CN103555717A - Functional molecular markers of related genes of sweetness and sourness characters of muskmelon and application of markers - Google Patents
Functional molecular markers of related genes of sweetness and sourness characters of muskmelon and application of markers Download PDFInfo
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Abstract
The invention discloses functional molecular markers of related genes of sweetness and sourness characters of muskmelon and applications of the markers. According to the markers and the applications of the markers, by utilizing a Super-BAS technique, the functional molecular markers of the related genes of the sweetness and sourness characters of the muskmelon are provided; related gene sequences of the sweetness and sourness characters of flavor muskmelons are confirmed; a functional molecular marker of the related gene of the sweetness character, namely SLAF18745-S01 and three functional molecular markers of the related gene of the sourness character, namely SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 are successfully developed on the basis; conventional phenotypic selection is changed into direct genotypic selection through sweetness and sourness marker assisted selection, so that the functional molecular markers have the important application value and significance on the improvement of the sweetness and sourness characters of the muskmelons.
Description
Technical field
The present invention relates to agricultural biological technical field.Specifically, the present invention relates to technical field a kind of and melon fruit sweet taste, tart flavour trait related gene functional molecular marker and application thereof.
Background technology
Muskmelon (
cucumis melo L.) belong to Curcurbitaceae Cucumis, be a kind of important garden crop, cultivated area and the output of China's muskmelon all rank first in the world.Muskmelon is a kind of high polymorphic species, the genotype that contains various different taste of fruit.Ripe melon fruit sugar, acid constituents and content thereof have important impact to fruit interior quality, are the important indicators that determines taste of fruit.The variation of the local flavor such as sweet, sour is a complicated process, is subject to impact and the regulation and control of a series of genes involveds.Location muskmelon sweet taste, tart flavour trait related gene development functionality mark, provide thinking for improving melon fruit quality trait Breeding Efficiency and muskmelon molecular breeding.
In existing muskmelon breeding practice, to local flavor proterties such as sweet taste, tart flavours, be indirectly genotype to be selected by phenotype mostly, cause breeding cycle long, efficiency is low, is restricting the process of melon variety improvement.Marker assisted selection, especially can greatly improve the efficiency of selection to the selection of the functional molecular marker associated with objective trait, accelerates genetic improvement.
In prior art, the molecule marker that functional molecular marker forms as functional single nucleotide polymorphism (SNP) site in the functional gene motif with phenotypic correlation exploitation.The exploitation of functional label must possess following two conditions: (1) has candidate gene the known allelic sequence information of definite function; (2) in a plurality of materials, objective trait is investigated, target gene is carried out to sequential analysis, in conjunction with proterties and gene order information, carry out the association analysis based on linkage disequilibrium (LD).Functional gene location is the basis that functional molecular marker is excavated, and functional gene is located conventional method at present has based on traditional genetic map location and cluster segregation analysis (BSA).
javier Mdeng (2009), based on traditional genetic map, muskmelon sugar, acid shape have been carried out to QTL location, but the cycle of the method is long, density is low, cost is high; And can fast, effectively find and the closely linked molecule marker of target gene based on conventional cluster segregation analysis (BSA), but mixed pond number is limited, is generally no more than 10, and association analysis false positive probability is large, and mark density is low.The selected gene type evaluation of development in recent years and cluster compartment analysis (
superbSA), utilize
sLAF-seq(
specific Location Amplified Fragments seqthe order-checking of specific position amplified fragments), the specific fragment that selection is evenly distributed on whole genome and avoids tumor-necrosis factor glycoproteins region carries out high depth order-checking, by the difference of different genotype frequency of occurrences in two mixed ponds of SNP mark relatively, determines the molecule marker being closely related with proterties.
superbSA molecule marker density is high, and the mixed pond of 30-200 individuality has guaranteed effect and the accuracy of the assignment of genes gene mapping.Meanwhile, because the pleomorphism site in SLAF label has been positioned on genome, can use primer-design software to carry out design of primers, development function mark according to genomic position.The molecular marking technique that these grew up in recent years, by molecular marker assisted selection, traditional Phenotypic Selection can be changed into direct Select gene type and all have great importance, but so far there are no carries out research and the report of muskmelon sweet taste tart flavour trait related gene functional molecular marker aspect.
Summary of the invention
Object of the present invention is intended to for the deficiencies in the prior art, utilizes
super-BAS technology, a kind of muskmelon sweet taste tart flavour trait related gene functional molecular marker is provided, determined local flavor muskmelon sweet taste, tart flavour trait related gene sequence, and a sweet taste trait related gene functional molecular marker SLAF18745-S01 and three each and every one tart flavour trait related gene functional molecular marker SLAF36334-S02 have successfully been developed on this basis, SLAF50072-S03 and SLAF31212-S04, and the application that traditional Phenotypic Selection is changed into direct Select gene type by sweet taste tart flavour marker assisted selection is for muskmelon sweet taste, the improvement of tart flavour proterties and improvement have important using value and meaning.
The present invention is achieved through the following technical solutions: by sweet taste, the extreme colony of tart flavour proterties are carried out based on simplify gene order-checking (
sLAFselected gene type evaluation-seq) and cluster compartment analysis (
super-BAS), in conjunction with bioinformatics method location and sweet taste, the associated candidate region closely of tart flavour proterties, acquired character correlation candidate gene, for this candidate gene, develop the functional molecular marker of muskmelon sweet taste, tart flavour character gene, reach the sweet tart flavour strain of seed selection, shorten breeding cycle, improve and select the good technique effect of accuracy rate.
The present invention specifically provides a kind of muskmelon sweet taste tart flavour trait related gene functional molecular marker, comprises the following steps acquisition.
(1) identify male parent, female parent, the F of " No. four, local flavor " muskmelon
1and F
2colony's individual plant taste of fruit.
(2) to " No. four, local flavor " muskmelon male parent, female parent, F
2sweet not sour, sweetless, the acid sweetless individual plant DNA mixed Chi Jianku of equivalent respectively again neither of acid only only in colony, simplifies gene order-checking, obtains SLAF label, and SLAF label is carried out to mononucleotide (SNP) polymorphism analysis.
(3) according to polymorphism mark and sweet taste, the cognation location sweet taste of tart flavour proterties, ，Bing Dui candidate region, tart flavour proterties candidate region gene, carry out functional annotation.
(4) according to the SLAF sequence label of the functional gene of location, design primer, exploitation sweet taste, the functional mark of tart flavour proterties.
Further, the present invention
in detaila kind of muskmelon sweet taste tart flavour trait related gene functional molecular marker is provided, has comprised the following steps acquisition.
(1) male parent, female parent, the F to " No. four, local flavor " muskmelon
1and F
2for the sweet tart flavour of each individual plant fruit by apparatus measures in conjunction with Taste evaluation, obtain that only acid is sweetless, sweet not sour, not only not only sweet, neither acid but also sweetless 4 kinds of local flavors of acid only.
(2) by CTAB method, extract male parent, female parent, F
1and F
2for each individual plant genomic dna, respectively balanced mix male parent, female parent, sweetless, sweet not sour, acid sweetless each individual plant genomic dna again neither only of acid only, form 5 mixed ponds, respectively 5 mixed pond genomes being carried out to enzyme cuts, the sequence dna fragment of the same site of each each individual plant of mixed pond, through pcr amplification, order-checking, Blat comparison software enters cluster, Cap snp software detection single nucleotide polymorphism, obtains the SLAF label of polymorphism.
(3) according to the cognation of polymorphism mark and sweet taste, tart flavour proterties, use ratio correlation method to position.Calculate respectively colony neither acid between sweetless ac and sweet not sour ab and neither between acid but also sweetless ac and sour sweetless aa, derive from not only the difference ratio of different genotype, obtain difference mark.The difference mark associated with sweet taste, tart flavour proterties compared by blat software, on parents' genome, locate, according to mark physical location, extract the gene at mark place, compare with Swissprot and Kegg database, obtain and sweet taste proterties, annotation of gene function that tart flavour proterties is relevant, pick out the label in the SLAF of functional gene exon.
(4) according to the polymorphic sequence of the polymorphic sequence of label SLAF18745 location and a sweet taste proterties correlation function gene M ELO3C011944T1 place in exon, 3 tart flavour proterties correlation function gene M ELO3C020002T1, MELO3C025794T1, MELO3C026251T1 place label SLAF36334, SLAF50072, SLAF51212, utilize Primer Primer5.0 software design primer, with male parent, female parent, F
1, F
2only sweet not sour, only acid sweetless, neither acid again sweetless material be material, carry out pcr amplification, develop a sweet taste trait related gene functional molecular marker SLAF18745-S01 and three tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04.
The corresponding DNA sequence dna of functional molecular marker of a sweet taste, three tart flavour trait related genes is respectively.
The DNA sequence dna of SLAF18745-S01
tgggaccagttcctcattctttctcagcagttcacttgccatcgtatctgctggaaaatgcagaagactacaaattccttttgcctgggaattgcatacgagagagtggctgatctctaactttggcgtctacctccctgtgaaggttagttgcgatgtagcttgtctgacacctcgtggaccttccatctcgatagttgttgaccaatatgcagtcaaccgtggtggtcttttgcctccagataagct
The DNA sequence dna of SLAF18745-S02
catagctggtgtctggatatctctaatgtatatgttatttatgtgtgaggttggattttgagatca
The DNA sequence dna of SLAF18745-S03
caaagccgctggactactcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggggtcagcttggacctgaaattgcaagtttgactcaattgcgtactattgatttgacaaccaacgatttctctggtgaaattccttatgggattggtaactGtacccatttagagttcttggatctctctttcaaccgatttggtg
SLAF18745-S04
cacccttcaacctaatcttgttccacaaggagtaatacatgattcatttgtactatgtatgcttcgaaagaagattttgaaacaacacaaaagagcaccatgtaccagaaaagagcatctaattcctaaacgcaaaagagtggaaggacaaatttccattgatagagttgaaaacgaatgtttcgtcaatacgagacagttttcagaactacttttgaatggatgcgatttggaagaacaatctcaacttctttttcaggaaagcggtcatcaa
In the present invention, in a sweet taste trait related gene functional molecular marker SLAF18745-S01, special primer name is called SLAF_No.1, and sequence is.
5'TGGGACCAGTTCCTCATT 3'
5'AGCTTATCTGGAGGCAAA 3'
In the present invention, in three tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04, special primer title is respectively.
SLAF_No.2 distinguished sequence is,
5'CATAGCTGGTGTCTGGAT 3'
5' GATCTCAAAATCCAACCT 3'
SLAF_No.3 distinguished sequence is,
5'CAAAGCCGCTGGACTACTCAC 3'
5' CACCAAATCGGTTGAAAG 3'
SLAF_No.4, sequence is respectively:
5'ACACCCTTCAACCTAATC 3'
5' TTGATGACCGCTTTCCTG 3'。
Simultaneously, the invention provides the application of muskmelon sweet taste trait related gene functional molecular marker, be about to muskmelon sweet taste trait related gene functional molecular marker SLAF18745-S01 for the application of the sweet taste genotype detection of melon variety or strain, specifically comprise the following steps.
(1) with Muskmelon Inbred Line " elderly person for whom a birthday celebration is being held " and other muskmelons, hybridize and multiply to F
2more than generation.
(2) to the single plant of muskmelon obtaining by step (1), extract genomic dna, in the genomic DNA of Detection and Extraction, whether there is the molecule marker mutually chain with sweet taste proterties correlation function gene M ELO3C011944T1; As pleasantly sweet proterties functional molecular marker SLAF36334-S01 occurs, whether prediction Muskmelon Plants has sweet taste.
Further, the invention provides the application of muskmelon tart flavour trait related gene functional molecular marker, be tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 for the application of the tart flavour genotype detection of melon variety or strain, specifically comprise the following steps.
(1) with Muskmelon Inbred Line " sour-sweet melon " and other muskmelons, hybridize and multiply to F
2more than generation.
(2) to the single plant of muskmelon obtaining by step (1), extract genomic dna, in the genomic DNA of Detection and Extraction, whether have the molecule marker mutually chain with tart flavour proterties correlation function gene M ELO3C020002T1, MELO3C025794T1, MELO3C026251T1; If any tart flavour proterties functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04, occur, whether prediction Muskmelon Plants has tart flavour.
By implementing the concrete summary of the invention of the present invention, can reach following effect.
(1) the present invention utilizes super-BAS technology only to use the time of four months, obtain functional molecular marker and the Fine Mapping region of muskmelon sweet taste, tart flavour proterties, cycle is short, cost is low, efficiency is high, can be development functionality molecule marker in other species important successful case is provided, also for molecule marker in molecular breeding, phyletic evolution, germ plasm resource evaluation provides a kind of important development approach.
(2) the present invention obtains a sweet taste trait related gene functional molecular marker SLAF18745-S01 and three tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 first, for the molecular marker assisted selection of muskmelon quality trait gene provides important mark basis.By application melon fruit sweet taste, tart flavour trait related gene functional molecular marker, the molecule marker of detection and sweet taste, the tart flavour linkage of characters, predict sweet taste, the tart flavour of melon fruit, rapid screening goes out the kind of sweet tart flavour or strain for muskmelon quality breeding.
accompanying drawing explanation
Fig. 1 is shown as sweet taste trait related gene functional molecular marker SLAF18745-S01 to parents, the amplification figure in sweet not sour, sweetless not sour material only.In figure, M is that 500bp Ladder DNA, 1 is that sweet not sour individual plant, 3,4,8 is not only sweetless but also not sour individual plant for " elderly person for whom a birthday celebration is being held ", 2 for " sour-sweet melon ", 5,6,7,9,10.
Fig. 2 is shown as tart flavour trait related gene functional molecular marker SLAF36334-S02 to the amplification figure in parents, only sour sweetless, sweetless not sour material.In figure, M be 500bp Ladder DNA, 1 for " elderly person for whom a birthday celebration is being held ", 2 for " sour-sweet melon ", 4,5,7,8,10 for sour sweetless individual plant, 3,6,9 be only not only sweetless but also not sour individual plant.
Fig. 3 is shown as tart flavour trait related gene functional molecular marker SLAF50072-S03 to the amplification figure in parents, only sour sweetless, sweetless not sour material.In figure, M be 500bp Ladder DNA, 1 for " elderly person for whom a birthday celebration is being held ", 2 for " sour-sweet melon ", 4,7,8,9,10 for sour sweetless individual plant, 3,5,6 be only not only sweetless but also not sour individual plant.
Fig. 4 is shown as tart flavour trait related gene functional molecular marker SLAF31212-S04 to the amplification figure in parents, only sour sweetless, sweetless not sour material.In figure, M be 500bp Ladder DNA, 1 for " elderly person for whom a birthday celebration is being held ", 2 for " sour-sweet melon ", 4,5,7,8,9,10 for sour sweetless individual plant, 3,5 be only not only sweetless but also not sour individual plant.
Fig. 5 is shown as sweet taste trait related gene functional molecular marker SLAF18745-S01 at F
1, F
2the stability diagram of individual plant.In figure, M is 500bp Ladder DNA, and 1,2,3,4 is not only sweetless but also not sour individual plant, and 5,6 is sweet not sour individual plant only, and 7,8 is F
1individual plant, 9 is sour sweetless individual plant only, 10 is F
2in not only acid but also sweet individual plant.
Fig. 6 is shown as tart flavour trait related gene functional molecular marker SLAF36334-S02 at F
1, F
2the stability diagram of individual plant.In figure, M is 500bp Ladder DNA, and 1,2 is not only sweetless but also not sour individual plant, 3,4 sweet not sour individual plants, and 5,6 is sour sweetless individual plant only, 7,8 is F
1individual plant, 9,10 is F
2in not only acid but also sweet individual plant.
Fig. 7 is shown as tart flavour trait related gene functional molecular marker SLAF50072-S03 at F
1, F
2the stability diagram of individual plant.In figure, M is 500bp Ladder DNA, and 1 is F
1individual plant, 2 is sweet not sour individual plant only, 3,4 is F
2in not only acid but also sweet individual plant, 5 is not only sweetless but also not sour individual plant, 6,7,8,9,10 is sour sweetless individual plant.
Fig. 8 is shown as tart flavour trait related gene functional molecular marker SLAF31212-S04 at F
1, F
2the stability diagram of individual plant.In figure, M is 500bp Ladder DNA, and 1 is F
1individual plant, 2 is sweet not sour individual plant, 3,4,5 is F
2in not only acid but also sweet individual plant, 6 is sweet not sour individual plant, 7,8,10 is sour sweetless individual plant, 9 is not only sweetless but also not sour individual plant.
Embodiment
Below, for embodiment, the present invention is described, still, the present invention is not limited to following embodiment.
In the present invention, equipment, reagent and information analysis software have:
PAL-1 sugar degree measuring instrument (Japanese ATATO company), the portable PH meter of STARTER300 (Ohaus Instrument (Shanghai) Co., Ltd.), TC-512 type pcr amplification instrument (Britain Techne company), JY3000 type electrophoresis apparatus (Jun Yi east, Beijing electrophoresis equipment company limited), JY-SPE type electrophoresis chamber (Jun Yi east, Beijing electrophoresis equipment company limited), the digital gel imaging instrument (Jun Yi east, Beijing electrophoresis equipment company limited) of JY04S-3C type.XhoI, MseI endonuclease, enzyme cutting buffering liquid 4 and build storehouse reagent (U.S. New Englang Biolabs company), purification kit (German QIAGEN company).Special primer is developed Taq enzyme used, dNTPs is all purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Primer Primer5.0 primer-design software, the short sequence alignment software of blat, blast comparison software, Cap snp detect software, can obtain by general biological website, biotech company.
Sweet taste trait related gene functional molecular marker SLAF18745-S01 and three tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 special primer utilize Primer Primer5.0 software design.
All reagent of selecting in the present invention, instrument and data message analysis software are all well known selecting, but do not limit enforcement of the present invention.
embodiment mono-: the acquisition of muskmelon sweet taste tart flavour trait related gene functional molecular marker
Muskmelon sweet taste tart flavour trait related gene functional molecular marker obtains and comprises the following steps:
(1) the sugared Muskmelon Inbred Line of height " elderly person for whom a birthday celebration is being held " is hybridized for male parent with Muskmelon Inbred Line " sour-sweet melon " for maternal, obtain hybrid F
1i.e. " No. 4, local flavor "; By hybrid F
1self-pollination obtains F
2for colony, to Parent, F
1and each F
2sweet tart flavour for individual plant fruit identifies in conjunction with trial test by apparatus measures, obtains that only acid is sweetless, sweet not sour, not only not only sweet, neither acid but also sweetless 4 types of acid only.
(2) by CTAB method, extract the genomic dna of every part of material in (1), to 5 strain male parents, 5 strains are maternal and sour sweetless, sweet not sour, the acid genomic dna balanced mix of sweetless each 50 individual plants again neither only only, form 5 mixed ponds, respectively 5 mixed pond genomes being carried out to enzyme cuts, the sequence dna fragment of the same site of each sample in mixed pond, through pcr amplification, order-checking, Blat comparison software carries out cluster, Cap snp software detection single nucleotide polymorphism to order-checking read, obtains the SLAF label of polymorphism.
(3) according to the cognation of polymorphism mark and sweet taste, tart flavour proterties, use ratio correlation method to position.Select the pure and mild mark of a/b>3 in not sour sweetless mixed pond or b/B>3 to carry out association; Calculate respectively colony neither acid between sweetless ac and sweet not sour ab and neither between acid but also sweetless ac and sour sweetless aa, derive from not only the difference ratio of different genotype, obtain difference mark.The difference mark associated with sweet taste, tart flavour proterties compared by blat software, on parents' genome, locate, according to mark physical location, extract the gene at mark place, compare with Swissprot and Kegg database, obtain and sweet taste proterties, annotation of gene function that tart flavour proterties is relevant, pick out the label in the SLAF of functional gene exon.
(4) according to the polymorphic sequence of the label SLAF18745 at sweet taste proterties correlation function gene M ELO3C011944T1 place location and in exon, 3 tart flavour proterties correlation function gene M ELO3C020002T1, MELO3C025794T1, the place label SLAF36334 of MELO3C026251T1, SLAF50072, the polymorphic sequence of SLAF51212, utilize Primer Primer5.0 software design primer, to only sweet not sour, only acid is sweetless, acid sweetless material genomic dna again neither, carry out pcr amplification, develop a sweet taste trait related gene functional molecular marker SLAF18745-S01 and three tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04.
embodiment bis-: the Super-BSA based on simplifying gene order-checking (SLAF-Seq)
(1) by CTAB method, extract 5 strain male parents, 5 strains female parents, F
2colony is sweet not sour, sweetless, acid sweetless each 50 each individual plant genomic dnas of individual plant again neither of acid only only, and balanced mix forms 5 mixed ponds respectively.
(2)
sLAF-seq library construction and order-checking.
The one, simplified design and genomic dna enzyme are cut: utilize enzyme to cut forecasting software and respectively the mixed pond of (1) 5 genomic dna of step is carried out after GC content, tumor-necrosis factor glycoproteins and gene characteristic analysis, utilize XhoI+ MseI endonuclease that the genomic dna of mixing is cut into 450-500bp fragment; The enzyme system of cutting is genomic dna 500ng, the New Englang Biolabs damping fluid 41L of company, and each 0.12L of XhoI, MseI, mends ddH
2o to 50L, mixes after reagent prepares, 37℃15h， QIAGEB company test kit purifying.
The 2nd, by the reparation of 5' end: reaction system is purifying sample DNA 30L, containing the T4 DNA of 10 mM ATP, connect damping fluid 10L, 10 mM dNTP mixed solution 4L, T4 DNA polysaccharase 5L, Klenow enzyme 1 L, T4 PNK5L, mend ddH
2o to 45L, mixes after reagent prepares, 20 ℃ of 30min, and reaction finishes with QIAGEN test kit, to purify afterwards, 33L EB back dissolving.
The 3rd, 3' end is added to A: reaction system is purifying sample DNA 32L in above-mentioned steps, Klenow damping fluid 5L, 1 mM dATP 10L, Klenow Exo-3L, benefit ddH
2o to 50L, 37 ℃ of 30min.
The 4th, connect solexa sequence measuring joints: reaction system is that in above-mentioned steps, purification of samples DNA 10L, 2 * DNA connect damping fluid 25L, solexa sequence measuring joints 10L, DNA ligase 5L, mend ddH
2o to 50L, reaction finishes with QIAGEN test kit, to purify afterwards, 30L EB back dissolving.
The 5th, adopt electrophoresis to cut glue: prepare in advance 2% low melting-point agarose and complete blob of viscose; After adding 6 x Loading buffer 6L to mix upper step purifying after product, add and prepare in blob of viscose, add 10L ladder, 120V 60min.QIAGEN glue reclaims test kit and reclaims object fragment.
The 6th, through PCR, react: increase starting template amount, reach machine order-checking aequum; Reactions steps is step 5) purifying sample DNA 8L, PCR primer PE 2.0, PCR primer PE 1.0 each 1.5L, Phusion DNA polysaccharase 20L, ddH
2o 9L; Response procedures is 98 ℃ of denaturation 30s; 98 ℃ of sex change 40s, 65 ℃ of annealing 30s, 72 ℃ are extended 30s, 10-12 circulation; 72 ℃ are extended 5min; Reaction finishes with QIAGEN test kit, to purify afterwards, 30L EB back dissolving.
The 7th, upper machine order-checking: the sample that front step is handled well carries out accurate quantification (Qubit), then carries out bridge-type PCR on chip (flow cell) surface, makes DNA fragmentation amplification for unique DNA bunch; After unique DNA bunch long getting well, flow cell is moved in Hi-Seq, check order.
(3) SLAF label obtains: genome is cut to process through enzyme and interrupted as a plurality of small segments, each fragment is a marker site, same site reads sequence is used Blat software to carry out similarity cluster, selecting the genotype that the degree of depth is greater than more than 10x is oligogene type, final acquisition 46,087 SLAF label, the ensemble average degree of depth reaches 161.81x.
(4) polymorphism analysis: to the SLAF label obtaining, according to the difference between number of alleles and gene order, first with the comparison of blat software, then find pleomorphism site with call snp software, obtain altogether 4480 polymorphism marks.
embodiment tri-: muskmelon sweet taste, tart flavour trait related gene excavate
(1) by taste of fruit is carried out to Instrument measuring in conjunction with Taste evaluation, maternal for sweet not sour, male parent only be sour sweetless a, F
1for not only acid but also sweet, F
2only sweet not sour 130 strains in colony, only sweetless 77 strains of acid, neither acid sweetless 60 strains again, not only acid but also sweet 233 strains, wherein, sweet: sweetless=2.65:1, acid: not sour=1.63:1.In conjunction with parental trait and F
1proterties and F
2the separated ratio of proterties, sweet and acid is dominant character, sweet taste deflection qualitative character, tart flavour deflection quantitative character, the hybrid model that design parent is AAbbxaaBB.
(2) to sweet taste, tart flavour proterties, use ratio correlation method to position.Select neither acid again in sweetless mixed pond the pure and mild mark of a/b>3 or b/B>3 carry out association.Neither acid is not only sweetless to calculate respectively the ac(of colony) and ab(only sweet not sour) between and neither acid but also sweetless of ac() and aa(only acid is sweetless) between derive from the difference ratio of different genotype, obtain respectively relevant to sweet taste proterties 114, tart flavour proterties 215 the difference marks of being correlated with; Wherein, continuous 3 regions that meet above the mark place of segregation ratio are associated region, obtain 6 sweet taste Candidate regions, in region, have 13 difference marks, in Table 1,23 tart flavour Candidate region, in region, have 48 difference marks, in Table 2.
Table 1: sweet taste proterties candidate region and region interpolation heterolabeling.
Table 2: tart flavour proterties candidate region and region interpolation heterolabeling.
(3) the difference mark associated with sweet taste, tart flavour proterties compared by software blat, according to mark physical location, the gene at mark place is extracted, compare with Swissprot and Kegg database, in 13 sweet taste proterties difference marks, 1 is positioned on parents' genome and is in the exon of functional gene; In 48 tart flavour proterties difference marks, 3 are positioned on parents' genome and are in the exon of functional gene, in Table 3.
Table 3: the sweet taste of location, tart flavour proterties correlation function gene.
| SLAF label | Gene I/D | Position on karyomit(e) | Gene function | Associated proterties |
| SLAF18745 | MELO3C011944T1 | 1899138-1901813 | N-acetylglucosaminyltransferase III | Sugariness shape is associated |
| SLAF36334 | MELO3C020002T1 | 2986302-2987086 | Mus musculus squamous cell carcinoma antigen 2 (Scca2) gene, | Acid shape is associated |
| SLAF50072 | MELO3C025794T1 | 196788-200201 | receptor-like protein kinase-like | Acid shape is associated |
| SLAF51212 | MELO3C026251T1 | 595039-596189 | NAC transcription factor 29-like | Acid shape is associated |
embodiment tetra-: the exploitation of muskmelon sweet taste, tart flavour trait related gene functional molecular marker
The polymorphic sequence of the SLAF label by 1 sweet taste proterties correlation function gene that step (3) in embodiment tri-is obtained and 3 tart flavour proterties correlation function genes, utilize Primer Primer5.0 software respectively to design 1 pair of primer, primer sequence is in Table 4, take male parent, female parent, only sweet not sour, only acid sweetless, neither acid again sweetless individual plant be material, carry out pcr amplification, to develop sweet taste, the functional mark of tart flavour proterties.PCR system 25L, containing 100 ng μ L
– 1template DNA 1 μ L, 10 * buffer, 2.5 μ L, 2.5 mmol L
– 1dNTP 2 μ L, 10 μ mol L
– 1forward and reverse primer each 1 μ L, 5 U μ L
– 1taq DNA enzyme 0.3 L, ddH
2o 17.2 μ L.PCR response procedures is 94 ℃ of denaturation 5 min; 94 ℃ of sex change 50s, 45-60 ℃ of annealing 1 min, 72 ℃ are extended 1.5 min, 35 circulations; 72 ℃ are extended 10min.With 6%PAGE glue silver, dye detection amplified production; Will be only maternal, only sweet not sour and male parent, only in sour sweetless individual plant, have amplified production, fragment is respectively 249bp, 66bp, 283bp, 274bp, in Table 5, and neither in the sweetless individual plant of acid, there is no the mark of amplified production respectively as sweet taste, the functional mark of tart flavour proterties.Result successful development sweet taste proterties related functionality molecule marker SLAF18745-S01 referring to each and every one tart flavour trait related gene functional molecular marker SLAF36334-S02 of accompanying drawing 1, three referring to accompanying drawing 2, SLAF50072-S03 referring to accompanying drawing 3 and SLAF31212-S04 referring to accompanying drawing 4.
embodiment five: sweet taste, the functional mark of tart flavour trait related gene are to F
1
, F
2
individual plant detects
Further to F
1, F
2individual plant material carries out pcr amplification, the sweet taste that checking obtains, the stability of tart flavour trait related gene functional molecular marker.Sweet taste trait related gene functional molecular marker SLAF18745-S01 is at F
1and F
2in only sweet not sour, not only in acid but also sweet material, amplified 249bp band, and F
2in neither acid again sweetless, only do not occur in sour sweetless material, referring to accompanying drawing 5; Tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03, SLAF31212-S04 are just at F
1and F
2in only acid sweetless, not only in acid but also sweet material, amplified 66bp, 283bp, 274bp band, and F
2in neither acid again sweetless, only do not occur in sweet not sour material, referring to accompanying drawing 6, accompanying drawing 7, accompanying drawing 8, illustrate that 4 functional molecular markers are stable.
embodiment six: the application of sweet taste, the functional mark of tart flavour trait related gene
Take the F that self-mating system " elderly person for whom a birthday celebration is being held ", " sour-sweet melon " be parent
2, F
3progeny population after planting, get plant cotyledon or blade and extract genomic dna, with primer SLAF_No.1, SLAF_No.2, SLAF_No.3, SLAF_No.4, DNA is carried out to pcr amplification and electrophoresis detection, amplification and detection method are the same, the plant with sweet tart flavour can amplify respectively 249bp, 66bp, 283bp, 274bp band, and both sweetless not sour plant can not amplify band at same position.From extracting genomic dna to obtaining qualification result, in laboratory, only need to complete 1-2 working days.
In existing muskmelon quality breeding practice, first to collect the parent with different taste of fruit genes, carrying out a series of hybridization backcrosses and sets up colony, after the local flavor proterties such as sweet taste, tart flavour such as are mainly at the fruit maturation, identify one by one, from being seeded into fruit maturation, at least need more than 65 days, cause the seed selection cycle long, efficiency is low, and cost is high.By a sweet taste trait related gene functional molecular marker SLAF18745-S01 and three tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03, SLAF31212-S04, detect the sweet tart flavour of muskmelon, can be in laboratory to seedling stage individual plant carry out the genotype identification of sweet tart flavour, both saved production cost and greatly improved the efficiency of selecting, having accelerated the genetic improvement of muskmelon quality trait.
Show the primer sequence of 4:1 sweet taste, 3 tart flavour functional molecular markers
Show the DNA sequence dna of 5:1 sweet taste, 3 tart flavour functional molecular markers
| Mark title | The DNA sequence dna of functional label (5'-3') | Length (bp) |
| SLAF18745-S01 | tgggaccagttcctcattctttctcagcagttcacttgccatcgtatctgctggaaaatgcagaagactacaaattccttttgcctgggaattgcatacgagagagtggctgatctctaactttggcgtctacctccctgtgaaggttagttgcgatgtagcttgtctgacacctcgtggaccttccatctcgatagttgttgaccaatatgcagtcaaccgtggtggtcttttgcctccagataagct | 249 |
| SLAF36334-S02 | catagctggtgtctggatatctctaatgtatatgttatttatgtgtgaggttggattttgagatca | 66 |
| SLAF50072-S03 | caaagccgctggactactcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggggtcagcttggacctgaaattgcaagtttgactcaattgcgtactattgatttgacaaccaacgatttctctggtgaaattccttatgggattggtaactGtacccatttagagttcttggatctctctttcaaccgatttggtg | 283 |
| SLAF31212-S04 | cacccttcaacctaatcttgttccacaaggagtaatacatgattcatttgtactatgtatgcttcgaaagaagattttgaaacaacacaaaagagcaccatgtaccagaaaagagcatctaattcctaaacgcaaaagagtggaaggacaaatttccattgatagagttgaaaacgaatgtttcgtcaatacgagacagttttcagaactacttttgaatggatgcgatttggaagaacaatctcaacttctttttcaggaaagcggtcatcaa | 274 |
In sum, the present invention utilizes super-BAS technology only to use the time of four months, obtain and muskmelon sweet taste, tart flavour proterties functional molecular marker and Fine Mapping region, cycle is short, cost is low, efficiency is high, can be development functionality molecule marker in other species important successful case is provided, also for molecule marker in molecular breeding, phyletic evolution, germ plasm resource evaluation provides a kind of important development approach.
By the campaign of the various embodiments described above, confirmed that the present invention obtains a sweet taste trait related gene functional molecular marker SLAF18745-S01 and three tart flavour trait related gene functional molecular marker SLAF36334-S02 first, SLAF50072-S03 and SLAF31212-S04, by application melon fruit sweet taste, tart flavour trait related gene functional molecular marker, reproducible between different generations, easy and simple to handle, for the molecular marker assisted selection of muskmelon quality trait gene provides important mark basis, can be easy, be applied to fast breeding practice.
Organization Applicant
Street: No. 403, Nanchang road
City: Urumqi City
State: Xinjiang
Country: China
PostalCode : 830091
PhoneNumber : 0991-4539658
FaxNumber : 0991-4539658
<110> OrganizationName: Xinjiang Agricultural Sciences institute hami melon research centre
Application Project
<120> Title: a kind of muskmelon sweet taste tart flavour trait related gene functional molecular marker and application
<130> AppFileReference : Jordi Garcia-Masa.The genome of melon (Cucumis melo L.).PNAS, 2012, 109(29): 11872-11877.
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
<213> OrganismName : Cucumis melo
<400> PreSequenceString :
tgggaccagt tcctcattct ttctcagcag ttcacttgcc atcgtatctg ctggaaaatg 60
cagaagacta caaattcctt ttgcctggga attgcatacg agagagtggc tgatctctaa 120
ctttggcgtc tacctccctg tgaaggttag ttgcgatgta gcttgtctga cacctcgtgg 180
accttccatc tcgatagttg ttgaccaata tgcagtcaac cgtggtggtc ttttgcctcc 240
agataagct 249
<212> Type : DNA
<211> Length : 249
SequenceName : SLAF18745-S01
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
tgggaccagt tcctcatt 18
<212> Type : DNA
<211> Length : 18
SequenceName : SLAF_No.1 Forward
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
agcttatctg gaggcaaa 18
<212> Type : DNA
<211> Length : 18
SequenceName : SLAF_No.1 Reverse
SequenceDescription :
Sequence
<213> OrganismName : Cucumis melo
<400> PreSequenceString :
catagctggtgtctggatatctctaatgtatatgttatttatgtgtgaggttggattttgagatca
<212> Type : DNA
<211> Length : 66
SequenceName : SLAF36334-S02
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
catagctggt gtctggat 18
<212> Type : DNA
<211> Length : 18
SequenceName : SLAF_No.2 Forward
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
gatctcaaaa tccaacct 18
<212> Type : DNA
<211> Length : 18
SequenceName : SLAF_No.2 Reverse
SequenceDescription :
Sequence
<213> OrganismName : Cucumis melo
<400> PreSequenceString :
caaagccgct ggactactca cacctctttt gtccctgttt ggaatgcctc tcattccact 60
ccctgttctt gggctgggca cacctctttt gtccctgttt ggaatgcctc tcattccact 120
ccctgttctt gggctggggg tcagcttgga cctgaaattg caagtttgac tcaattgcgt 180
actattgatt tgacaaccaa cgatttctct ggtgaaattc cttatgggat tggtaactgt 240
acccatttag agttcttgga tctctctttc aaccgatttg gtg 283
<212> Type : DNA
<211> Length : 283
SequenceName : SLAF50072-S03
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
caaagccgct ggactactca c 21
<212> Type : DNA
<211> Length : 21
SequenceName : SLAF_No.3 Forward
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
caccaaatcg gttgaaag 18
<212> Type : DNA
<211> Length : 18
SequenceName : SLAF_No.3 Reverse
SequenceDescription :
Sequence
<213> OrganismName : Cucumis melo
<400> PreSequenceString :
cacccttcaa cctaatcttg ttccacaagg agtaatacat gattcatttg tactatgtat 60
gcttcgaaag aagattttga aacaacacaa aagagcacca tgtaccagaa aagagcatct 120
aattcctaaa cgcaaaagag tggaaggaca aatttccatt gatagagttg aaaacgaatg 180
tttcgtcaat acgagacagt tttcagaact acttttgaat ggatgcgatt tggaagaaca 240
atctcaactt ctttttcagg aaagcggtca tcaa 274
<212> Type : DNA
<211> Length : 274
SequenceName : SLAF31212-S04
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
acacccttca acctaatc 18
<212> Type : DNA
<211> Length : 18
SequenceName : SLAF_No.4 Forward
SequenceDescription :
Sequence
<213> OrganismName: artificial sequence
<400> PreSequenceString :
ttgatgaccg ctttcctg 18
<212> Type : DNA
<211> Length : 18
SequenceName : SLAF_No.4 Reverse
SequenceDescription :
Claims (6)
1. a preparation method for muskmelon sweet taste tart flavour trait related gene functional molecular marker, is characterized in that, described preparation method comprises the following steps:
(1) the sugared Muskmelon Inbred Line of height " elderly person for whom a birthday celebration is being held " is hybridized for male parent with self-mating system " sour-sweet melon " for maternal, obtain hybrid F
1i.e. " No. 4, local flavor "; By hybrid F
1self-pollination obtains F
2for colony, to each F
2sweet tart flavour for individual plant fruit identifies in conjunction with trial test by apparatus measures, obtains that only acid is sweetless, sweet not sour, not only not only sweet, neither acid but also sweetless 4 kinds of local flavors of acid only;
(2) by CTAB method, extract male parent, female parent, F
1and F
2for each individual plant genomic dna, respectively balanced mix male parent, female parent, sweetless, sweet not sour, acid sweetless each individual plant genomic dna again neither only of acid only, form 5 mixed ponds, respectively 5 mixed pond genomes being carried out to enzyme cuts, the sequence dna fragment of the same site of each each individual plant of mixed pond, through pcr amplification, order-checking, Blat comparison software carries out cluster, Cap snp software detection single nucleotide polymorphism, obtains the SLAF label of polymorphism;
(3) according to the cognation of polymorphism mark and sweet taste, tart flavour proterties, use ratio correlation method to position: calculate respectively colony neither acid between sweetless ac and sweet not sour ab and neither between acid but also sweetless ac and sour sweetless aa, derive from not only the difference ratio of different genotype, obtain difference mark; The difference mark associated with sweet taste, tart flavour proterties compared by blat software, on parents' genome, locate, according to mark physical location, extract the gene at mark place, compare with Swissprot and Kegg database, obtain and sweet taste proterties, annotation of gene function that tart flavour proterties is relevant, pick out the label in the SLAF of functional gene exon;
(4) according to the polymorphic sequence of the label SLAF18745 at sweet taste proterties correlation function gene M ELO3C011944T1 place location and in exon, 3 tart flavour proterties correlation function gene M ELO3C020002T1, MELO3C025794T1, the place label SLAF36334 of MELO3C026251T1, SLAF50072, the polymorphic sequence of SLAF51212, utilize Primer Primer5.0 software design primer, with male parent, maternal, only sweet not sour, only acid is sweetless, neither acid again sweetless material be material, carry out pcr amplification, develop a sweet taste trait related gene functional molecular marker SLAF18745-S01 and three tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04.
2. the preparation method of muskmelon sweet taste tart flavour trait related gene functional molecular marker as claimed in claim 1, is characterized in that, the corresponding DNA sequence dna of functional molecular marker of a described sweet taste, three tart flavour trait related genes is respectively:
SLAF18745-S01
tgggaccagttcctcattctttctcagcagttcacttgccatcgtatctgctggaaaatgcagaagactacaaattccttttgcctgggaattgcatacgagagagtggctgatctctaactttggcgtctacctccctgtgaaggttagttgcgatgtagcttgtctgacacctcgtggaccttccatctcgatagttgttgaccaatatgcagtcaaccgtggtggtcttttgcctccagataagct
SLAF18745-S02
catagctggtgtctggatatctctaatgtatatgttatttatgtgtgaggttggattttgagatca
SLAF18745-S03
caaagccgctggactactcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggcacacctcttttgtccctgtttggaatgcctctcattccactccctgttcttgggctgggggtcagcttggacctgaaattgcaagtttgactcaattgcgtactattgatttgacaaccaacgatttctctggtgaaattccttatgggattggtaactGtacccatttagagttcttggatctctctttcaaccgatttggtg
SLAF18745-S04
Cacccttcaacctaatcttgttccacaaggagtaatacatgattcatttgtactatgtatgcttcgaaagaagattttgaaacaacacaaaagagcaccatgtaccagaaaagagcatctaattcctaaacgcaaaagagtggaaggacaaatttccattgatagagttgaaaacgaatgtttcgtcaatacgagacagttttcagaactacttttgaatggatgcgatttggaagaacaatctcaacttctttttcaggaaagcggtcatcaa。
3. a sweet taste trait related gene functional molecular marker SLAF18745-S01, is characterized in that, sweet taste trait related gene functional molecular marker SLAF18745-S01 special primer name is called SLAF_No.1, and sequence is:
5'TGGGACCAGTTCCTCATT 3'；
5'AGCTTATCTGGAGGCAAA 3'。
4. a tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04, it is characterized in that, tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04 special primer title are respectively SLAF_No.2, SLAF_No.3, SLAF_No.4, and its primer sequence is respectively:
5'CATAGCTGGTGTCTGGAT 3'；
5'GATCTCAAAATCCAACCT 3'；
5'CAAAGCCGCTGGACTACTCAC 3'；
5'CACCAAATCGGTTGAAAG 3'；
5'ACACCCTTCAACCTAATC 3' ；
5'TTGATGACCGCTTTCCTG 3'。
5. the application of a muskmelon sweet taste trait related gene functional molecular marker as claimed in claim 3, it is characterized in that, application by muskmelon sweet taste trait related gene functional molecular marker SLAF18745-S01 for the sweet taste genotype detection of melon variety or strain, specifically comprises the following steps:
(1) with Muskmelon Inbred Line " elderly person for whom a birthday celebration is being held " and other muskmelons, hybridize and multiply to F
2more than generation;
(2) to the single plant of muskmelon obtaining by step (1), extract genomic dna, in the genomic DNA of Detection and Extraction, whether there is the molecule marker mutually chain with sweet taste proterties correlation function gene M ELO3C011944T1; As pleasantly sweet proterties functional molecular marker SLAF36334-S01 occurs, whether prediction Muskmelon Plants has sweet taste.
6. the application of a muskmelon tart flavour trait related gene functional molecular marker as claimed in claim 4, it is characterized in that, tart flavour trait related gene functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04, for the application of the tart flavour genotype detection of melon variety or strain, specifically comprise the following steps:
(1) with Muskmelon Inbred Line " sour-sweet melon " and other muskmelons, hybridize and multiply to F
2more than generation;
(2) to the single plant of muskmelon obtaining by step (1), extract genomic dna, in the genomic DNA of Detection and Extraction, whether have the molecule marker mutually chain with tart flavour proterties correlation function gene M ELO3C020002T1, MELO3C025794T1, MELO3C026251T1; If any tart flavour proterties functional molecular marker SLAF36334-S02, SLAF50072-S03 and SLAF31212-S04, occur, whether prediction Muskmelon Plants has tart flavour.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212898A (en) * | 2014-09-05 | 2014-12-17 | 中国农业科学院麻类研究所 | Method for developing ramie genome SNP marks on large scale and primer developed by method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888400A (en) * | 2012-11-09 | 2013-01-23 | 扬州大学 | Long rachis elytrigia repens 7E chromosome specific molecular marker based on SLAF-seq development and application thereof |
| CN103088120A (en) * | 2012-11-29 | 2013-05-08 | 北京百迈客生物科技有限公司 | Large-scale genetic typing method based on SLAF-seq (Specific-Locus Amplified Fragment Sequencing) technology |
-
2013
- 2013-11-18 CN CN201310575600.6A patent/CN103555717B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888400A (en) * | 2012-11-09 | 2013-01-23 | 扬州大学 | Long rachis elytrigia repens 7E chromosome specific molecular marker based on SLAF-seq development and application thereof |
| CN103088120A (en) * | 2012-11-29 | 2013-05-08 | 北京百迈客生物科技有限公司 | Large-scale genetic typing method based on SLAF-seq (Specific-Locus Amplified Fragment Sequencing) technology |
Non-Patent Citations (3)
| Title |
|---|
| XIAOWEN SUN ET AL.: "SLAF-seq：An Efficient Method of Large-Scale De Novo SNP Discovery and Genotyping Using High-Throughput Sequencing", 《PLOS ONE》, vol. 8, no. 3, 19 March 2013 (2013-03-19), pages 58700 * |
| YANXIN ZHANG ET AL.: "Construction of a high-density genetic map for sesame based on large scale marker development by specific length amplified fragment(SLAF)sequencing", 《BMC PLANT BIOLOGY》, vol. 13, no. 141, 24 September 2013 (2013-09-24), pages 1 - 12 * |
| 张红 等: "甜瓜糖酸性状的遗传研究", 《园艺学报》, vol. 36, no. 7, 31 December 2009 (2009-12-31), pages 989 - 996 * |
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