CN107475417B

CN107475417B - Molecular marker related to oat β glucan content and application thereof

Info

Publication number: CN107475417B
Application number: CN201710863673.3A
Authority: CN
Inventors: 吴斌
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2017-09-22
Filing date: 2017-09-22
Publication date: 2020-04-14
Anticipated expiration: 2037-09-22
Also published as: CN107475417A

Abstract

The molecular marker is positioned on the 20D chromosome of oat, is derived from oat glucan synthase gene AsCslH, is positioned at the 123 th position from the 5' end in SEQ ID No.1, is an SNP marker with polymorphism of C or G, and can be obtained by amplifying primers shown in SEQ ID No. 2-4.

Description

Molecular marker related to oat β glucan content and application thereof

Technical Field

The invention relates to the technical field of crop molecular marker assisted breeding, in particular to a molecular marker related to oat β glucan content and application thereof.

Background

Oat is rich in nutrition, compared with crops such as wheat, rice, corn and the like, nutritional indexes such as protein, unsaturated fatty acid, vitamin, mineral elements and the like of oat are all in the front, so that oat is one of the best full-value nutritional foods in cereals, oat is also a recognized health Food, and oat products are confirmed to be health foods by the United states Food and drug administration.

Cellulose-like synthases like CSL are structurally similar to Cellulose synthase (CESA), Csl gene was originally found by alignment of Ces gene sequence to genomic sequence of model plant Arabidopsis thaliana. like CES protein, Csl gene encoded protein also has glycoside transferase (glycosylation transfer) domain, but differs from Ces gene, the most significant difference being that Csl gene has additional specific sequence, Csl gene comprises many members, is a huge gene family, but the function of many proteins encoded by Csl gene family members is still unclear at present. Csl gene family member is originally found by analysis of rice genome sequence by Hazen, however, function has not been determined yet, Doblin et al transferred cloned into Csl gene into CslH gene, which was observed to have been generated by glucan in Arabidopsis thaliana incapable of synthesizing glucan, which later on the study of polysaccharide production by polysaccharide synthesis, which was found to be responsible for polysaccharide synthesis by barley polysaccharide synthesis strain, which was found to be a large gene family of rice genome sequence, thus the role of polysaccharide synthesis by barley polysaccharide synthesis study, rice polysaccharide synthesis by rice strain.

Correlation analysis is an analysis method for correlating markers with target trait phenotypes based on linkage disequilibrium phenomena. The association analysis mainly comprises two strategies: i.e., genome-wide scan-based and candidate gene-based association analysis. In association analysis based on whole genome scanning, simultaneous detection of all genes of a large population of species with high-throughput markers distributed throughout the genome is required. In practical application, it is difficult to truly utilize large populations to perform high-throughput marker detection and analysis in plants due to the limitations of the number difference of markers on different chromosomes, the cost of genotype identification, heavy statistical analysis workload and other factors. From the biochemical point of view, the expression of a certain trait in an organism is almost the result of the joint action of various enzymatic reactions involving various proteins, so that the change of the activity of the protein caused by the difference of genes encoding related proteins can also cause the trait change. It is therefore feasible to discover functional allelic variation closely related to phenotypic variation by detecting variation of candidate genes in related populations.

Disclosure of Invention

The first purpose of the invention is to provide a molecular marker related to the glucan content of oat β.

The second purpose of the invention is to provide the application of the molecular marker.

It is a third object of the present invention to provide a method for identifying oat varieties with high β glucan content.

The invention aims to realize the technical scheme that an applicant collects 96 oat germplasm resources with different geographical sources, obtains a glucan synthase gene AsCslH (HQ128579) sequence of the oat germplasm resources by using a homologous cloning method as a related group of the research, discovers a variant ectopic site of the gene in 96 oat germplasm resources by virtue of multiple sequence comparison, combines the data of glucan content of 96 oat germplasm resources β at multiple points for years, takes the glucan synthase gene AsCslH as a candidate gene, performs association analysis on the candidate gene by using a general linear model and a mixed linear model of TASSEL software, detects a SNP site which is obviously related to the glucan content of oat β, is located on the 20D chromosome of oat, is derived from the oat glucan synthase gene AsCslH, is located on the 123 th position in SEQ ID No.1, and has two homozygous genotypes of C and G because of C and G in a test line, has a flanking sequence shown in SEQ ID No.1, has obvious allele marker content of glucan related to the oat germplasm resource C/C β, and has high content of glucan related molecular marker molecules of oat germplasm resources of C/G6335.

After screening the SNP site which is obviously related to the content of oat β glucan, based on the flanking sequence of the site, the applicant designs a specific primer pair which contains the SNP site and is used for detecting the oat glucan synthase gene AsCslH gene, the sequences of the primer pair are shown as follows:

upstream of primer 1 (F): 5'-TATCTTTACTTGGTCCCAGCAGC-3' (SEQ ID NO.2)

Primer 2 downstream (R): 5'-CATCAGCGGATCAGATTGCCAC-3' (SEQ ID NO.3)

Downstream of primer 3 (R): 5'-AGCGGATCAGATTGCCAG-3' (SEQ ID NO.4)

In the primers, the 5' ends of the primer 2(SEQ ID NO.3) and the primer 3(SEQ ID NO.4) are connected with different fluorescent groups.

The 5' end of the primer 2(SEQ ID NO.3) is connected with a FAM fluorescent group;

the 5' end of the primer 3(SEQ ID NO.4) is connected with a HEX fluorescent group.

In the above set of primers, the molar ratio of the primer 2, the primer 3 and the primer 1 is 2: 2: 5.

the invention provides application of the SNP molecular marker in oat breeding.

The invention provides application of the SNP molecular marker in breeding of β oat with high glucan content.

The invention provides application of the SNP molecular marker in screening β oat varieties with high glucan content.

The invention provides a specific primer pair for detecting SNP molecular markers related to oat β glucan content, which consists of three primers, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 2-4.

The invention provides application of the specific primer pair in oat germplasm resource improvement.

The invention provides a method for identifying oats with high β glucan content, which comprises the following steps:

(1) extracting genome DNA of the oat to be detected;

(2) taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by using the specific primer pair;

(3) if the 123 th position of the segment shown in SEQ ID NO.1 in the amplification product is C, the oat to be detected is high β glucan content oat.

The invention provides a kit containing the specific primer pair and used for detecting oat with high β glucan content.

The invention provides application of the kit in screening oat varieties with high β glucan content.

The invention provides a method for detecting the genotype of an oat glucan synthase gene AsCslH, which comprises the steps of carrying out PCR amplification on oat genomic DNA to be detected by using the primer pair, and if a fragment of an SNP (single nucleotide polymorphism) mark C marked in SEQ ID NO.1 can be amplified, indicating that the oat to be detected has the gene AsCslH with high β glucan content.

To further verify the effectiveness of the developed marker, the inventor randomly selects 24 oat varieties in the related population, uses DNA as a template, utilizes competitive Allele Specific PCR (Kompetitive Allele Specific PCR), namely KASP technology, and carries out SNP detection on a sample to be detected based on an SNpline genotyping technology platform of LGC Genomics company, and the result shows that the SNP locus successfully carries out genotyping on the glucan synthase gene AsCslH of the 24 oat varieties (figure 2).

Meanwhile, the inventor uses the DNA of 190 families in an oat β glucan RIL group (red 38 multiplied by summer naked oats) constructed in the early stage as a template to carry out SNP detection on the site (figure 3). The result shows that in the RIL group, the SNP site has obvious correlation with the glucan content (figure 4) and is basically consistent with the correlation analysis result of the AsCslH gene in 96 oat germplasm resources from different sources.

According to the invention, by combining the sequence diversity of the AsCslH gene in oat germplasm resources of different geographical sources, the internal relation between the gene and the glucan content of oat β is disclosed by using a candidate gene association analysis strategy, obvious functional sites in the gene are discovered, and the gene is used as a genetic marker to be applied to oat molecular breeding, so that the important significance is provided for improving the oat quality.

The method has the beneficial effects that a candidate gene association analysis method is applied, a molecular marker locus which is obviously associated with a specific character can be quickly and accurately detected, an SNP locus which is obviously associated with the glucan content of the oat β exists in the AsCslH locus on the 20D chromosome of the oat, the explanation phenotype variation is 3.08%, and the SNP locus can be used as a genetic marker for breeding the oat with high β glucan content and has higher application value.

Drawings

Figure 1 is the frequency of β glucan content distribution in the oat population using the correlation analysis.

FIG. 2 is a SNP analysis of 24 oat germplasm resource species based on KASP technique.

FIG. 3 shows SNP analysis of 190 families in RIL population based on KASP technique.

FIG. 4 is a comparison of the glucan content of oat β for individuals with the two genotypes "C/C" and "G/G" in the RIL population (P.ltoreq.0.01). The families with the "C/C" and "G/G" genotypes in the RIL population are 101 and 86, respectively.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified; the technical means used in the examples are conventional means well known to those skilled in the art; the percentages in the following examples are by weight.

Example 1 obtaining of molecular markers of oat β glucan synthase gene AsCslH gene significantly correlated with β glucan content and determination of detection primers thereof

The molecular marker of the oat β glucan synthase gene AsCslH gene and β glucan content which are obviously related is obtained by the following method:

1) 96 oat germplasm resources from different geographical sources were collected and established as relevant populations for mapping. The population has abundant genetic diversity.

2) β glucose content determination of related group seeds, 2016, planting the collected materials in Changping test base of China academy of agriculture, Zhangkou academy of agriculture, Changji test station of Xinjiang academy of agriculture, normal experimental management, no special treatment, using β glucose content analysis of the harvested seeds, using β glucose content determination of the harvested seeds, using standard glucose content determination Method (AACC Method32-23) published by the American Association of cereals (AACC), according to the instruction of commercial kit produced by Megazyme, the Method is briefly described as follows, adding 50% ethanol and sodium phosphate buffer to the ground samples, shaking after boiling water bath for 2min, adding 0.2mL lichenase (10U) after the test tube is recovered to 50 ℃, strongly shaking, incubating for 1h at 50 ℃, adding 5mL sodium acetate buffer, mixing, centrifuging for 10min, sucking 0.1mL supernatant into the test tube, adding 0.1mL sodium acetate (10U), strongly shaking for 1 min, incubating for 1h at 50 ℃, adding 10 g-10 min, adding 10 g of glucose (A) to obtain a glucose absorbance of glucose concentration, wherein the glucose concentration is calculated by using a formula of glucose oxidase (A-10 mg) after the glucose concentration is calculated as 10min, and the absorbance of glucose concentration of glucose is calculated by the blank reagent after the concentration of glucose is calculated as 10.10 mg-10 min, wherein the concentration of glucose (A) after the glucose is calculated as 10 mg-10 min, the concentration of glucose in the test tube, the glucose concentration of glucose is calculated as 10min, and the test tube, the value of glucose concentration of glucose is calculated as 10min, the value of glucose is calculated as shown by the value of glucose after.

3) Cloning and sequencing of the oat AsCslH gene. Degenerate primers (P1: 5 '-CTGCGAGGCGTGGTTCG/ACCTTC-3'; P2: 5'-AGGTGATG/ACGTTGCATCCTGTG-3') were designed to amplify oat cDNA based on the sequence of the conserved region of CslH gene of other species as deposited in GenBank; and recovering PCR products through electrophoresis, sequencing the recovered products on an ABI 3730 sequencer, comparing and analyzing sequencing results in an NCBI database, and determining the obtained fragments as target gene fragments.

Designing specific primers according to the cloned gene segments, amplifying upstream and downstream flanking sequences of the AsCSLH gene by using a genome walking technology, specifically performing the method according to the specification of a genome Walker Universal Kit (Clontech) Kit, performing enzyme digestion on genome DNA by using different blunt-end endonucleases, and then connecting the digested genome DNA with a linker in the Kit to build a library. Primer AP1 provided using the kit: 5'-GTAATACGACTCACTATAGGGC-3' and specific primers (PWU 1: 5'-CGTTACTCATCACGCCCGACACCCTTG-3'; PWD 1: 5'-GTCAGGAGGTACTTAGTTGCCGTCGTT-3') were subjected to PCR amplification with 7 cycles of 94 ℃ for 25s at 72 ℃ for 3min, 32 cycles of 94 ℃ for 25s at 67 ℃ for 3min, and 7min at 67 ℃ for the last cycle. Taking the PCR product as a template after being diluted by 50 times, and taking the PCR product as a template by the ratio of AP 2: 5'-CACCGGCCTTGAAGTTATGGTAGTGTT-3' and specific primers (PWU 2: 5'-CGTTACTCATCACGCCCGACACCCTTG-3'; PWD 2: 5'-TCTGCCTACTCACAAACCAGTCCTTCT-3') as primers for nested amplification, and the reaction procedure is as above. And (4) converting, identifying, sequencing and splicing the amplification product. A primer (F: 5'-GCATTCGGTCACGCTTCCACTA-3'; R: 5'-GAGACAGCCGTGCGCTAAGAGT-3') is designed according to the splicing result, and the corresponding sequence of the CslH gene in the oat genome is amplified (Wu bin, Zhang Zong. cloning and characteristic analysis of the oat glucan synthase gene AsCSLH. Proc. Rev. Proc. for crops, 2011,37(4): 723-728).

Taking oat genome DNA of the related population as a template, and carrying out PCR amplification by using Pyrobest high fidelity polymerase (Takara Shuzo) according to the following procedures: pre-denaturation at 94 ℃ for 3 min; at 95 ℃ for 10s, at 56 ℃ for 30s, at 68 ℃ for 3min, for 35 cycles; finally, extending for 5min at 68 ℃; storing at 4 ℃. Sequencing the amplified product by ABI 3730 sequencer. And (4) re-amplifying the sample with the sequencing peak image having the hybrid peak or the set of peaks, and carrying out sequencing verification. And splicing sequencing results, performing multi-sequence joint matching comparison, and performing local manual adjustment to obtain 27 SNPs and 4 InDel.

4) And (3) performing association analysis by combining the phenotype data of the content of the glucan in the oat germplasm resource β in the step 2) and the molecular data in the step 3) by respectively using a General Linear Model (GLM) and a Mixed Linear Model (MLM) of association analysis software TASSEL, wherein the allele frequency threshold is set to be 0.05, and as a result, the SNP locus in the gene is found to be significantly related to the content of the glucan in the oat β, the SNP molecular marker is located on the 20D chromosome of the oat, is derived from the oat glucan synthase gene AsCslH, is located at the 123 th position in SEQ ID No.1, and has the polymorphism of C or G, the detection P values of the GLM model and the MLM model are respectively 0.0087 and 0.0036, and the interpretable phenotypic variation is respectively 2.38% and 3.04%.

Example 2 application experiment of the molecular marker of the invention on the content of oat β glucan

1) Detection of molecular markers in a portion of oat varieties. The specific method comprises the following steps: randomly selecting 24 oat varieties, taking oat genome DNA as a template, taking specific primers (SEQ ID NO.2, 3 and 4) of flanking sequences of the site as primers, and using the three primers at a molar ratio of 5:2: 2. The competitive allele specificity PCR technology is utilized to carry out SNP detection on a sample to be detected, the result shows that the site specificity primer successfully completes genotyping on a material to be detected, the result is shown in figure 2, 24 parts of self-bred lines are divided into two genotypes of 6 parts of C/C and 16 parts of G/G on the site, and the accuracy of correlation analysis detection results and primer design is further proved.

2) The specific method comprises the steps of screening and identifying a variety 'summer hulless oat' with high β glucan content and a variety 'red 38' with low β glucan content respectively, constructing an RIL population comprising 190 families by taking the 'summer hulless oat' and the 'red 38' as parents, taking all families and amphiphilic DNA in the population as templates, taking specific primers (SEQ ID NO.2, 3 and 4) of flanking sequences of the SNP loci as primers, carrying out PCR amplification on a reaction system and a process which are the same as those in experiment 1 in example 2, and obtaining a result shown in figure 3.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. A molecular marker related to the glucan content of oat β, wherein the molecular marker is derived from oat glucan synthase gene AsCslH, is located at position 123 in SEQ ID No.1, and has a polymorphism of C or G.

2. The molecular marker of claim 1, wherein the detection is performed by a primer having a nucleotide sequence as set forth in SEQ ID No. 2-4.

3. Use of a primer for detecting the molecular marker of claim 1 or 2 in breeding β oats with high glucan content.

4. Use of a primer for detecting the molecular marker of claim 1 or 2 for screening β oat varieties with high glucan content.

5. The specific primer pair for detecting the molecular marker related to the oat β glucan content is characterized by consisting of three primers, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 2-4.

6. The use of the specific primer pair of claim 5 in germplasm resources improvement related to oat β glucan content.

7. A method for identifying oats with a high β glucan content, comprising the steps of:

(1) extracting genome DNA of the oat to be detected;

(2) performing PCR amplification reaction by using the DNA extracted in the step (1) as a template and using the specific primer pair as described in claim 5;

8. The kit for detecting the oat with high β glucan content, which comprises the specific primer pair of claim 5, wherein the molar ratio of the primers shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 is 5:2: 2.

9. Use of the kit of claim 8 for screening oat varieties having a high β glucan content.