CN105603096A - Co-dominance InDel molecular marker for identifying single embryo and multiple embryos of citrus and application thereof - Google Patents
Co-dominance InDel molecular marker for identifying single embryo and multiple embryos of citrus and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of citrus genomics and discloses a co-dominance InDel molecular marker for identifying single embryo and multiple embryos of citrus and application thereof. By carrying out depth re-sequencing on whole genome DNA of 107 materials including tangelos and oranges, whole genome sequence information is obtained, by means of comparing and screening, the gene sequence information in single-embryo and multi-embryo candidate areas is obtained, the sequence with the highest relevance degree to phenotype is screened out, and the sequence is an InDel sequence of 6bp. Accordingly, a set of simple, fast and co-dominant PCR markers are developed, and primers are used for carrying out PCR amplification on CK F/R, INS F/R and DEL F/R, so that the co-dominance InDel molecular marker for identifying the single embryo and the multiple embryos of the citrus is obtained. Finally, the marker is utilized for testing loci in 115 single-plant natural groups and 100 single-plant hybridized groups. The marker has the biggest advantages that cost is low, accuracy is high, and embryonic early selection can be achieved during the citrus seedling stage.
Description
Technical field
The present invention relates to oranges and tangerines genomics field, refer to particularly a kind of codominance of differentiating oranges and tangerines list embryo and polyembryonyInDel molecular labeling and application thereof, this molecular labeling can be used as oranges and tangerines list/polyembryony breeding marker assisted selection, for differentiating mandarin orangeTangerine list/polyembryony kind provides new molecular labeling.
Background technology
Polyembriony is very general in citrus, and seed of oranges and tangerines of polyembryony type can produce 2-10 embryo, according toReport that 42 embryos appear at most in a seed. Polyembriony is a kind of special Apomixis, and it and zoogamy are (singlyEmbryo) there is obvious difference, do not experience meiosis and fertilization process, develop into embryo by body cell (nucellar cell). Polyembryony processBe vegetative propagation mode, offspring is actually asexual the copying (Clonalcopy) of parent. Polyembryony proterties is wide in Orange ProducingGeneral utilization, the offspring who especially produces by apomixia in stock breeding germinate and emergence rate is high, nursery stock evenly sturdy,Neat and consistent, virus-free and convenient management. How polyembryony characteristic is imported to crop and be subject to breeding scholar's concern always, especially solidDetermine hybrid vigour aspect and there is wide application potential. If female parent is hybrid major clique, offspring is by this persistence base soBecause of type. China is in leading in the world aspect rice heterosis utilization, and Academician YUAN Long-ping has proposed rice heterosis utilizationTrilogy: " three line method " to " two line method ", again to " one is method ", wherein " once being " refers to utilize apomixia to fix hybridAdvantage.
The qualification of polyembryony and single embryo is generally to examine under a microscope by naked eyes, exists time-consuming, effort, accuracy rate notThe problems such as height, but be divided into the exploitation from molecular labeling about oranges and tangerines polyembryony proterties, make slow progress for many years. Most important reasonBe the shortage that is limited by genomic information, molecular labeling is distant from polyembryony site, be difficult to effective exploitation be divided into from pointSub-mark.
Summary of the invention
Object of the present invention provides a kind of codominance InDel molecular labeling of differentiating oranges and tangerines list embryo and polyembryony and shouldWith, its early diagnosis for oranges and tangerines embryo and correlation molecule breeding provide a kind of simple, quick and effective supplementary breeding method.
For achieving the above object, a kind of codominance InDel molecule mark of differentiating oranges and tangerines list embryo and polyembryony provided by the inventionNote, the nucleotide sequence of described codominance InDel molecular labeling is respectively as SEQIDNO:1, SEQIDNO:2 and SEQIDShown in NO:3.
For obtaining the primer pair of above-mentioned molecular labeling, described primer pair is respectively:
Primer pair CKF/R
Forward primer 5'-CGTTCTTCAGACCCTTTCAG-3',
Reverse primer 5'-TGTCCTTTGTCCCATCCTTC-3';
Primer pair INSF/R
Forward primer 5'-GTATCCTAAAAATATAATCTTTATG-3',
Reverse primer 5'-ATCAATTTGTCCTTTGTCCC-3';
Primer pair DELF/R
Forward primer 5'-CAAAGATTATTGATATGGGAAAG-3',
Reverse primer 5'-CCTCGATGTACCTAACGATT-3'.
The present invention also provides a kind of preparation of the above-mentioned codominance InDel molecular labeling that is applicable to the qualification of oranges and tangerines list polyembryonyMethod, comprises the following steps:
1) utilize 107 oranges and tangerines materials that complete early stage all to carry out the gene order-checking that the degree of depth is about 30X, comprehensively existing rightThe hereditary positioning result of polyembryony proterties, choosing No. 4 chromosomal 2.355-3.83M regions of sweet orange is oranges and tangerines list polyembryony correlation candidateRegion; Use blastn, in 107 parts of materials, find respectively the corresponding homology segment order of this candidate's section in each genomeRow;
2), according to the oranges and tangerines classification under material, the order-checking reads of 107 materials is compared respectively respectively with BWA softwareFrom genomic candidate's section; Utilize ICORN2 software, with annotated 208 genes out in sweet orange genome candidate regionFor reference sequences, obtain 208 gene orders of each material candidate region;
3) utilize MEGA software, check one by one the comparison result of multisequencing, according to single polyembryony phenotype situation of material, statisticsThe ratio of the various base/haplotypes in site, and manually check, filter out the sequence polymorphisms the highest with phenotype relevance degreeProperty, find that InDel difference in polyembryony and single embryo material of a 6bp is obvious, this result is the list based on sequencing result predictionPolyembryony linked marker;
4) according to above sequence information, designed experimental verification, InDel sequence TTTATG has been developed as to codominance PCR markNote, is respectively the insertion the called after INS-specific that detect this InDel, detects disappearance the called after DEL-of InDelSpecific, and contrast called after CK, comprehensive three's result, directly judges the gene in each this site of material from glue figureType.
As preferred version: described step 4) concrete steps are as follows:
1) the INS-specific primer that design forward primer 3 ' end is TTTATG, as follows:
Primer pair INSF/R
Forward primer 5'-GTATCCTAAAAATATAATCTTTATG-3',
Reverse primer 5'-ATCAATTTGTCCTTTGTCCC-3';
Its PCR product is shown in SEQIDNO:2, the existence that this 6bp base of specific detection is inserted;
2) design forward primer is crossed over the DEL-specific primer of this InDel site and disappearance TTTATG sequence, as followsShown in:
Primer pair DELF/R
Forward primer 5'-CAAAGATTATTGATATGGGAAAG-3',
Reverse primer 5'-CCTCGATGTACCTAACGATT-3';
Its PCR product is shown in SEQIDNO:3, and this site of specific detection lacks the situation of this 6bp base;
3) in this InDel upstream end design forward primer, downstream design reverse primer, the CK primer that partners, following instituteShow:
Primer pair CKF/R
Forward primer 5'-CGTTCTTCAGACCCTTTCAG-3',
Reverse primer 5'-TGTCCTTTGTCCCATCCTTC-3';
Its PCR product is shown in SEQIDNO:1, and comprehensive three's result, directly judges this position of each material from glue figureThe genotype of point.
The present invention also provides the application of a kind of above-mentioned molecular labeling in the qualification of oranges and tangerines list polyembryony, utilizes codominanceInDel molecular labeling is being differentiated the embryo of 115 oranges and tangerines materials.
The present invention also provides a kind of above-mentioned Auele Specific Primer to reflect at oranges and tangerines embryo to CKF/R, INSF/R and DELF/RApplication in fixed, utilizes 3 primer pairs to carry out PCR qualification HB shaddock (single embryo) × Fairchild mandarin orange (polyembryony) hybrid Population 100 strainsOffspring.
Meaning of the present invention
Genetic marker, as the objective performance of hereditary difference between bion or colony, mainly refers to that those can clearly reflectThe biological characteristic of biologic polymorphism. Along with the fast development of biotechnology, the kind of genetic marker obtained rapidly development andAbundant, successively there is morphological markers, cytological marker, protein labeling and DNA marker. DNA molecular marker and other marksNote technology is compared, have that quantity is abundant, polymorphism is high, be subject to environmental condition and stage of development affect little, detection means simple, rapidlyEtc. advantage. Therefore, DNA marker, as a kind of desirable molecular labeling, extensively meets the tendency and vegeto-animal molecular biology research,In research aspect the assignment of genes gene mapping and clone, the Origin of Species and domestication, bringing into play more and more important effect. In recent years, withThe development of high throughput sequencing technologies, molecular markers development technology has also obtained rapidly development. Utilize high-throughput techniques exploitationPCR-based technology, the molecular labeling that simple to operate, using value is large, can be the assignment of genes gene mapping of controlling oranges and tangerines Main Agronomic CharactersBreeding lays the foundation with molecular marker assisted selection.
Based on the molecular labeling lnDel (Insertion/Deletion of new generation of complete genome DNA sequence exploitationPolymorphisms) insertion and deletion mark, refers to the difference in full genome in two kinds of parents, another parent relatively, in one of them parent's genome, have the nucleotides of some to insert or disappearance. According to insertion and deletion in genomeSite, designs the PCR primer in some these insertion and deletion sites of increasing, and Here it is InDel mark is a kind of special in genomeTwo equipotential gene genetic marks of type, show as the small fragment DNA sequence dna that inserts or lacked different sizes in genome, belong toIn codominant marker, there is the advantages such as good stability and polymorphism.
Quick, an effective way are provided by whole genome sequence information excavating for identifying single polyembryony. Due to InDelDimorphism, either-or, in genome screening, often only need the analysis of +/-, and need not analyze the length of fragment. InDelBe considered to the most promising third generation molecular labeling after RFLP and SSR, in genetic map construction, the proterties of plant fieldRelated molecular marker exploitation and Basic of Biology research is application fast.
Pcr amplification aim sequence and agarose electrophoresis are to differentiate the simplest and the most direct method of InDel. Special according to aim sequence designOpposite sex primer, taking the genomic DNA of Different Individual as template, increases by same PCR reaction system, to obtained amplificationProduct directly carries out agarose electrophoresis, carefully check each individuality whether have object band just can find out this aim sequence survey eachBetween genes of individuals group, whether there is InDel.
Beneficial effect of the present invention is:
The present invention has successfully obtained the codominance InDel molecular labeling of oranges and tangerines embryos, uses this mark to carry out oranges and tangerines embryoDiscriminating, can overcome tradition and rely on naked eyes to differentiate time-consuming, the effort of embryo, the shortcoming that accuracy rate is not high; Simultaneously can be in hybridizationOffspring early stage (in 1 year) carries out molecular labeling diagnosis, and traditional breeding method is waited for when fruit result produces seed demand 3-8Between, by contrast, this molecular labeling be applied to assistant breeding have the time early, advantage that cost is low. This mark can be applied toDifferent target in breeding practice, carries out stock breeding and can utilize this label screening polyembryony material, and sieve is cross-breedingMenu embryo material, thus the breeding efficiency of oranges and tangerines improved.
Brief description of the drawings
Fig. 1 is primer pair CKF/R, INSF/R and DELF/R design diagram.
In figure, left side glue figure is result example, single/how to represent respectively that phenotype is single embryo/polyembryony. Fig. 2: primer pair CKF/R,Primer pair INSF/R and primer pair DELF/R be the amplification in 115 variety genome DNAs of oranges and tangerines natural population respectively.
In figure: the vertical flower of 1 representative tangerine (how assorted); 3 represent the wild tangerine in Dao County (how assorted); 6 represent the wild tangerine tertia in Dao County (how assorted); 7Represent the wild tangerine in Chongyi (how assorted); 9 represent red skin acid tangerine (how assorted); The sliding skin tangerine of 11 representative (how assorted); 12 represent ponkan (how assorted); 13Represent Huangyan local early (how assorted); 14 represent satsuma orange (how assorted); 16 represent Dao County-3 (how assorted); 21 represent that citrus reticulata“Chachi” is (manyAssorted); 22 represent rock sugar tangerine (how assorted); 25 represent south, Changsha tangerine (how assorted); 26 represent red tangerine (how assorted); 27 represent that bitter orange is (manyAssorted); 28 represent bitter orange (how assorted); 29 represent bitter orange (how assorted); 30 represent bitter orange (how assorted); 31 represent bitter orange (how assorted); 33 generationsShow dark orange (how assorted); 34 represent brocade orange (how assorted); 35 represent Kurt Hamrin (how assorted); 36 represent peach leaf orange (how assorted); 37 representativesChina's navel (how assorted); 38 representative snow mandarin oranges (how assorted); 39 represent Xia Cheng (how assorted); 40 represent blood orange (how assorted); 41 represent that crystal sugar orange is (manyAssorted), 2 represent the wild tangerine of India (single embryo); 15 represent Nanfeng orange (single embryo); 32 represent amber sweet orange (single embryo); 55 represent NingboGold mandarin orange (single embryo); 56 represent mountain gold mandarin orange (single embryo); 57 represent Fructus Aurantii (single embryo); 58 represent Ke Liman fourth (single embryo); 59 representatives are fertileMandarin orange (single embryo); 60 represent Weir gold (single embryo); (single embryo) is shown in clearly in 61 representatives, and 62 represent Linwu bitter orange (single embryo); 63 represent purple skinShaddock (single embryo); 64 represent Huajuhong (single embryo); 65 represent shatian pomelo (single embryo); 66 Dai Biao Guanxi small stream honey shaddocks (single embryo); 67 representativesAnacidity shaddock (single embryo); 68 represent Ji'an shaddock (single embryo); 69 represent Ruili shaddock (single embryo); 70 represent Hua Nonghong shaddock (single embryo);71 represent high spot shaddock (single embryo); 72 represent Guilin shaddock (single embryo); 73 represent Wanbai pummelo (single embryo); 74 represent that Chongqing shaddock is (singleEmbryo); 75 represent Zhejiang shaddock (single embryo); 76 represent Ma Jiayou (single embryo); 78 representative element tangerines (single embryo); 79 represent big hill mandarin orange(single embryo), 80 represent the large wing orange of little Hua (single embryo); 84 represent Ichang papeda 2 (single embryo); 85 represent Ichang papeda 3 (single embryo); 88 representativesCitron (single embryo); 90 represent citron (single embryo); 92 represent citron (single embryo); 94 represent citron (single embryo); 95 represent that Buddha's hand is (singleEmbryo); 96 represent Fructus Aurantii 2 (single embryo); 97 represent Fructus Aurantii 3 (single embryo); 98 represent Amo (single embryo); 99 represent Cpo (single embryo); 103 generationsTable Sdi (single embryo); 106 represent that wine cake strangles (single embryo); 107 representative howl shell thorns (single embryo); 83 represent Ichang papeda (single embryo); 110 generationsTable A ce (single embryo); 114 represent Aro (single embryo); 4 represent big hill tangerine (how pure); 8 represent big hill tangerine (sharp leaf) (how pure); 17Represent a year tangerine (how pure); 23 represent Yangshan tangerine (how pure); 5 represent the wild tangerine smooth bark 2 in Dao County (how assorted); 20 represent that the sweet tangerine of Ming Liu is (manyAssorted); 18 represent Ma Shui tangerine (how assorted); 19 represent granulated sugar tangerine (how assorted); 43 represent sieve navel (how assorted); 42 representatives are (how assorted) from generation to generation;46 represent chicken tail grape fruit (how assorted); 45 represent Flame (how assorted); 47 represent StarRuby (how assorted); 51 represent that citron is (manyAssorted); 49 represent Lisbon lemon (how assorted); 48 represent Muli-lemon (how assorted); 50 represent Mexico's Lay covers (how assorted); 124 generationsTable DYC (single embryo); 125 represent JF (single embryo); 126 represent KM (single embryo); 127 represent LS1 (single embryo); 128 represent TK (single embryo);129 represent XC (single embryo); 130 represent YJ (single embryo); 131 represent YL (single embryo); 132 represent ZY (single embryo); 133 represent that XZ1 is (singleEmbryo); 86 represent JY4 (single embryo); 87 represent JY5 (single embryo); 89 represent JY8 (single embryo); 91 represent JY28 (single embryo); 115 representativesCDSJ (single embryo); 116 represent GDMM (single embryo); 117 represent HDGKZ (single embryo); 118 represent JMPG1 (single embryo); 119 representativesJMPG2 (single embryo); 120 represent SND (single embryo); 121 represent WNNL (single embryo); 122 represent WNSMW (single embryo); 123 represent WSD(single embryo); M represents 1KbMarker (clip size is followed successively by 250,500,750,1000,1500,2000bp etc.).
Fig. 3: primer pair CKF/R, primer pair INSF/R and primer pair DelF/R are respectively HB shaddock and Fairchild mandarin orangeAmplification in 110 strain filial generation genomic DNAs.
In figure: H represents HB shaddock; F represents Fairchild mandarin orange; Other numeral is the individual plant of H and F filial generation; M representative100bpMarker (clip size is followed successively by 100,200,300,400,500bp etc.).
Detailed description of the invention
In order to explain better the present invention, further illustrate main contents of the present invention below in conjunction with specific embodiment, butContent of the present invention is not only confined to following examples.
The qualification of embodiment 1 tangerine, shaddock, Cheng Deng115Ge natural population bill of materials polyembryony
1, prepare the genomic DNA of the oranges and tangerines such as tangerine, shaddock, orange, citron, This-problem lemon
In the present embodiment, adopt the CTAB method of improvement to extract the genomic DNA of the oranges and tangerines such as tangerine, shaddock, orange, citron, This-problem lemon;2, utilize the genomic DNA of primer pair CKF/R, INSF/R and the above-mentioned all oranges and tangerines of DelF/R Amplification Analysis;
Above-mentioned primer pair is respectively:
Primer pair CKF/R
Forward primer 5'-CGTTCTTCAGACCCTTTCAG-3',
Reverse primer 5'-TGTCCTTTGTCCCATCCTTC-3';
Primer pair INSF/R
Forward primer 5'-GTATCCTAAAAATATAATCTTTATG-3',
Reverse primer 5'-ATCAATTTGTCCTTTGTCCC-3';
Primer pair DELF/R
Forward primer 5'-CAAAGATTATTGATATGGGAAAG-3',
Reverse primer 5'-CCTCGATGTACCTAACGATT-3'.
PCR reaction system is as follows: 7 μ lPCRMix (purchased from Hubei Jing Mao company), 100ngDNA, forward and reverse primer(synthesizing in raw work) each 0.2 μ M, ddH2O is supplemented to final volume 15 μ l. Thermal circulation parameters is: 95 DEG C of 5min; 95 DEG C of 30sec, 58DEG C 30sec, 72 DEG C of 45sec, 35 circulations; 72 DEG C of 10min, 1 circulation; 4 DEG C of preservations, reaction is at S1000ThermalOn CyclerPCR instrument, complete. Amplified production 1.0% agarose gel electrophoresis on Horizontal electrophoresis tank detects, and uses 1xTAE slowRush liquid (0.04MTris-acetate, 0.001MEDTA, pH8.0), voltage 8V/cm, electrophoresis 30min. Electrophoresis is complete, gelImaging system (UVP) preservation of taking pictures.
Above-mentioned primer pair CKF/R, INSF/R and DelF/R amplified production in oranges and tangerines genomic DNA is respectivelyThe single bright band of 550bp, 340bp, 120bp left and right. What have 550bp, 120bp two strip-types is single embryo, has 550bp, 340bp twoIsozygotying for polyembryony of strip-type, what have 550bp, 340bp, 120bp tri-strip-types is polyembryony heterozygosis (experimental result is shown in Fig. 2).
The qualification of embodiment 2HB shaddock and the single polyembryony of Fairchild mandarin orange 100 strain filial generations
1, the genomic DNA of preparation HB shaddock and Fairchild mandarin orange 100 strain filial generations
In the present embodiment, adopt the CTAB method of improvement to extract the gene of HB shaddock and Fairchild mandarin orange 100 strain filial generationsGroup DNA.
2, utilize the genome of above-mentioned primer pair CKF/R, INSF/R and the above-mentioned all oranges and tangerines of DelF/R Amplification AnalysisDNA。
PCR reaction system is as follows: 7 μ lPCRMix (purchased from Hubei Jing Mao company), 100ngDNA, forward and reverse primer(synthesizing in raw work) each 0.2 μ M, ddH2O is supplemented to final volume 15ul. Thermal circulation parameters is: 95 DEG C of 5min; 95 DEG C of 30sec, 58DEG C 30sec, 72 DEG C of 45sec, 35 circulations; 72 DEG C of 10min, 1 circulation; 4 DEG C of preservations, reaction is at S1000ThermalOn CyclerPCR instrument, complete. Amplified production 1.0% agarose gel electrophoresis on Horizontal electrophoresis tank detects, and uses 1xTAE slowRush liquid (0.04MTris-acetate, 0.001MEDTA, pH8.0), voltage 8V/cm, electrophoresis 30min. Electrophoresis is complete, and gel becomesPicture system (UVP) preservation of taking pictures.
Above-mentioned primer pair CKF/R, INSF/R and DelF/R amplified production in oranges and tangerines genomic DNA is respectivelyThe single bright band of 550bp, 340bp, 120bp left and right. Have 550bp, 120bp two strip-types for single embryo, have 550bp, 340bp,120bp tri-strip-types for polyembryony heterozygosis (experimental result is shown in Fig. 3).
Other unspecified part is prior art. Although above-described embodiment has been made detailed retouching to the present inventionState, but it is only the present invention part embodiment, instead of whole embodiment, people can also according to the present embodiment withoutUnder creative prerequisite, obtain other embodiment, these embodiment belong to protection domain of the present invention.
Claims (6)
1. a codominance InDel molecular labeling of differentiating oranges and tangerines list embryo and polyembryony, is characterized in that: described codominance InDelThe nucleotide sequence of molecular labeling is respectively as shown in SEQIDNO:1, SEQIDNO:2 and SEQIDNO:3.
2. for obtaining the primer pair of molecular labeling described in claim 1, it is characterized in that: described primer pair is respectively:
Primer pair CKF/R
Forward primer 5'-CGTTCTTCAGACCCTTTCAG-3',
Reverse primer 5'-TGTCCTTTGTCCCATCCTTC-3';
Primer pair INSF/R
Forward primer 5'-GTATCCTAAAAATATAATCTTTATG-3',
Reverse primer 5'-ATCAATTTGTCCTTTGTCCC-3';
Primer pair DELF/R
Forward primer 5'-CAAAGATTATTGATATGGGAAAG-3',
Reverse primer 5'-CCTCGATGTACCTAACGATT-3'.
3. described in claim 1, be applicable to a preparation method for the codominance InDel molecular labeling of oranges and tangerines list polyembryony qualification, itsBe characterised in that: comprise the following steps:
1) utilize 107 oranges and tangerines materials that complete early stage all to carry out the gene order-checking that the degree of depth is about 30X, comprehensive existing to polyembryonyThe hereditary positioning result of proterties, choosing No. 4 chromosomal 2.355-3.83M regions of sweet orange is oranges and tangerines list polyembryony correlation candidate districtTerritory; Use blastn, in 107 parts of materials, find respectively the corresponding homology segment order of this candidate's section in each genomeRow;
2), according to the oranges and tangerines classification under material, the order-checking reads of 107 materials is compared respectively to base separately with BWA softwareBecause of candidate's section of group; Utilize ICORN2 software, taking annotated 208 genes out in sweet orange genome candidate region as ginsengExamine sequence, obtain 208 gene orders of each material candidate region;
3) utilize MEGA software, check one by one the comparison result of multisequencing, according to single polyembryony phenotype situation of material, statistics siteThe ratio of various base/haplotypes, and manually check, filter out the sequence polymorphism the highest with phenotype relevance degree, send outInDel difference in polyembryony and single embryo material of an existing 6bp is obvious, and this result is that the single polyembryony based on sequencing result prediction connectsLock mark;
4) according to above sequence information, designed experimental verification, InDel sequence TTTATG has been developed as to codominance PCR mark, pointWei not detect insertion the called after INS-specific of this InDel, detect disappearance the called after DEL-of InDelSpecific, and contrast called after CK, comprehensive three's result, directly judges the gene in each this site of material from glue figureType.
4. be applicable to the preparation method of the codominance InDel molecular labeling of oranges and tangerines list polyembryony qualification according to claim 3, its featureBe: described step 4) concrete steps: the INS-specific primer that 1) design forward primer 3 ' end is TTTATG, following instituteShow:
Primer pair INSF/R
Forward primer 5'-GTATCCTAAAAATATAATCTTTATG-3',
Reverse primer 5'-ATCAATTTGTCCTTTGTCCC-3';
Its PCR product is shown in SEQIDNO:2, the existence that this 6bp base of specific detection is inserted;
2) design forward primer is crossed over the DEL-specific primer of this InDel site and disappearance TTTATG sequence, as follows:
Primer pair DELF/R
Forward primer 5'-CAAAGATTATTGATATGGGAAAG-3',
Reverse primer 5'-CCTCGATGTACCTAACGATT-3';
Its PCR product is shown in SEQIDNO:3, and this site of specific detection lacks the situation of this 6bp base;
3) at this InDel upstream end design forward primer, downstream design reverse primer, the CK primer that partners, as follows:
Primer pair CKF/R
Forward primer 5'-CGTTCTTCAGACCCTTTCAG-3',
Reverse primer 5'-TGTCCTTTGTCCCATCCTTC-3';
Its PCR product is shown in SEQIDNO:1, and comprehensive three's result directly judges each this site of material from glue figureGenotype.
5. the application of molecular labeling claimed in claim 1 in the qualification of oranges and tangerines list polyembryony, is characterized in that: utilize aobvious altogetherProperty InDel molecular labeling at the embryo of differentiating 115 oranges and tangerines materials.
An Auele Specific Primer claimed in claim 2 to CKF/R, INSF/R and DelF/R in the qualification of oranges and tangerines embryoApplication, it is characterized in that: utilize 3 primer pairs to carry out the single embryo × Fairchild mandarin orange of PCR qualification HB shaddock polyembryony hybrid Population100 strain offsprings.
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| CN201610105440.2A CN105603096B (en) | 2016-02-25 | 2016-02-25 | Codominant InDel molecular marker for identifying single embryo and multiple embryos of citrus and application thereof |
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| CN201610105440.2A CN105603096B (en) | 2016-02-25 | 2016-02-25 | Codominant InDel molecular marker for identifying single embryo and multiple embryos of citrus and application thereof |
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| CN105603096B CN105603096B (en) | 2020-01-14 |
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| CN108660246A (en) * | 2018-05-21 | 2018-10-16 | 华中农业大学 | One group of horse family's shaddock InDel molecular labeling and its application in Citrus Cultivars seedling early stage distinguishes tertia horse man shaddock |
| CN109161610A (en) * | 2018-11-07 | 2019-01-08 | 黑龙江大学 | It is a kind of detect beet seed graininess SSR molecular marker BvRE016 and its application |
| CN109207629A (en) * | 2018-11-07 | 2019-01-15 | 黑龙江大学 | It is a kind of detect beet seed graininess SSR molecular marker BvRE049 and its application |
| CN109266776A (en) * | 2018-10-24 | 2019-01-25 | 华中农业大学 | Utilize the kit and method of InDel Marker Identification citrus shaddock hybrid generation |
| CN109321676A (en) * | 2018-11-07 | 2019-02-12 | 黑龙江大学 | It is a kind of detect beet seed graininess SSR molecular marker BvRE105 and its application |
| CN110273018A (en) * | 2019-05-29 | 2019-09-24 | 广西壮族自治区农业科学院 | Fertile mandarin orange SSR molecular marker and its application |
| CN110669863A (en) * | 2019-10-30 | 2020-01-10 | 华中农业大学 | Citrus mitochondria InDel molecular marker and application thereof |
| CN111286556A (en) * | 2020-04-03 | 2020-06-16 | 江西省农业科学院园艺研究所 | Method for identifying variety of golden orchid pomelo based on whole genome InDel marker |
| CN114438249A (en) * | 2022-02-22 | 2022-05-06 | 江西省农业科学院园艺研究所 | Primer group, kit and identification method for identifying cultivation types of valium odoratum honey pomelos |
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| CN108660246B (en) * | 2018-05-21 | 2021-01-22 | 华中农业大学 | InDel molecular markers of Ma (Citrus paradisi) pomelos and application of InDel molecular markers in early-stage differentiation of rough-peel Ma pomelos of citrus varieties |
| CN109266776A (en) * | 2018-10-24 | 2019-01-25 | 华中农业大学 | Utilize the kit and method of InDel Marker Identification citrus shaddock hybrid generation |
| CN109207629B (en) * | 2018-11-07 | 2021-06-29 | 黑龙江大学 | SSR molecular marker BvRE049 for detecting granularity of beet seeds and application thereof |
| CN109321676A (en) * | 2018-11-07 | 2019-02-12 | 黑龙江大学 | It is a kind of detect beet seed graininess SSR molecular marker BvRE105 and its application |
| CN109207629A (en) * | 2018-11-07 | 2019-01-15 | 黑龙江大学 | It is a kind of detect beet seed graininess SSR molecular marker BvRE049 and its application |
| CN109161610A (en) * | 2018-11-07 | 2019-01-08 | 黑龙江大学 | It is a kind of detect beet seed graininess SSR molecular marker BvRE016 and its application |
| CN109161610B (en) * | 2018-11-07 | 2021-06-29 | 黑龙江大学 | SSR molecular marker BvRE016 for detecting granularity of beet seeds and application thereof |
| CN109321676B (en) * | 2018-11-07 | 2021-06-29 | 黑龙江大学 | SSR molecular marker BvRE105 for detecting granularity of beet seeds and application thereof |
| CN110273018A (en) * | 2019-05-29 | 2019-09-24 | 广西壮族自治区农业科学院 | Fertile mandarin orange SSR molecular marker and its application |
| CN110669863A (en) * | 2019-10-30 | 2020-01-10 | 华中农业大学 | Citrus mitochondria InDel molecular marker and application thereof |
| CN111286556A (en) * | 2020-04-03 | 2020-06-16 | 江西省农业科学院园艺研究所 | Method for identifying variety of golden orchid pomelo based on whole genome InDel marker |
| CN114438249A (en) * | 2022-02-22 | 2022-05-06 | 江西省农业科学院园艺研究所 | Primer group, kit and identification method for identifying cultivation types of valium odoratum honey pomelos |
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