CN105802961A - Molecular marker and use thereof - Google Patents
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Abstract
The invention discloses a molecular marker and a use thereof. The molecular marker has insertion/deletion length polymorphism of a nucleotide sequence shown in the formula of SEQ ID NO: 1. The molecular marker is closely related to muskmelon pulp color characters and can be effectively used for muskmelon molecular marker-assisted breeding.
Description
Technical field
The present invention relates to molecular marker and application thereof.Specifically, the present invention relates to the molecular marker that Fructus Melo pulp colour is relevant, for detecting primer pair and the test kit of this molecular marker, previous molecular labelling, primer pair, the test kit purposes in Fructus Melo breeding, detects method and the Fructus Melo auxiliary breeding means of Fructus Melo pulp colour character.
Background technology
Fructus Melo (CucumismeloL.) is Cucurbitaceae Cucumis, is a kind of important garden crop, has higher commercial value and economic worth.China's melon wilt history of existing more than 3000 year, is one of country cultivating Fructus Melo the earliest in the world, is also the country that melon wilt area is maximum in the world, yield is the highest at present.The fruit of Fructus Melo is fragrant and sweet good to eat, is that the tradition of China's general eating of the numerous both urban and rural residents eats fruit raw.Along with the raising of expanding economy and living standards of the people, the domestic demand to Fructus Melo is growing.Adding up according to FAO (Food and Agriculture Organization of the United Nation) (FAO), Fructus Melo has been enter into the row of the big important fruit in the world ten.Owing to its color has concurrently, contain again various compositions necessary to human nutrition: carbohydrate, protein, fat, calcium, phosphorus, ferrum, carotene, VC, VB, VB2, Vpp etc., especially VC content exceedes tens times of the animal foodstuffs such as milk simultaneously.The sarcocarp sweet in the mouth of Fructus Melo, cold, sliding, have quench the thirst, effect of heat clearing away and restlessness relieving, diuresis；Its Pedicellus Melo can be used as medicine, name Pedicellus Melo.Fructus Melo is of many uses, and except can also being processed into except directly eating, melon is dry, melon dried meat, melon beans etc..In world wide, its status is even higher than Citrullus vulgaris.
Along with the multiformity of growth in the living standard and fruit variety, domestic consumer is also more and more higher to the requirement of melon and fruit quality.Melon and fruit breeding is more and more diversified, with meeting the market requirement.As Fructus Melo quality has crisp, crisp, soft, soft and succulence, soft and few juice etc., Fructus Melo sarcocarp has the colors such as snow-white, orange red, emerald green, can select according to the hobby of different regions consumer.
In existing Fructus Melo breeding practice, being indirectly genotype is selected by Phenotype mostly to Fructus Melo Main Agronomic Characters, cause breeding cycle long, efficiency is low, governs the process of melon variety improvement.Marker assisted selection, the functional molecular marker especially with objective trait association carries out assisted Selection, it is possible to traditional Phenotypic Selection is changed into direct genotype and selects such that it is able to greatly improve the efficiency of breeding, accelerate genetic improvement.And find and the important closely linked molecular marker of economical character, it is by the basis of molecular marker assisted selection (also known as molecular breeding) and map based cloning.Thus, exploitation has the molecular marker of the important character gene of China's characteristic and carries out assisted selection research, significant.
Color is one of mark primary directly perceived evaluating food quality.Gorgeous color and luster not only gives the enjoyment of U.S., and, pigment itself has again abundant nourishing healthy and immunologic function.During young Fructus Melo, sarcocarp is all green.Along with the growth promoter of fruit, time ripe, green day is decrescence few, so that being wholly absent, manifests salmon pink or white, and only sarcocarp green person still keeps quite green.The composition of pigment content has larger difference with melon variety difference.Sarcocarp salmon pink person's carotenoid content is higher, is generally 8-10 μ g/g fresh weight, and high person reaches 19-23.15 μ g/g fresh weight；The Fructus Melo of pulp white, carotenoid content is few, is generally 0.1-0.3 μ g/g fresh weight, and minority reaches 0.5 μ g/g fresh weight；Sarcocarp green person, carotenoid content is between 0.59-6.88 μ g/g fresh weight.Based on the biological immune function of carotenoid, sarcocarp salmon pink Fructus Melo has caused the most attention of vast breeding and grower.Thus, to find and the Fructus Melo closely linked DNA marker of pulp colour character, the selection-breeding for Fructus Melo pulp colour character improvement and sarcocarp salmon pink Fructus Melo is most important.
But, present stage and the Fructus Melo closely linked molecular marker of pulp colour gene, still have to be excavated.
Summary of the invention
It is contemplated that at least solve one of technical problem of existence in prior art.For this, it is an object of the present invention to propose a kind of relevant to Fructus Melo pulp colour character, it is possible to be effective to the molecular marker of Fructus Melo selection-breeding.
It should be noted that, the present invention is based on the following work of inventor and completes: inventor is with Fructus Melo " gold honey six " for object of study, by building breeding population, colony's individual plant is carried out RAD order-checking, the gene information analysis that gene type etc. are deep, in conjunction with phenotype authentication information, (genome sequencing based on current Fructus Melo completes with genome sequence to utilize biology information technology association important character, inventor have selected the exploitation utilizing Fructus Melo whole genome sequence information guiding SNP marker), quickly located the gene/QTL site controlling pulp colour character, and successfully develop molecular marker closely linked with it, it is thus possible to the molecular mark for Fructus Melo provides theoretical foundation.
According to an aspect of the present invention, the invention provides the molecular marker that a kind of Fructus Melo pulp colour is relevant.According to embodiments of the invention, described molecular marker shows as the insertion/deletion length polymorphism of nucleotide sequence shown in SEQIDNO:1.According to embodiments of the invention, nucleotide sequence shown in SEQIDNO:1 following (114bp):
TCTTTATCAGGGGTGGAAATTGCGGAGAGATTCGCCTTATTTGGAACTTCCTCCAA TTTGATTAACTTCTTAACGGACCAGCTCCAGCAGTCGACTGCCATGGCAGCCAAGA AT (SEQIDNO:1).Wherein, the molecular marker of the present invention is 3.574cM with the genetic linkage distance of Fructus Melo pulp colour gene.
According to embodiments of the invention, there is nucleotide sequence shown in SEQIDNO:1 and the individuality isozygotied in described molecular marker site, show as sarcocarp salmon pink；Nucleotide sequence shown in disappearance SEQIDNO:1 and the individuality isozygotied in described molecular marker site, show as pulp white；There is nucleotide sequence shown in SEQIDNO:1 and the individuality in described molecular marker site heterozygosis, show as sarcocarp salmon pink.
Inventor have found that, by whether the genomic DNA (its source is unrestricted, for instance can extract from the seed of Fructus Melo, plant etc.) of detection Fructus Melo has above-mentioned molecular marker, it is possible to effectively determine its pulp colour character.Specifically, as previously mentioned, the melon fruit without described molecular marker is pulp white, the melon fruit with this molecular marker is sarcocarp salmon pink, thus, detect when Fructus Melo does not have this molecular marker, then can determine Fructus Melo pulp white to be measured, and when Fructus Melo has this molecular marker, then can determine Fructus Melo sarcocarp salmon pink to be measured.Further, when adopting the specific primer for this molecular marker that Fructus Melo genomic DNA sample to be measured is carried out pcr amplification and detected through gel electrophoresis, by the bar number of purpose band and length, can effectively determine the pulp colour character of Fructus Melo to be measured, specifically, when purpose band is one, and when described purpose band is about 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured；When purpose band is one, and when described purpose band is about 896bp, it may be determined that Fructus Melo pulp white to be measured；When purpose band is two, and during the length of described purpose band respectively about 896bp and 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured.Thus, inventors determined that, the pulp colour character of the molecular marker of the present invention and Fructus Melo is closely related, it is possible to be effective to the molecular mark of Fructus Melo.And then can according to requirement to pulp colour in actual breeding, Fructus Melo breeding material is carried out Seedling selection, it is effectively improved efficiency and the accuracy of breeding further, improves the genetic level of Fructus Melo reproductive population such that it is able to select Fructus Melo improved seeds accurately and efficiently.Such as, Fructus Melo genomic DNA to be measured is expanded by the specific primer utilizing this molecular marker, gel electrophoresis, amplified production purpose band is selected to be one and be about the sample to be tested of 1010bp, can easily screen the Fructus Melo obtaining sarcocarp salmon pink, such that it is able to directly apply to molecular breeding practice.
Additionally, according to some embodiments of the present invention, utilize the molecular marker of the present invention to carry out Fructus Melo molecular mark, there is early screening, saving time, the advantage that with low cost, accuracy is high.
According to a further aspect in the invention, present invention also offers the primer pair of a kind of molecular marker for detecting the foregoing present invention.According to embodiments of the invention, described primer pair has the nucleotide sequence shown in SEQIDNO:2-3.Specifically, the sequence of the primer pair of the present invention is as follows:
5 '-GACTAAGTGCTGTTGAGTGT-3 ' (SEQIDNO:2)；
5 '-TGAACCTGTACGTCCTAGTT-3 ' (SEQIDNO:3).
According to embodiments of the invention, utilize described primer pair that Fructus Melo genomic DNA to be measured carries out pcr amplification, and detect the length of amplified fragments, as the long 1010bp of described amplified fragments, described Fructus Melo to be measured has the insertion of nucleotide sequence shown in SEQIDNO:1, shows as sarcocarp salmon pink；As the long 896bp of described amplified fragments, nucleotide sequence shown in described Fructus Melo to be measured disappearance SEQIDNO:1, show as pulp white；When described amplified fragments is 896bp and 1010bp two kinds, the described molecular marker site heterozygosis of described Fructus Melo to be measured, show as sarcocarp salmon pink.
According to embodiments of the invention, utilize the preferred agarose gel electrophoresis of gel electrophoresis, detect the length of described amplified fragments.
It is surprisingly found by the inventors that, the fragment at the above-mentioned of the Fructus Melo to be measured molecular marker place relevant to pulp colour character can be carried out pcr amplification by primer pair effectively that utilize the present invention, and then can effectively realize the detection to this molecular marker by order-checking or electrophoresis, it is determined that whether Fructus Melo to be measured has this molecular marker.Such as, during by electrophoresis detection, according to the quantity of amplified production purpose band and length, namely can effectively determine the pulp colour character of Fructus Melo to be measured.Specifically, utilize above-mentioned primer pair, when Muskmelon Plants to be measured is carried out pcr amplification and detected through gel electrophoresis, when purpose band is one, and when described purpose band is about 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured；When purpose band is one, and when described purpose band is about 896bp, it may be determined that Fructus Melo pulp white to be measured；When purpose band is two, and during the length of described purpose band respectively about 896bp and 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured.And the amplified production of 896bp sequence of disappearance compared with the amplified production of 1010bp is the molecular marker of the present invention, thus the molecule labelled series of the present invention is arranged in the amplified production of 1010bp.Thus, for detecting the primer pair of the molecular marker of the foregoing present invention, it is possible to be effective to the molecular mark of Fructus Melo, and then early stage can be assisted to realize short time, low cost, high accuracy ground selection-breeding Fructus Melo improved seeds.
It should be noted that, those skilled in the art know, in sequence shown in above-mentioned SEQIDNO:2 and SEQIDNO:3,1～10 base can be increased respectively at its 5 ' end or 3 ' ends, the base type increased can be determined according to the base type in the region that matches with SEQIDNO:2 and SEQIDNO:3 on Fructus Melo genomic DNA foundation basepairing rule, the primer pair thus obtained essentially identical with the amplified production of SEQIDNO:2 and SEQIDNO:3 (DNA sequence between upstream and downstream primer is identical).Therefore, 5 ' ends or 3 ' ends at SEQIDNO:2 and SEQIDNO:3 increase by 1～10 base respectively and can expand the primer pair obtaining essentially identical DNA fragmentation, are included in the primer pair of the present invention.
According to another aspect of the invention, present invention also offers a kind of test kit for detecting foregoing molecular marker.According to embodiments of the invention, this test kit comprises: be described previously for the primer pair of the molecular marker of the detection present invention.Namely the test kit of the present invention comprises the primer pair with the nucleotide sequence shown in SEQIDNO:2-3.According to embodiments of the invention, utilize the primer pair comprised in the test kit of the present invention, can effectively realize the detection of the molecular marker that the above-mentioned of Fructus Melo to be measured is relevant to pulp colour character, determine whether Fructus Melo to be measured has this molecular marker, and then can effectively determine the pulp colour character of Fructus Melo to be measured.Specifically, for instance when utilizing above-mentioned primer pair that Fructus Melo genomic DNA to be measured is carried out pcr amplification and detected through gel electrophoresis, when purpose band is one, and when described purpose band is about 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured；When purpose band is one, and when described purpose band is about 896bp, it may be determined that Fructus Melo pulp white to be measured；When purpose band is two, and during the length of described purpose band respectively about 896bp and 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured.Thus, the test kit of the molecular marker for detecting the foregoing present invention of the present invention, it is possible to be effective to the molecular mark of Fructus Melo, and then early stage can be assisted to realize short time, low cost, high accuracy ground selection-breeding Fructus Melo improved seeds.
In accordance with a further aspect of the present invention, present invention also offers the molecular marker of the foregoing present invention, primer pair or test kit, the purposes in Fructus Melo selection-breeding.As previously mentioned, by can be used in the reagent of the molecular marker relevant to Fructus Melo pulp colour character of the detection present invention, such as aforesaid primer pair or comprise the test kit etc. of this primer pair, it is possible to effectively determine whether Fructus Melo genomic DNA to be measured has above-mentioned molecular marker.Such as when adopting the specific primer for this molecular marker that Fructus Melo genomic DNA to be measured is carried out pcr amplification and detected through gel electrophoresis, quantity according to amplified production purpose band and length, can effectively determine the pulp colour character of Fructus Melo to be measured such that it is able to effectively auxiliary Fructus Melo selection-breeding.
And then, according to a further aspect in the invention, present invention also offers a kind of method detecting Fructus Melo pulp colour character.According to embodiments of the invention, Fructus Melo to be measured is carried out the detection of foregoing molecular marker by the method, in order to determine the pulp colour character of Fructus Melo to be measured.Specifically, can pass through can be used in the reagent of the molecular marker relevant to Fructus Melo pulp colour character of the detection present invention, such as aforesaid primer pair or comprise the test kit etc. of this primer pair, the genomic DNA of Fructus Melo to be measured is carried out pcr amplification, gel electrophoresis, thus according to the quantity of amplified production purpose band and length, it is possible to effectively determine the pulp colour character of Fructus Melo to be measured.Wherein, as it was previously stated, utilize above-mentioned primer pair or test kit, when Muskmelon Plants to be measured is carried out pcr amplification and detected through gel electrophoresis, specifically, when purpose band is one, and when described purpose band is about 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured；When purpose band is one, and when described purpose band is about 896bp, it may be determined that Fructus Melo pulp white to be measured；When purpose band is two, and during the length of described purpose band respectively about 896bp and 1010bp, it may be determined that Fructus Melo sarcocarp salmon pink to be measured.Thus, the method utilizing the detection Fructus Melo pulp colour character of the present invention, Fructus Melo pulp colour character can be detected quickly, efficiently and accurately, and then the molecular mark of Fructus Melo can be effective to such that it is able to auxiliary early stage realizes short time, low cost, high accuracy ground selection-breeding Fructus Melo improved seeds.
It addition, the method for detection Fructus Melo pulp colour character according to the above embodiment of the present invention can also have following additional technical characteristic:
According to embodiments of the invention, the method for the detection Fructus Melo pulp colour character of the present invention farther includes: utilize foregoing primer pair or test kit, described Fructus Melo genomic DNA to be measured is carried out pcr amplification；The length of detection amplified fragments；And the length based on described amplified fragments, it is determined that the pulp colour character of described Fructus Melo to be measured, wherein, as the long 1010bp of described amplified fragments, described Fructus Melo to be measured is sarcocarp salmon pink；As the long 896bp of described amplified fragments, described Fructus Melo to be measured is pulp white；When described amplified fragments is 896bp and 1010bp two kinds, the described molecular marker site heterozygosis of described Fructus Melo to be measured, sarcocarp salmon pink.Thereby, it is possible to whether the detection Fructus Melo to be measured of efficiently and accurately has the molecular marker of the present invention, and then effectively can determine the pulp colour character of Fructus Melo to be measured based on testing result.
According to embodiments of the invention, extract the method obtaining Fructus Melo genomic DNA to be measured and be not particularly limited, it is possible to adopt any of genome DNA extracting method or test kit to carry out.Some concrete examples according to the present invention, adopt the genomic DNA of cetyl trimethylammonium bromide method (CTAB method) extracting Fructus Melo to be measured.Thereby, it is possible to effectively obtain the genomic DNA that quality is good, purity is high, it is simple to subsequent step carries out.
According to embodiments of the invention, utilize the preferred agarose gel electrophoresis of gel electrophoresis, detect the length of described amplified fragments.Thus, convenient and swift.
Inventor have found that, the method for the detection Fructus Melo pulp colour of the present invention can be effective to the molecular mark of Fructus Melo such that it is able to auxiliary early stage realizes short time, low cost, high accuracy ground selection-breeding Fructus Melo improved seeds.
According to another aspect of the invention, present invention also offers a kind of Fructus Melo auxiliary breeding means.According to embodiments of the invention, the method includes the method by foregoing detection Fructus Melo pulp colour character, detects described molecular marker, in order to determine the pulp colour character of described Fructus Melo to be measured.Thus, the Fructus Melo auxiliary breeding means of the present invention is utilized, it is possible to effectively determine the pulp colour character of Fructus Melo, and then be capable of short time, low cost, high accuracy ground selection-breeding Fructus Melo improved seeds.
It should be noted that the molecular marker relevant to Fructus Melo pulp colour of the present invention and application thereof have the advantage that
(1) molecular marker that Fructus Melo pulp colour character provided by the invention is relevant is not by the restriction of the growth stage of Fructus Melo, can be used for the early stage selection-breeding of Fructus Melo, can remarkably promote the breeding process of Fructus Melo；
(2) method of the detection Fructus Melo such as molecular marker that the Fructus Melo pulp colour character shown in SEQIDNO:1 is correlated with, accurately and reliably, easy to operate；
(3) detection of the Fructus Melo such as molecular marker that the Fructus Melo pulp colour character shown in SEQIDNO:1 is correlated with, the marker assisted selection for Fructus Melo growth traits provides scientific basis.
The additional aspect of the present invention and advantage will part provide in the following description, and part will become apparent from the description below, or is recognized by the practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage are from conjunction with will be apparent from easy to understand the accompanying drawings below description to embodiment, wherein:
Fig. 1 shows that the DNA of primer pair parent and first familiar generation that utilization has nucleotide sequence shown in SEQIDNO:2-3 carries out the electrophoresis detection result figure of the amplified production of pcr amplification according to one embodiment of the invention；
Fig. 2 shows that the DNA utilizing the generation of the primer pair part F2 with nucleotide sequence shown in SEQIDNO:2-3 individual carries out the electrophoresis detection result figure of the amplified production of pcr amplification according to one embodiment of the invention.
Detailed description of the invention
Embodiments of the invention are described below in detail.The embodiments described below is illustrative of, and is only used for explaining the present invention, and is not considered as limiting the invention.Unreceipted concrete technology or condition in embodiment, technology or condition described by the document in this area are (such as outstanding with reference to J. Pehanorm Brooker etc., " the Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can pass through city available from conventional products, for instance can purchase from Illumina company.
Embodiment 1F2 builds for segregating population and character investigation
(1) material
The male parent " K3-92-1 " of the Fructus Melo " gold honey No. six " provided with Baofeng, Xinjiang Zhong Ye company limited and maternal " K1-7 " are for material, and structure F2 is for segregating population.
Male parent " K3-92-1 " is selfing line, the long 21.6cm of average leaf, mean blade width 29.3cm, pachydermia netted melon, pale yellow skin, fine and closely woven reticulate pattern, without covering stricture of vagina, sarcocarp salmon pink, the long base of a fruit, average single melon quality 2.4kg, fruit shape index 1.55, edge soluble solid content 7.4%, center soluble solid content 11.9%.
Excellent single melon system that maternal " K1-7 " is Kazakhstan one farm variety " Caput Caprae seu ovis " selfed breeding, the long 24.1cm of average leaf, mean blade width 34.5cm, thick-skinned melon, melon is relatively big, and fruit stem end is yellow green skin, areola end is golden yellow skin, and thin rare reticulate pattern, without covering stricture of vagina, pulp white, the short base of a fruit, average single melon quality 3.0kg, fruit shape index 1.52, edge soluble solid content 5.1%, center soluble solid content 7.5%.
(2) informative population
With K1-7 (P1) for maternal, carry out hybridizing (for salmon pink, maternal pulp colour is white to male parent pulp colour) with K3-92-1 (P2) for male parent, it is thus achieved that first-filial generation (F1), F2 colony, totally 500 individual plants is obtained after strict self-pollination.
(3) character investigation
In the fruit maturation phase, according to " Germplasm Resources of Cucumis Melo L Description standard and data standard " (Ma Shuanwu etc., 2006) description, the pulp colour of each individual plant of investigation P1, P2 and F2 colony, according to phenotypic evaluation result, calculate segregation ratio, use MicrosoftExcel2003 software to carry out data statistic analysis, use SPSS17.0 that result is carried out Chi-square statistic.
Character survey result: have 114 strain pulp colours in F2 colony for white, 325 strain pulp colours are salmon pink (wouldn't distinguish shade, in Table 1), and Chi-square statistic shows, the segregation ratio of pulp colour is about 3:1, meets the law of segregation of Mendelian one pair of genes.Therefore, Fructus Melo pulp colour gene is the qualitative trait of Dominant gene, and wherein pulp colour salmon pink shows as dominant character.
Table 1 Fructus Melo F2Colony's pulp colour trait segregation ratio
χ2 0.05,1=3.84
Embodiment 2:SNP labeled analysis and genetic map are drawn
1, SNP marker analysis and genetic map build
1.1SNP labeled analysis
Parent " K1-7 " (P1) in Example 1, the tender leaf of each individual plant of " K3-92-1 " (P2) and F2 colony, extract genomic DNA by CTAB (cetyl trimethylammonium bromide) method of improvement, specific as follows:
Extract the genomic DNA of Parent, F1 generation and F2 generation individuality respectively by CTAB method, concrete grammar is as follows:
(1) weigh the fresh blade of 1.0g, shred and put into mortar, with adding 3mL1.5 × CTAB after liquid nitrogen grinding, grind to form in the centrifuge tube that homogenate proceeds to 15mL, in mortar, then add 1mL1.5 × CTAB flushing proceed to again in centrifuge tube.In 65 DEG C of water-bath 30min after mixing, period slowly shakes up frequently.
Wherein 1.5 × CTAB formula following (1L):
| CTAB | 15g |
| The Tris.Cl (pH is 8.0) of 1mol/L | 75mL |
| The EDTA of 0.5mol/L | 30mL |
| NaCl | 61.4g |
Add deionized water and be settled to 1L, before using, add the mercaptoethanol of final concentration of 0.2% (2ml).
(2) it is cooled to room temperature, adds equal-volume chloroform/isoamyl alcohol (24:1), mix gently, become bottle green to subnatant.
(3) the centrifugal 10min of 4200rpm, moves on to upper water new 15mL centrifuge tube mutually, adds the dehydrated alcohol of 2 times of volume pre-coolings, mix static 5min.Place 30min in-20 DEG C and precipitate DNA.
(4) the centrifugal 10min of 4200rpm, discards supernatant, adds 1mL75% washing with alcohol and precipitates 1 time, is inverted centrifuge tube dry DNA, adds 200 μ LTE dissolving DNAs.
(5) genomic DNA is detected with the agarose gel of 0.8%.
(6) the genomic DNA individual Parent obtained and F1 generation, F2 generation is stored in-20 DEG C standby.
Then, take 1 μ g genomic DNA, with restricted enzyme, it is interrupted at random, electrophoresis reclaims the DNA fragmentation of 400-600bp, what be then based on Hiseq2000 high-flux sequence platform builds storehouse strategy, building the genomic library of each sample, after then being mixed by various kinds Ben Wenku, (each sample carries the label mutually do not allowed) carries out sequencing analysis by Agilent2100 biological analyser and Hiseq2000 high-flux sequence platform.
nullAccording to identifying that sequence label obtains the order-checking reads of each individuality，Use the SOAP2.20 software (can be referring to: LiR.etal.，2009a，SOAP2:animprovedultrafasttoolforshortreadalignment.Bioinformatics，By referring to being incorporated by herein)，(can be referring to: Garcia-Masetal. for reference genome with Fructus Melo DHL92,2012，Thegenomeofmelon(CucumismeloL.).，By referring to being incorporated by herein)，Carry out alignment，Result SAMtools0.1.8、RealSFS0.983 software (can be referring to: Lietal.，2009b，SNPdetectionformassivelyparallelwhole-genomeresequencing.，By referring to being incorporated by herein) conversion、Analyze，Identify each site，Then result is filtered，Filter criteria is 50≤degree of depth≤2500，Site mutation probability >=95%，Obtain the SNP collection of high credibility.Integrate as reference with SNP, be filtrated to get the genotype of parent and colony.
The structure of 1.2 genetic maps
Substantial amounts of SNP causes being difficult to each labelling is built Genetic linkage map, simpler effectively according to the continuous SNP Bin combined gene constructed Bin collection of illustrative plates energy.
Genotype according to parent Yu colony, selects with every 15 SNP for a window, and slide a SNP every time, it is determined that the genotype of each window and the exchange site of each individuality, obtains the Bin genotype of each individuality.Ready Bin genotype data, use MSTMap software, graphing method selects Regressionmapping algorithm, mapping function selects Kosambi ' s function to build genetic map (can be referring to: Wuetal., 2008, Efficientandaccurateconstructionofgeneticlinkagemapsfrom theminimumspanningtreeofagraph., by referring to being incorporated by herein).Chromosome sequence number name chromosome is added, as chr01 represents No. 1 chromosome with abbreviation chr.
Wherein, the result that sequencing result is analyzed comparison is as shown in table 2, remove an active non-anchor sequence, originally and 44722 SNP marker being detected altogether in F2 colony parents, average each SNP length is 8.21Kb, and the SNP quantity on every chromosome is different, minimum with the SNP on o.11 chromosome, being 1894, the SNP on No. 6 chromosomes is maximum, reaches 5865；SNP distribution on each chromosome is from 1SNP/4.7Kb (No. 3 chromosomes) to 1SNP/4.714.5Kb (No. 11 chromosomes).After combined, raw 2277 the Bin fragments of common property in F2 colony, fragment length from 20Kb to 4.83Mb not etc., average length is 138.95kb, construct the Bin genetic map that total genetic distance is 1296.174cM, No. 10 chromosome maps are from the shortest (77.997cM), and No. 1 chromosome map is from the longest (143.278cM).In the genotype that whole F2 colony is whole, have 25.36% from male parent, 24.67% from female parent, 49.96% is heterozygous, and 3 kinds of genotype ratio in total genotype segregation ratio close to 1:2:1 is described.
The distribution of SNP, Bin on table 2 genetic map
| Chromosome | SNP | Bin | The genetic distance (cM) |
| chr01 | 3319 | 244 | 143.278 |
| chr02 | 3743 | 169 | 109.473 |
| chr03 | 5675 | 275 | 114.580 |
| chr04 | 3056 | 191 | 134.128 |
| chr05 | 4150 | 204 | 111.275 |
| chr06 | 5865 | 253 | 125.153 |
| chr07 | 4623 | 179 | 100.867 |
| chr08 | 2984 | 160 | 101.795 |
| chr09 | 3554 | 186 | 101.119 |
| chr10 | 2764 | 108 | 77.997 |
| chr11 | 1894 | 127 | 86.461 |
| chr12 | 3095 | 181 | 90.048 |
| Total | 44722 | 2277 | 1296.174 |
3, gene mapping
Application winQTLCart2.5 software, adopts CIM (composite interval mapping method) to carry out gene mapping.Specifically: the individual phenotype according to F2 colony, similar to male parent type character is designated as a, and similar to maternal type character is designated as b, and character occupy and is designated as h between male parent and female parent.Obtain all individual phenotypic data, individual phenotypic data is compared with the genotype data obtained before, pulp colour character is carried out gene mapping.Result shows, pulp colour gene mapping is in No. 9 chromosome 20433942bp～20573889bp intervals, and length is about 139kb, and additive effect is-0.2402, and phenotypic variation explanation rate is 0.0479.
4, molecular markers development
Parent all carries out full-length genome resurvey sequence (10X), according to genome sequencing result, utilize the SOAP software (can referring to application link: http://soap.genomics.org.cn/, by referring to being incorporated by herein) comparison order-checking reads, then find, with SOAPsv, the molecular marker that Fragment Differential is bigger, it is simple to distinguish with gel electrophoresis and differentiate.
As a result, select the labelling M092078 (nucleotide sequence shown in SEQIDNO:1) of close pulp colour gene place section as candidate.
The nucleotide sequence of molecular marker M092078 (114bp) is as follows:
TCTTTATCAGGGGTGGAAATTGCGGAGAGATTCGCCTTATTTGGAACTTCCTCCAA TTTGATTAACTTCTTAACGGACCAGCTCCAGCAGTCGACTGCCATGGCAGCCAAGA AT (SEQIDNO:1).
Embodiment 3: the pulp colour relevance verification of molecular marker
Fructus Melo pulp colour related molecular marker M092078 (nucleotide sequence shown in SEQIDNO:1) determined in embodiment 2 is verified, specific as follows:
Designing primer for above-mentioned molecular marker, primer sequence is as follows:
Forward primer: 5 '-GACTAAGTGCTGTTGAGTGT-3 ' (SEQIDNO:2)；
Reverse primer: 5 '-TGAACCTGTACGTCCTAGTT-3 ' (SEQIDNO:3).
Utilize above-mentioned primer, detected the polymorphism verifying this labelling and amplification stability by pcr amplification and agarose gel electrophoresis.
Specifically, the male parent, female parent, F1 generation or the genomic DNA in F2 generation that extract in embodiment 2, for template, utilize above-mentioned amplimer to carry out pcr amplification, wherein,
PCR reaction system is as follows:
| Sterilized water | 20.2μl |
| 10*Buffer is (containing Mg2+) | 2.5μl |
| dNTPs(25mM) | 0.15μl |
| Taq enzyme (5U/ μ l) | 0.15μl |
| Forward primer | 0.5μl |
| Reverse primer | 0.5μl |
| Template | 1.0μl |
| Cumulative volume | 25μl |
PCR response procedures is as follows:
94 DEG C of denaturations 5 minutes；94 DEG C of degeneration 30 seconds, anneal 30 seconds for 60 DEG C, and 72 DEG C extend 40 seconds, run 35 circulations；Last 72 DEG C extend 3 minutes.Pcr amplification product can 4 DEG C of preservations.
Then, each pcr amplification product taking part and carries out agarose gel electrophoresis detection, result is shown in Fig. 1 and Fig. 2.As it is shown in figure 1, the amplified fragments that maternal amplified production is 896bp size, male parent amplified production is the amplified fragments of 1010bp size, and first familiar generation then contains the two amplified fragments simultaneously.Fig. 2 shows the electrophoresis detection result of the amplified production of part F2 generation individuality, the individual plant of pulp white all amplifies the band of 896bp, the individual plant of sarcocarp salmon pink all amplifies the band of 1010bp, and wherein the individual plant of part sarcocarp salmon pink is heterozygosis, amplifies above-mentioned two band.Thus, it was demonstrated that this molecular marker M092078 (nucleotide sequence shown in SEQIDNO:1) has polymorphism between Parent, this molecular marker and Fructus Melo pulp colour character are closely related.
Then, utilize 3730 sequenators that each amplified production is checked order, result, the amplified band of pulp white individual plant in maternal and F2 generation, having lacked one section of sequence with male parent and F2 compared with the amplified band of sarcocarp salmon pink individual plant, this sequence is aforesaid molecular marker M092078 (nucleotide sequence shown in SEQIDNO:1).
Wherein, male parent amplified fragments sequence (1010bp) is as follows:
nullTGTGATTCTGATTTGCAGCCTTACTAATTAAAAAGTGTTCAACAAAATGGAGGCTCCTCTTTTGGATGAGACGGTGGAGGGGGCAGTTGATTACAAGGGCCGCCCAGTCTGCAGATTCAATTCCGGCGGGTGGAGATCCGCCTCCCTCATCATTGGTAAATCATCAAATCATCATTACTCTCTTCTCTTTCTTCTCTTTCGCTATCCAATTTTTGTACCCTCTTTATCAGGGGTGGAAATTGCGGAGAGATTCGCCTTATTTGGAACTTCCTCCAATTTGATTAACTTCTTAACGGACCAGCTCCAGCAGTCGACTGCCATGGCAGCCAAGAATGTCAATGCCTGGTCTGGTACCGCGGCCTTGCTACCTTTACTCGGCGCCTTTCTCGCTGATTGCTTCCTTGGACAATACCGTACCATTGTTCTTTTCACTGCTCTTTATGTGCTGGTTCTTAAACCACTTTATGCTTTTACACTTTTATTGCTTTTCTTTTCCAAATCCGAACTATATAAAATCTCACAAGACTATGCAAGTTATTAGCTCGGGATCTGGAAATTTCAAATAGTTTTCTCTTCATAAATTGAAACTTTCAAAATATTCACTGTCACTTTTTCCTTTTGTTGTTTCTGGAATTTAGGGCCTTGGGTTGTTGACCTTGTCTGCAGCATTACCTTCTCTCGGCATTTCTGCCTGCCAACAGACTGAAAAATTCCTGCCATGTTCCCCCAATCTAGTCCAAGTAATTTTGTTTTTCTTCTCCTTATATCTGGTGGCATTTGCTAAAGGGGGACATGAGCCTTGCATCCAAGCTTTCGGAGCCGACCAATTTGATGAACAGCATCCAGAAGAGAGAAAAGCTAAAAGCTCTTTCTTCAATTGGTGGTACTTGGGTATCTCTTTGGGAACCCTTTCACTATTATATCATGAGCTATGTGCAGATACTCAGTGAGTCTGGTTTGGATCTGTATGCATGCTTTGACTGTAGTATCTGCTGACAGACAGGGCAAAA ( SEQIDNO：4 )，
Maternal amplified fragments sequence (896bp) is as follows:
nullTGTGATTCTGATTTGCAGCCTTACTAATTAAAAAGTGTTCAACAAAATGGAGGCTCCTCTTTTGGATGAGACGGTGGAGGGGGCAGTTGATTACAAGGGCCGCCCAGTCTGCAGATTCAATTCCGGCGGGTGGAGATCCGCCTCCCTCATCATTGGTAAATCATCAAATCATCATTACTCTCTTCTCTTTCTTCTCTTTCGCTATCCAATTTTTGTACCCGTCTTTATGTGCTGGTTCTTAAACCACTTTATGCTTTTACACTTTTATTGCTTTTCTTTTCCAAATCCGACGACTATATAAAATCTCACAAGACTATGCAAGTTATTAGCTCGGGATCTGGTAATTCAGGATTATTTTGATTCCTGTTCTTTCTCGCAAGTACTTAATAGCTGGAAACAACTGGAAATTTCAAATAGTTTTCTCTTCATAAATTGAAACTTTCAAAATATTCACTGTCACTTTTTCCTTTTGTTGTTTCTGGAATTTAGGGCCTTGGGTTGTTGACCTTGTCTGCAGCATTACCTTCTCTCGGCATTTCTGCCTGCCAACAGACTGAAAAATTCCTGCCATGTTCCCCCAATCTAGTCCAAGTAATTTTGTTTTTCTTCTCCTTATATCTGGTGGCATTTGCTAAAGGGGGACATGAGCCTTGCATCCAAGCTCTCGGAGCCGACCAATTTGATGAACAGCATCCAGAAGAGAGAAAAGCTAAAAGCTCTTCTTCAATTGGTGGTACTTGGGTATCTCTTTGGGAACCCTTTCAACTATTAATATCATGAGCTATGTGCAAGATAACCTCAGTTGGAGTCTTGGGTTTGGAATTCCTTGTATTGCAATGGCTTTTGGACTTGTAGTATTCTTGCTGGACAATACAAAAAGAGTGATGATCATCCAA ( SEQIDNO：5 ).
In the description of this specification, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means in conjunction with this embodiment or example describe are contained at least one embodiment or the example of the present invention.In this manual, the schematic representation of above-mentioned term is not necessarily referring to identical embodiment or example.And, the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiments or example.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: these embodiments can being carried out multiple change, amendment, replacement and modification when without departing from principles of the invention and objective, the scope of the present invention is limited by claim and equivalent thereof.
Claims (10)
1. the molecular marker that a Fructus Melo pulp colour character is relevant, it is characterised in that described molecular marker shows as the insertion/deletion length polymorphism of nucleotide sequence shown in SEQIDNO:1.
2. molecular marker according to claim 1, it is characterised in that there is nucleotide sequence shown in SEQIDNO:1 and the individuality isozygotied in described molecular marker site, show as sarcocarp salmon pink；Nucleotide sequence shown in disappearance SEQIDNO:1 and the individuality isozygotied in described molecular marker site, show as pulp white；There is nucleotide sequence shown in SEQIDNO:1 and the individuality in described molecular marker site heterozygosis, show as sarcocarp salmon pink.
3. the primer pair being used for test right requirement molecular marker described in 1 or 2, it is characterised in that described primer pair has the nucleotide sequence shown in SEQIDNO:2-3.
4. primer pair according to claim 3, it is characterised in that
Utilizing described primer pair that Fructus Melo genomic DNA to be measured carries out pcr amplification, and detect the length of amplified fragments, as the long 1010bp of described amplified fragments, described Fructus Melo to be measured has the insertion of nucleotide sequence shown in SEQIDNO:1, shows as sarcocarp salmon pink；When described amplified fragments is long, nucleotide sequence shown in described Fructus Melo to be measured disappearance SEQIDNO:1, show as pulp white；When described amplified fragments is 896bp and 1010bp two kinds, the described molecular marker site heterozygosis of described Fructus Melo to be measured, show as sarcocarp salmon pink,
Optionally, utilize the preferred agarose gel electrophoresis of gel electrophoresis, detect the length of described amplified fragments.
5. the test kit being used for test right requirement molecular marker described in 1 or 2, it is characterised in that comprise:
Primer pair described in claim 3 or 4.
6. the test kit described in the molecular marker described in claim 1 or 2, the primer pair described in claim 3 or 4 or claim 5, the purposes in Fructus Melo breeding.
7. the method detecting Fructus Melo pulp colour character, it is characterised in that
Fructus Melo to be measured is carried out the detection of molecular marker described in claim 1 or 2, in order to determine described Fructus Melo pulp colour character to be measured.
8. method according to claim 7, it is characterised in that described method includes:
Utilize the primer pair described in claim 3 or 4 or the test kit described in claim 5, described Fructus Melo genomic DNA to be measured is carried out pcr amplification；
The length of detection amplified fragments；And
Length based on described amplified fragments, it is determined that the pulp colour character of described Fructus Melo to be measured,
Wherein, as the long 1010bp of described amplified fragments, described Fructus Melo sarcocarp salmon pink to be measured；As the long 896bp of described amplified fragments, described Fructus Melo pulp white to be measured；When described amplified fragments is 896bp and 1010bp two kinds, the described molecular marker site heterozygosis of described Fructus Melo to be measured, sarcocarp salmon pink.
9. method according to claim 8, it is characterised in that utilize the preferred agarose gel electrophoresis of gel electrophoresis, detect the length of described amplified fragments.
10. a Fructus Melo auxiliary breeding means, it is characterised in that described method includes:
By the method described in any one of claim 7-9, test right requires the molecular marker described in 1 or 2, in order to determine Fructus Melo pulp colour character to be measured.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106834529A (en) * | 2017-04-05 | 2017-06-13 | 东北农业大学 | A kind of molecular labeling for indicating and identifying muskmelon orange flesh proterties and its primer and application |
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| CN108676905A (en) * | 2018-05-29 | 2018-10-19 | 中国农业科学院郑州果树研究所 | Watermelon yellowish pink major gene resistance and its InDel molecular labelings and application |
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| CN110273020A (en) * | 2019-06-27 | 2019-09-24 | 华中农业大学 | For distinguishing the SNP marker and application of citrus summer orange and common sweet orange |
| CN110273020B (en) * | 2019-06-27 | 2021-10-22 | 华中农业大学 | SNP molecular marker for distinguishing summer orange and common sweet orange of citrus and application thereof |
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