CN115011728B - Molecular markers and application of the function of the peanut endocarp color regulatory gene AhLAC - Google Patents

Abstract

The present invention provides a molecular marker and application of the function of the peanut endocarp color regulatory gene AhLAC, wherein the molecular marker is A05.114993389, located at 114,993,389bp of peanut chromosome 5, and the molecular marker site is an InDel insertion and deletion marker. Based on the mutation type of the sequence of the peanut endocarp color regulatory gene AhLAC, the present invention develops the KASP molecular marker of A05.114993389 (InDel site), and the typing results of the marker in the natural population material of peanut are completely linked to the endocarp color, proving the accuracy of the marker. The KASP marker can quickly and accurately obtain the endocarp color information of the peanut material, and can be applied to peanut molecular assisted marker breeding.

Description

Molecular marker for functions of peanut inner seed coat color regulating gene AhLAC and application

Technical Field

The invention relates to development and application of a functional molecular marker of a peanut kernel coat color regulating gene AhLAC, belonging to the field of molecular biology.

Background

Peanuts (Arachis hypogaea l.; 2n=4x=40) are important oil crops with total world yield up to 4765 ten thousand tons, next to soybeans, canola and sunflower. Anthocyanin composition is a factor in determining peanut seed coat color. Anthocyanin is used as a secondary metabolic substance of plants, widely exists in flowering plants, is a safe antioxidant, and has health care values of protecting eyesight, preventing protein saccharification and the like. With the enhancement of health care consciousness, anthocyanin is favored by more and more people, and research on anthocyanin synthesis routes and cultivation of anthocyanin-rich crop varieties become one of hot spots of research.

Seed coat color is an important descriptive trait that distinguishes different peanut varieties. Peanut seed coat color trait description is divided into exocarp color and endocoat color. At present, peanut exocarp colors are divided into a single color type and a multicolor type, wherein the single color type exocarp colors comprise five types of white, brown, pink, red and purple, while the multicolor type exocarp colors comprise red and white variegates, purple stripes on brown, white spots on red and the like. The colors of the inner skin are divided into two types, white and yellow. Yellow is a separate yellow from deep yellow. The seed coats have great influence on the sensory evaluation of peanut products, and the deep color peanut seed coats have great influence on the flavor. The bitter taste of peanut is closely related to the color of the seed coat, and the darker the color of the seed coat, the more the bitter taste is.

In plants, browning reactions are generally the oxidation of phenolic compounds to give quinone compounds, which are unstable molecules that tend to form yellowish-brown polymers. These reactions can be catalyzed by polyphenol oxidase enzymes such as laccase (laccase), catechol oxidase (catechol oxidase), tyrosinase (tyrosinase), and peroxidase (peroxidases). Laccase enzymes are widely found in eukaryotes (higher plants, fungi and insects) and prokaryotes. They are generally less specific for reducing substrates and are capable of oxidizing diphenols, monophenolic ascorbic acid. In transgenic corn, laccase gene expression is associated with corn kernel browning and limited germination.

Competitive allele-specific PCR (Kompetitive ALLELE SPECIFIC PCR, KASP) allows for accurate bi-allele determination of SNPs and InDels at specific sites in a broad range of genomic DNA samples, even some complex genomic DNA samples. Compared with the TaqMan bicolor marked probe method, the Massarray molecular weight array technology and the Affymetrix SNP chip, the KASP technology has higher flexibility, lower reagent cost and at least twice the data volume obtained by the same cost. Therefore, the QTL localization of the regulatory genes of peanut coat color is performed by utilizing peanut recombinant inbred line populations, and molecular markers are developed based on target genes, so that the method can be applied to peanut breeding.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide development and application of a functional molecular marker of a peanut inner seed coat color regulating gene AhLAC.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the functional molecular marker of the peanut endoplasmic reticulum color regulating gene AhLAC is A05.114993389, which is positioned at 114,993,389bp of peanut chromosome 5, and the molecular marker locus is InDel InDel marker;

wherein, the sequence 200bp before and after the wild type molecular marker locus is:

CCTGGTGACTTTTTTCCTTGCTCAAGAGAAGGTATGCTGTATGCATGAAGATTTCATTTCAAGCATGCTCAAATGTTCTTTGATTTTAGAATCTCTAACTGCATCAATTCTTTTTGTTTTTTCACAGAAACATTCAAGTTTAATGTAGATTTTGGCAAGACTTATCTGCTGAGGATTGTGAATGCTGCAATGAATGTAGTTCTCTTTTTCTCTGTTTACAAACACAGTCTCACTGTTGTTGGTGCTGATGCTGCCTACACCGAGCCATTAACAAGAGACTTCATATGCATAGCTCCAGGACAATCACTTGATGTTTTGTTGCATGCAAATCAAGAACCTGATCACTATTACATGGCTGCAAGAGCATATTCAAGTTCCTCTATTCTTCCTTTTGATAACA;

The mutant is inserted 214bp sequence, the inserted 214bp sequence is arranged in square brackets, and the sequences of 200bp before and after the molecular marker locus are as follows:

CCTGGTGACTTTTTTCCTTGCTCAAGAGAAGGTATGCTGTATGCATGAAGATTTCATTTCAAGCATGCTCAAATGTTCTTTGATTTTAGAATCTCTAACTGCATCAATTCTTTTTGTTTTTTCACAGAAACATTCAAGTTTAATGTAGATTTTGGCAAGACTTATCTGCTGAGGATTGTGAATGCTGCAATGAATGTAGT[TCTCTTTTTGGCAACTACTCCTATGAAGATGCCAAAAACATCTTTTTATGATGATCTTTGTTTAAAAAGTGTAACTTATTTGTTTAGCAACACTTTAAATAAAGATAACACTTTTACTTTAAATAAAATCAAACCCTACAATCTATCATCTAATGGTCAAAATGAAATATCTTCATATGATGACAATCATAAAATCTTCATTGGAGTAGCCACC]TCTCTTTTTCTCTGTTTACAAACACAGTCTCACTGTTGTTGGTGCTGATGCTGCCTACACCGAGCCATTAACAAGAGACTTCATATGCATAGCTCCAGGACAATCACTTGATGTTTTGTTGCATGCAAATCAAGAACCTGATCACTATTACATGGCTGCAAGAGCATATTCAAGTTCCTCTATTCTTCCTTTTGATAACA.

a KASP primer combination for amplifying the functional molecular marker, the KASP primer combination comprising:

qIIC_A05_F1:5’-GCTCAGAGAAAAAGAGAACTACATTC-3’,

qIIC_A05_F2:5’-ATTGACCATTAGATGATAGATTGTAG-3’,

qIIC_A05_com:5’-CAAGAGAAGGTATGCTGTATGCA-3’。

the method for identifying the color of the peanut inner seed coat by utilizing the functional molecular marker comprises the following steps:

(1) Extracting DNA of a peanut sample to be identified, detecting genotype data of the peanut sample to be identified by using the KASP primer combination of claim 2, and carrying out genotyping on the InDel locus of A05.114993389 of the peanut sample to be identified by using a SNPLine genotyping platform;

(2) If the parting result of the A05.114993389 site is mutant (MITE: MITE), the color of the seed coats in the peanut sample to be identified is white, and if the parting result of the A05.114993389 site is wild type (-: -), the color of the seed coats in the peanut sample to be identified is yellow.

The identification in the step (1) adopts a PCR technology, and a PCR amplification program comprises 94 ℃ pre-denaturation for 15min, 94 ℃ denaturation for 20s,61 ℃ to 55 ℃ extension for 1min,10 cycles, 94 ℃ denaturation for 20s,55 ℃ extension for 1min,26 cycles and 10 ℃ preservation.

The functional molecular marker is applied to color identification of peanut inner seed coats.

The functional molecular marker is applied to molecular breeding of peanuts.

The invention has the beneficial effects that:

The invention constructs a high-density linkage map by utilizing a recombinant inbred line population taking far impurities 9102 and wt09-0023 as parents, and locates to a QTL (qIIC _A05) for regulating and controlling the color of the peanut inner seed coat, wherein the physical map position corresponding to qIIC _A05 is chr05:114741295-115061124, the interval size is 319.8Kb, the LOD value is 186.5-444.4, and the genetic contribution rate is 75.8% -94.6%. The QTL positioning is reported for the first time, and fills the blank of peanut inner seed coat research.

The invention obtains the target gene AhLAC for regulating and controlling the color of the inner seed coat based on qIIC _A05 interval, clones the homologous gene mutant of the gene transformed arabidopsis thaliana, and the color of the seed coat of a positive plant is darker than that of the yellow seed coat of the mutant, thus proving that the AhLAC gene has laccase activity. The gene for regulating and controlling the color of the peanut inner seed coat is cloned for the first time, and fills the blank of the research of the inner seed coat regulating and controlling gene.

Based on mutation type of sequence of peanut inner seed coat color regulating gene AhLAC, KASP molecular marker of A05.114993389 (InDel locus) is developed, and parting result of the marker in peanut natural population material is completely linked with inner seed coat color, thus proving accuracy of the marker. The KASP marker can rapidly and accurately obtain the color information of the inner seed coat of the peanut material, and can be applied to peanut molecular auxiliary marker breeding.

Drawings

FIG. 1 is a plot of the endoplasmic reticulum color frequency of example 1 "far impurities 9102×wt09-0023" RIL population progeny material.

FIG. 2 alignment of the sequences of the AhLAC gene wild type (AhLAC-WT) and mutant (AhLAC-Mu) in example 2.

FIG. 3 comparison of the color of seed coats over-expressed with the AhLAC gene in Arabidopsis mutant attt, 35S in example 2.

Wherein 35S is that the seed coat of AhLAC positive strain is dark yellow, the seed coat of Arabidopsis mutant attt is pale yellow, and the seed coat of wild Arabidopsis is yellow brown.

FIG. 4 KASP verification of the A05.114993389InDel site in example 3 in the far heterozygous 9102 and wt09-0023 RIL populations.

Wherein, the left graph shows KASP primer genotyping results, red near the X-axis and blue near the Y-axis, wherein the genotype is MITE, the MITE spot is red, and the genotype is-: -spot is blue.

FIG. 5 KASP verification of the A05.114993389InDel site in 334 parts of natural population material in example 3.

Wherein the left graph shows KASP primer genotyping results, the point close to the X axis is red, the point close to the Y axis is blue, the point at the middle of the quadrant is green, the genotype is MITE, the point of MITE is red, the genotype is-: -the point of MITE is blue, and the genotype is MITE: -the point of MITE is green.

Detailed Description

The following describes the embodiments of the present invention in further detail with reference to examples. Unless otherwise specified, the instruments and equipment involved in the examples are conventional instruments and equipment, the reagents involved are commercially available conventional reagents, and the test methods involved are conventional methods.

Example 1 acquisition of seed coat color Main QTL

(1) The far heterozygous 9102 and wt09-0023 Recombinant Inbred (RIL) populations were sequenced using a simplified genomic sequencing technique, with the two parents sequencing more than 20× deep and the 521 offspring families sequencing 5× deep. Genotyping data of parents and offspring were obtained with reference to cultivar peanut genome information (Arachis hypogaea. Cv. Tifrener V1.0). The two parents obtain 9 ten thousand polymorphic SNP with homozygous genotypes. SNP markers with deletion rates exceeding 10% in all offspring are filtered, SNP markers with approximate genetic linkage positions are filtered through QTL ICIMAPPING V4.1.1 software, and finally the rest markers are mapped by JoinMap5.0 software, so that a high-quality and high-density genetic map containing 20 linkage groups is constructed, the number of the markers in the map is 5120, and the average genetic distance is 0.62cM (shown in table 1).

TABLE 1 RIL linkage group information for high-density linkage map of group

(2) The inventors investigated the endoplasmic reticulum color of the RIL population progeny of the distal impurities 9102 and wt09-0023 in 2017, 2019 and 2020. Wherein RIL group (521 families and two parents) is planted in Liaoning sand control and utilization research institute test base of Liaoning Fuxin city in 2017, and 2 times of repetition are set. And 2019 and 2020 are planted on Henan modern agricultural research and development bases of Henan agricultural sciences of Henan province of New county city of Henan province, and 2 times of repetition are set. In 2021, a natural population consisting of 334 peanut germplasm is planted in a Henan modern agriculture research development base, and 2 times of repetition are set. When investigating the color of the peanut inner seed coat, 20 kernels are selected from each family or material, and the seed coat is required to be full without disease infection. The seed coat color was recorded by cutting the seed coat, with a yellow mark of 2 and a white mark of 1 (as shown in fig. 1).

(3) QTL mapping was performed on the genetic map and phenotype data using ICIMAPPING software, and found to be qIIC _a05 mapped in a 3 year environment. qIIC A05 is located on chromosome 5, and the corresponding physical map position is Chr05:114741295-115061124, interval size is 319.8Kb, LOD value is 186.5-444.4, and genetic contribution rate (PVE) is 75.8% -94.6%. (as shown in Table 2).

TABLE 2 QTL localization results of endoplasmic reticulum color in RIL populations of far impurities 9102 and wt09-0023

(4) Genes and functions annotated in QTL interval

Genes annotated in the qIIC _a05 interval (chromosome 5 114741295bp to 115061124 bp) were obtained with reference to peanut cultivar genome information (Arachis hypogaea. Cv. Tifluner V1.0). According to genes related to the regulation of seed coat color published by Arabidopsis thaliana, it was found that 5 genes among the 32 genes in this interval might be related to the regulation of endoplasmic coat color, including 1 laccase gene (arahy.0C6ZNN) and 4 oxidoreductase family proteins (arahy.7FUC5Y, arahy.I6RXR7, arahy.QAUQ3D and arahy.85VNG6). Genomic peanut cultivars Tifrunner and lion head rabbet have been reported to differ in endoplasmic color, tifrunner being yellow and lion head rabbet being white. The genome sequences of the above 5 genes are intercepted from Tifrunner and lion head enterprise genomes for comparison, and the laccase gene in Tifrunner genome is found to have an InDel marker compared with the laccase gene in the lion head enterprise genome, namely, 214bp is deleted at 114993389bp of chromosome 5 of Tifrunner genome compared with the lion head enterprise genome. The other 4 oxidoreductase family proteins were found to be unchanged, and the laccase gene was determined to be the target gene for controlling the endoplasmic reticulum color for further validation. (as shown in Table 3).

TABLE 3 Table 3

Example 2 cloning and functional verification of genes of interest regulating the color of the Endocarpium

And respectively extracting total seed coat RNA of the yellow and white inner seed coat material by using a plant RNA extraction kit, and converting the total seed coat RNA into first-strand cDNA. The wild-type (AhLAC-WT) and mutant (AhLAC-Mu) sequences of the primer clone AhLAC gene were designed (see FIG. 2).

The upstream sequence of the primer used was 5'-TGTTAGACAGCCAGAAATTTC-3' (SEQ ID NO. 3),

The downstream sequence is 5'-CCCTATTTTTGCATAAAAGAAAGTA-3' (SEQ ID NO. 4).

Mutation occurs at 711bp of AhLAC gene CDs, and a 214bp MITE (the same miniature inverted-repeat transposable element) sequence is inserted, so that protein translation is terminated in advance.

The wild type sequence was over-expressed in the Arabidopsis Mutant, 35S is shown by AhLAC positive transgenic plants (35S in FIG. 3: ahLAC) with a darker color than the yellow seed coat of the Mutant [ see Mutant (atlac/attt 10) in FIG. 3 ], the seed coat of the Mutant atlac (attt) was pale yellow, 35S is shown by AhLAC positive transgenic plants with a dark yellow color, and the seed coat of the wild type Arabidopsis [ see WT (Col-0) in FIG. 3 ] was yellow brown, demonstrating that the AhLAC gene had laccase activity. 35S AhLAC the seed coat colour of the plant did not return to that of the wild type, possibly in relation to the promoter of the over-expression vector.

The promoter of the overexpression vector used in this study was the 35S promoter of cauliflower mosaic virus (CaMV). The CaMV35S promoter, which is a constitutive promoter, can promote gene expression in all tissues, and is persistent and does not exhibit space-time specificity. Therefore, 35S:: ahLAC may be expressed less in the seed coat than under the control of the AtLAC (AtTT) own promoter, resulting in a seed coat color of the 35S:: ahLAC positive plant that does not recover to wild type levels.

Example 3 verification of Endocarpium color functional molecular markers in Natural populations

(1) Based on the wild type and mutant sequences of AhLAC genes, 1 set of KASP primers was developed.

Extracting DNA of the RIL group peanut sample to be identified, and detecting genotype data of the RIL group peanut sample to be identified through KASP primer.

The primer sequences are as follows:

qIIC_A05_F1:5’-GCTCAGAGAAAAAGAGAACTACATTC-3’(SEQ ID NO.5),

qIIC_A05_F2:5’-ATTGACCATTAGATGATAGATTGTAG-3’(SEQ ID NO.6),

qIIC_A05_com:5’-CAAGAGAAGGTATGCTGTATGCA-3’(SEQ ID NO.7)。

KASP primer mix: the total volume was 100. Mu.L, of which 12. Mu.L was qIIC-A05-F1 primer (100. Mu.M concentration), 12. Mu.L was qIIC-A05-F2 primer (100. Mu.M concentration), 30. Mu.L was qIIC-A05-com primer (100. Mu.M concentration) and 46. Mu.L was double distilled water.

The LGC SNPLINE genotyping platform and the matched reagent consumable used in the invention are purchased from LGC company in England.

The PCR reaction was carried out by adding 1. Mu.L of template DNA (concentration: 20-50 ng/. Mu.L), drying, and adding 1. Mu.L of a mixture of 1X KASP MASTER Mix and KASP primer, wherein the volume of KASP primer in the mixture was about 1.4%, which was accomplished by a Meridian ² micro-scale dispenser.

The PCR amplification was performed in a water bath thermal cycler with the PCR amplification procedure of 94℃for 15min, 94℃for 20s,61℃to 55℃for 1min,10 cycles with a temperature decrease of 0.6℃for each cycle, 94℃for 20s,55℃for 1min,26 cycles, and 10℃for storage. The KASP reaction products were read for fluorescence data using a microplate reader PHERASTAR, and the fluorescence results were automatically converted into a pattern.

Genotyping the InDel locus of A05.114993389 of the peanut sample of the RIL population to be identified by using a SNPLine genotyping platform.

The authenticity of the A05.114993389InDel site in the far heterozygous 9102 and wt09-0023 RIL populations was verified using SNPLine genotyping platform (LGC) (see left in FIG. 4). If the typing result of the site A05.114993389 is mutant (MITE: MITE), the inner seed coat color of the child material of the RIL group appears white, if the typing result of the site A05.114993389 is wild type (-: -), the inner seed coat color of the child material of the RIL group appears yellow, the genotyping result of 490 families in the RIL group is completely consistent with the inner seed coat color (see right of FIG. 4), wherein the genotyping result of 31 families is heterozygous, and the inner seed coat color of the child is separated from yellow and white.

(2) And detecting natural population materials by utilizing a AhLAC gene functional molecular marker, wherein the genotyping result is completely linked with the endoplasmic reticulum color.

The research group selects 334 cultivated germplasm from different sources to form natural population (including farmhouse seeds, foreign introduction and domestic bred varieties) to investigate the color of the inner seed coat and detect the AhLAC gene functional molecular markers. Of 334 parts of natural population materials, 147 parts of continuous flowering subspecies materials and 187 parts of alternate flowering subspecies materials. There were 78 parts of the bead variety (var. Vulgaris), 26 parts of the open-branch variety (var. Fastigiata), 43 parts of the intermediate type material (irregularis-type), 79 parts of the close-branch variety (var. Hypogaea), 12 parts of the fine-hair variety (var. Hirsuta) and 96 parts of the intermediate type material (irregularis-type) in the alternate subspecies material.

Extracting DNA of a natural population peanut sample to be identified, detecting genotype data of the natural population peanut sample to be identified through KASP primers, and genotyping InDel locus of A05.114993389 of the natural population peanut sample to be identified through SNPLine genotyping platform.

334 Natural population materials were tested using the KASP primer at the a05.114993389indel site to obtain genotype data (see left in fig. 5) for linkage analysis with the endoplasmic phenotype. The results show that if the typing result at the A05.114993389 locus is mutant (MITE: MITE), the endoplasmic reticulum color of the natural population material appears white, and if the typing result at the A05.114993389 locus is wild type (-: -), the endoplasmic reticulum color of the natural population material appears yellow, and the genotyping result of the natural population material is completely consistent with the endoplasmic reticulum color (see right in FIG. 5).

The genotyping result of the natural population material with 2 parts of material A05.114993389InDel locus is not consistent with the color of the inner seed coat, the genotype is wild type (-: -), but the color of the inner seed coat is white. The exodermis of these 2 parts of material is also white, probably because no procyanidins accumulate in both the exodermis and the exodermis due to the mutation of genes upstream of the anthocyanin synthesis pathway, so laccase genes function normally, but the endoepithelium remains white due to the absence of substrate.

In combination with the verification results of the far-mixed 9102 and wt09-0023RIL population and natural population materials, the InDel mark A05.114993389 is completely linked with the color of the testa, the color of the testa of the peanut material appears yellow when the genotype is wild (-: -), and the color of the testa of the peanut material appears white when the genotype is mutant (MITE: MITE), thus proving the accuracy of the mark.

Wherein the InDel marker A05.114993389 is positioned at 114993389bp of peanut chromosome 5,

The sequence 200bp before and after the site A05.114993389 of the wild molecular marker is:

CCTGGTGACTTTTTTCCTTGCTCAAGAGAAGGTATGCTGTATGCATGAAGATTTCATTTCAAGCATGCTCAAATGTTCTTTGATTTTAGAATCTCTAACTGCATCAATTCTTTTTGTTTTTTCACAGAAACATTCAAGTTTAATGTAGATTTTGGCAAGACTTATCTGCTGAGGATTGTGAATGCTGCAATGAATGTAGTTCTCTTTTTCTCTGTTTACAAACACAGTCTCACTGTTGTTGGTGCTGATGCTGCCTACACCGAGCCATTAACAAGAGACTTCATATGCATAGCTCCAGGACAATCACTTGATGTTTTGTTGCATGCAAATCAAGAACCTGATCACTATTACATGGCTGCAAGAGCATATTCAAGTTCCTCTATTCTTCCTTTTGATAACA(SEQ ID NO.1);

the mutant is inserted 214bp sequence, and the sequence 200bp before and after the site A05.114993389 of the molecular marker is (the inserted 214bp sequence is in square brackets):

CCTGGTGACTTTTTTCCTTGCTCAAGAGAAGGTATGCTGTATGCATGAAGATTTCATTTCAAGCATGCTCAAATGTTCTTTGATTTTAGAATCTCTAACTGCATCAATTCTTTTTGTTTTTTCACAGAAACATTCAAGTTTAATGTAGATTTTGGCAAGACTTATCTGCTGAGGATTGTGAATGCTGCAATGAATGTAGT[TCTCTTTTTGGCAACTACTCCTATGAAGATGCCAAAAACATCTTTTTATGATGATCTTTGTTTAAAAAGTGTAACTTATTTGTTTAGCAACACTTTAAATAAAGATAACACTTTTACTTTAAATAAAATCAAACCCTACAATCTATCATCTAATGGTCAAAATGAAATATCTTCATATGATGACAATCATAAAATCTTCATTGGAGTAGCCACC]TCTCTTTTTCTCTGTTTACAAACACAGTCTCACTGTTGTTGGTGCTGATGCTGCCTACACCGAGCCATTAACAAGAGACTTCATATGCATAGCTCCAGGACAATCACTTGATGTTTTGTTGCATGCAAATCAAGAACCTGATCACTATTACATGGCTGCAAGAGCATATTCAAGTTCCTCTATTCTTCCTTTTGATAACA(SEQ ID NO.2).

Sequence listing

<110> Academy of agricultural sciences in Henan province

<120> Molecular marker of peanut inner seed coat color regulating gene AhLAC function and application

<130> Molecular biology

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 400

<212> DNA

<213> Artificial sequence ()

<400> 1

cctggtgact tttttccttg ctcaagagaa ggtatgctgt atgcatgaag atttcatttc 60

aagcatgctc aaatgttctt tgattttaga atctctaact gcatcaattc tttttgtttt 120

ttcacagaaa cattcaagtt taatgtagat tttggcaaga cttatctgct gaggattgtg 180

aatgctgcaa tgaatgtagt tctctttttc tctgtttaca aacacagtct cactgttgtt 240

ggtgctgatg ctgcctacac cgagccatta acaagagact tcatatgcat agctccagga 300

caatcacttg atgttttgtt gcatgcaaat caagaacctg atcactatta catggctgca 360

agagcatatt caagttcctc tattcttcct tttgataaca 400

<210> 2

<211> 614

<212> DNA

<213> Artificial sequence ()

<400> 2

cctggtgact tttttccttg ctcaagagaa ggtatgctgt atgcatgaag atttcatttc 60

aagcatgctc aaatgttctt tgattttaga atctctaact gcatcaattc tttttgtttt 120

ttcacagaaa cattcaagtt taatgtagat tttggcaaga cttatctgct gaggattgtg 180

aatgctgcaa tgaatgtagt tctctttttg gcaactactc ctatgaagat gccaaaaaca 240

tctttttatg atgatctttg tttaaaaagt gtaacttatt tgtttagcaa cactttaaat 300

aaagataaca cttttacttt aaataaaatc aaaccctaca atctatcatc taatggtcaa 360

aatgaaatat cttcatatga tgacaatcat aaaatcttca ttggagtagc cacctctctt 420

tttctctgtt tacaaacaca gtctcactgt tgttggtgct gatgctgcct acaccgagcc 480

attaacaaga gacttcatat gcatagctcc aggacaatca cttgatgttt tgttgcatgc 540

aaatcaagaa cctgatcact attacatggc tgcaagagca tattcaagtt cctctattct 600

tccttttgat aaca 614

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tgttagacag ccagaaattt c 21

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence ()

<400> 4

ccctattttt gcataaaaga aagta 25

<210> 5

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 5

gctcagagaa aaagagaact acattc 26

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 6

attgaccatt agatgataga ttgtag 26

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 7

caagagaagg tatgctgtat gca 23

Claims (6)

1. The functional molecular marker of the peanut endoplasmic reticulum color regulating gene AhLAC is characterized in that the molecular marker is A05.114993389, is positioned at 114,993,389bp of peanut chromosome 5, and is an InDel InDel marker;

wherein, the sequence 200bp before and after the wild type molecular marker locus is:

CCTGGTGACTTTTTTCCTTGCTCAAGAGAAGGTATGCTGTATGCATGAAGATTTCATTTCAAGCATGCTCAAATGTTCTTTGATTTTAGAATCTCTAACTGCATCAATTCTTTTTGTTTTTTCACAGAAACATTCAAGTTTAATGTAGATTTTGGCAAGACTTATCTGCTGAGGATTGTGAATGCTGCAATGAATGTAGTTCTCTTTTTCTCTGTTTACAAACACAGTCTCACTGTTGTTGGTGCTGATGCTGCCTACACCGAGCCATTAACAAGAGACTTCATATGCATAGCTCCAGGACAATCACTTGATGTTTTGTTGCATGCAAATCAAGAACCTGATCACTATTACATGGCTGCAAGAGCATATTCAAGTTCCTCTATTCTTCCTTTTGATAACA;

The mutant is inserted 214bp sequence, the inserted 214bp sequence is arranged in square brackets, and the sequences of 200bp before and after the molecular marker locus are as follows:

CCTGGTGACTTTTTTCCTTGCTCAAGAGAAGGTATGCTGTATGCATGAAGATTTCATTTCAAGCATGCTCAAATGTTCTTTGATTTTAGAATCTCTAACTGCATCAATTCTTTTTGTTTTTTCACAGAAACATTCAAGTTTAATGTAGATTTTGGCAAGACTTATCTGCTGAGGATTGTGAATGCTGCAATGAATGTAGT[TCTCTTTTTGGCAACTACTCCTATGAAGATGCCAAAAACATCTTTTTATGATGATCTTTGTTTAAAAAGTGTAACTTATTTGTTTAGCAACACTTTAAATAAAGATAACACTTTTACTTTAAATAAAATCAAACCCTACAATCTATCATCTAATGGTCAAAATGAAATATCTTCATATGATGACAATCATAAAATCTTCATTGGAGTAGCCACC]TCTCTTTTTCTCTGTTTACAAACACAGTCTCACTGTTGTTGGTGCTGATGCTGCCTACACCGAGCCATTAACAAGAGACTTCATATGCATAGCTCCAGGACAATCACTTGATGTTTTGTTGCATGCAAATCAAGAACCTGATCACTATTACATGGCTGCAAGAGCATATTCAAGTTCCTCTATTCTTCCTTTTGATAACA.

2. a KASP primer combination for amplifying the functional molecular marker of claim 1, wherein the KASP primer combination comprises:

qIIC_A05_F1:5’-GCTCAGAGAAAAAGAGAACTACATTC-3’,

qIIC_A05_F2:5’-ATTGACCATTAGATGATAGATTGTAG-3’,

qIIC_A05_com:5’-CAAGAGAAGGTATGCTGTATGCA-3’。

3. The method for identifying the color of the peanut kernel coat by using the functional molecular markers as claimed in claim 1, which comprises the following steps:

(1) Extracting DNA of a peanut sample to be identified, detecting genotype data of the peanut sample to be identified by using the KASP primer combination of claim 2, and carrying out genotyping on the InDel locus of A05.114993389 of the peanut sample to be identified by using a SNPLine genotyping platform;

(2) If the parting result of the A05.114993389 site is mutant (MITE: MITE), the color of the seed coats in the peanut sample to be identified is white, and if the parting result of the A05.114993389 site is wild type (-: -), the color of the seed coats in the peanut sample to be identified is yellow.

4. The method of claim 3, wherein the step (1) is performed by PCR technique, and the PCR amplification procedure is 94℃for 15min, 94℃for 20s,61℃to 55℃for 1min,10 cycles, 94℃for 20s,55℃for 1min,26 cycles, and 10℃for preservation.

5. The use of the functional molecular marker according to claim 1 for identifying the color of peanut seed coats.

6. The use of the functional molecular marker of claim 1 in molecular breeding of peanuts.