CN116814839B - Molecular marker AhyCs1 closely linked with peanut seed coat color speckles and application thereof - Google Patents

Abstract

The invention discloses a molecular marker AhyCs1 that is closely linked to the colorful markings on peanut seed coat and its application. It belongs to the field of agricultural biotechnology. The specific primer pair of the molecular marker AhyCs1 includes the positive sequence shown in SEQ ID No. 1-2. Forward and reverse primers. The molecular marker AhyCs1 of the present invention can be used to identify peanuts with colorful striped seed coats. The seed coat color of the next generation of harvested materials can be determined in advance by detecting the DNA of peanut seeds and leaves, thereby improving breeding efficiency. On the one hand, it is helpful for fine positioning of genes. , isolation and cloning. On the other hand, it has important application value for molecular breeding and quality improvement of peanuts.

Description

A molecular marker AhyCs1 closely linked to the colorful markings on peanut seed coat and its application

Technical field

The invention relates to the field of agricultural biotechnology, and in particular to a molecular marker AhyCs1 that is closely linked to the colorful markings on peanut seed coats and its application.

Background technique

Peanuts are an important oil crop and cash crop in my country. The annual planting area of peanuts in my country is about 70 million acres, and the output is about 17 million tons. Peanuts are rich in fatty acids, proteins, trace elements such as calcium, iron, zinc, and selenium, and vitamin E. They are also rich in functional active substances such as resveratrol, which have important health care effects. In my country, peanuts are also known as the "immortality fruit", and their effects are recorded in the "Compendium of Materia Medica", "Xinglin Medicine", "Luchuan Materia Medica" and "National Collection of Chinese Herbal Medicine" in the Ming Dynasty. In foreign countries, a large number of studies have also shown that peanuts have a significant role in preventing and treating cardiovascular diseases and treating malnutrition. For example, research in The New England Journal of Medicine shows that eating nuts every day can reduce death from heart disease by 29%, and even eating peanuts twice a week can reduce the risk by 24% (Bao et al .2013). In Africa, peanut-based peanut butter with a specially formulated formula has become the United Nations' main therapeutic ready-to-eat nutritional food for the treatment of child malnutrition. Since 2004, Project Peanut Butter has cured 1 million malnourished children.

Peanut seed coat, also known as peanut red coat, is an important carrier of peanut nutrients. Peanut seed coat is rich in natural pigments, especially anthocyanins, which have various biological effects such as inhibiting free radicals and antioxidants (Zhang Yufang, 2003). In addition, the peanut seed coat is also called peanut red coat. It is a traditional Chinese medicine that has the effect of stopping bleeding and treating hemophilia. The peanut red coat pigment is a pure natural pigment, non-toxic and safe, and is a rare antioxidant natural pigment on the market. The seed coat colors of peanuts are mainly pink and red. Peanut varieties with colorful seed coats are rare and generally have low yields. The coloring depth of peanut seed coat is closely related to the accumulation of anthocyanins, and the colorful spots of peanut seed coat contain more anthocyanins. Anthocyanins have various biological effects such as antioxidant and inhibiting the accumulation of free radicals. In recent years, the market demand for colorful seed coat peanuts has been increasing. However, currently there are only a few varieties of colorful spots such as "Yunnan Colorful Peanut". There are no colorful peanut varieties in major peanut producing areas such as Henan and Shandong. The yield of existing colorful peanut varieties is lower than that of conventional varieties. Therefore, cultivating high-yield colorful seed coat peanuts is the fundamental way to meet market demand.

The seed coat of peanut is also developed from the integument. Compared with other traits, the seed coat color can only appear after another generation (Zhao et al. 2020). In addition, peanuts are a crop that "flowers above ground and bears fruit underground", and the color of the seed coat cannot be counted until harvested. The above characteristics have greatly limited the traditional breeding methods to cultivate peanut varieties with colorful seed coats. Molecular marker-assisted breeding focuses on genotype identification. For the colorful seed coat trait, by cutting off part of the cotyledons of the seeds and detecting the genotype of the cotyledons, the seed coat color of the next generation of harvested materials can be determined in advance (Qiu Jingjing et al., 2018) , it is also possible to determine the seed coat color of contemporary harvest materials in advance by detecting the genotype of contemporary leaves, which can accelerate the process of breeding new dark-colored peanut varieties and improve breeding efficiency. However, there have been no reports on the genes and molecular markers related to the colorful seed coat of peanut.

Contents of the invention

The purpose of the present invention is to provide a molecular marker AhyCs1 that is closely linked to the colorful markings on the peanut seed coat and its application, so as to solve the problems existing in the above-mentioned prior art. The molecular marker of the present invention can effectively identify the color of the colorful markings on the seed coat of peanuts. , the seed coat color of the next generation of harvest materials can be determined in advance by detecting peanut seed and leaf DNA, which improves breeding efficiency and contributes to the fine positioning, isolation and cloning of genes. It is also important for molecular breeding and quality improvement of peanuts. application value.

In order to achieve the above objects, the present invention provides the following solutions:

The present invention provides a molecular marker AhyCs1 that is closely linked to the colorful spots on peanut seed coat. The specific primer pair of the molecular marker AhyCs1 includes:

The primer whose nucleotide sequence is shown in SEQ ID No. 1;

The nucleotide sequence of the primer is shown in SEQ ID No. 2.

The invention also provides a method for identifying the color of peanut seed coat using the molecular marker AhyCs1, which includes the following steps:

The DNA of the peanut leaves or seeds to be tested is extracted as a template, and the specific primer pair is used to perform PCR amplification. The amplified product is then detected by gel electrophoresis, and the seed coat color of the peanut to be tested is judged based on the gel electrophoresis results.

Furthermore, if a characteristic band of 233 bp appears in the gel electrophoresis result, it is determined that the seeds of the peanut to be tested and its progeny are colorful striped bark.

Furthermore, if the gel electrophoresis result only shows a characteristic band of 233bp in size, the seeds of the peanut to be tested and its progeny are judged to have obvious colorful stripes; if the gel electrophoresis result shows both characteristic bands of 229bp and 233bp in size. If there is a band, it is determined that the seeds of the peanut to be tested and its offspring are light-colored striped skins; if the gel electrophoresis result only shows a characteristic band of 229 bp, it is determined that the seeds of the peanut to be tested and its offspring are not colorful stripes. Striped leather.

Further, the PCR system for PCR amplification is: 1 μL of DNA template, 0.5 μL of forward primer and reverse primer, 0.5 μL of dNTPmix, 2.0 μL of 10×Taq Buffer, 2.0 μL of MgCl ₂ , 0.20 μL of Taq enzyme, and water to 20 μL.

Further, the PCR program of the PCR amplification is: pre-denaturation at 95°C for 3 minutes; 35 cycles of 94°C for 30s, 56°C for 30s, and 72°C for 20s; and extension at 72°C for 5 minutes. .

Further, the gel electrophoresis detection includes 8% non-denaturing polyacrylamide gel electrophoresis.

The present invention also provides a kit for identifying color spots on peanut seed coat, including a specific primer pair shown in SEQ ID No. 1-2.

The present invention also provides an application of the molecular marker AhyCs1, which is used in peanut breeding and/or peanut quality improvement.

Further, it is used to identify peanuts with colorful markings on the seed coat.

The invention discloses the following technical effects:

The present invention provides a molecular marker AhyCs1 that is closely linked to the colorful striped seed coat of peanuts, which can be used to identify peanuts with colorful striped seed coats. The seed coat color of the next generation of harvest materials can be determined in advance by detecting the DNA of peanut seeds and leaves. Improve breeding efficiency. In addition, peanut is an allotetraploid, and the alleles of the A and B subgenomes are highly homologous. It is difficult for many markers to effectively distinguish the A and B subgenomes. However, the molecular markers of the present invention were developed through many scientific experiments and explorations. Screened, the results are reliable and have high credibility.

The molecular marker of the present invention is a simple PCR marker with simple technical requirements. Compared with the CAPS molecular marker, it does not need to go through steps such as enzyme digestion, purification, recovery, etc., and identification can be achieved directly through PCR amplification and electrophoresis. There are no requirements for instrument operation. It is also relatively low, can be operated using conventional instruments for conventional experiments, and is more easily accepted by people.

The molecular marker of the present invention can effectively identify the color of the colorful striped seed coat of peanuts. On the one hand, it is helpful for the fine positioning, isolation and cloning of genes. On the other hand, it has important application value for molecular breeding and quality improvement of peanuts.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the preliminary positioning of the genes controlling the color of peanut's colorful seed coat through whole-genome resequencing combined with cluster segregation analysis; A: Using segregated populations to construct extreme pools for BSA-seq analysis; B: Linked markers to the colorful seed coat Distribution on peanut genome;

Figure 2 shows the fine mapping of the genes controlling the colorful seed coat of peanut and the development of closely linked molecular markers;

Figure 3 shows the verification results of the molecular marker AhyCs1 in peanut materials of different colors;

Figure 4 shows the sampling of peanut seeds and the germination test of peanut seeds after sampling; A: Intact seed control; B: Seeds after cutting cotyledons; C: Germination status of uncut cotyledons; D: Germination status after cutting cotyledons;

Figure 5 shows the authenticity identification of the hybrid F1 generation using the molecular marker AhyCs1; the boxes are true hybrid offspring;

Figure 6 is a breeding flow chart of molecular marker-assisted recurrent selection and some new high-yielding colorful seed coat peanut lines obtained through molecular marker screening.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range, and any other stated value or value intermediate within a stated range, is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, 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 the 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 invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples of the present invention are exemplary only.

The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.

Example 1 Positioning of genes that control the color of peanut’s colorful striped seed coat and design of molecular marker AhyCs1 that is closely linked to peanut’s colorful striped seed coat

In order to locate and control the colorful seed coat color gene of peanut, the inventor used the peanut variety (female parent) G06G with solid color (pink) seed coat (from the Shandong Provincial Crop Germplasm Resource Bank) and the peanut variety (father) with colorful seed coat. This) colorful peanut (QCHS, from Shandong Crop Germplasm Resource Bank) was crossed to construct a segregating population. Genetic analysis showed that the presence or absence of colorful spots is controlled by a single gene.

Through the phenotypic statistics of the F2 and F3 generations of colored seed coats, 30 offspring lines with significant colored spots and 30 solid color (pink) were selected to construct extreme pools of colored seed coats and pink seed coats, respectively. The parent G06G and Colorful Peanut were subjected to whole-genome resequencing together, and the sequencing volumes of the parent and extreme pools were 32Gb and 92Gb respectively (Figure 1A). Cluster segregation analysis (BSA) was used to analyze the resequencing data, and the distribution of candidate linked SNP sites on the peanut genome was calculated. It was found that there are a large number of SNP sites on chromosome 6 (Chr.06) (Figure 1 B), indicating that the gene controlling the color of the peanut seed coat may be located on chromosome 6.

Based on the initial mapping results of BSA-seq, markers were further developed within the candidate interval, genotype detection was performed in all strains of the segregating population, and a local high-density genetic linkage map of the candidate interval was constructed. Combining the phenotypic statistical results of the seed coat color of each strain, through linkage analysis and comparative genomics analysis, the key genes controlling the color of the peanut colorful stripe seed coat were located and locked within the 24745546-49876762 bp interval on chromosome 6 (Figure 2 ). At the same time, the inventor found that the marker AhyCs1 is closely linked to the color of the colorful seed coat, and the specific primers of AhyCs1 were used to verify it in different color germplasm materials (Figure 3). From Figure 3, it can be seen that the colorful seed coat (deep color and Peanuts with obvious colorful spots and stripes all have characteristic bands of 233 bp. Peanuts with intermediate seed coats (light-colored striped skin) have characteristic bands of 229 and 233 bp at the same time. Peanuts with pink testa all have characteristic bands of 229 bp. It is proved that the marker AhyCs1 is an effective marker for judging the color of peanut seed coat.

The specific primer pair of AhyCs1 includes forward primer: 5'-GTATTGAAGAAAGCTCGGAATT-3' (SEQ ID No. 1) and reverse primer: 5'-AGCGGTGAAGAGGAAGATGA-3' (SEQ ID No. 2).

Example 2 Using the molecular marker AhyCs1 to rapidly cultivate a new high-yielding peanut variety with colorful seed coats

By using the molecular marker AhyCs1 for selection and combined with backcross breeding, ordinary peanuts (recurrent parents) can be improved into new peanut varieties with colorful seed coats in about 3 years, and more than 97% of the genetic background of the recurrent parents can be retained. On the premise of retaining most of the excellent traits of the original peanut varieties, the directional improvement of the seed coat color from pink to colorful seed coats has been achieved.

Taking the common peanut variety Huayu 23 (HY23, from the Shandong Crop Germplasm Resource Bank) as the recurrent parent, and the colorful peanut variety Qicai Peanut (QCHS) as the donor male parent, the specific steps for improving the color of the colorful peanut seed coat are Steps include:

(1)Hybridization

Hybridization was carried out with HY23 as the female parent (reincarnation parent) and the colorful seed coat peanut variety QCHS as the male parent. The hybridization method is as follows: the female parent HY23 begins to be emasculated a few days after it blooms, usually after 16:00 every day. Use the thumb and middle finger of your left hand to pinch the base of the flower bud, hold the tweezers in your right hand to gently open the calyx, flag petals, and wing petals, then use the index finger and thumb of your left hand to press the opened petals, and then use the tweezers to gently press the keel petals. Use tweezers to remove the anthers of the 8 stamens once or several times at the curved back of the flower to expose the stamens. Do not damage the stigma of the pistil, and then push the keel flap back to its original position with your fingers. The emasculated flowers were artificially pollinated between 5:00 and 9:00 on the second day. Before pollination, collect the flowers of the male parent QCHS, and then use tweezers to squeeze out the pollen of the male parent flower. During pollination, hold the emasculated flower with the index and middle fingers of your left hand, and gently squeeze the keel petals with your right thumb or tweezers. , so that the stigma of the pistil is exposed, and then dip the tip of the tweezers into pollen and apply it to the stigma.

(2) Identification of the authenticity of hybrid F ₁

The molecular marker AhyCs1 was used to identify the authenticity of the harvested hybrid F ₁ generation. Methods as below:

Sampling: Harvest all pods from the mother plant and, after drying, number all harvested seeds. Then, use a scalpel to remove part of the seed coat, and then remove part of the cotyledon tissue (about 30 mg), put it into a 1.5 mL centrifuge tube, and put magnetic beads at the same time. The remaining peanut seeds are stored in cold storage and will be planted in fields after testing. Through experiments, it was proved that the germination rate of peanut seeds was not affected after part of the tissue was removed (Figure 4).

DNA extraction: Extract DNA from peanut seeds to be tested. The specific method is as follows:

(1) Quickly cool the 1.5mL centrifuge tube containing peanut tissue with liquid nitrogen, and then grind it;

(2) Preheat CTAB extraction solution (2% CTAB, 1.4mol/LNaCl, 20mmo/L EDTA (pH8.0), 100mmol/LTris-HCl (pH8.0), 2% pvp-40) in a 65°C water bath ;

(3) Estimate the mass of the sample tissue, add 700 μL of preheated CTAB extraction solution to every 200 mg of sample, mix quickly, and incubate at 65°C for 10 to 30 minutes, mixing 2 to 5 times during the period;

(4) Add 1 times the volume of phenol/chloroform/isoamyl alcohol (volume ratio 12:12:1) and mix evenly;

(5) Centrifuge at room temperature at 12,000 rpm for 10 minutes;

(6) Transfer the supernatant to a new centrifuge tube;

(7) Repeat steps (4) to (6) with chloroform/isoamyl alcohol (volume ratio 24:1);

(8) Add 0.7 times the volume of -20°C pre-cooled isopropyl alcohol, mix by inverting, and leave at room temperature for 10 minutes;

(9) Centrifuge at 12,000 rpm at room temperature for 15 minutes;

(10) Pour off the supernatant and wash the pellet 2 to 3 times with 500 μL of 70% ethanol pre-cooled at -20°C;

(11) After the precipitation is dried, dissolve the DNA in 50 μL of deionized water or TE, and place it at -20°C for later use.

(12) Take 5 μl of dissolved DNA, add 45 μL of deionized water, and mix to prepare peanut genomic DNA for later use.

PCR reaction and electrophoresis detection: Use the specific primer pair of AhyCs1 (as shown in SEQ ID No. 1 and SEQ ID No. 2) to conduct molecular marker detection on the parents and all F1 hybrids. According to the electrophoresis results, including the male and female parents This specific band is a true hybrid.

The PCR amplification reaction system is as follows:

The total amplification volume is 20 μL:

DNA template 20-30ng/μL 1μL, specific primer pair 0.5pmol/μL 0.5μL each, 10mM dNTP mix 0.5μL, 10×Taq Buffer 2.0μL, 25mM MgCl ₂ 2.0μL, Taq enzyme 5U/μL 0.20μL, add water to 20μL .

PCR reaction conditions: pre-denaturation at 95°C for 3 minutes; 35 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 20 seconds; extension at 72°C for 5 minutes.

PCR amplification products were detected using 8% nondenaturing polyacrylamide gel (Acr:Bis=39:1) electrophoresis.

Among them, the method for preparing 8% denatured polyacrylamide gel is as follows;

Add 3 μL of indicator, that is, loading buffer (containing 50mM Tris-HCl pH 8.0, 50mM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanine, 50% glycerol) to 10 μL of amplification product;

The electrophoresis buffer system is 1×TBE (90mM Tris-borate pH 8.3, 2mM EDTA), electrophoresis at 120V for about 4 hours.

The preparation of 30ml of 8% non-denaturing polyacrylamide gel is shown in Table 1:

Table 130ml 8% non-denaturing polyacrylamide gel preparation

Silver staining detection, the method is as follows:

a. Stain with 500 mL of 0.1% silver nitrate solution for 15-20 minutes.

b. Rinse quickly with deionized water for about 15 seconds.

c. Developer (1000mL deionized water + 20g NaOH + 0.5g Na ₂ CO ₃ , 1.5mL formaldehyde added immediately) develops color, and shake continuously until the DNA band is clearly visible.

d. Rinse with tap water.

e. Scan and take photos.

As shown in Figure 5, the offspring with both paternal and maternal bands were detected by electrophoresis as true hybrids.

(3) Backcrossing and screening of offspring

The breeding program using molecular marker-assisted recurrent selection involves two seasons per year, and the entire cycle takes about three years plus one generation (Figure 6). First, ordinary high-yielding and high-quality peanuts are used as the female parent (recurrent parent, pink seed coat), and true hybrid F ₁ is used as the male parent for hybridization. The hybridization method is the same as above. The harvested BC ₁ F ₁ is again detected using the AhyCs1 molecular marker. To retain the offspring with paternal-specific bands, the methods of DNA extraction and molecular marker detection are the same as above. Backcrossing and screening were performed 4 times continuously to obtain the BC ₄ F ₁ generation, and then self-crossing was performed, and pure offspring were selected for breeding testing and variety registration.

Since the seed coat develops from the integument, the seed coat color is inherited from the mother. Compared with other traits, the seed coat color only appears after another generation. Therefore, there is great blindness in screening and cultivating colorful seed coat peanuts with the naked eye. Using the markers provided by the present invention combined with the method of backcrossing recurrent selection, the breeding efficiency can be improved, and germplasm innovation of colorful seed coat peanuts can be achieved in about three years (Figure 6). More efficient than traditional methods.

The above-described embodiments only describe preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All modifications and improvements shall fall within the protection scope determined by the claims of the present invention.

Claims (5)

1. A method for identifying the color of peanut seed coat using a specific primer pair of the molecular marker AhyCs1, which is characterized by comprising the following steps: Extract the DNA of peanut leaves or seeds to be tested as a template, use the specific primer pair of the molecular marker AhyCs1 to perform PCR amplification, and then conduct gel electrophoresis detection of the amplified product, and determine the seed coat of the peanut to be tested based on the gel electrophoresis results. color; If the gel electrophoresis result only shows a characteristic band of size 233bp, then the seeds of the peanut and its progeny to be tested are determined to have obvious colorful stripes; if the result of gel electrophoresis shows both characteristic bands of 229bp and 233bp, then It is determined that the seeds of the peanut to be tested and its progeny are light-colored striped bark; if the gel electrophoresis result only shows a characteristic band of 229 bp size, it is determined that the seeds of the peanut to be tested and its progeny are not colored striped bark; The specific primer pair of the molecular marker AhyCs1 is: The primer whose nucleotide sequence is shown in SEQ ID No. 1; The nucleotide sequence of the primer is shown in SEQ ID No. 2. 2. The method according to claim 1, characterized in that the PCR system of the PCR amplification is: 1 μL of DNA template, 0.5 μL of forward primer and reverse primer, 0.5 μL of dNTPmix, 2.0 μL of 10×TaqBuffer, MgCl ₂ 2.0 μL, Taq enzyme 0.20 μL, add water to 20 μL. 3. The method according to claim 1, characterized in that the PCR program of the PCR amplification is: pre-denaturation at 95°C for 3 minutes; 94°C for 30s, 56°C for 30s, 72°C for 20s, 35 cycles; 72°C extension 5 minutes. 4. The method of claim 1, wherein the gel electrophoresis detection includes 8% non-denaturing polyacrylamide gel electrophoresis. 5. An application of a pair of specific primers using the molecular marker AhyCs1 described in claim 1 in identifying peanuts with colorful markings on the seed coat, characterized in that the specific primers using the molecular marker AhyCs1 are used to perform PCR on the peanut DNA to be tested Amplify, and then conduct gel electrophoresis detection on the amplified product, and judge the seed coat color of the peanut to be tested based on the gel electrophoresis results; If the gel electrophoresis result only shows a characteristic band of size 233bp, then the seeds of the peanut and its progeny to be tested are determined to have obvious colorful stripes; if the result of gel electrophoresis shows both characteristic bands of 229bp and 233bp, then The seeds of the peanut to be tested and its progeny are judged to be light-colored striped bark; if the gel electrophoresis result only shows a characteristic band of size 229bp, it is determined that the seeds of the peanut to be tested and its progeny are not colored striped bark.