CN112646925A

CN112646925A - Corn ear position high-correlation ZmRzf gene SNP molecular marker and application

Info

Publication number: CN112646925A
Application number: CN202110064297.8A
Authority: CN
Inventors: 胡军; 潘洪玉; 丁韦辰; 甘会云; 于汇琳; 张鑫生; 唐心龙; 王世壮; 王寒; 王鹏葳; 陈逸飞; 金天
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-01-18
Filing date: 2021-01-18
Publication date: 2021-04-13
Anticipated expiration: 2041-01-18
Also published as: CN112646925B

Abstract

The invention discloses a corn ear height related ZmRzf gene SNP molecular marker and application, belongs to the technical field of crop genetic breeding molecular marker application, and particularly relates to SNP molecular markers of 3 ear height related ZmRzf genes on a corn chromosome 8 and application thereof, wherein the three markers are respectively positioned at the Chr8:156959358, Chr8:156959642 and Chr8: 156959657. The SNP molecular marker is obtained from the whole genome association analysis of a corn inbred line population, and the nucleotide sequences of the SNP molecular marker are respectively as follows: as shown in SEQ ID N01 and SEQ ID N02, the SNP marker can be used for auxiliary selection of high traits of maize ear positions in a breeding process, can improve the selection accuracy and quickens the process of variety breeding.

Description

Corn ear position high-correlation ZmRzf gene SNP molecular marker and application

Technical Field

The invention belongs to the technical field of preparation of crop molecular markers, and particularly relates to an SNP molecular marker related to the maize tassel branch number trait and application thereof.

Background

The corn has high yield and wide application, and is an important grain crop, feed crop, energy resource crop and economic crop in our country. The method for improving the planting density is a main way for high-yield cultivation of the corn at present. However, the high density and the high spike position are easy to cause colony lodging and stalk breakage under adverse meteorological conditions. For example, in the summer of 2020, the major production area of spring corn in northeast China causes large-area serious quality reduction and yield reduction due to typhoon and rainstorm weather. The ear height refers to the distance between the corn ear growing node and the ground, the ear height is an important property for determining the lodging resistance of the corn, the higher the growing node of the corn ear is, the higher the center of gravity of the corn is, and the lodging resistance of the corn variety can be better improved by reducing the ear height under the precondition of utilizing the favorable property of medium or medium-high stalk. The high panicle position is a continuously distributed, typical, quantitative trait controlled by multiple genes, and the generalized and narrow heritability of the trait is high, which indicates that a major gene which greatly contributes to the trait plays a role.

At present, a plurality of QTL regions and candidate genes related to spike height are determined by a QTL positioning and candidate gene method, but the QTL positioning region generally has a large span and usually has a few centimorgans (cM), the positioned region comprises a large number of candidate genes, and the fine positioning of the genes is difficult. The candidate gene method is to directly select genes which may affect the trait from a known or potential gene library based on the existing background knowledge of life science, but has the disadvantage that a new gene cannot be identified.

Therefore, the development of the auxiliary selection marker of the high trait of the maize ear position in the breeding process of the maize improves the selection accuracy, accelerates the breeding process of the variety and has important significance for the molecular marker auxiliary breeding related to the high trait of the maize ear position and the genetic improvement of the related trait.

Disclosure of Invention

One of the objects of the present invention is to provide an SNP molecular marker associated with a maize-like ear height.

The development method of the SNP molecular marker related to the maize-like ear height provided by the invention comprises the following steps: by using a whole genome association analysis method, SNP molecular markers related to the ear height character are searched and used as auxiliary selection markers of the ear height character in the corn breeding process, so that the selection accuracy is improved, and the variety breeding process is accelerated.

The development process of the SNP molecular marker of the invention is as follows:

step (1), 431 maize inbred lines with different genetic relationships are used for constructing a GWAS analysis group, maize seedlings are taken as samples for extracting Genome DNA after the group is planted in a field, and the high trait of the maize ear position is investigated after maize pollination and used as phenotype data of Genome-wide Association Study (GWAS). The genome DNA is connected with a proper connector after enzyme digestion to construct a genome sequencing library, and the library is subjected to double-end sequencing (PE150) by adopting an Illumina Nova Seq 6000 sequencing platform, wherein the read length (Reads) is2 multiplied by 150 bp. And performing quality control and filtering on the generated sequencing data, and comparing the data qualified in quality control with a B73 reference genome sequence to obtain SNP information in the genome of the colony.

And (2) performing quality control on the SNP data obtained in the step (1), wherein the quality control standard is MAF <0.05, the deletion rate is greater than 0.8, HW (Hadi-Weinberg index) >0.0001, and the SNP/InDel information in the VCF file is annotated by using genome structure annotation data (GTF file) of software Snp Eff (version 4.3t), namely whether the influence on the gene coding protein can be caused, including the mutation type and mutation position of the SNP, the mutation type and effect of amino acid and the like. The line size trait was correlated using the CMLM model (compressed mixed linear model) in the software GAPIT (3.1.0). In the correlation analysis, the optimal number of PCs in the GWAS model is found based on the model selection of Bayesian Information Criterion (BIC) so as to fit the optimal character factor.

And (3) using 549211 qualified SNPs obtained in the step (2) for genome-wide association analysis, and when the P value of the detected SNP is less than 10-5, the SNP is considered to reach the significance of the genome level, and after the genome-wide association analysis, 3 SNP loci which meet the conditions are detected from the intron region of the ZmRzf gene and are respectively located at the positions of No. 8 chromosome Chr8:156959358, Chr8:156959642 and Chr8:156959657 of the corn.

And (4) performing group comparison on samples with different genotypes of the significant loci detected in the step (3):

the difference between T/T and (C/C + C/T) at the Chr8:156959358 sites is extremely obvious (P is less than 0.0001), the difference of the mean value among groups is 10.637cm, and genotype T/T is an extremely obvious molecular marker of the low spike position; genotypes C/C and C/T are extremely significant molecular markers of high spike positions;

the difference between G/G and (A/A + A/G) at the Chr8:156959642 sites is very significant (P <0.0001), and the difference between the groups is 10.215 cm. Genotype G/G is a very significant molecular marker at the low ear position, and genotypes A/A and A/G are very significant molecular markers at the high ear position;

the difference between G/G and (A/A + A/G) at the Chr8:156959657 sites is extremely obvious (P is less than 0.0001), the difference of the mean value between groups is 10.637cm, the genotype G/G is an extremely obvious molecular marker at the low spike position, and the genotypes A/A and A/G are extremely obvious molecular markers at the high spike position.

By utilizing the technical measures, 3 SNP markers are finally obtained and are respectively positioned on the No. 8 chromosome Chr8:156959358, Chr8:156959642 and Chr8:156959657 of the corn:

is positioned at the Chr8:156959358 position of the No. 8 chromosome of the corn, the nucleotide sequence of the gene is shown as SEQ ID N01, and the base Y at the 159 position of the sequence is C or T;

the nucleotide sequence of the chromosome is shown as SEQ ID N02 at the position of Chr8:156959642 of the No. 8 chromosome of the corn, and the base R at the position 143 of the sequence is A or G;

is located at the position of Chr8:156959657 of the No. 8 chromosome of corn, the nucleotide sequence of the gene is shown as SEQ ID N02, and the base R at the 158 th position of the sequence is A or G.

The invention also aims to provide application of the SNP molecular marker in molecular marker assisted breeding related to high trait at maize ear position.

The SNP marker is positioned at the Chr8:156959358 position of the 8 th chromosome of the corn, and the genotype T/T is a very significant molecular marker of the low ear position; genotypes C/C and C/T are very significant molecular markers of high spike positions.

The SNP marker located at the Chr8:156959642 position of the 8 th chromosome of the corn, the genotype G/G is a very significant molecular marker of the low ear position, and the genotypes A/A and A/G are very significant molecular markers of the high ear position.

The SNP marker located at the Chr8:156959657 position of the 8 th chromosome of the corn, the genotype G/G is a very significant molecular marker of the low ear position, and the genotypes A/A and A/G are very significant molecular markers of the high ear position.

The invention researches the Association of the polymorphic locus and the character in the population by Genome-wide Association Study (GWAS), and further researches the function of an unknown gene, which is a very effective way.

Zinc finger (Zinc finger) proteins are widely distributed in eukaryotic genomes and play an important role, and are rich in Zinc-binding domains consisting of cysteine and histidine residues and a class of proteins which are well conserved in sequence. The zinc finger proteins can be divided into various types such as C2H2, C2HC, C3HC4, C4HC3, C4, C6 according to the number and positions of cysteine and histidine residues, wherein C and H respectively represent Cys and His. Many transcription factors are finger-like zinc finger proteins that play an important role in gene regulation, and zinc fingers regulate gene expression at the transcriptional and translational levels by sequence-specific binding to target molecules DNA, RNA, DNA-RNA, and binding to themselves or other zinc finger proteins. Plays an important role in degradation of proteins, repair of DNA, migration of cells, differentiation of cells, development of embryos, and the like.

Ring Zinc Finger domains (RING Finger domains) are a class of Zinc Finger domains that include 40-60 amino acid residues with the motif Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-Cys-X2-Cys-X4-48-Cys-X2-Cys, X represents any amino acid residue, and Cys and His are sometimes interchangeable. Ubiquitination refers to the process of classifying proteins in cells and specifically modifying target proteins by ubiquitin (a class of low molecular weight proteins) molecules under the action of a series of special enzymes. Ubiquitination, i.e., covalent modification of proteins by the addition of ubiquitin, relies on a series of enzymes to act on proteins, and finally facilitates the transfer of ubiquitin from the E2 enzyme to the target protein by E3 ligase. The most common E3 ligase contains a zinc finger domain called the loop, and although the important role of the zinc finger domain in ubiquitin transfer is widely recognized, its molecular mechanism is still unclear at present. Ubiquitination plays an important role in the metabolism and function of proteins, and is involved in many important vital activities such as cell cycle, proliferation, apoptosis, differentiation, and metastasis.

The invention discovers that 3 sites of a ZmRzf gene (Zea mays RING Finger Domain, Zm00001d011636) are greatly and obviously associated with the high trait of the maize ear, wherein the 3 sites are located at the position of chromosome 8 Chr8:156959358 of maize, the nucleotide sequence is shown as SEQ ID N01, and the base Y of the 159 site of the sequence is C or T, so that the genetic polymorphism and the high trait of the ear of the maize inbred line to be tested are greatly and obviously different; the nucleotide sequences of the genes are shown in SEQ ID N02 at the positions of chromosome 8 Chr8:156959642 and Chr8:156959657 of the maize, and the base groups R at the 143 th site and the 158 th site of the sequences are A or G, so that the genetic polymorphism and the ear position high character of the inbred line population of the maize to be tested are very obviously different. The marker is used as an auxiliary selection marker for high trait of the ear position in the corn breeding process, so that the selection accuracy is improved, and the variety breeding process is accelerated.

Drawings

FIG. 1 is a histogram of high trait frequency distribution at the ear position of a corn study population;

FIG. 2 is a (partial) electrophoretogram for detection of genomic DNA from maize;

FIG. 3 is a Manhattan plot of maize ear position high trait whole genome association analysis;

FIG. 4 QQ plots of genome-wide association analysis of the maize ear height trait;

FIG. 5 is a box plot of spike-position high-order number distribution of different genotype samples at sites chr8:156959358 of ZmRzf gene;

FIG. 6 is a box plot of spike-position high-order distribution of different genotype samples at sites chr8:156959642 of ZmRzf gene;

FIG. 7 is a box plot of spike height distribution of different genotype samples at the site of chr8:156959657 of ZmRzf gene.

The specific implementation mode is as follows:

example 1

1.1 construction of GWAS population

The corn whole genome association analysis population comprises 431 inbred lines with different genetic relationships. All inbred lines were provided by the college of plant science of Jilin university and planted in the base of teaching and research experiments of the college of plant science of Jilin university (Green park of Changchun city, Jilin province). The cell arrangement follows the random block design, the block is repeated for 3 times, the single row of cells has the row length of 3m, the row spacing of 65 cm and the plant spacing of 20 cm, and the field management measures are the same as the field management measures.

1.2 Collection of samples and investigation of phenotypic data

Collecting 3 seedlings at the 5-6 leaf stage after the emergence of the maize seedlings in the residential area, cleaning the seedlings, and putting the seedlings in a refrigerator at the temperature of-20 ℃ for freezing storage. The Ear Height (EH) of representative 5 plants was investigated 20 days after maize pollination in each plot, the mean of triplicates of the blocks was used as phenotypic data for GWAS analysis, and the data was collated using Excel 2013 software.

1.3 extraction and detection of maize genomic DNA

(1) Grinding 50-100mg of maize seedlings into powder by using liquid nitrogen, transferring the powder into a 1.5ml centrifuge tube, adding 400ul of Buffer PCL and 8ul of beta-mercaptoethanol, and shaking and uniformly mixing. Water bath at 65 deg.c for 45min until the sample is completely lysed.

(2) Adding 200ul Buffer PP, fully reversing and mixing uniformly, placing in a refrigerator at-20 ℃ for 5min, centrifuging at room temperature of 10000rpm for 5min, and transferring the supernatant (500 plus 550ul) into a new 1.5ml centrifuge tube. If the supernatant is turbid, equal volume of chloroform can be added and mixed evenly, and the supernatant is obtained by centrifugation at 12000 rpm.

(3) Adding equal volume of isopropanol, reversing for 5-8 times to mix thoroughly, and standing at room temperature for 2-3 min. Centrifuge at 10000rpm for 5min at room temperature, and discard the supernatant.

(4) Adding 1ml 75% ethanol, rinsing by inversion for 1-3min, centrifuging at 10000rpm for 2min, and discarding the supernatant. Repeating the above operation once, uncovering the cover, and inverting for 5-10min at room temperature until the residual ethanol is completely volatilized.

(5) The obtained DNA was dissolved in 50-100ul of TE Buffer. The extracted DNA can be immediately subjected to the next experiment or stored at-20 ℃.

(6) DNA integrity was checked on 1% agarose gel (200V electrophoresis for 30min) and the concentration of DNA sample was quantified by Qubit2.0.

1.4 results of the study

The mean value of the maize ear position high character samples of the inbred line population is 61.82cm, the median is 60.40cm, the average deviation is 11.77cm, the range is 83.84cm, the standard deviation is 14.91cm, the coefficient of variation is 0.241, the 95% confidence interval is 60.407-63.23cm, and the distribution condition of the times of the ear position high character is shown in figure 1.

After the Genomic DNA of the maize seedling is extracted by a Rapid Plant Genomic DNA Isolation Kit, RNA is cracked and digested by a lysis solution, and impurities such as protein and the like are eluted by an organic phase to obtain pure DNA, and the DNA sample is qualified by electrophoresis detection (10 ng/ul) and can be used for constructing a Genomic library, wherein the electrophoresis detection result is shown in figure 2.

Example 2

2.1 construction of sequencing libraries

(1)200ng of genome DNA is cut by restriction enzyme EcoRI, and magnetic beads are purified and recovered after the digestion is completed.

(2) And connecting the purified DNA after enzyme digestion with a T4 DNA Ligase to Barcode adapters PI, purifying and recovering a magnetic bead connecting product, and recording a qualified P1 primer label.

(3) All samples were mixed in the required equal proportions to give a total DNA mix, which was fragmented using covaris220, the fragmented DNA having a length of about 200-500 bp.

(4) After End Repair & dA-labeling, the ligation product was ligated to Adaptor P2 and the ligation product was recovered by magnetic bead purification.

(5) Amplifying and enriching a joint connection product by using a KAPA 2G Robust PCR Kit, purifying and sorting PCR reaction products by using magnetic beads, detecting the quality of the constructed library PCR purification product by using 2% agarose gel electrophoresis, and performing second-generation sequencing on qualified PCR products.

2.2 simplified genome sequencing (Restriction site-associated DNA sequencing, RAD)

(1) Double-ended sequencing (PE150) was performed using the Illumina NovaSeq 6000 sequencing platform with a read length (Reads) of 2X 150 bp.

(2) Quality control and filtering of output data: and predicting the error occurrence probability of base detection, and if the base number with the quality score (Q-score) of low quality (Q is less than or equal to 5(E)) accounts for more than half of the whole read, removing the read and simultaneously removing the tag sequence for sample identification.

(3) And (3) performing stack clustering analysis on all samples by using STACKS-1.08(http:// creskolab. uoregon. edu/STACKS), and detecting to obtain the Tag sequence and SNP information of each sample.

(4) Data alignment and SNP-Calling: BWA software (version 0.7.17-r1188) is adopted to carry out genome alignment, and data qualified by quality control is aligned with a B73 reference genome sequence.

2.3 genotype testing and GWAS

(1) And (3) mutation detection and screening: repeats were marked using Picard software, sequencing bams by SamTools (1.9), and calling SNPs by BcfTools (1.9).

(2) SNP quality control: the quality control software is Vcf Tools (0.1.16) for SNP, the quality control standard is MAF <0.05, deletion rate >0.8, HW (Hadi-Weinberg index) > 0.0001.

(3) SNP annotation software Snp Eff (version 4.3t) utilizes genome structure annotation data (GTF file) to annotate SNP/InDel information in VCF file, namely whether the gene coding protein can be influenced or not, including the mutation type and mutation position of SNP, the mutation type and effect of amino acid and the like.

(4) GWAS: correlation analysis was performed on the traits using the CMLM model (compressed mixed linear model) in the software GAPIT (3.1.0).

2.4 results of the study

After SNP quality control, 549211 qualified SNPs are obtained for whole genome association analysis, and the optimal PCs number in the GWAS model is found based on model selection of Bayesian Information Criterion (BIC) during association analysis so as to fit the optimal character factor. The GWAS analysis results of the maize ear position high traits are shown in figures 3 and 4, figure 3 is a Manhattan graph of the correlation analysis results, the X axis is the position of each SNP on a genome, and the Y axis is the negative logarithm of the base 10 of the P value of each SNP site under the CMLM model. FIG. 4 is a QQ plot of a correlation analysis, with the Y-axis being the negative logarithm to base 10 of the observed P-values, and the X-axis being the negative logarithm to base 10 of the expected observed P-values assuming that the P-values obey a uniform [0,1] distribution;

when the P value of the detected SNP is less than 10^-5When the SNP is considered to reach the significance of the genome level, 3 SNP sites are detected in the intron region of the ZmRzf gene and meet the conditions, the three sites are respectively located at 156959358, 156959642 and 156959657 of the No. 8 chromosome of corn, the bioinformatics function annotation shows that the three sites belong to a RING zinc finger domain protein superfamily gene (RING zinc finger domain protein), the existing research result shows that the E3 ligase contains a RING zinc finger domain, the E3 ligase can promote the ubiquitin to be transferred from the E2 enzyme to the target protein, and the ubiquitination of the protein participates in a plurality of important life activities such as cell cycle, proliferation, apoptosis, differentiation, transfer and the like, and the result is detailed in Table 1.

TABLE 1 Association analysis of 3 loci of Zm00001d011636 gene

Example 3 comparison between groups to detect distinct genotypes at significant sites

3.1 comparison between groups at the Chr8:156959358 sites of ZmRzf Gene

The ZmRzf gene Chr8:156959358 locus comprises three genotypes, namely C/C, T/T, C/T, and a box diagram of the high distribution of the spike positions of samples with different genotypes is shown in a figure 5; the median of the C/C group is 66.94cm, the lower quartile Q1 is 60.89cm, and the upper quartile Q3 is 81.39 cm; the median of the T/T group is 58.33cm, the lower quartile Q1 is 50.11cm, and the upper quartile Q3 is 69.22 cm; the median C/T is 68.48cm, the lower quartile Q1 is 59.67cm, and the upper quartile Q3 is 72.66 cm.

The T test results of the differences of the phenotype mean values among different genotype groups are shown in table 2, and the differences between C/C and T/T groups are very significant (P <0.0001) and the differences of the mean values among the groups are 10.084cm as can be seen from table 2; the difference between T/T and (C/C + C/T) is very significant (P <0.0001), and the mean difference between groups is 10.637 cm. Therefore, genotype T/T is a very significant molecular marker with a lower ear height, and genotypes C/C and C/T are very significant molecular markers with a higher ear height.

TABLE 2 comparison of the mean height of panicle positions at the Chr8:156959358 sites of the ZmRzf Gene between groups

3.2 comparison between ZmRzf Gene Chr8:156959642 site groups

The ZmRzf gene Chr8:156959642 locus comprises three genotypes, namely G/G, A/A, A/G, and a box diagram of high trait distribution of spike positions of samples with different genotypes is shown in figure 6; the median of the G/G group is 58.17cm, the lower quartile Q1 is 49.87cm, and the upper quartile Q3 is 68.56 cm; the median of the A/A group is 68.31cm, the lower quartile Q1 is 61.31cm, and the upper quartile Q3 is 79.95 cm; the median of the A/G group was 64.10cm, the lower quartile Q1 was 59.67cm, and the upper quartile Q3 was 76.78 cm.

The t test result of the phenotype mean difference among different genotype groups is shown in table 3, and the difference between A/A and G/G groups is very significant (P <0.0001) and the mean difference between the groups is 9.489cm as shown in table 3; the difference between A/G and G/G groups is very obvious (P is 0.0006), and the difference between the groups is 11.919 cm; the difference between G/G and (A/A + A/G) is very significant (P <0.0001), and the mean difference between groups is 10.215 cm. Therefore, genotype G/G is a very significant molecular marker for the lower ear height, and genotypes A/A and A/G are very significant molecular markers for the higher ear height.

TABLE 3 comparison of the mean height of panicle positions at the Chr8:156959642 sites of the ZmRzf Gene between groups

3.3 comparison between ZmRzf Gene Chr8:156959657 site groups

The ZmRzf gene Chr8:156959657 locus comprises three genotypes, namely G/G, A/A, A/G, and a box diagram of high trait distribution of spike positions of samples with different genotypes is shown in figure 7; the median of the G/G group is 58.45cm, the lower quartile Q1 is 50.03cm, and the upper quartile Q3 is 69.33 cm; the median of the A/A group is 68.31cm, the lower quartile Q1 is 61.72cm, and the upper quartile Q3 is 82.06 cm; the median A/G was 64.10cm, the lower quartile Q1 was 58.34cm, and the upper quartile Q3 was 72.67 cm.

The t test results of the differences of the phenotype mean values among different genotypes are shown in table 4, and the differences between A/A and G/G are very significant (P <0.0001), and the difference of the mean values among groups is 11.023 cm; the difference between A/G and G/G is obvious (P is 0.0194), and the difference between the groups is 9.149 cm; the difference between G/G and (A/A + A/G) is very significant (P <0.0001), and the mean difference between groups is 10.637 cm. Therefore, genotype G/G is a very significant molecular marker for the lower ear height, and genotypes A/A and A/G are very significant molecular markers for the higher ear height.

TABLE 4 comparison of the mean height of panicle positions at the Chr8:156959657 sites of the ZmRzf Gene between groups

Sequence listing

<110> Jilin university

<120> corn ear position high-correlation ZmRzf gene SNP molecular marker and application

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tcaatacacg ggtcacggct gtaccaccga tatcattgtg cctgttaagg caccggttga 300

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Claims

1. An SNP molecular marker related to the high trait of the maize ear position is characterized in that the SNP molecular marker is located at the position of chromosome 8 Chr8:156959358 of maize, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID N01, and the base Y at the 159 th position of the sequence is C or T.

2. An SNP molecular marker related to the high trait of the maize ear position is characterized in that the SNP molecular marker is located at the position of chromosome 8 Chr8:156959642 of maize, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID N02, and the base R at the position 143 of the SNP molecular marker is A or G.

3. An SNP molecular marker related to the high trait of the maize ear position is characterized in that the SNP molecular marker is located at the position of chromosome 8 Chr8:156959657 of maize, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID N02, and the base R at the 158 th position of the sequence is A or G.

4. The application of the SNP molecular marker of claim 1 in the auxiliary selection of high traits at the maize ear position, wherein genotype T/T is a very significant molecular marker at the low ear position; genotypes C/C and C/T are very significant molecular markers of high spike positions.

5. The application of the SNP molecular marker of claim 2 in the auxiliary selection of high traits at the maize ear position, wherein genotype G/G is a very significant molecular marker at the low ear position, and genotypes A/A and A/G are very significant molecular markers at the high ear position.

6. The application of the SNP molecular marker of claim 3 in the auxiliary selection of high traits at the maize ear position, wherein genotype G/G is a very significant molecular marker at the low ear position, and genotypes A/A and A/G are very significant molecular markers at the high ear position.