A molecular marker ahytt8 closely linked to peanut white seed coat and application thereof

Abstract

The application discloses a molecular marker AhyTT8 closely linked to peanut white seed coat and an application thereof, and belongs to the field of agricultural biotechnology. The AhyTT8 comprises: (1) AhyTT8_all, specific primers are a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer as shown in SEQ ID NO:2; (2) AhyTT8_02 and AhyTT8_12, specific primers are forward primers with nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:5 and reverse primers as shown in SEQ ID NO:4 and SEQ ID NO:6. By using the molecular marker provided in the application, it can be determined whether the white seed coat peanut is caused by a TT8 mutation, the seed coat color of the next generation of harvested materials can be determined in advance by detecting peanut DNA, the breeding efficiency is improved, and new germplasm resources can be quickly obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, and in particular to a molecular marker AhyTT8 closely linked to peanut white seed coat and its application. BACKGROUND

[0002] Peanut is an important oil crop and economic crop in China, and is an important source of vegetable oil and protein. Due to the different types and contents of anthocyanins in peanut seed coat, peanuts present different colors such as pink, red, purple, black and white, greatly enriching the types of peanuts in the market. The oil extracted from white peanut is light yellow and transparent, and the color does not change after heating treatment, without the need for decolorization treatment. This characteristic can improve the quality of peanut oil and reduce production costs, which is of great significance to peanut food processing (Zhuang Jianwei, 1998). There are few existing excellent white seed coat peanut varieties, and breeding white seed coat peanuts with high yield, high oil and resistance to various biological and abiotic stresses is the fundamental way to meet market demand. However, the mechanism of white seed coat formation in peanuts is not clear, which seriously hinders the molecular breeding process of white seed coat peanuts.

[0003] There are relatively few studies on the genetics of peanut white seed coat at home and abroad. In early literature, peanut white seed coat color was found to be controlled by two recessive genes (Higgins, 1940; Hammons, 1963). Studies in Arabidopsis thaliana have shown that mutations in multiple genes such as TT8, TT2, and WD40 can cause the seed coat color to change to transparent or light yellow. Studies on multiple species including Camelina sativa, Brassica napus, and Nicotiana tabacum have shown that mutations in the TT8 gene can cause the seed coat color to change from brown to yellow, and the total fatty acid (FA) content to increase (Cai, 2024; Tian, 2020; Zhai, 2020). Based on previous studies, it is inferred that multiple genes in peanuts determine seed coat color, and whether TT8 is associated with peanut white seed coat traits needs further exploration.

[0004] Cultivated peanuts are allo-tetraploid, originating from the domestication of the wild tetraploid species Arachis monticola (AABB, 2n = 4x = 40). Due to having both AA and BB genomes, a single gene mutation may not be enough to change its traits. At the same time, peanut seed coat develops from the integument, and compared to other traits, peanut seed coat color needs to be observed after one generation, which greatly limits the use of traditional breeding methods to breed white seed coat peanuts. Molecular marker-assisted breeding is mainly based on genotypic identification. For white seed coat traits, a portion of the cotyledon can be cut off, and the genotype of the cotyledon can be detected to determine the seed coat color of the next generation of harvested materials in advance, which can shorten the breeding cycle of white peanut new varieties and greatly improve breeding efficiency.

[0005] With the continuous decline in the cost of next-generation sequencing (NGS) technology, high-density SNP (Single Nucleotide Polymorphisms) molecular markers have gradually replaced traditional molecular marker methods such as RAPD, RFLP, AFLP and SSR. By combining the advantages of traditional BSA (Bulked Segregant Analysis) technology and second-generation sequencing, BSA-Seq technology compares the DNA of two groups of populations with different phenotypes (white seed coat peanuts and pink seed coat peanuts) by using high-density SNP molecular markers, thereby efficiently identifying genes or genetic markers related to the target traits.

[0006] The above research lays a foundation for the identification of peanut white seed coat determining genes and molecular breeding. Due to the low linkage of the previously developed markers with the white seed coat color trait, these markers are difficult to accurately and conveniently apply to peanut genetics and molecular breeding research. SUMMARY

[0007] The purpose of the present application is to provide a molecular marker AhyTT8 closely linked to peanut white seed coat and its application, to solve the problems existing in the prior art. The molecular marker AhyTT8 is closely linked to peanut white seed coat, and can be used for identifying white seed coat peanuts, peanut breeding and distinguishing different gene-determined white seed coat peanut germplasm, which is beneficial to quickly obtain new germplasm resources.

[0008] To achieve the above purpose, the present application provides the following scheme:

[0009] The present application provides a molecular marker AhyTT8 closely linked to peanut white seed coat, which comprises the molecular marker shown in any one of the following:

[0010] (1) AhyTT8_all; the specific primers of AhyTT8_all are the forward primer with the nucleotide sequence shown in SEQ ID NO: 1 and the reverse primer with the nucleotide sequence shown in SEQ ID NO: 2;

[0011] (2) AhyTT8_02 and AhyTT8_12; the specific primers of AhyTT8_02 are the forward primer with the nucleotide sequence shown in SEQ ID NO: 3 and the reverse primer with the nucleotide sequence shown in SEQ ID NO: 4, and the specific primers of AhyTT8_12 are the forward primer with the nucleotide sequence shown in SEQ ID NO: 5 and the reverse primer with the nucleotide sequence shown in SEQ ID NO: 6;

[0012] The molecular marker AhyTT8 is located in the region of 97,427,114-97,427,397 of chromosome 2 and the region of 114,045,338-114,045,687 of chromosome 12.

[0013] The application further provides a method for identifying the color of peanut seed coat closely linked to the molecular marker AhyTT8, comprising the following steps:

[0014] The specific primers of the AhyTT8_all are used for PCR amplification with the peanut DNA to be detected as a template; the amplification product is detected by gel electrophoresis, if a single 350bp band appears, the band is recovered from the gel;

[0015] The DNA recovered from the gel is sequenced, and the sequencing result is compared with the peanut reference genome, and the color of the peanut seed coat to be detected is determined according to the comparison result.

[0016] The AhyTT8_all is located in the region of 97,427,114-97,427,397 of chromosome 2 and the region of 114,045,338-114,045,68712 of chromosome 12.

[0017] Preferably, if the sequencing result shows that the sequence is mutated from “GGATCARCT” to “GGATTAACT”, it is determined that the next generation of seeds of the peanut to be detected is white seed coat.

[0018] Optionally, the reaction system of the PCR amplification is as follows: 1 μL of DNA template, 1 μL of forward primer and reverse primer respectively, 10 μL of 2xApexHF FS PCR Master Mix, and water to 20 μL.

[0019] Optionally, the reaction program of the PCR amplification is as follows: pre-denaturation at 94℃ for 5min; 94℃ for 30s, 55℃ for 30s, 72℃ for 30s, 35 cycles; and extension at 72℃ for 7min.

[0020] The application further provides a kit for identifying the white seed coat peanut closely linked to the molecular marker AhyTT8, comprising the specific primers of the molecular marker AhyTT8.

[0021] The molecular marker AhyTT8 is located in the region of 97,427,114-97,427,397 of chromosome 2 and the region of 114,045,338-114,045,68712 of chromosome 12.

[0022] The application further provides the application of the detection product of the molecular marker AhyTT8 in identifying the white seed coat peanut closely linked to the molecular marker AhyTT8.

[0023] The application also provides application of the detection product of the molecular marker AhyTT8 in peanut breeding.

[0024] The application also provides application of the detection product of the molecular marker AhyTT8 in distinguishing different gene-determined white seed coat peanut germplasm.

[0025] Optionally, the detection product comprises specific primers for detecting the molecular marker AhyTT8; the specific primers comprise any one of the following primer pairs:

[0026] (1) a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2;

[0027] (2) a forward primer with a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 4, and a forward primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 6.

[0028] The application discloses the following technical effects:

[0029] The application provides a molecular marker AhyTT8 for identifying white seed coat peanuts, wherein the molecular marker AhyTT8 comprises any one of the following molecular markers: (1) AhyTT8_all; the specific primers of the AhyTT8_all are a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2; (2) AhyTT8_02 and AhyTT8_12; the specific primers of the AhyTT8_02 are a forward primer with a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 4, and the specific primers of the AhyTT8_12 are a forward primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 6. By using the molecular marker provided in the application, it can be determined whether the white seed coat peanut is caused by a TT8 mutation, and the seed coat color of the next generation of harvested materials can be determined in advance by detecting the DNA of cotyledons, breeding efficiency is improved, and new germplasm resources can be quickly obtained.

[0030] The application also provides a method for identifying white seed coat peanuts in linkage with the molecular marker AhyTT8 by using the molecular marker AhyTT8, which is simple in technology and easy to operate, simple in molecular marker, and can be identified by PCR amplification, agarose gel electrophoresis and Sanger sequencing, in addition, the instrument is simple and easy to operate, the instrument precision is not required to be too high, and the conventional instrument of conventional experiments can be operated, and is more easy to be accepted by people. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0032] Figure 1 AhyTT8_02 (MP3D3D) in white seed coat;

[0033] Figure 2 AhyTT8_12 (26781N) in white seed coat;

[0034] Figure 3 Primer position designed for TT8 gene nonsense mutation site;

[0035] Figure 4 AhyTT8_all verification result map in F2 offspring;

[0036] Figure 5 AhyTT8_all verification result map in peanut white seed coat varieties and other color seed coat varieties;

[0037] Figure 6 Sanger sequencing results of PCR products obtained by using AhyTT8_all primers in 6 white seed coat peanuts. DETAILED DESCRIPTION

[0038] The various illustrative embodiments of the present application will now be described in detail below. The detailed description is made with reference to the drawings, in which like reference numerals refer to like elements throughout the several views. The detailed description illustrates by way of example not by way of limitation the principles of the application. This description will clearly enable one skilled in the art to practice the application, and the only practical function of this description is to enable one skilled in the art to practice the application.

[0039] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated value or stated range, and any other stated value or stated range within the stated range, is also included within the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0040] 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 application pertains unless otherwise specifically defined herein. Although 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 application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0041] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0042] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0043] Example 1: Mapping of the gene controlling peanut white seed coat color and design of molecular marker AhyTT8 tightly linked to peanut white seed coat

[0044] To map the gene controlling peanut white seed coat color, the inventors previously used a white seed coat peanut variety (female parent, FZ001) and a pink seed coat peanut variety (male parent, Jihua No. 8 (JH8)) to cross and construct a segregation population FJ (FZ001 x Jihua No. 8). As shown in Table 1, genetic analysis showed that the seed coat color of peanut is controlled by two pairs of recessive genes.

[0045] Table 1: Population construction and genetic analysis

[0046]

[0047] The white seed coat extreme material pool, pink seed coat extreme material pool, male parent Jihua No. 8, and female parent FZ001 of the F2 generation of the FJ population were subjected to whole genome resequencing using the BSA-seq method, with a total sequencing amount of 260 Gb. A total of 201,491 high-quality SNP sites were detected between the parents, covering 20 chromosomes of peanut. Bioinformatics analysis showed that the white seed coat candidate gene was located at Chr02:90M-102M and Chr12:110Mb-120M. Within these two intervals, the TT8 genes MP3D3D and 26781N both had nonsense mutations. Figure 1 and Figure 2

[0048] ​Therefore, the inventor designed primers SEQ ID NO: 1 and SEQ ID NO: 2 (see Table 2) according to the Tifrunner Version 1 reference genome sequence at the mutation site. The primers were simultaneously aligned to chromosome 2 97,427,114-97,427,397 (284bp) and chromosome 12 114,045,338-114,045,687 (350bp). The pink seed coat peanut JH8 had two bands of 284bp and 350bp, and the white seed coat peanut had only one band of 350bp. The marker was named AhyTT8_all (as shown in Table 1). Figure 3 )。

[0049] It was found by FJ population PCR and agarose gel electrophoresis that the primers were closely linked to the white seed coat trait of peanuts. The F2 generation of white seed coat peanuts had only one band of 350bp (as shown in Table 2). Figure 4 ) Since the primer AhyTT8_all cannot distinguish between chromosome 2 and chromosome 12 of FZ001, a pair of chromosome 2 specific primers SEQ ID NO: 3 and SEQ ID NO: 4 and a pair of chromosome 12 specific primers SEQ ID NO: 5 and SEQ ID NO: 6 (see Table 2) were designed, named AhyTT8_02 and AhyTT8_12 (as shown in Table 1). Figure 3 )。

[0050] Table 2 Primer sequences

[0051]

[0052] Example 2 Rapid breeding of high-yield white seed coat peanut varieties using molecular marker AhyTT8

[0053] 1. Experimental object

[0054] The seed coat color of FZ001 peanut variety is white, and the seed coat color of Jihua No. 8 peanut variety is pink. Jihua No. 8 is a high-oleic acid and high-yield peanut variety developed by Shandong Agricultural Academy of Sciences. Jihua No. 8 was used as the research object to genetically improve it to obtain white seed coat traits while maintaining its high-oleic acid and high-yield traits.

[0055] 2. Experimental steps

[0056] (1) Hybridization

[0057] Pink-skinned peanut Jihua No. 8 was used as the female parent, and white-skinned peanut FZ001 was used as the male parent for hybridization.

[0058] The hybridization method is as follows: at the initial flowering stage of peanuts, the flowers of the female parent Jihua No. 8 are removed to make the hybridization time of a combination relatively concentrated; the male organs in the orange yellow flower buds of the female parent are removed after 5 pm every day during the full flowering period of the female parent. The flower has one large banner petal, two wing petals and one hard keel petal. The base of the flower bud is gently pinched with the thumb and middle finger of the left hand, and the flower is gently pried from the outside to the inside with tweezers. The 8 stamens and anthers are gently removed with tweezers once or more times, without damaging the stigma of the pistil. Then the keel petal is restored with fingers, and a label is inserted for counting the number. The dehiscing flowers are artificially pollinated at 6-7 am the next morning; before pollination, the flowers of the male parent FZ001 are collected, then the pollen of the male parent flowers is squeezed out with tweezers, and the pollen is smeared on the stigma of the female parent with tweezers.

[0059] (2) Identification of true and false hybrid F1

[0060] The true and false of the harvested hybrid F1 generation are identified by using molecular markers AhyTT8_02 and AhyTT8_12, and the method is as follows.

[0061] Material taking: all the pods grown on the female parent plant are dried, the shells are peeled off, the seeds are numbered, then part of the seed coat is gently cut off with a blade, and part of the cotyledon tissue (about 30 mg) is scraped off and put into a 2 mL centrifuge tube for DNA extraction, PCR and agarose gel electrophoresis of the product. The AhyTT8_02 marker has two bands of 284 bp and 350 bp, which is true hybrid seed. Then the detected true peanut hybrid seeds are stored in a cold storage for planting in the field in the planting season.

[0062] (1) DNA extraction

[0063] The improved CTAB method is used for genomic DNA extraction of peanut tissue, and the specific method is as follows.

[0064] a) Put peanut cotyledon tissue and a steel ball into a 2 mL centrifuge tube respectively, mark them, and then freeze them in liquid nitrogen. Then put the centrifuge tube into a pre-cooled grinder for grinding.

[0065] b) Take out the centrifuge tube, quickly open the lid, add 600 μL of 65 ℃ preheated 2×CTAB (containing RNase), and shake gently;

[0066] c) Put it into a 65 ℃ water bath for 25 min, and mix slowly 3-5 times during the period;

[0067] d) After cooling, 750 μL chloroform:isopropyl alcohol (24:1) was added, inverted 2-3 times to mix thoroughly, until the solution became a milky turbidity, and left to stand at room temperature for 10 min.

[0068] e) After centrifugation at 12000 rpm for 10 min at room temperature, the supernatant was gently pipetted into a centrifuge tube containing 600 μL pre-cooled isopropyl alcohol, inverted gently to mix thoroughly for 30 s, until a white flocculent DNA precipitate appeared, which was placed at -20°C for 30 min;

[0069] f) Centrifugation at 12000 rpm for 10 min, the supernatant was discarded, and the DNA precipitate was not poured out, then 500 μL of 75% pre-cooled ethanol at -20°C was added to the precipitate, and the precipitate was washed 2-3 times;

[0070] g) Centrifugation at 12000 rpm for 10 min, the liquid was discarded, and the DNA precipitate was left to dry at room temperature;

[0071] h) 50 μL ddH2O was added to the centrifuge tube, and left to stand overnight at room temperature until the DNA was completely dissolved, and stored at -20°C for use.

[0072] (2) PCR reaction and electrophoresis detection

[0073] The parent and all F1 hybrids were detected by molecular markers using specific primers shown in SEQ ID NO: 3-6, and according to the electrophoresis results, those containing parent-specific bands were true hybrids.

[0074] The PCR amplification reaction system is shown in Table 3.

[0075] Table 3 20 μL PCR amplification reaction system

[0076]

[0077] The PCR amplification reaction conditions were as follows: pre-denaturation at 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; and extension at 72°C for 7 min.

[0078] The PCR amplification products were detected by 1.5% agarose gel electrophoresis.

[0079] The electrophoresis buffer system was 1×TAE, and the electrophoresis was performed at 120 V for about 40 min. 100 mL of 1.5% agarose gel was prepared as shown in Table 4.

[0080] Table 4 Preparation of 1.5% agarose gel

[0081]

[0082] The agarose gel electrophoresis method was as follows:

[0083] a) Rinse the gel mold and comb with distilled water, place it on the gel plate, and seal the edges of the mold. Install the comb.

[0084] b) Accurately weigh 1.5 g of agarose dry powder into a triangular flask, then add 100 mL of 1 × TAE electrophoresis buffer.

[0085] c) Place the triangular flask in the microwave oven to melt. After cooling for a few moments, add a drop of fluorescent dye and gently rotate to thoroughly mix the gel solution. Then pour the mixed gel solution into the electrophoresis tank and let it stand until it solidifies.

[0086] d) After standing at room temperature for 30-45 minutes, until the gel is completely coagulated, carefully pull out the comb and place the gel in the electrophoresis tank.

[0087] e) Pour enough electrophoresis buffer into the electrophoresis tank to completely cover the surface of the gel by about 1 mm. If there are air bubbles in the sample wells, try to remove them.

[0088] f) Add 10 times the volume of loading buffer to the DNA sample, mix it well, and then use a gun to slowly add the mixture to the submerged gel sample well.

[0089] g) Turn on the power, with the red electrode as the positive electrode and the black electrode as the negative electrode. Remember that the DNA sample moves from the negative electrode to the positive electrode (i.e., the end closest to the sample well is negative). Usually set the voltage to 120V and run the electrophoresis for 40 minutes.

[0090] h) According to the position of the indicator in the gel, determine whether to terminate the electrophoresis.

[0091] i) After the electrophoresis is complete, turn off the power, use the gel imager to observe the electrophoresis bands and their positions, and compare them with the nucleic acid molecular weight marker to determine the size of the amplified product.

[0092] 3. Screening of offspring

[0093] Using the conventional breeding combined with molecular marker screening breeding program, there are two seasons per year, and the whole cycle needs about three years plus generations. Specifically, Jihua No. 8 is used as the female parent, and the F1 true hybrid offspring is used as the male parent for hybridization, the hybridization method is the same as above, and the harvested BC1F1 is detected again using AhyTT8_02 and AhyTT8_12 molecular markers. The seeds with the parent-specific band (350 bp) for AhyTT8_02 marker and the nonsense mutation site for AhyTT8_12 marker after PCR product sequencing continue to be backcrossed. Through continuous backcrossing and screening for 4 times, BC4F1 generation is obtained, then selfing is performed, and the homozygous offspring is selected for test and variety registration.

[0094] Compared with the traditional method, the breeding efficiency can be greatly improved by using the method of marker combined with backcross selection provided by the application, and the cultivation of white seed coat, high-yield peanut variety can be realized in about 3 years.

[0095] Since the color of peanut seed coat appears after one generation, there is a great blindness in screening and cultivating white peanuts by naked eye, and the identification by the molecular marker screening method of the application can greatly shorten the screening time and reduce the land for planting, thereby laying a foundation for rapid cultivation of excellent new varieties by using molecular breeding means.

[0096] Example 3: Identification of whether the mutation of TT8 gene occurs in other white seed coat peanut germplasm by using molecular marker AhyTT8

[0097] 1. Experimental object

[0098] A plurality of white seed coat peanut germplasms were collected in the laboratory, in addition to FZ001, including BZZ, XY1, PDCS-B, 23F8-258, KNB, RHBYKQ and HN4. Previous studies in Arabidopsis thaliana showed that the mutation of multiple genes can lead to the occurrence of white seed coat. The molecular marker AhyTT8 can be used to distinguish whether these white seed coat peanuts are caused by the mutation of AhyTT8 transcription termination.

[0099] 2. Experimental steps

[0100] 2.1 DNA extraction

[0101] The extraction method is the same as that in Example 2.

[0102] 2.2 PCR amplification and electrophoresis detection

[0103] The forward primer (as shown in SEQ ID NO: 1) and the reverse primer (as shown in SEQ ID NO: 2) of AhyTT8 were used for molecular marker detection of the selected varieties.

[0104] The PCR amplification conditions and electrophoresis detection are the same as those in Example 2.

[0105] 2.3 Gel recovery

[0106] The agarose block containing the target DNA fragment was cut, and the Omega D2500-02 gel recovery kit was used for gel recovery according to the instructions.

[0107] 2.4 Sanger sequencing

[0108] The gel recovery product was sent to a sequencing company for Sanger sequencing.

[0109] Through the results of PCR and agarose gel electrophoresis of different seed coat color peanuts, it was found that in addition to FZ001, the other four white seed coat peanuts Bai Zhenzhu, Xueyu No. 1, Pingdu Color-White and 23F8-258 also only had a 350 bp band (such as Figure 5 ), and the sequencing results showed that the sequence was mutated from "GGATCARCT" to "GGATTAACT" (R represents A / G, such as Figure 6 ). While Kaikangbai only had a 284 bp band, Rihua Baiyuyi Kangqing and Henan No. 4 had two 284 bp and 350 bp bands (such as Figure 5 ), and the sequencing results of Kaikangbai showed that no nonsense mutation occurred (such as Figure 6 ), and the white seed coat traits of the three white seed coat peanuts may be determined by other mechanisms. The results showed that the production of white seed coat in peanuts may be determined by multiple genes respectively, which proved the diversity of white seed coat peanut germplasm. Among the randomly selected 10 non-white seed coat peanuts, there was no individual with only a 350 bp single band, which further proved the high linkage of this molecular marker with white seed coat.

[0110] The above-described embodiments are only descriptions of the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for identifying a color of seed coat of peanut closely linked to a molecular marker AhyTT8, characterized in that, The method comprises the following steps: Taking the peanut DNA to be tested as a template, performing PCR amplification by using a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer as shown in SEQ ID NO: 2; performing gel electrophoresis detection on the amplification product, and if a single 350 bp band appears, recovering the band from the gel; If the sequencing result shows that the sequence of the amplification product contains "GGATTAACT", it is determined that the next-generation seeds of the peanut to be tested are white seed coats.

2. The method of claim 1, wherein, The reaction system of the PCR amplification is as follows: 1 μL of DNA template, 1 μL of the forward primer and the reverse primer respectively, 10 μL of 2×ApexHF FS PCR Master Mix, and water added to 20 μL.

3. The method of claim 1, wherein, The reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 5 min; 35 cycles of 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 30 s, and extension at 72 ℃ for 7 min.

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