CN116970733B - Molecular marker primer for detecting aegilops tenuis 7M chromosome and application thereof - Google Patents
Molecular marker primer for detecting aegilops tenuis 7M chromosome and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker primer for detecting a aegilops on top 7M chromosome and application thereof, belonging to the field of molecular genetics, wherein the molecular marker primer comprises a nucleotide sequence shown in SEQ ID NO:1-20, 10 pairs of primers shown. The invention utilizes SLAF-seq technology to sequence the two-body substitution system of Easter paraquat, chinese spring and common wheat-Easter paraquat 7M, obtains the specific sequence of 7M chromosome by sequence comparison analysis, and designs 10 pairs of specific molecular marker primers of Easter paraquat 7M chromosome based on the sequence. The markers can be used for detecting 7M chromosomes in a wheat background, and plants with the 7M chromosomes can be rapidly screened, so that molecular auxiliary breeding of the wheat with the blood margin of aegilops on the top of the Chinese silvergrass or the excellent properties is facilitated, and the selection efficiency is improved.
Description
Technical Field
The invention relates to the field of molecular genetics, in particular to a molecular marker primer for detecting a capricorn-grass 7M chromosome and application thereof.
Background
The wheat kindred plants have wide geographic distribution, strong environmental adaptability and rich genetic diversity, and have great potential for improving the yield, quality, disease resistance and stress resistance of the wheat. Aegilops comosa (Aegilops comosa sm.in. Ibth.et Sm., 2n=2x=14, mm) is close to common wheat in relation, is one of important diploid species of Aegilops, contains abundant excellent genes for resisting wheat diseases, stress tolerance and quality improvement, and can transfer the excellent genes or characters to common wheat through sexual hybridization so as to realize genetic improvement of the wheat.
A single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refers to a polymorphism in the DNA sequence at a particular nucleotide position in the genomic DNA due to changes in transitions, transversions, insertions, deletions, etc. High throughput sequencing techniques can achieve dense coverage of SNPs without reference genomes. The specific site amplified fragment sequencing technology (Specific Length Amplified Fragment Sequencing, SLAF-seq) developed based on high-throughput sequencing has the characteristics of low cost and high throughput, and is a set of simplified genome sequencing technology. Can be widely applied to crops such as rice, wheat, rape and the like.
The development of molecular markers that can detect and track exogenous genetic material is of great importance in wheat breeding. The current conventional methods for developing markers are time consuming, expensive and not highly accurate, such as using 200 RAPD primers to screen 3 Huashan new wheat straw genome specific repeats. The SLAF-seq technology has been successfully applied to the efficient development of molecular markers of various wild kindred seeds of wheat, such as elytrigia elongata, wheatgrass, new wheatgrass in Huashan, and the like. However, the development of a aegilops on the top-awamori 7M chromosome specific molecular marker has not been used.
Disclosure of Invention
The invention aims to provide a molecular marker primer for detecting a 7M chromosome of aegilops on top of the invention and application thereof, so as to solve the problems in the prior art, and provides a 10-pair specific molecular marker primer for the 7M chromosome of aegilops on top of the invention, wherein the markers can be used for detecting the 7M chromosome in a wheat background, and plants with the 7M chromosome can be rapidly screened, so that molecular auxiliary breeding with the blood margin of aegilops on top of the invention or wheat with excellent properties is facilitated, and the selection efficiency is improved.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a molecular marker primer for detecting a aegilops on top-miscanthus 7M chromosome, which comprises a nucleotide sequence shown in SEQ ID NO:1-20, 10 pairs of primers shown.
The invention also provides a kit for detecting the aegilops on top-miscanthus 7M chromosome, which comprises the molecular marker primer.
The invention also provides a method for detecting the wheat introgression line containing the aegilops on the top-miscanthus 7M chromosome, which comprises the following steps:
extracting genome DNA of a sample to be detected as a template, and utilizing a nucleotide sequence shown as SEQ ID NO:1-20, performing PCR amplification on the PCR amplification product, and performing gel electrophoresis on the PCR amplification product, and judging whether the sample to be detected contains the aegilops horn 7M chromosome according to the existence of electrophoresis bands.
Further, if an electrophoresis band appears and the band size is 500bp, the sample to be detected contains a capricorn kaempferi 7M chromosome; if the electrophoresis band is not provided, the sample to be detected does not contain the aegilops on the top of the chromosome 7M.
Further, the reaction system for PCR amplification comprises: 2 XTaq Master Mix 10 mu L, ddH 2 O7. Mu.L, 1. Mu.L of the front primer, 1. Mu.L of the rear primer and 1. Mu.L of the DNA.
Further, the reaction procedure of the PCR amplification includes: pre-denaturation at 94℃for 3min; denaturation at 94℃for 20s, annealing at 58℃for 20s, elongation at 72℃for 25s,35 cycles; the extension is maintained at 72 ℃ for 3min, and the temperature is kept at 12 ℃.
The invention also provides application of the molecular marker primer or the kit in identifying whether wheat contains a aegilops on the top of the aegilops on the 7M chromosome.
The invention also provides application of the molecular marker primer or the kit in wild wheat resource utilization, genetic improvement of wheat or molecular breeding of wheat.
The invention also provides application of the molecular marker primer or the kit in the resource utilization of the aegilops on the top of the aegilops on the bottom of the application of the molecular marker primer or the kit in the genetic material detection of the aegilops on the top of the application of the molecular marker primer or the kit in the application of the application.
The invention discloses the following technical effects:
the invention utilizes SLAF-seq technology to sequence the two-body substitution system of Easter paraquat, chinese spring and common wheat-Easter paraquat 7M, and obtains the specific sequence of 7M chromosome by sequence comparison analysis, based on which 10 pairs of specific molecular marker primers of Easter paraquat 7M chromosome are designed. The markers can be used for detecting 7M chromosomes in a wheat background, and plants with the 7M chromosomes can be rapidly screened, so that molecular auxiliary breeding of the wheat with the blood margin of aegilops on the top of the Chinese silvergrass or the excellent properties is facilitated, and the selection efficiency is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of the amplified bands of 7M chromosome specific molecular marker 7M-SF-93 in various materials; 1: aegilops tenuis PI 551070;2: the tetraploid wheat-aegilops acrosilverus is artificially synthesized into a diploid STM (Langdon/PI 551070); 3: NAL-33 (common wheat-Easter pratensis 7M (7A) diad substitution line); 4: NAL-42 (common wheat-Easter pratensis 7M (7B) monomer substitution line); 5: NAL-43 (common wheat-Easter pratensis 7M (7D) monomer substitution line); 6: NAL-44 (common wheat-Easter pratensis 7M monomer addition line); 7: langdon;8: CSph2a;9: BZ 1313;10: CN 16;
FIG. 2 is a PCR band with 16 molecular markers in 4 different aegilops on top populations; m1: PI 551070; m2: PI 551059; m3: PI 551061; m4: PI 551062;
FIG. 3 is a graph showing 16 molecular marker analyses of aegilops tauschii different common wheat-aegilops tauschii different staining systems and aegilops tauschii materials of different genomes; 1M in a: different dyeing system NAL-35 containing Naemorhedi 1M; 2M: different dyeing system NAL-38 containing Naemorhedi 2M; 3M: different dyeing system NAL-39 containing Naemorhedi 3M; 5M: different dyeing system NAL-40 containing aegilops on top 5M; 7M: different dyeing system NAL-33 containing Naemorhedi 7M; m in b: diploid aegilops tenuis PI 554419 containing M genome; u: diploid aegilops parviflora CIae 29 containing the U genome; d: diploid arthrodesis AS 60 containing D genome; c: diploid aegilops tenuis PI 551139 containing the C genome;
FIG. 4 shows 74 7M (7B) monomer substitution lines F 2 Molecular marker analysis of the selfing population and the use of the FISH probe psc119.2/pTa535, pTa 71/(CTT) 12 Verifying a result; a:7M-SF-9 molecular marker analysis results; b-d, e1, f1 and g1: FISH probe psc119.2/pTa535 assay; e2 F2 and g2: FISH probe pTa 71/(CTT) 12 Analyzing the result; wherein a: B1-B74; b: b16; c: b37; d: b45; e1-e2: b9; f1-f2: b33; g1-g2: B58.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The common wheat-Aegilops acremonium metastain system part material used in the following examples is described in document "Zuo et al (2022) Identification and characterization ofwheat-Aegilops comosa 7M (7A) disomic substitution lines with stripe rust andpowdery mildew resistance. Plant disease.106:2663-2671 "and" Zuo et al (2023) Disomic 1M (1B) Triticum aestivum-Aegilops comosa substitution line with favorable protein properties. Journal ofAgricultural and Food chemistry.71:7258-7267", other materials are available from Sichuan university of agriculture, wheat research. Wherein, the genealogy source of the common wheat-aegilops tenuis 7M (7A) two-body substitution system NAL-33 is Langdon/PI 551070// CSph2a///BZ 1313//// CN 16.PI 551070 is Easter parada, langdon is tetraploid wheat, and CSph2a, BZ 1313 and CN 16 are hexaploid wheat. The two-body substitution system is obtained by hybridizing a hybrid chromosome of aegilops on the top of the Chinese aegilops on the side of the tetraploid wheat with different hexaploid wheat after natural doubling. Different aegilops verrucosa materials comprise PI 551059, PI 551061 and PI 551062, aegilops verrucosa with different genomes comprise aegilops verrucosa PI 551139 containing C genome, aegilops parviflora CIae 29 containing U genome and Artemisia rupestris AS 60 containing D genome, different common wheat-aegilops verrucosa heterodyeing systems comprise different materials containing aegilops verrucosa 1M, 2M, 3M, 5M and 7M, and the selfing segregation population is F of 7M (7B) chromosome monomer substitution NAL-42 selfing 2 。
The early research of the invention shows that the 7M chromosome of the aegilops on the top can contain excellent genes for resisting stripe rust and powdery mildew, and the stripe rust and powdery mildew resistance of the background wheat can be improved. But currently there is a lack of technical means to detect the aegilops on the top in the wheat background 7M chromosome. The following studies were carried out for this purpose:
example 1 development of a specific molecular marker for the 7M chromosome of Easter pratensis
1. Extraction and purification of genomic DNA
The genome DNA of the aegilops on the top and the common wheat-aegilops on the top 7M (7A) two-body substitution system and the Chinese spring are respectively extracted by using a CTAB method, and the specific operation steps are as follows:
(1) Cutting young leaves into small sections by using sterilized scissors, and placing the small sections in a 2ml EP pipe containing 1 steel ball;
(2) Then freezing in liquid nitrogen for 10min, and grinding with a high-flux plant tissue grinder for 1min with the frequency of about 21Hz;
(3) After grinding the sample, adding 600 mu L of 2 XCTAB extracting solution preheated to 65 ℃ into an EP tube, rapidly vibrating and uniformly mixing, and then placing in a 65 ℃ water bath for 1-2h, and lightly shaking every 10-15min to fully react;
(4) Taking out the sample, cooling to room temperature, adding equal volume of chloroform/isoamyl alcohol (24:1), and gently mixing upside down until the supernatant is milk-shaped;
(5) Centrifuging at 12000rpm for 10min, collecting 500 μl supernatant, placing in 1.5ml EP tube, adding 500 μl pre-cooled isopropanol at-20deg.C, slightly reversing, mixing, placing in a refrigerator at-20deg.C for 1-2 hr, picking out precipitated DNA with toothpick, washing with 70% and 100% ethanol for 1 time respectively until the DNA appears white, and air drying in a fume hood;
(6) After the alcohol had completely volatilized, an appropriate amount of 1×TE at pH 8.0 was added to the EP tube, the DNA concentration was measured with a Nanodrop DNA concentration detector, and the DNA mass was detected with 1% agarose electrophoresis.
2. Obtaining specific sequence tags based on SLAF-seq technology
Sequencing of genomic DNA of Naemorhedi PI 551070, emammalia niruri 7M (7A) two-body substitution NAL-33 and China spring (completed by Beijing Baimeike Biotech Co., ltd.) was performed by SLAF-seq method to obtain sequence tags of each sample. SLAFs sequenced by PI 551070 and NAL-33 are clustered with China spring respectively, and sequences which are not gathered together are subjected to BLAST, so that SLAFs with sequence similarity exceeding 90% are left. And then comparing with the nucleic acid and protein sequences of the Chinese spring reference genome and other species, and leaving the sequence with the similarity of 0. The number of specific sequence tags of the finally obtained 7M chromosome is 58.
3. Development of aegilops tenuis 7M chromosome specific molecular marker
By clustering and comparing SLAF-seq sequences obtained from different materials, 58 7M specific candidate SLAF s sequence tags are obtained, and primers are further designed by using on-line software (https:// www.ncbi.nlm.nih.gov/tools/prime r-blast/index. Cgilink_LOC=blastHome) of NCBI website, so that 30 pairs are successfully designed. Primers were synthesized at the same company as the Optimago, inc. of the Optimago, and purified by HAP (High affinity purification). PCR amplification is carried out on primers in diploid apocynum venetum, tetraploid wheat-apocynum venetum amphidiploid, tetraploid and hexaploid wheat parents and common wheat-apocynum venetum introgression lines containing 7M chromosomes (an amplification system and a program are shown in tables 1 and 2), 16 pairs of primers are obtained, target strips are arranged in apocynum venetum diploid, tetraploid wheat-apocynum venetum amphidiploid and common wheat-apocynum venetum 7M chromosome introgression lines, no strips are arranged in tetraploid and hexaploid wheat parents, the primers are used as 7M chromosome specific candidate molecular markers, wherein FIG. 1 is a PCR strip of 7M chromosome specific molecular markers 7M-SF-93 in apocynum mandshurdle and an apocynum venetum 7M-containing metachromatin systems, and 1: aegilops tenuis PI 551070;2: the tetraploid wheat-aegilops acrosilverus is artificially synthesized into a diploid STM (Langdon/PI 551070); 3: NAL-33 (common wheat-Easter pratensis 7M (7A) diad substitution line); 4: NAL-42 (common wheat-Easter pratensis 7M (7B) monomer substitution line); 5: NAL-43 (common wheat-Easter pratensis 7M (7D) monomer substitution line); 6: NAL-44 (common wheat-Easter pratensis 7M monomer addition line); 7: langdon;8: CSph2a;9: BZ 1313;10: CN 16. Amplification in different aegilops on the top material showed that 16 pairs of primers all had identical bands (FIG. 2). Further carrying out PCR amplification in a common wheat-top Chinese aegilops penetrating line containing different M chromosomes (1M, 2M, 3M, 5M and 7M) and aegilops containing different genomes to obtain a target strip only in the common wheat-top Chinese aegilops penetrating line containing 7M chromosomes and the top Chinese aegilopsThe 10 pairs of primers of the band (Table 3) served as 7M chromosome-specific molecular markers (FIG. 3). Randomly selecting one from 10 markers, and selfing F in 7M (7B) monomer substitution NAL-42 2 And carrying out PCR amplification on the population, randomly selecting 3 single plants containing target bands and no target bands for FISH analysis, and verifying the stability of the molecular marker. As shown in FIG. 4, the random selection marker 7M-SF-9 was used to PCR amplify the F2 population of the 7M monomer substitution NAL-42 selfing, and a total of 74 individuals (B1-B74), 3 individuals B16, B37 and B45 without target bands and 3 individuals B9, B33 and B58 with target bands were randomly selected, respectively, using the FISH probe pSc119.2/pTa535 (B-d, e1, F1 and g1 in FIG. 4), pTa 71/(CTT) 12 (e 2, f2 and g2 in FIG. 4) the results of the FISH analysis show that B16, B37 and B45 do not contain 7M chromosome, and B9, B33 and B58 do not contain 7M chromosome, which indicates that the molecular marker primer designed by the invention has accurate identification and stability.
4. PCR reaction
PCR amplification is carried out by taking a common wheat-aegilops verrucosa heterodye system containing 7M chromosomes, a parent aegilops verrucosa, tetraploid wheat-aegilops verrucosa amphidiploid, tetraploid and hexaploid wheat as templates and according to primers designed by a specific sequence of the aegilops verrucosa chromosome obtained by sequencing. The PCR reaction system and procedure are shown in tables 1 and 2.
TABLE 1 PCR reaction System
| PCR reaction component | Volume (mu L) |
| 2 XTaq Master Mix (Nuo Wei Zan) | 10 |
| ddH 2 O | 7 |
| Primer F (10. Mu.M) | 1 |
| Primer R (10. Mu.M) | 1 |
| DNA(150ng/μL) | 1 |
| Totals to | 20 |
TABLE 2 PCR reaction procedure
5. Agarose gel electrophoresis and PCR reaction product analysis
The PCR amplified product was electrophoresed on a 2% agarose gel at 120V constant pressure for 20-30min with 1 XTAE as electrophoresis buffer. After the electrophoresis was completed, the result was observed. If the amphidiploids of common wheat-aegilops verrucosa, aegilops verrucosa and tetraploid wheat-aegilops verrucosa have amplified bands and tetraploid and hexaploid wheat have no amplified bands, the marker is considered to be a candidate aegilops verrucosa chromosome specific molecular marker. And then verifying the repeatability, the specificity and the stability of the markers in different aegilops verrucosa materials, common wheat-aegilops verrucosa heterogenic dyeing systems containing different M chromosomes, aegilops verrucosa of different genomes and separated groups of 7M chromosomes to obtain the specific molecular markers of the 7M chromosomes of the aegilops verrucosa. The 10 7M chromosome specific molecular markers have good accuracy, reliability and specificity. The 10 pairs of molecular marker primers specific to the 7M chromosome of aegilops on the top are shown in Table 3. FIG. 1 shows the amplification result of the molecular marker 7M-SF-93, and shows that the 500bp band can be amplified in the common wheat-Easter paraquat 7M related heterodyeing system, easter paraquat and tetraploid wheat-Easter paraquat amphidiploid, but the amplified band is not in the tetraploid and hexaploid wheat.
TABLE 3.10 Easter pratensis 7M chromosome specific molecular markers
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (3)
1. A method for detecting a wheat introgression line containing a aegilops on a 7M chromosome, comprising the steps of:
extracting genome DNA of a sample to be detected as a template, carrying out PCR amplification by using a molecular marker primer, carrying out gel electrophoresis on a PCR amplification product, and judging whether the sample to be detected contains a aegilops on the basis of the existence of an electrophoresis band or not;
if an electrophoresis band appears and the band size is 500bp, the sample to be detected contains a capricorn 7M chromosome; if the electrophoresis strip does not exist, the sample to be detected does not contain a capricorn 7M chromosome;
the molecular marker primer is a nucleotide sequence shown in SEQ ID NO:1-2, SEQ ID NO:3-4, SEQ ID NO:5-6, SEQ ID NO:7-8, SEQ ID NO:9-10, SEQ ID NO:11-12, SEQ ID NO:13-14, SEQ ID NO:15-16, SEQ ID NO:17-18 and SEQ ID NO:19-20, and one or more of the 10 primer pairs shown.
2. The method of claim 1, wherein the reaction system for PCR amplification comprises: 2 XTaq Master Mix 10 mu L, ddH 2 O7. Mu.L, 1. Mu.L of the front primer, 1. Mu.L of the rear primer and 1. Mu.L of the DNA.
3. The application of a molecular marker primer in the identification of whether wheat contains a aegilops horn 7M chromosome is characterized in that the molecular marker primer has a nucleotide sequence shown in SEQ ID NO:1-2, SEQ ID NO:3-4, SEQ ID NO:5-6, SEQ ID NO:7-8, SEQ ID NO:9-10, SEQ ID NO:11-12, SEQ ID NO:13-14, SEQ ID NO:15-16, SEQ ID NO:17-18 and SEQ ID NO:19-20, and one or more of the 10 primer pairs shown.
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