and 16_1 as the primer, and the third lane is the lane M, which shows DL2000 marker;
FIG. 3 is a diagram showing the alignment results of the sequences of the PH-04-413 loci in the SNP markers of the female parent and the male parent in the example of the present application, wherein the PH-04-413 loci are in the bin markers from the 18023413bp to the 21734440bp of the fourth chromosome, specifically, the PH-04-413 loci are at the 18023413bp position of the fourth chromosome sequence, the female parent base is C, and the male parent base is T.
Detailed Description
The application provides SNP markers of a bin marker region related to the plant height of millet, detection primers and application thereof. The SNP marker PH-04-413 tightly linked with the height of the millet plant is positioned in bin markers from 18023413bp to 21734440bp of a fourth chromosome, and is specifically positioned at 18023413 bp.
In this application, Bin means Bin mapping (i.e., Bin map), and the minimum unit (Recombination Bin) constituting a Recombination event is obtained using all Recombination site information in the mapped population. The obtained bin is used as a marker for subsequent linkage map construction and QTL positioning.
In the present application, a genetic map (genetic map or linking map) refers to a linear arrangement of relative positions between markers constructed on the basis of the recombination rate between genetic markers. Sequencing parent and filial generation populations of the mapping population based on a high-throughput resequencing technology, and taking a certain number of continuous SNPs as a basis for judging recombination sites of the filial generation to obtain a whole genome physical recombination map of each filial generation. Using all Recombination site information in the mapped population, the minimum unit (Recombination Bin) and Bin map (i.e., Bin map) that constitute the Recombination event are obtained and the resulting bins are used as markers for subsequent linkage map construction and QTL mapping.
The application provides male parent 'Ji Zhang Gu No. I' and female parent 'A2' materials of millet, F1 generation materials of hybridization of the male parent and the female parent, and 441 parts of self-bred 13 generation F2 group materials. Planting all the materials in Hainan, and recording and sorting the character data of plant height and the like. And after carrying out degenerate genome re-sequencing on each material, completing comparison and reference genome splicing to obtain the SNP polymorphic marker. And obtaining related SNP markers through plant height trait data, and verifying the SNP markers through clone sequencing.
The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Example high throughput sequencing and SNP marker information analysis
This example uses the male parent "Ji Zhang Gu I" and female parent "A2" material, the F1 generation material of the hybridization of the two, and the F2 group 441 parts material of the selfing 13 generation, i.e. recombination selfing line (RILs). Planting all the materials in Hainan, and recording and sorting the character data of plant height and the like. And after carrying out degenerate genome re-sequencing on each material, completing comparison and reference genome splicing to obtain the SNP marker. And (3) obtaining related SNP markers by analyzing plant height character data, and verifying the SNP markers by clone sequencing.
In this example, genomic DNA of each sample was extracted by RADSeq method, and a total of 444 samples including 441 RILs, 2 parents and 1F 1 individuals were obtained. Performing quality detection on the extracted genome DNA, performing enzyme digestion on the qualified DNA, recovering DNA fragments through electrophoresis, adding a joint for cluster preparation, and finally performing machine sequencing.
The specific steps of this example are as follows:
(1) weighing 1.0g of fresh leaves, shearing, putting into a mortar, grinding with liquid nitrogen, adding 3mL of 1.5 xCTAB, grinding into homogenate, transferring into a 15mL centrifuge tube, adding 1mL of 1.5 xCTAB into the mortar for washing, transferring into the centrifuge tube, uniformly mixing, and slowly shaking at intervals in a water bath at 65 ℃ for 30 min.
Wherein 1L of a 1.5 × CTAB formulation comprises: 15g of CTAB, 1mol/L of Tris.Cl 75mL with the pH value of 8.0, 0.5mol/L EDTA 30mL and 61.4g of NaCl, and adding deionized water to fix the volume to 1L; before use, about 2mL of mercaptoethanol was added to a final concentration of 0.2%.
(2) After the water bath is finished, cooling to room temperature, adding equal volume of chloroform/isoamylol, and gently mixing uniformly until the subnatant turns into dark green; wherein the volume ratio of chloroform to isoamyl alcohol is 24: 1.
(3) Centrifuging at 4200rpm for 10min, transferring the upper aqueous phase to a new 15mL centrifuge tube, adding 2 times volume of precooled absolute ethanol, mixing and standing for 5 min. The DNA was precipitated by standing at-20 ℃ for 30 min.
(4) Centrifuging at 4200rpm for 10min, discarding the supernatant, adding 1mL of 75% ethanol to wash the precipitate 1 time, inverting the centrifuge tube to dry the DNA, and adding 50. mu.L of TE to dissolve the DNA.
(5) The concentration of DNA was measured and adjusted to 20 ng/. mu.L with water.
(6) And digesting by adopting PstI enzyme to break the genome DNA, wherein the digestion reaction system of the PstI enzyme is 30 mu L and comprises the following steps: genomic DNA 20. mu. L, PstI enzyme 1. mu.L, 10 XPstI buffer 3. mu. L, Nuclease-free water 6. mu.L. After the enzyme digestion reaction system is prepared, incubation is carried out for at least 1 hour at the constant temperature of 37 ℃.
(7) And (3) performing a joint reaction, namely adding a sequencing joint to the broken genomic DNA fragment, wherein the joint reaction system is 40 mu L and comprises the following steps: 30 μ L of PstI digestion product, 1 μ L of sequencing linker, 10 Xligation buffer 1 μ L, T4DNA ligase 2 μ L, rATP 0.4.4 μ L, ddH2O 5.6μL。
(8) mu.L of each reaction product was taken and added to a new centrifuge tube, one set of 12 samples each, and the total volume of each set was 12. mu.L.
(9) Electrophoresis was performed on a 3% recovery gel for 1h, and the 300-and 700 bp-sized fragment was excised after EB staining. Gel purification was performed with QIAquick Kit and the recovered product was dissolved in 30. mu.L of EB solution.
(10) And (3) carrying out PCR reaction, carrying out PCR amplification on the cut and recovered fragments by adopting a universal primer of a sequencing adaptor, wherein the PCR reaction system is 25 mu L and comprises: 10 XBuffer 2.5 uL, 25mM dNTPs 0.25 uL, 5U/. mu.L Hi-Fi enzyme 0.25 uL, 10 uM sequencing adaptor forward primer 0.5 uL, 10 uM sequencing adaptor reverse primer 0.5 uL, gel cutting recovery product 20 uL, sterile water 1 uL.
The PCR reaction conditions were 94 ℃ pre-denaturation for 5min, followed by 10 cycles: denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 40 seconds, and extension at 72 ℃ for 3min after the cycle is finished.
(11) And (5) magnetic bead purification to complete library construction.
The specific purification method comprises the following steps: first, 1.2-fold volume of magnetic beads was added to the PCR product, and the mixture was allowed to stand for 10 min. Then, the mixture was adsorbed on a magnetic frame, and the supernatant was removed. Then, 500. mu.L of 70% ethanol was added to wash twice. After drying to dryness on a dry heat instrument, 15 μ L of EB solution was added and dissolved for 5 min. Finally, the supernatant was transferred to a 1.5mL centrifuge tube by adsorption on a magnetic rack.
(12) And (5) performing on-machine sequencing after the detection reaches the on-machine standard.
As a result: enzyme digestion, library building and sequencing are carried out on 444 samples, 75.99Gb raw data are obtained, and 171.54Mb of each individual is averaged. The sequencing sequences were aligned to the reference genome with an average alignment of 86.326%, an average coverage of 8.226%, and an average sequencing depth of 3.655 ×.
Wherein, 444 samples comprise 441 RILs, 2 parents and 1F 1 individuals; the reference genome in this example, Yugu genome, can be obtained from the following website:
https://www.ncbi.nlm.nih.gov/genome/？term＝Setaria+italica+(foxtail+millet)。
33771 SNP markers with polymorphism between parents are obtained by sequencing and information analysis. Selecting a plurality of SNPs as a window according to a window sliding method, sliding one SNP every time to determine the genotype of each window and the crossover site of each individual, and generating a bin genotype and a bin map. According to the bin genotype data, MSTMap software is used for constructing a genetic map, 2022 bins are positioned on 9 chromosomes, and then the genetic map data are introduced into MapChart software to integrate a genome genetic linkage map.
And carrying out QTL analysis on the plant height character phenotype of the millet by using the constructed high-density genetic map of the millet and adopting composite interval mapping analysis (CIM). QTL detection adopts 5% of total significance level, and according to 500 times of arrangement test results, critical LOD value of QTL analysis of plant height trait phenotype data is determined, and prediction bin interval and SNP locus information related to plant height are obtained through analysis.
The result shows that the SNP marker related to the height trait of the millet plant is positioned in 82.7 centiMorgan (cM), 82 th bin to 83 th bin (namely, chr4_ bin82-chr4_ bin83) of the fourth chromosome of the genetic linkage map, namely, the bin marker of the 18023413bp to 21734440bp of the fourth chromosome; the bases of the SNP markers related to the millet plant height character are all C or T; the PH-04-413 locus is also in the region, and is specifically located at the 18023413bp position of the fourth chromosome sequence. By using a composite interval mapping method, the total significance level of 5% is adopted, the LOD value detected by QTL is 3.2792, the phenotypic variation interpretation rate is 2.76%, and the Additive effect value (A) is 2.0272.
EXAMPLE two SNP marker validation
And (3) comparing the reference genome according to the predicted bin marker and the SNP locus to obtain a gene sequence of a related region, selecting about 300bp before and after the SNP locus, designing and developing SNP marker primers, and performing PCR amplification by taking male parent and female parent material DNAs as templates. Selecting the amplification products with normal amplification of the primers and the composite predicted size of the PCR amplification products to carry out gel cutting recovery, sequencing, and selecting the marker primers with the SNP locus difference of the male parent material amplification gene sequences and the female parent material amplification gene sequences. And (3) carrying out SNP marker primer PCR amplification and result sequencing by taking F1 generation and F2 generation materials as templates, and selecting a marker primer which accords with genotyping and is related to phenotypic traits.
Firstly, according to the sequencing result after PCR product recovery, the marker of the difference of the SNP loci of the amplified gene sequences of the male parent material and the female parent material is selected. The method comprises the following specific steps:
(1) according to the steps (1) to (4) in example one, male and female genomic DNAs were extracted by CTAB method, respectively.
(2) The extracted male parent and female parent genomic DNAs were detected by 0.8% agarose gel electrophoresis, and the detection results are shown in FIG. 1, wherein the second lane, lane labeled 1, represents the male parent "Ji Zhang Gu No.' genomic DNA, and the third lane, lane labeled 2, represents the female parent" A2 "genomic DNA. Therefore, the male parent and the female parent genome DNA can be successfully extracted.
(3) Storing the extracted male parent and female parent genome DNA at-20 ℃ for later use.
(4) And respectively taking the extracted male parent genomic DNA and the extracted female parent genomic DNA as templates, and amplifying the genomic DNA by adopting the primer combination of 16_1F and 16_ 1R. Wherein, the upstream primer 16_1F is a sequence shown in Seq ID No. 2; the downstream primer 16_1R is a sequence shown as Seq ID No. 3;
Seq ID No.2：5’-AAACGAGCAACGGGTAGGC-3’
Seq ID No.3：5’-GGAAAGACAGCAGGAACAGAG-3’。
PCR reaction 25. mu.L, comprising: 10 XBuffer 2.5 uL, 25mM dNTPs 0.15 uL, 5U/. mu.L Taq enzyme 0.15 uL, 10 uM 16_1F upstream primer 0.5 uL, 10 uM 16_1R downstream primer 0.5 uL, genomic DNA1.0 uL, sterile water 20.2 uL.
The PCR reaction conditions were 94 ℃ pre-denaturation for 5min, followed by 35 cycles: denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 40 seconds, and extension at 72 ℃ for 3min after the cycle is finished.
The PCR amplification product was purified and stored at 4 ℃. A portion of each PCR amplification product was detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 2. In FIG. 2, the first lane, designated A16-1, shows a PCR amplification product using genomic DNA of the female parent "A2" as a template, and the second lane, designated 316-1, shows a PCR amplification product using genomic DNA of the male parent "Jizhuanggu No. one" as a template. The results in FIG. 2 show that the primers 16_1F and 16_1R can amplify the genomic DNA of the male and female parents to target fragments that are expected.
Sequencing the male parent and female parent genome DNA amplification products respectively, wherein the sequencing sequence of the male parent amplification product obtained by amplifying the primers 16_1F and 16_1R is shown as Seq ID No.1, and the PH-04-413 site is 163 th base from the 5' end in the sequence shown as Seq ID No. 1. The sequencing sequence alignment results of the male parent amplification product and the female parent amplification product are shown in FIG. 3, wherein the base marked with gray background represents the base at the PH-04-413 locus, the female parent base is C, and the male parent base is T.
Then, screening according to the plant height data of the samples: the height of the male parent plant is 60cm, and the height of the female parent plant is 45 cm. Among 441 samples, the minimum value was 11cm, and the maximum value was 82 cm; of the 50 samples with the minimum plant height, the number of samples identical to the maternal SNP is more than 65%; of the 50 samples with the largest plant height, the number of samples identical to the paternal SNP was more than 65%.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Zhang Kouzhou agricultural science institute (alpine crop research institute in Hebei province)
<120> SNP marker related to millet plant height trait, detection primer and application thereof
<130> 17I25634
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 304
<212> DNA
<213> PH-04-413 site sequence
<400> 1
acgagcaacg ggtaggcttg ggcgtcgtgg tggggctagt ggagctgcgg gcggacggag 60
acgcgaatgg aaacggcggc gtgggtgttg gccggcggcg gcgcttgctc gctcgagctc 120
tgctcctgtc gacgtaaaac ggccgggtga aaaggataag cgtggggata gcaacgtcgt 180
ggtctttcac cgtgccacgt ccagcttggc tcgtggtaga cgacgtggcg cagctatggc 240
cgtcgcgctg gccggcatgc gcgagagccg gcggcggcac ggctcttcct gttgcctctg 300
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
aaacgagcaa cgggtaggc 19
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence
<400> 3
ggaaagacag caggaacaga g 21
Claims (3)
1. The application of the SNP marker in predicting the height of a millet plant, early identifying the height of the millet plant or molecular-assisted screening and breeding in the aspect of the height of the millet plant is characterized in that: the sequence of the SNP marker is shown as Seq ID No.1, and the 163 st base from the 5' end of the sequence is C or T; the SNP marker is positioned in bin markers from 18023413bp to 21734440bp of a fourth chromosome and is tightly linked with the height of a millet plant.
2. Use according to claim 1, characterized in that: for SNP markers, a composite interval mapping method is utilized, the total significance level of 5% is adopted, the LOD value detected by QTL is 3.2792, and the interpretation rate of phenotypic variation is 2.76%.
3. A method for predicting the plant height of millet is characterized by comprising the following steps: comprises the steps of carrying out PCR amplification on the genomic DNA of millet to be predicted by adopting a primer pair, obtaining the base condition of the SNP marker in claim 1 or 2 according to the sequencing result of the PCR amplification product, and predicting the plant height of the millet according to the base condition of the SNP marker;
the upstream primer of the primer pair is a sequence shown by Seq ID No.2, and the downstream primer is a sequence shown by Seq ID No. 3;
Seq ID No.2：5’-AAACGAGCAACGGGTAGGC-3’
Seq ID No.3：5’-GGAAAGACAGCAGGAACAGAG-3’。
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