• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    The present disclosure relates to the technical field of genetic analysis, specifically to a CAPS marker closely linked to gene related to synthesis of P3G and C3G in colored barley. Also provided are primers for detecting the marker. The genetic distance between the marker of the present disclosure and the PBG.ant—2H locus is 0.0 CM. Therefore, it is possible to more accurately screen out the barley varieties or lines With major effect QTL for synthesis of P3G and C3G. Applying it to early generation selection of barley breeding can further improve the accuracy of selection in breeding.
    公开号:NL2027031A
    申请号:NL2027031
    申请日:2020-12-03
    公开日:2021-08-11
    发明作者:Liu Chunjie；Mei Shiyong；Liu Chunji
    申请人:Inst Of Bast Fiber Crops Chinese Academyof Agricultural Sciences；
    IPC主号:
    专利说明:

    [0001] [0001] The present invention relates to the technical field of genetic analysis, specifically to a CAPS molecular marker closely linked to gene related to synthesis of P3G and C3G in colored barley.BACKGROUND
    [0002] [0002] Colored barley is rich in anthocyanins and has important potential values to human health, and is an important material for the development of functional foods and health nutrition products. And its antioxidant and antimutagenic effects can be detected not only in barley grains, but also in its fermentation broth. Studies have shown that different anthocyanins have different functions, therefore different health care values, and the anthocyanin composition of barley of different colors and even of barley of similar colors may be quite different. Peonidin-3-glucoside (P3G) and Cyanidin-3-glucoside (C3G) are ubiquitous important anthocyanins. Therefore, research on P3G and C3G synthetic genes is of great significance to the quality improvement of barley and the screening or breeding of germplasm.
    [0003] [0003] Compared with methods such as morphological identification and cytological identification, molecular marker-assisted selection has the advantages of simple operation, short cycle and accurate results, etc. In previous studies, Insertion-deletion (InDel) markers closely linked to the P3G and C3G synthetic gene in colored barley were found. Although the detection method of the InDel marker is relatively simple, the InDel marker obtained by the previous screening is far away from the quantitative trait locus (QTL), and in the follow-up study, no molecular markers having shorter genetic distance to the main QTL were found.
    [0094] [0094] CAPS, ie. cleaved amplified polymorphic sequence, is based on PCR amplification and restriction enzyme digestion techniques, which can be used to detect SNP sites occurring at the same position in the genome of different individuals within a genetic population. This method has the advantages of good stability, high polymorphism, and simple typing system. But so far, no reports have been found about the sequence information of CAPS marker closely linked to P3G and C3G synthetic gene/genetic site in barley.SUMMARY
    [0005] [0005] In view of this, the technical problem to be solved by the present disclosure is to provide a CAPS marker closely linked to gene related to synthesis of P3G and C3G in colored barley, and the genetic distance between the marker and the PBG.ant-2H locus is 0.0 cM. PBG.ant-2H is a QTL conferring anthocyanin production, wherein “PBG” stands for “purple pericarp barley grain” and “ant” for “anthocyanin” (Xiao-Wei Zhang et al., PLoS ONE 12(8): e0183704).
    [0006] [0006] The present disclosure provides a CAPS marker closely linked to the main effect QTL, PBG.ant-2H, for synthesis of P3G and C3G in barley. The marker is named BACS2H and its nucleotide sequence is shown in SEQ ID NO: 1.
    [0008] [0008] The present disclosure also provides a primer set for detecting the molecular marker, which is composed of the two primers of the nucleotide sequence shown in SEQ ID NOs: 2 to 3. {0009] The marker, detection primer and detection method provided by the present disclosure can be applied to early generation selection of barley breeding, and it can be carried out in the early stage of barley plant growth {the inheritance of grain color of many barley materials has maternal effects, and their grain color can only be observed dozens of days after flowering, so the experimental period is long), and it can greatly reduce the workload of breeding and screening, shorten the breeding cycle, accelerate the breeding speed, and reduce the breeding cost, It has the advantages of simple operation, low cost and short cycle.
    [0010] [0010] Use of the primer set of the present disclosure in the breeding of colored barley.
    [0011] [0011] The presence or absence of the CAPS marker closely linked to PBG.ant-2H actually and directly determines the presence or absence, and high or low, of the content of the two anthocyanins. The presence or absence of this genetic locus can be identified by detecting the marker of the present disclosure.
    [0012] [0012] The color barley breeding in the present disclosure is to determine the presence or absence of the marker closely linked to the genetic locus of PBG.ant-2H based on the results of the enzyme digestion after amplification, and then predict the presence or absence, and high or low, of the content of anthocyanins P3G and C3G in the barley grains.
    [0013] [0013] A kit for detecting the main effect QTL for synthesis of anthocyanins P3G and C3G in barley comprises the primer set of the present disclosure.
    [0014] [0014] The kit further comprises restriction endonuclease Mfe 1.
    [0015] [0015] The kit further comprises a marker, which contains three DNA molecules with lengths of 1496 bp, 1329 bp and 167 bp.
    [0016] [0016] The kit of the present disclosure further comprises PCR amplification reagents. The PCR amplification reagents include dNTP, MgCl, and Tag polymerase.
    [0017] [0017] The kit of the present disclosure further comprises an enzyme digestion buffer, and the digestion buffer is Mfe 1 buffer, which contains Tris-HCI, MgCl, NaCl, DTT and BSA.
    [0018] [0018] The present disclosure further provides a method for detecting the major effect QTL for synthesis of anthocyanins P3G and C3G in barley, which comprises steps of:
    [0019] [0019] using genomic DNA extracted from a barley leave as a template, carrying out PCR amplification with the primer set of the present disclosure, digesting the amplified products with restriction enzyme Mfe 1, and determining the presence or absence of the main effect QTL for synthesis of anthocyanins P3G and C3G in barley based on the digested products.
    [0020] [0020] In the embodiment of the present disclosure, the barley leave is a young leave of barley. Specifically, it is an early leave of barley plants. The genomic DNA extraction method is CTAB (cetyl rimethylammonium bromide) method.
    [0021] [0021] In the embodiment of the present disclosure, each 25 pL of the PCR amplification system includes: H;O and 10~100 ng of genomic DNA, 0.4 pmol/L forward primer, 0.4 pmol/L reverse primer, 3 mmol/L MgCl,, I mmol/L dNTPs and 2.5 U Tag DNA polymerase.
    [0022] [0022] In the embodiment of the present disclosure, the PCR amplification procedure is:
    [0023] [0023] denaturation at 95°C for 5 minutes;
    [0024] [0024] denaturation at 94°C for | min,
    [0025] [0025] annealing at 60°C for | min, | 35 cycles;
    [0026] [0026] extension at 72°C for | min,
    [0027] [0027] final extension at 72°C for 10 min.
    [0028] [0028] In the embodiment of the present disclosure, the criterions of the determining is:
    [0029] [0029] when the number of fragments in the digested products is 3, the plant is determined to have PBG.ant-2H, and the genetic site that controls the synthesis of P3G and C3G in barley is heterozygous;
    [0030] [0030] when the number of fragments in the digested products is 1, the plant is determined to have PBG.ant-2H, and the genetic site that controls the synthesis of P3G and C3G in barley is homozygous; and
    [0031] [0031] when the number of fragments in the digested products is 2, the plant is determined to have no main effect QTL for synthesis of the anthocyanins P3G and C3G in barley.
    [0032] [0032] By detecting the marker of the present disclosure, the presence or absence of the PBG.anr-2H genetic locus can be identified, and then the presence or absence, and high or low, of the content of two anthocyanins in the barley grains is determined.
    [0033] [0033] The present disclosure provides a CAPS marker closely linked to the main effect QTL for synthesis of P3G and C3G in barley, and detection primers for the marker. The marker provided by the present disclosure has a genetic distance to the PBG.ant-2H locus of 0.0 cM.
    [0034] [0034] Figure 1 shows the BACS2H designed based on the SNP of the anthocyanin-related gene Ra near PBG.ant-2H between the parents, wherein GAal represents the Ra gene fragment in the parent Gairdner, and 49al represents the Ra gene fragment in the parent Russia68.
    [0035] [0035] Figure 2 shows the results of polymorphism detection between BACS2H of the parents, 5 wherein M represents the DNA molecular weight standard DL5000, G represents the parent Gairdner, and R represents the parent Russia68.
    [0036] [0036] Figure 3 shows using the BACS2H marker to identify genotype of population, G represents the parent Gairdner, R represents the parent Russia68, and 1-14 represent the population samples numbered 1-14 respectively.
    [0037] [0037] Figure 4 shows the analysis of the correlation between the BACS2H marker and P3G and C3G contents by single marker method. Among them, Figure A represents the correlation analysis between the BACS2H marker and C3G content, Figure B represents the correlation analysis between the BACS2H marker and P3G content; A and a represent alleles from the purple parent RUSSIA68 and the colorless parent Gairdner, respectively.DETAILED DESCRIPTION
    [0038] [0038] The present disclosure provides a CAPS marker closely linked to gene related to synthesis of P3G and C3G in colored barley. A person having ordinary skill in the art can learn from the content of the present disclosure and appropriately improve the process parameters to achieve this. It should be particularly pointed out that all similar substitutions and modifications are obvious to a person having ordinary skill in the art, and they are all deemed to be included in the present disclosure. The method and application of the present disclosure have been described through the preferred embodiments. 1t is obvious that relevant persons can make modifications or appropriate changes and combinations to the methods and applications of the present disclosure to realize and apply the technology of the present disclosure without departing from the content, spirit and scope of the present disclosure.
    [0039] [0039] The test materials used in the present disclosure are all commercially available in the market. {0040] The present disclosure will be further illustrated according to the following examples.
    [0041] [0041] The purple barley RUSSIA68 involved in the following examples was obtained from USDA-ARS (http://www.ars-grin.gov), and the colorless barley Gairdner was obtained from the Seed Bank of the Division of Plant Industry of the Commonwealth Science and Industry Organization of Australia. The standard anthocyanin P3G and C3G were purchased from PhytoLab; PCR reagents such as Tag, MgCl, and NTPs were purchased from Bioline; the restriction endonuclease Mfe 1 was purchased from NEB. Unless otherwise specified, other reagents were purchased from Sigma.
    [0042] [0042] The formula of solutions are as follows:
    [0043] [0043] CTAB buffer, 0. Imol/L Tris-HCl (PH8.0) + 1% CTAB (m/v) + 0.7% NaCl (m/v) + 10mmol/L EDTA;
    [0044] [0044] Anthocyanin extraction buffer, Methanol: 1M HCI = 85:15;
    [0045] [0045] Solution B, formic acid: acetonitrile: H,O = 0.1: 90: 9.9;
    [0046] [0046] Solution A, formicacid: H,O = 0.1: 99.9.
    [0048] [0048] According to the main effect QTL PBG.ant-2H for synthesis of P3G and C3G in colored barley grains reported in the literature (Xiao-Wei Zhang, Qian-Tao Jiang, Yu-Ming Wei, et al. Inheritance analysis and mapping of quantitative trait loci (QTL) controlling individual anthocyanin compounds in purple barley (Hordeum vulgare L.) grains. Plos one, 2017, 12: e0183704), based on the genome information of the cultivated barley Hordeum vulgare cv. Morex, gene prediction analysis was performed on the sequences on both sides of PBG.ant-2H, potential genes related to anthocyanin synthesis pathway were selected, SNP sites that can be used for CAPS design were screened, PCR amplification primer pairs at both ends or inside the candidate genes were designed using Primer Premier 5 software.
    [0049] [0049] The genomic DNA was extracted by CTAB method from young leaves of purple parent RUSSIA68 and colorless parent Gairdner. PCR amplification was performed with the primers designed in the above step using the genomic DNA of the two parents as templates. {0050] The sequences of PCR amplification primers for BACS2H marker are as follows:
    [0051] [0051] SEQ ID NO: 2: TTTTTGCCTCCTTGCTTGCTCTAA (5-3) {0052] SEQ ID NO: 3: GCATGTGAGCTACACAAGATGACAAT (5-37)
    [0054] [0054] The PCR products were cut with restriction endonuclease Mfel, and the reaction system was: 1 ug of DNA, 1 ul of restriction endonuclease, 1 ul of 10x buffer, and H-O was added until the total system was 10 ui. The digested products were detected by 2% agarose electrophoresis, the electrophoresis buffer was 1x TBE, and the electrophoresis power was constant at 50 W. f0055] By sequence analysis, gene prediction, amplification and sequencing, a gene Ra related to anthocyanin synthesis was found near PBG.ant-2H, and this gene has a SNP site for CAPS design between the two parents (as shown in Figure 1). Using BACS2H primers, highly specific fragments can be amplified, and after the cleavage of restriction endonuclease Mfel and electrophoresis detection, significant differences in banding patterns between the two parents can be observed (as shown in Figure 2). Example 2 Test of BACS2H marker in detection of main effect QTL PBG.ant-2H for synthesis of P3G and C3G in barley
    [0056] [0056] The grains of the F2 population to be verified were collected and placed in a ventilated box at 28°C for two weeks until the test. Based on the literature (Abdel-Aal and Hucl 2007), anthocyanins from grains was extracted using an improved method. The specific operation process was as follows: the grains were pulverized by a ball mill, and sieved with a 0.2 mm screen to obtain flour powders. The anthocyanin extract buffer was added to the flour at a ratio of 10:1 (V/M), mixed well, and the pH was adjust to 1. The mixture was shaken and mixed on a shaker at 4°C at 250 rpm for 24 h, and then centrifuged at 10,000 g for 25 min. The supernatant was transferred to a clean centrifuge tube as the sample to be tested. The standard anthocyanin was dissolved and serially diluted with the anthocyanin extract buffer. 10 pL of sample or standard was injected into Shimadzu Nexera High performance liquid chromatography (HPLC) system, and then passed Kinetex C18 1.7 um separation column (Phenomenex 2.1 mm X 100 mm), the flow rate was
    [0058] [0058] Single marker analysis (SMA) was performed using mapping software MapQTL 5.0 (Van Ooijen 2004) according to the phenotype/trait data obtained in step 1 and the population plant genotype data obtained in step 2. The results are shown in Figure 4. The genetic locus indicated by the BACS2H marker had a very significant correlation with the contents of anthocyanin P3G and C3G in barley, and the BACS2H marker is closely linked to the PBG.ant-2H locus with a genetic distance of 0.0 cM. The genetic distance between the previously discovered BAID2H marker and the PBG.ant-2H locus is greater than 3 cM.
    [0059] [0059] The above are only the preferred embodiments of the present disclosure. 1t should be pointed out that, for a person having ordinary skill in the art, a number of improvements and modifications can be made without departing from the principle of the present disclosure. These improvements and modifications should also be deemed to be the protection scope of the present disclosure.SEQLTXT
    SEQUENCE LISTING <110> INSTITUTE OF BAST FIBER CROPS, CHINESE ACADEMY OF
    AGRICULTURAL SCIENCES <120> CAPS MOLECULAR MARKER CLOSELY LINKED TO GENE RELATED TO SYNTHESIS OF P3G AND C3G IN COLORED BARLEY <130> CP0O020-NL-0358 <150> CN 201911233028.9 <151> 2019-12-05 <160> 3 <170> PatentIn version 3.3 <210> 1 <211> 1496 <212> DNA <213> Hordeum vulgare Russia 68 <400> 1 tttttgcctc cttgcttgct ctaacttcac ctcgtctgca tcatgcatat gaaatgaagg 60 aaggaaataa tatggtaatg gcgctaccaa tagttcgtcc gagccaggaa gaaccgccga 120 cggggaagca attcagctac cagctcgctg ccgctgtgag gagcatcaac tggagctacg 180 ccatattctg gtccatttca accagccgtc cagggtaggg agtgcatcaa attgatgatc 240 acttggcact ggccgtttcc tttcctctta tgatccgttt gttatgtagg gtactgacct 300 ggaaggacgg gttctacaac ggcgagataa agacgaggaa ggtcaccagc tcggcggacc 360 tcaccgccga ccagctcctc ctgcagagga gcgagcagct gcgggagctc taccagtccc 420 tcctgtccgg ccagtgcgac caccggggga ggcgcccggc cgccgcgctc tCgCCggagg 480 acctcgggga cgccgaatgg tactacgccg tctgcatgag ctatgccttc cgccctggcc 540 aagggtatgt actactccgt agtatgcttc aagaacaact ccactgatcc ataacggccg 600 atgcctgcgc acctacagta gttagcaatc atcttatctt gataagataa gatttacttt 660 gctagagagt gaaaactaca acgtttgcta tttcgtcatc aggttgccag gcagaagctt 720 tgcgagcaac gagcctgttt ggctgtgcaa tgctcagtgc gcagacacca aaactttcca 780 gcgctcgctc ttagcgaagg tacgtttgat tgcaccgccc gagtgtctcg caagcctcac 840 tctcatgtag tccctccgta aataaatgga gtataagagt gcttagatca ctacttagtg 900 Pagina 1
    SEQLTXT atataaacgc ttttatgttc ctttacgaag ggagtactat ctgcctaata tgtacccgat 960 cttgactaat accatgttgt ctactgtgta cttttggcgc acatactata tctgtcggct 1020 aaatcagacg acgtctatcc aggttagcta gcgcatgtca tgcaccctat atctgtgtat 1080 actgttcatg tttctgaccc cagctgcacg ggtgacaata ttttttcttc agacggtcgc 1140 ctgcattccc ttgatgggtg gtgtgcttga gctcgggacg acagataccg tgagttttcc 1200 tcgcatcaag cacatgatat tcacttacgg tttaaaatta aacggaaaac tcactattag 1260 taacgctttg tctcatcaag cacatcgaaa ttgcaaaatt taacccgggt tccatggagc 1320 cttgttgtta agaaaaacac aacagaagtt gtgagcacct atatatttcc gttcagtaca 1380 tcttgccata tgtgggggta gacaacaaag acaaatatgc ggatctacaa atagccaata 1440 atagatgtgc tagtatttcc aaaaccacac attgtcatct tgtgtagctc acatgc 1496 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 tttttgcctc cttgcttgct ctaa 24 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gcatgtgagc tacacaagat gacaat 26 Pagina 2
    权利要求:
    Claims (9)
    [1]
    1. A spliced amplified polymorphic sequence (CAPS) marker closely associated with a major effect quantitative property (QTL) locus, PBG.ant-2H, for synthesis of peonidin-3-glucoside (P3G) and cyanidin-3 -glucoside (C3G) in barley with the nucleotide sequence shown in SEQ ID NO: | is displayed.
    [2]
    A primer set for detecting the marker according to claim 1, consisting of two primers of the nucleotide sequence shown in SEQ ID NOs: 2 to 3.
    [3]
    Use of the primer set according to claim 2 in the breeding of colored barley.
    [4]
    A kit for detecting the main effect QTL for the synthesis of anthocyanins P3G and C3G in barley grains, comprising the primer set according to claim 2.
    [5]
    The kit of claim 4, further comprising restriction endonuclease Mfe I.
    [6]
    The kit of claim 4 or 5, further comprising a DNA marker, wherein the DNA marker comprises three DNA molecules having lengths of 1496 bp, 1329 bp and 167 bp.
    [7]
    A method for detecting the main effect QTL for the synthesis of anthocyanins P3G and C3G in barley, comprising: using genomic DNA extracted from a barley leaf as a template, performing PCR amplification with the primer set according to claim 3 , processing the amplified products with the restriction enzyme Mfe I, and determining the presence or absence of the main effect QTL for the synthesis of anthocyanins P3G and C3G in barley based on the processed products.
    [8]
    The method of claim 7, wherein the barley leaf is a young barley leaf.
    [9]
    The method according to claim 7 or 8, wherein the criterion of determining is: if the number of fragments in the digested products is 3, the plant is determined to be PBG. ant-2H and that the genetic site controlling the synthesis of P3G and C3G in barley is heterozygous if the number of fragments in the digested products is 1, the plant is determined to have PBG.ant-2H and that the genetic site containing regulates the synthesis of P3G and C3G in barley, is homozygous; and if the number of fragments in the digested products is 2, it is determined that the plant has no major effect QTL for the synthesis of the anthocyanins P3G and C3G in barley.
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    同族专利:
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN201911233028.9A|CN110872634B|2019-12-05|2019-12-05|CAPS molecular marker closely linked with P3G and C3G synthetic genes in colored barley|
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