Molecular marker AhyTT8 closely linked with white seed coat of peanut and application of molecular marker AhyTT8

Abstract

The invention discloses a molecular marker AhyTT8 closely linked with white seed coats of peanuts and application of the molecular marker AhyTT8, and belongs to the technical field of agricultural biology. The AhyTT8 comprises the following components: (1) AhyTT8all, wherein specific primers 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) AhyTT802 and AhyTT812, wherein the specific primers are a forward primer with nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 5 and a reverse primer with nucleotide sequences as shown in SEQ ID NO: 4 and SEQ ID NO: 6. By utilizing the molecular marker provided by the invention, whether the white seed coat peanut is caused by TT8 mutation can be determined, and the seed coat color of a next-generation harvested material can be determined in advance by detecting the DNA of the peanut, so that the breeding efficiency is improved, and a new germplasm resource can be quickly obtained.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, in particular to a molecular marker AhyTT8 tightly linked to peanut white seed coat and application thereof. Background Art

[0002] Peanut is an important oil and cash crop in my country, and an important source of vegetable oil and protein. Due to the different types and contents of anthocyanins in the seed coat of peanuts, it appears in different colors such as pink, red, purple, black and white, which greatly enriches the types of peanuts in the market. The oil extracted from white-skinned peanuts is light yellow and transparent, and the color remains unchanged after heating, and no bleaching treatment is required. 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 high-quality white-skinned peanut varieties available. Cultivating white-skinned peanuts with high yield, high oil content and resistance to various biological and abiotic stresses is the fundamental way to meet market demand. However, the mechanism of the formation of white seed coat traits in peanuts is still unclear, which seriously hinders the molecular breeding process of white seed coat peanuts.

[0003] There are relatively few studies on the inheritance of white seed coat in peanuts at home and abroad. In early literature, the white seed coat color of peanuts was found to be controlled by two sets of recessive genes (Higgins, 1940; Hammons, 1963). Studies in Arabidopsis have shown that mutations in multiple genes such as TT8, TT2, and WD40 can change the seed coat color to transparent or light yellow. Studies on multiple species including Camelinasativa, 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 increase the total fatty acid (FA) content (Cai, 2024; Tian, ​​2020; Zhai, 2020). According to previous studies, there should also be multiple genes in peanuts that determine the seed coat color. Whether TT8 is associated with the white seed coat trait of peanuts needs further exploration.

[0004] Cultivated peanuts are allotetraploids, originating from the domestication of the wild tetraploid species Arachis monticola (AABB, 2n=4x=40). Since they have two sets of genomes, AA and BB, mutations in a single gene may not be sufficient to change their traits. At the same time, the seed coat of peanuts is developed from the integuments. Compared with other traits, the color of the seed coat of peanuts needs to appear in alternate generations. This characteristic greatly limits the use of traditional breeding methods to cultivate white seed coat peanuts. Molecular marker-assisted breeding is mainly based on the identification of genotypes. For the white seed coat trait, the seed coat color of the next generation of harvested materials can be determined in advance by cutting off part of the cotyledons of the seeds and testing the genotype of the cotyledons, which can shorten the breeding cycle of new white peanut varieties and greatly improve breeding efficiency.

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

[0006] The above research has laid the foundation for the identification of genes determining white seed coat in peanuts and molecular breeding. However, due to the low linkage between the previously developed markers and the white seed coat color trait, these markers are difficult to accurately and conveniently apply to peanut genetics and molecular breeding research. Summary of the invention

[0007] The purpose of the present invention is to provide a molecular marker AhyTT8 tightly linked to the white seed coat of peanut and its application to solve the problems existing in the above-mentioned prior art. The molecular marker AhyTT8 is tightly linked to the white seed coat of peanut and can be used to identify white seed coat peanuts, peanut breeding and distinguish white seed coat peanut germplasm determined by different genes, which is conducive to quickly obtaining new germplasm resources.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a molecular marker AhyTT8 tightly linked to peanut white seed coat, wherein the molecular marker AhyTT8 includes any of the following molecular markers:

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

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

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

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

[0014] Using the peanut DNA to be tested as a template, PCR amplification is performed using the specific primers of AhyTT8_all; the amplified product is detected by gel electrophoresis, and if a single 350 bp band appears, the band is recovered by gel electrophoresis;

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

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

[0017] Preferably, if the sequencing result shows that the sequence mutates from "GGATCARCT" to "GGATTAACT", it is determined that the next generation seeds of the peanut to be tested have white seed coat.

[0018] Optionally, the reaction system for PCR amplification is: 1 μL of DNA template, 1 μL of forward primer and 1 μL of reverse primer, 10 μL of 2×ApexHF FS PCR Master Mix, and water is added to 20 μL.

[0019] Optionally, the reaction procedure of the PCR amplification is: 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.

[0020] The present invention also provides a kit for identifying white seed coat peanuts tightly linked to the molecular marker AhyTT8, comprising specific primers for the molecular marker AhyTT8;

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

[0022] The present invention also provides application of the detection product of the molecular marker AhyTT8 in identifying white seed coat peanuts that are tightly linked to the molecular marker AhyTT8.

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

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

[0025] Optionally, the detection product includes specific primers for detecting the molecular marker AhyTT8; the specific primers include primer pairs shown in any one of the following:

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

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

[0028] The present invention discloses the following technical effects:

[0029] The present invention provides a molecular marker AhyTT8 that can identify white seed coat peanuts, and the molecular marker AhyTT8 includes any of the following molecular markers: (1) AhyTT8_all; the specific primer of AhyTT8_all is 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; the specific primer of AhyTT8_02 is a forward primer with a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer as shown in SEQ ID NO: 4, and the specific primer of AhyTT8_12 is a forward primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse primer as shown in SEQ ID NO: 6. Using the molecular marker provided by the present invention, it can be determined whether the white seed coat peanut is caused by the TT8 mutation, and the seed coat color of the next generation of harvested materials can be determined in advance by detecting the DNA of the cotyledons, thereby improving the breeding efficiency and facilitating the rapid acquisition of new germplasm resources.

[0030] The present invention also provides a method for identifying white seed coat peanuts closely linked to the molecular marker AhyTT8 by using the molecular marker AhyTT8. The method is simple in technology and easy to operate. It uses simple molecular markers and can achieve identification through PCR amplification, agarose gel electrophoresis and Sanger sequencing. In addition, the instrument is simple and easy to operate, and the accuracy of the instrument does not need to be too high. It can be operated using conventional instruments in conventional experiments, and is more easily accepted by people. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 It is the nonsense mutation site of AhyTT8_02 (MP3D3D) in the white seed coat;

[0033] Figure 2 It is the nonsense mutation site of AhyTT8_12 (26781N) in the white seed coat;

[0034] Figure 3 Design primer positions for both ends of the nonsense mutation site in the TT8 gene;

[0035] Figure 4 This is a diagram showing the validation results of the molecular marker AhyTT8_all in F2 progeny;

[0036] Figure 5 This is a diagram showing the validation results of the molecular marker AhyTT8_all in peanut white seed coat varieties and other colored seed coat varieties;

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

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

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

[0044] In order to locate the gene controlling the white seed coat color of peanuts, the inventors previously used a white seed coat peanut variety (female parent, FZ001) and a pink seed coat peanut variety (male parent, Jihua 8 (JH8)) to cross and construct a segregating population FJ (FZ001×Jihua 8). As shown in Table 1, genetic analysis showed that the seed coat color of peanuts is controlled by two pairs of recessive genes.

[0045] Table 1 Population construction and genetic analysis

[0046]

[0047] The whole genome resequencing of the F2 generation of the FJ population white seed coat extreme material mixed pool, pink seed coat extreme material mixed pool, male parent Jihua 8, and female parent FZ001 was performed using the BSA-seq method. The total sequencing volume was 260Gb, and a total of 201,491 high-quality SNP sites were detected between the parents, covering 20 chromosomes of peanut. After bioinformatics analysis, the candidate genes for white seed coat were located at Chr02:90M-102M and Chr12:110Mb-120M. In these two intervals, nonsense mutations occurred in the TT8 gene MP3D3D and 26781N ( Figure 1 and Figure 2 ).

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

[0049] The results of FJ population PCR and agarose gel electrophoresis showed that this pair of primers was closely linked to the white seed coat trait of peanuts. The white seed coat peanuts of the F2 generation had only one band of 350 bp (e.g. Figure 4 Since primer AhyTT8_all could not distinguish chromosome 2 and chromosome 12 of FZ001, a pair of chromosome 2-specific primers SEQ ID NO: 3 and SEQ ID NO: 4 and chromosome 12-specific primers SEQ ID NO: 5 and SEQ ID NO: 6 (see Table 2) were designed and named AhyTT8_02 and AhyTT8_12, respectively (see Table 2). Figure 3 ).

[0050] Table 2 Primer sequences

[0051]

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

[0053] 1. Experimental subjects

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

[0055] 2. Experimental steps

[0056] (1) Hybridization

[0057] Hybridization was carried out with pink-skinned peanut Jihua No. 8 as the female parent and white-skinned peanut FZ001 as the male parent.

[0058] The hybridization method is as follows: In the early flowering period of peanuts, there are fewer flowers. In order to make the hybridization time of a combination relatively concentrated, the flowers of the female parent Jihua No. 8 are removed; emasculation begins in the flowering period of the female parent, generally after 5 pm every day, the stamens in the orange-yellow unopened buds of the female parent are removed. The flower consists of a large flag petal, two wing petals and a harder keel petal. Use the thumb and middle finger of the left hand to gently pinch the base of the bud, and use the tweezers in the right hand to gently open the flower from the outside to the inside. Use the tweezers to gently remove the 8 stamens and anthers once or several times, without damaging the stigma of the pistil, and then restore the keel petal with your fingers, and insert a mark to count the number. The emasculated flowers are artificially pollinated at 6-7 am the next day; before pollination, collect the flowers of the male parent FZ001, and then use tweezers to squeeze out the pollen of the male parent's flowers. When pollinating, use tweezers to dip the pollen and apply the pollen to the stigma of the female parent.

[0059] (2) Identification of the authenticity of hybrid F1

[0060] The authenticity of the harvested hybrid F1 generation was identified using molecular markers AhyTT8_02 and AhyTT8_12 as follows.

[0061] Materials collection: After all the pods grown from the mother plant are collected and dried, the seeds are numbered after the shells are peeled, and then part of the seed coat is gently cut off with a blade, and part of the cotyledon tissue (about 30 mg) is scraped and placed in a 2 mL centrifuge tube for DNA extraction, PCR and agarose gel electrophoresis of the product are performed. The AhyTT8_02 marker with two bands of 284 bp and 350 bp is a true hybrid seed. The tested true peanut hybrid seeds are then placed in a cold storage for storage and planted in the field in the planting season.

[0062] (1) DNA extraction

[0063] The genomic DNA was extracted from peanut tissue using the modified CTAB method. The specific method is as follows.

[0064] a) Take peanut cotyledon tissue and a steel ball and place them in a 2 mL centrifuge tube, mark it and freeze it 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 2× CTAB (containing RNase) preheated at 65°C, and shake gently;

[0066] c) Place in a 65°C water bath for 25 minutes, slowly inverting and mixing 3-5 times;

[0067] d) After cooling, add 750 μL of chloroform:isoamyl alcohol (24:1), invert for 2-3 minutes to mix thoroughly until the solution becomes milky and turbid, and let stand at room temperature for 10 minutes.

[0068] e) After centrifugation at 12000 rpm for 10 min at room temperature, gently aspirate the supernatant with a pipette and transfer it into a centrifuge tube containing 600 μL of pre-cooled isopropanol. Gently invert and mix for 30 seconds until a white flocculent precipitate of DNA appears, and place it at -20°C for 30 minutes;

[0069] f) Centrifuge at 12000 rpm for 10 min, discard the supernatant to prevent the DNA precipitate from pouring out, then add 500 μL of -20°C pre-cooled 75% ethanol to the precipitate and wash the precipitate 2 to 3 times;

[0070] g) Centrifuge at 12000 rpm for 10 min, discard the liquid, keep the DNA precipitate, and dry it indoors;

[0071] h) Add 50 μL ddH2O to the centrifuge tube and leave at room temperature overnight until the DNA is completely dissolved. Store at -20°C for future use.

[0072] (2) PCR reaction and electrophoresis detection

[0073] The specific primers shown in SEQ ID NO: 3-6 were used to perform molecular marker detection on the parents and all F1 hybrids. According to the electrophoresis results, the hybrids containing the specific bands of the male and female parents were true hybrids.

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

[0075] Table 3 20μL PCR amplification reaction system

[0076]

[0077] PCR amplification reaction conditions: 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; 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 1.5% agarose gel preparation

[0081]

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

[0083] a) Rinse the gelatin mold and comb with distilled water, place them on the gelatin plate, seal the edges of the mold, and install the comb.

[0084] b) Accurately weigh 1.5 g of agarose powder and add it to a conical flask, then add 100 mL of 1×TAE electrophoresis buffer.

[0085] c) Heat the flask in a microwave oven to melt. After cooling for a while, add a drop of fluorescent dye and gently swirl to mix the gel solution thoroughly. Then pour the mixed gel solution into the electrophoresis tank and let it stand to solidify.

[0086] d) Let stand at room temperature for 30 to 45 minutes until the gel is completely solidified, then carefully remove the comb and place the gel in the electrophoresis tank.

[0087] e) Pour enough electrophoresis buffer into the electrophoresis tank to completely cover the gel surface by about 1 mm. If there are 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 into the immersed gel loading well.

[0089] g) Turn on the power supply, the red electrode is the positive electrode, and the black electrode is the negative electrode. Remember that the DNA sample migrates from the negative electrode to the positive electrode (that is, the end close to the sample well is the negative electrode). Usually set the voltage to 120V and perform electrophoresis for 40 minutes.

[0090] h) Determine whether to terminate electrophoresis based on the position of the indicator in the gel.

[0091] i) After electrophoresis is completed, turn off the power supply, use a gel imager to observe the electrophoresis band and its position, and compare it with the nucleic acid molecular weight standard Marker to determine the size of the amplified product.

[0092] 3. Screening of offspring

[0093] The breeding scheme using conventional breeding combined with molecular marker screening has two seasons each year, and the entire cycle requires about three years of generation. Specifically, Jihua No. 8 is used as the female parent, and the F1 generation true hybrid offspring is used as the male parent for hybridization. The hybridization method is the same as above, and the harvested BC1F1 is tested again using AhyTT8_02 and AhyTT8_12 molecular markers. The AhyTT8_02 marker retains the male-specific band (350bp), and the seeds with nonsense mutation sites after sequencing the PCR product of the AhyTT8_12 marker continue to be backcrossed. By performing backcrossing and screening four times in a row, the BC4F1 generation can be obtained, and then after self-pollination, the homozygous offspring are selected for seed testing and variety registration.

[0094] Compared with the traditional method, the marker combined backcross selection method provided by the present invention can greatly improve the breeding efficiency and realize the breeding of white seed coat and high-yield peanut varieties within about 3 years.

[0095] Since the color of the peanut seed coat can only be revealed in alternate generations, there is a great deal of blindness in the screening and cultivation of white peanuts by naked eyes. However, the present invention uses a molecular marker screening method for identification, which can greatly shorten the screening time and reduce the planting land, laying a foundation for the rapid cultivation of excellent new varieties using molecular breeding methods.

[0096] Example 3 Using the molecular marker AhyTT8 to identify whether the TT8 gene of other white seed coat peanut germplasms has mutated

[0097] 1. Experimental subjects

[0098] The laboratory has collected multiple white seed coat peanut germplasms, including White Pearl (BZZ), Snow Jade No. 1 (XY1), Pingdu Color-White (PDCS-B), 23F8-258, Kainong White (KNB), Rihua White Jade Anti-Green (RHBYKQ) and Henan No. 4 (HN4) in addition to FZ001. Previous studies in Arabidopsis have shown that mutations in 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 all caused by AhyTT8 transcription termination mutations.

[0099] 2. Experimental steps

[0100] 2.1 DNA extraction

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

[0102] 2.2 PCR amplification and electrophoresis detection

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

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

[0105] 2.3 Rubber cutting and recycling

[0106] The agar block containing the target DNA fragment was cut out and gel-extracted using the Omega D2500-02 gel extraction kit according to the instructions.

[0107] 2.4 Sanger sequencing

[0108] The gel recovery products were sent to a sequencing company for Sanger sequencing.

[0109] Through the results of PCR and agarose gel electrophoresis of peanuts with different seed coat colors, it was found that except for FZ001, the other four white seed coat peanuts, Baizhuzhu, Xueyu No. 1, Pingdu Color-White and 23F8-258, also had only one band of 350 bp (such as Figure 5 ), and the sequencing results showed that the sequence mutated from "GGATCARCT" to "GGATTAACT" (R represents A / G, such as Figure 6 ). However, Kainongbai has only one band of 284bp, while Rihuabaiyukangqing and Henan No. 4 have two bands of 284bp and 350bp (such as Figure 5 ), and the sequencing results of Kainongbai showed that no nonsense mutations occurred (such as Figure 6 ), the white seed coat traits of these three white seed coat peanuts may be determined by other mechanisms. The results show that the production of white seed coat in peanuts may be determined by multiple genes, proving the diversity of white seed coat peanut germplasm. Among the 10 randomly selected non-white seed coat peanuts, there was no individual with only a single band of 350bp, further proving the high linkage of this molecular marker with white seed coat.

[0110] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A molecular marker AhyTT8 tightly linked to peanut white seed coat, characterized in that: The molecular marker AhyTT8 includes any of the following molecular markers: (1) AhyTT8_all; the specific primers of AhyTT8_all are a forward primer having 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; the specific primers of AhyTT8_02 are a forward primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer as shown in SEQ ID NO: 4, and the specific primers of AhyTT8_12 are a forward primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse primer as shown in SEQ ID NO: 6; The molecular marker AhyTT8 is located in the region 97,427,114-97,427,397 of chromosome 2 and in the region 114,045,338-114,045,687 of chromosome 12.

2. A method for identifying the peanut seed coat color tightly linked to the molecular marker AhyTT8, characterized in that: The following steps are involved: Using the peanut DNA to be tested as a template, PCR amplification is performed using the specific primers of AhyTT8_all described in claim 1; the amplified product is detected by gel electrophoresis, and if a single 350 bp band appears, the band is recovered by gel electrophoresis; The DNA recovered from the gel is sequenced, and the sequencing results are compared with the peanut reference genome, and the seed coat color of the peanut to be tested is determined according to the comparison results; The AhyTT8_all is located in the region 97,427,114-97,427,397 of chromosome 2 and in the region 114,045,338-114,045,68712 of chromosome 12.

3. The method according to claim 2, characterized in that If the sequencing result shows that the sequence mutates from "GGATCARCT" to "GGATTAACT", it is determined that the next generation seeds of the peanut to be tested have white seed coat.

4. The method according to claim 2, characterized in that The reaction system of the PCR amplification is: 1 μL of DNA template, 1 μL of forward primer and 1 μL of reverse primer, 10 μL of 2×ApexHF FS PCR Master Mix, and water is added to 20 μL.

5. The method according to claim 2, characterized in that The reaction procedure of the PCR amplification is: 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.

6. A kit for identifying white seed coat peanuts tightly linked to the molecular marker AhyTT8, characterized in that: A specific primer comprising the molecular marker AhyTT8 according to claim 1; The molecular marker AhyTT8 is located in the region 97,427,114-97,427,397 of chromosome 2 and in the region 114,045,338-114,045,68712 of chromosome 12.

7. Use of the detection product of the molecular marker AhyTT8 as claimed in claim 1 in identifying white seed coat peanuts that are tightly linked to the molecular marker AhyTT8.

8. Use of the detection product of the molecular marker AhyTT8 as claimed in claim 1 in peanut breeding.

9. Use of the detection product of the molecular marker AhyTT8 as claimed in claim 1 in distinguishing white seed coat peanut germplasm determined by different genes.

10. The use according to any one of claims 7 to 9, characterized in that: The detection product includes specific primers for detecting the molecular marker AhyTT8; the specific primers include primer pairs shown in any one of the following items: (1) a forward primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 2; (2) A forward primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer as shown in SEQ ID NO: 4, and a forward primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse primer as shown in SEQ ID NO: 6.

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