Functional molecular marker of a related gene Aft for synthesis of solanum lycopersicum anthocyanin and application thereof

[0103] Example 1. The cloning of tomato Aft gene and the discovery of its allelic variant

[0104] 1. Structure analysis of R2R3-MYB gene cluster linked to Aft gene

[0105] Existing studies have shown that the Aft gene is linked to a cluster of R2R3-MYB genes on chromosome 10. According to the tomato genome data SL2.50 version released by the SGN database (website: http://solgenomics.net/), the R2R3-MYB gene cluster linked to the Aft gene on tomato chromosome 10 was analyzed.

[0106] See the analysis results figure 1. The results showed that there are 4 R2R3-MYB genes in the R2R3-MYB gene cluster, which are Solyc10g086250 gene, Solyc10g086260 gene, Solyc10g086270 gene and Solyc10g086290 gene in order. The Solyc10g086250 gene is a reported AN2 gene (Meng X, Wang JR, Wang GD, Liang XQ, LiX-D, Meng QW. (2015) An R2R3-MYB gene, LeAN2, positively regulated the thermo-tolerance in transgenic tomato. J Plant Physiol 175:1-8.), Solyc10g086260 gene is the reported ANT1 gene (Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, MatheisN, Schuster DK, Menasco DJ, Wagoner W, Lightner J, Wagner DR. (2003). Activation tagging in tomato identifies a transcriptional regulator of anthocyaninbiosynthesis, modification, and transport. Plant Cell 15(8): 1689-703.).

[0107] 2. Analysis of the expression level of four genes in the R2R3-MYB gene cluster

[0108] 1. Extract the total RNA from the peel of Indigo Rose, and then reverse transcribed to obtain cDNA.

[0109] 2. Using the cDNA obtained in step 1 as a template, detect the relative expression of the gene to be tested (Solyc10g086250 gene, Solyc10g086260 gene, Solyc10g086270 gene or Solyc10g086290 gene) by fluorescent quantitative PCR (Actin2 gene is used as internal reference gene, Actin2 gene is Solyc11g005330 gene) . The fluorescence quantitative PCR reaction system is loaded according to the instructions of the SYBR Premix Ex Taq II quantitative PCR kit, and the reaction conditions are also carried out in accordance with the recommendations of the kit.

[0110] The primers for identifying the Solyc10g086250 gene are 5’-CCAGGAAGGACAGCAAACGA-3’ and

[0111] 5'-CGAGGATGAGAACGAGGACG-3'. The primers for identifying the Solyc10g086260 gene are

[0112] 5’-CGGAAGGACAGCTAACGATG-3’ and 5’-GCAATGTTCCTCCTCGTCCAA-3’. Identification

[0113] The primers of Solyc10g086270 gene are 5’-GAAGGAGCAGGAGGAGGTGTA-3’ and

[0114] 5'-ACCATCACTTTGTCCTTGTTGC-3'. The primers for identifying the Solyc10g086290 gene are

[0115] 5'-TGCCAAGACATTGGGAGTGAG-3' and 5'-CCAGAGCAAAGTCACCTCTCTT-3'. The primers for identifying the Actin2 gene are 5'-TTGCTGACCGTATGAGCAAG-3' and 5'-GGACAATGGATGGACCAGAC-3'.

[0116] See the results of the experiment figure 2. The results showed that only the Solyc10g086290 gene was expressed in the peel of Indigo Rose, and it was speculated that the Solyc10g086290 gene might be the Aft gene.

[0117] 3. Using CRISPR/Cas9 gene editing technology to determine tomato Aft gene

[0118] Use CRISPR/Cas9 gene editing technology to edit the Solyc10g086290 gene of Indigo Rose. The sgRNA target sequence is 5'-AGACATTGGGAGTGAGAAA-3'. The starting vector is the pKSE401 vector. The vector construction method and the method of obtaining homozygous target plants are all described in the following documents: Xing, HL, Dong, L., Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, and Chen ,QJ(2014).A CRISPR/Cas9toolkit for multipleplex genome editing in plants.BMC plant biology14:327.

[0119] See the sequencing results of Solyc10g086290 gene homozygous targeted plants image 3 (WT is Indigo Rose, #1 is Solyc10g086290 gene homozygous target plant lacking 4bp (hereinafter referred to as homozygous target plant #1), #2 is Solyc10g086290 gene homozygous target plant lacking 2bp (hereinafter referred to as homozygous target plant #2)). Homozygous targeting plant #1 and homozygous targeting plant #2 have missing bases that are not multiples of 3, leading to frameshift mutations and unable to translate normal Solyc10g086290 protein.

[0120] Observe the fruit phenotypes of Indigo Rose, homozygous targeting plant #1 and homozygous targeting plant #2. See the results of the experiment Figure 4 : Indigo Rose's green-ripening stage fruits are purple-black, while the green-ripening stage fruits of Homozygous Targeting Plant #1 and Homozygous Targeting Plant #2 are green.

[0121] The total anthocyanin content of Indigo Rose, homozygous targeting plant #1 and homozygous targeting plant #2 were detected. See the results of the experiment Figure 5 (WT is Indigo Rose): The total anthocyanin content in the peel of Indigo Rose is 7.05 mg/g fresh weight, and the total anthocyanin content in the peel of the homozygous target plant #1 is 0.06 mg/g fresh weight. The content of total anthocyanin in the peel of Heda Target Plant #2 was 0.09 mg/g fresh weight.

[0122] The above results indicate that the anthocyanin accumulation phenotype of Indigo Rose depends on the Solyc10g086290 gene. Therefore, the Solyc10g086290 gene was identified as the Aft gene. The nucleotide sequence of the Aft gene in Indigo Rose is shown in sequence 1 in the sequence listing.

[0123] Based on the sequence analysis of a large number of tomato varieties, the inventors of the present invention found that there are two allelic variants of the Aft gene in tomato: one is shown in sequence 1 in the sequence list (named allele AFT), One is shown in sequence 2 in the sequence listing (named allele aft). Compared with allele aft, allele AFT has a 3bp deletion and a 6bp deletion. The 6bp deletion nucleotide sequence is 5'-AGGACC-3', which contains a recognition site for restriction enzyme AvaII (5'-GGWCC-3').

[0124] Based on the allelic variant form of the Aft gene, tomato is divided into three genotypes: genotype I (allele AFT homozygous), genotype II (allele AFT and allele aft heterozygous) and genotype III (Allele aft homozygous).

[0125] Example 2. Development of molecular markers and polymorphism detection

[0126] 1. Development of molecular markers

[0127] Based on the two allelic variants of the Aft gene, molecular markers were developed.

[0128] The nucleotide sequence of the amplified molecular marker is as follows:

[0129] Primer F: 5'-GGTCACTTATTGCTGGGAGA-3' (sequence 3 in the sequence table);

[0130] Primer R: 5'-CTCCATGTTGCATGGTTGTTG-3' (sequence 4 in the sequence listing).

[0131] 2. Polymorphism detection

[0132] A. Polymorphism detection one

[0133] The tomatoes to be tested are Indigo Rose, LA1996 or Alisa Craig.

[0134] 1. Use the plant DNA rapid extraction kit to extract the genomic DNA of the tomato to be tested.

[0135] 2. Using the genomic DNA of step 1 as a template, use the primer pair composed of primer F and primer R in step 1 to perform PCR amplification to obtain PCR amplification products.

[0136] PCR reaction system (20μL): Premix Taq DNA polymerase Mix 10μL, primer F (5μmol/L) and primer R (5μmol/L) each 0.8μL, test tomato genomic DNA (20ng/μL) 1.5μL and ddH 2 O6.9 μL.

[0137] PCR reaction conditions: 95°C for 5min; 94°C for 20s, 56°C for 20s, 72°C for 20s, 35 cycles; 72°C for 10min.

[0138] 3. Take part of the PCR amplification product obtained in step 2 and perform 1.5% agarose gel electrophoresis detection (the purpose is to detect whether it contains DNA bands).

[0139] 4. Take part of the PCR amplified product obtained in step 2 and cut it with the restriction enzyme AvaII to obtain the digested product; then subject the digested product to 1.5% agarose gel electrophoresis, and make the following judgments based on the electrophoresis result: If the digested product has band type A (showing one band, which is 483bp), the genotype of the tomato to be tested is genotype I; if the digested product has band type B (showing three bands, which are 483bp, 315bp and 177bp), then the test The genotype of tomato is genotype II; if the digested product has band type C (showing two bands, 315bp and 177bp), the genotype of the tomato to be tested is genotype III.

[0140] Enzyme digestion system (20μL): PCR amplification product 10μL, restriction enzyme AvaII 1μL, BufferR 2μL and ddH 2 O 7μL. BufferR is a component of the restriction enzyme AvaII.

[0141] Enzyme digestion reaction conditions: 37°C for 5h.

[0142] See the results of the experiment Image 6 (M is DL2000 DNA marker, AC is Alisa Craig, IR is Indigo Rose). The results showed that the genotype of Alisa Craig was genotype III, and the genotype of Indigo Rose and LA1996 was genotype I.

[0143] B. Polymorphism detection two

[0144] The tomatoes to be tested are Indigo Rose, LA1996 or Alisa Craig.

[0145] 1. Same as 1 in step A.

[0146] 2. Same as step 2 in step A.

[0147] 3. Same as 3 in step A.

[0148] 4. Take part of the PCR amplification products obtained in step 2 and sequence them by Beijing Ruibo Xingke Company. According to the sequencing results, make the following judgment: If the PCR amplification product contains the nucleotide sequence shown in sequence 5 in the sequence table and Does not contain the nucleotide sequence shown in sequence 6 in the sequence list, the genotype of the tomato to be tested is genotype I; if the PCR amplification product contains the nucleotide sequence shown in sequence 5 in the sequence list and the sequence list The nucleotide sequence shown in sequence 6 in the sequence, the genotype of the tomato to be tested is genotype II; if the PCR amplification product contains the nucleotide sequence shown in sequence 6 in the sequence list and does not contain the sequence in the sequence list For the nucleotide sequence shown in sequence 5, the genotype of the tomato to be tested is genotype III.

[0149] The experimental results are completely consistent with the experimental results in Polymorphism Detection 1.

[0150] The above results indicate that the molecular marker developed in step one has high polymorphism. The molecular marker can be used to distinguish tomato materials containing allele AFT homozygous, and, allele AFT and allele aft heterozygous, and and allele aft homozygous.

[0151] Example 3. Application of molecular markers

[0152] One, application one

[0153] The tomatoes to be tested are Indigo Rose, LA1996 or Alisa Craig.

[0154] 1. According to the method of polymorphism detection 1 in Example 2, the tomato to be tested is genotyped.

[0155] 2. Plant the tomatoes to be tested in the field and perform routine field management. After maturity, observe the phenotype of the fruits in the green ripening period and detect the content of total anthocyanins in the skin of the tomatoes to be tested.

[0156] The results showed that the peel of Indigo Rose had a large amount of anthocyanin accumulation, which was purple-black, and the total anthocyanin content was 7.05mg/g fresh weight; the peel of LA1996 had a small amount of anthocyanin accumulation, showing purple spots, total anthocyanin The content of Alisa Craig is 0.12mg/g fresh weight; the peel of Alisa Craig accumulates without anthocyanins, which is green, and the content of total anthocyanins is 0mg/g fresh weight.

[0157] Two, application two

[0158] Take Indigo Rose as the female parent and Alisa Craig as the male parent to make a hybrid F 1; Then hybrid F 1 Selfing, get F 2 Offspring. Put 14 F 2 The offspring is named AC×IR F 2 1 to AC×IR F 2 14.

[0159] The tomatoes to be tested are Indigo Rose, Alisa Craig, Hybrid F 1 And 14 F 2 Offspring.

[0160] 1. According to the method of polymorphism detection 1 in Example 2, the tomato to be tested is genotyped.

[0161] See the results of the experiment Figure 7 (M is DL2000 DNA marker, AC is Alisa Craig, IR is Indigo Rose, F 1 For hybrid F 1 , 1-14 is AC×IR F 2 1 to AC×IR F 2 14). The results showed that the genotype of Alisa Craig was genotype III, the genotype of Indigo Rose was genotype I, and the genotype of hybrid F 1 The genotype is genotype II, 14 F 2 The genotype of the offspring is genotype I, genotype II or genotype III.

[0162] 2. Plant each tomato to be tested in the field, and perform routine field management. After maturity, observe the phenotype of the fruit in the green ripening period and detect the total anthocyanin content in the peel of the tomato to be tested.

[0163] The experimental results are shown in Table 1.

[0164] Table 1

[0165]

[0166]

[0167] The above results indicate that the genotype I or genotype II tomato contains total anthocyanins, and the genotype III tomato does not contain total anthocyanins. Therefore, the molecular markers developed in Example 2 can be used to screen tomato varieties with anthocyanins or tomato varieties without anthocyanins.

[0168] <110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Beijing Academy of Agriculture and Forestry Sciences

[0169] <120> Functional molecular marker of tomato anthocyanin synthesis related gene Aft and its application

[0170] <160> 6

[0171] <170> PatentIn version 3.5

[0172] <210> 1

[0173] <211> 1341

[0174] <212> DNA

[0175] <213> Tomato (Solanum lycopersicum cv.Indigo Rose)

[0176] <400> 1

[0177] atgaatattg ccaagacatt gggagtgaga aaaggttcat ggactgaaga tgaagatatt 60

[0178] cttttgagga aatgtattga caagtatgga gaaggaaagt ggcatcttgt tccttttaga 120

[0179] gctggtaaag caaaattaag attttaattt tatgtatttt aaattttatg ataataatta 180

[0180] agttttaaat ttatgtagat tttaagtaaa atttgttaat gcaaaaatac tatttaggca 240

[0181] aaatctgtta gattatacta aatttccttt ttaagaaaag agaaacttac cttttgttgt 300

[0182] gatagtggcg tcccaaccta taactctagc atgaatagca tttcatgcct ccttttttat 360

[0183] tactgagtcg taaattaatt ttggtaggag tttacaagtt aatatatata tatatttgat 420

[0184] taattttttt agtttatata caatatctat gaaaaaatta ctaggttcgt tcaacccaca 480

[0185] aatccccact tactattatt tcacgtgatt atatgcaggt ctaaatagat gtcgaaagag 540

[0186] ttgtagactg aggtggttga attatctaag gccacatatc aagagaggtg actttgctat 600

[0187] ggatgaaata gatctcattt tgagacttca caagcttcta ggcaataggc aagtcagaaa 660

[0188] tttagttaaa agaaattcaa aaattattgt acatatatat tcacgaaaag aaacttttga 720

[0189] catacaaatt tgtgtacata ctagtcttcc gtatatatta tagtaagttg tctttgatgc 780

[0190] catattttta ttttcttttg gtttagatgg tcacttattg ctgggagact tccgggaaga 840

[0191] acagcaaacg atgtgaaaaa ctattggaac acacacctac acaagaagtt attaataact 900

[0192] cctcagatac aagagaataa gtacaataaa accctcaaga ttatcactga aagcactata 960

[0193] ctacgaccac gaccaagacc tcgacctcga acattctcaa gtgaaaataa tatttcttgg 1020

[0194] tgcactaaca atagtatgat cacaaacaca ttagacaaag atgacgaaca acgcaacaaa 1080

[0195] gaaatcgcag taaatatttg tgagaagcca acaagagaaa caccgtcatc gtctatagac 1140

[0196] gatgatggag ttaaatggtg gacaaattta ctggaaaatt ggaaagaatt tgaggaagaa 1200

[0197] gcaacagcag tattgaactt tgaggaagaa aataagttgt taccaaattt gttgtgtgag 1260

[0198] gaacataatt caacaaccat gcaacatgga gaaaatgatg acttttcagt tgatattgac 1320

[0199] ctatggaatc tatttaatta g 1341

[0200] <210> 2

[0201] <211> 1356

[0202] <212> DNA

[0203] <213> Tomato (Solanum lycopersicum cv.Alisa Craig)

[0204] <400> 2

[0205] atgaatattg ccaagacatt gggagtgaga aaaggttcat ggactgaaga tgaagatatt 60

[0206] cttttgagga aatgtattga caagtatgga gaaggaaagt ggcatcttgt tccttttaga 120

[0207] gctggtaaag cgaaattaag attttagttt tataaatttt aaattttatg ataataaata 180

[0208] agttctaaat ttatgtagat attaagtaat aatttgttaa tgcaaaaata atatttaggc 240

[0209] aaaatctatt agattatact aaatttcctt tttaagaaaa gagaaactta accttttgtt 300

[0210] gtgatagtgg cgtcccaacc tataactcta gcatgaatag catttcatgc ctcctttttt 360

[0211] attaccgagt cataaatcaa tttcgttagg agtttacaaa ttaatataca catatattta 420

[0212] gttaaatttt tttagttcat atataacatc taccaaaaaa attactggat tcgttcaatc 480

[0213] cacaaatccc cacttactat tatttcatgt gaatatatgc aggtctaaat agatgtcgaa 540

[0214] agagttgtag actgaggtgg ttgaattatc taaggccaca tatcaagaga ggtgactttg 600

[0215] ctctggatga aatagatctc attttgagac ttcacaagct tctaggcaat aggcaaatca 660

[0216] aaaatttcgt taaaaaatat ttaaaaatta ttgtacatat atatattcac gaaaagtaat 720

[0217] ttttgacata taaatttacg tacatactag tctctcgaat atattatagt aagttatctt 780

[0218] tgatgccata tttttatatt tttttggttt agatggtcac ttattgctgg gagacttcct 840

[0219] ggaagaacag caaacgatgt gaaaaactat tggaacacac acctacacaa gaagttatta 900

[0220] ataactcctc ctcagataca agagaataag tacaataata ccctcaagat tatcactgaa 960

[0221] agcactatac tacgaccacg accaagacca ggacctcaac ctcgaacctt ctcaagtgaa 1020

[0222] aataatattt cttggtgcac taacaatagt atgatcacaa acacattaga caaagatgac 1080

[0223] gaacaacaca acaaagaaat cgcagtaaat atttgtgaga agccaacaaa aaaaacaccg 1140

[0224] tcatcgtcta tagacgatga tggagttcaa tggtggacaa atttactgga aaattggaaa 1200

[0225] gaatttgagg aagaagcaac agcagtattg aactttgagg aagaaaataa gttgttgcca 1260

[0226] aatttgttgt atgaggaaca taattcaaca accatgcaac atggagaaaa tgatgacttt 1320

[0227] tcagttgata ttgacctatg gaatctattt aattag 1356

[0228] <210> 3

[0229] <211> 20

[0230] <212> DNA

[0231] <213> Artificial sequence

[0232] <220>

[0233] <223>

[0234] <400> 3

[0235] ggtcacttat tgctgggaga 20

[0236] <210> 4

[0237] <211> twenty one

[0238] <212> DNA

[0239] <213> Artificial sequence

[0240] <220>

[0241] <223>

[0242] <400> 4

[0243] ctccatgttg catggttgtt g 21

[0244] <210> 5

[0245] <211> 483

[0246] <212> DNA

[0247] <213> Artificial sequence

[0248] <220>

[0249] <223>

[0250] <400> 5

[0251] ggtcacttat tgctgggaga cttccgggaa gaacagcaaa cgatgtgaaa aactattgga 60

[0252] acacacacct acacaagaag ttattaataa ctcctcagat acaagagaat aagtacaata 120

[0253] aaaccctcaa gattatcact gaaagcacta tactacgacc acgaccaaga cctcgacctc 180

[0254] gaacattctc aagtgaaaat aatatttctt ggtgcactaa caatagtatg atcacaaaca 240

[0255] cattagacaa agatgacgaa caacgcaaca aagaaatcgc agtaaatatt tgtgagaagc 300

[0256] caacaagaga aacaccgtca tcgtctatag acgatgatgg agttaaatgg tggacaaatt 360

[0257] tactggaaaa ttggaaagaa tttgaggaag aagcaacagc agtattgaac tttgaggaag 420

[0258] aaaataagtt gttaccaaat ttgttgtgtg aggaacataa ttcaacaacc atgcaacatg 480

[0259] gag 483

[0260] <210> 6

[0261] <211> 492

[0262] <212> DNA

[0263] <213> Artificial sequence

[0264] <220>

[0265] <223>

[0266] <400> 6

[0267] ggtcacttat tgctgggaga cttcctggaa gaacagcaaa cgatgtgaaa aactattgga 60

[0268] acacacacct acacaagaag ttattaataa ctcctcctca gatacaagag aataagtaca 120

[0269] ataataccct caagattatc actgaaagca ctatactacg accacgacca agaccaggac 180

[0270] ctcaacctcg aaccttctca agtgaaaata atatttcttg gtgcactaac aatagtatga 240

[0271] tcacaaacac attagacaaa gatgacgaac aacacaacaa agaaatcgca gtaaatattt 300

[0272] gtgagaagcc aacaaaaaaa acaccgtcat cgtctataga cgatgatgga gttcaatggt 360

[0273] ggacaaattt actggaaaat tggaaagaat ttgaggaaga agcaacagca gtattgaact 420

[0274] ttgaggaaga aaataagttg ttgccaaatt tgttgtatga ggaacataat tcaacaacca 480

[0275] tgcaacatgg ag 492