CN107541553A - One group is used to distinguish BAC mark of the sesame 13 to chromosome - Google Patents

Abstract

The invention belongs to sesame genome technical field, and in particular to one group is used to distinguish BAC mark patent application matters of the sesame 13 to chromosome.This group of BAC mark includes 17 BAC information altogether, and BAC mark essential informations are specifically shown, and the double terminal base sequence information of BAC are specific as shown in SEQ ID NO.1 ~ 34.17 BAC marks provided herein are single specific site on chromosome, the specificity and uniqueness with site；It can be accurately identified using differentiating method provided herein and determine each bar chromosome of sesame, the research such as check and correction is finely schemed available for sesame high-density cells genetic map construction, Sesame group, with important scientific research theory significance and scientific research value, can be studied for follow-up sesame genomics, sesame breeding provides theories integration and establishes application foundation.

Description

One group is used to distinguish BAC mark of the sesame 13 to chromosome

Technical field

The invention belongs to sesame genome technical field, and in particular to one group is used to distinguish sesame 13 to chromosome BAC marks patent application matters.

Background technology

Sesame（Sesamum indicumL., 2n=26）, unique cultigen during category Pedaliaceae flax belongs to, and China The high-quality oil crops of important characteristic.Fat content is 50 ~ 60% in sesame seed, and oleic acid/linoleic acid ratio is about 1:1, therefore It is considered as one of ideal edible oil and fat of the mankind.In addition, also contain sesamol, sesamin and vitamin in sesame seed The natural anti-oxidation class material such as E, there is important work(in terms of the health cares such as reducing blood lipid, antibacterial anti-cancer and delaying human body caducity Effect.

Sesame belongs to microchromosome crop, and chromosome number is more, and form is similar, and it is thin to limit sesame to a certain extent The development of born of the same parents' genetics research.Since the eighties in last century so far, China's Scientific Research Workers have carried out sesame dyeing in succession Body number and morphological feature observation, chromosome band type analysis and caryotype research.In recent years, it is announcement sesame chromosome Group and genome signature, China's sesame researcher have carried out system research to sesame Chromosome Technique（Liu Yanyang etc., 2011, the optimizing research of sesame chromosome conven tional tabletting techniques key factor；Zhang Haiyang etc., 2012, sesame karyotype and Like nearly coefficient analysis）.

But because 13 pairs of chromosome morphologies of sesame are more similar, and chromosome sectioning quality and developmental stage etc. because Element can influence in situ hybridization result, therefore still lack at present a kind of relatively stable and accurately distinguish 13 pairs of sesame chromosomes And to the method for its precise designation.Also therefore, if can develop it is a set of can accurately distinguish sesame 13 to the stability of chromosome and The stronger molecular labeling of specificity, to promoting sesame genome and genomics development just to seem very necessary.

Since 2012, to coordinate Sesame group finely to scheme structure and important character functional genomics research, river Southern Shanxi Academy of Agricultural Sciences's sesame research center successively constructs sesame cultigen（Henan sesame No. 11）BAC and the BIBAC text of genome Storehouse.Then on the basis of the optimization of sesame Chromosome Technique is completed, sesame BAC-FISH technologies and chromosome piece are established Repeat BAC-FISH hybridization techniques（The patent of application reference number 2014103220094）, so as to the further depth for sesame chromosome Enter research and establish certain technical foundation.But in general, due to sesame Chromosome Identification, distinguish when be faced with stability and The particular/special requirement of accuracy, thus when carrying out correlative study using BAC-FISH technologies, still suffer from more technical barrier.

The content of the invention

The main object of the present invention is to provide one group and is used to distinguish specific b AC mark of the sesame 13 to chromosome, utilizes This group of BAC mark and existing BAC-FISH technologies, there is provided it is a kind of to distinguish method of the sesame 13 to chromosome, so as to be Sesame Theoretical and application foundation is established in the further further investigation of group.

Details are as follows for the technical solution used in the present invention.

One group is used to distinguish BAC mark of the sesame 13 to chromosome, and the BAC group echos include 17 BAC information, BAC altogether Mark shown in essential information table specific as follows：

It is to be understood that " G " represents that the probe is green fluorescent label in probe title, " R " represents that the probe is glimmering for red Signal；

The double terminal base sequence information of BAC are specific as shown in SEQ ID NO.1 ~ 34, i.e.,：

Using it is described be used to distinguishing sesame 13 differentiating method of the sesame 13 to chromosome is distinguished to the BAC mark groups of chromosome, Specifically include following operating procedure：

（One）It is prepared by sesame chromosome specimen

Using the sesame tip of a root as sample, root tip chromosomes piece sample is prepared；Specifically, with the 1.2 of No. 11 seeds of Henan sesame ~ The 1.8cm tips of a root prepare sesame chromosome specimen as sample；

When preparing sesame chromosome specimen, refer to wall Low Osmotic Method and prepared（It is prepared by Chen Ruiyang etc., plant chromosome sample Go wall, Low Osmotic Method and its meaning in cytogenetics, 1982）, or improved on the basis of wall Low Osmotic Method is removed, specifically press It is prepared by following steps：

（1）Take healthy sesame seed（For example with Henan sesame No. 11）, it is sowed at and is covered with the culture dish of wet filter paper, 21 DEG C of light culture length Root；

（2）The long tips of a root of 1.2 ~ 1.8cm are taken, 4 ~ 5mm tip of a root top of supply line is cut, is placed in glass bottle, adds 0.002M 8- In oxyquinoline, 21 DEG C of lucifuges pre-process 1h, and transfer cuts off tip of a root top of supply line into fixer（It is now with the current, no water beetle Alcohol：Glacial acetic acid=3:1）, 4 DEG C of fixed 1h；

（3）After distilled water embathes 2 ~ 3 times, 1mm or so places Meristernatic zone is cut, then every 10 Meristernatic zone add 20 μ L Mixed enzyme solution（2.5% cellulase and 2.5% pectase）, 35 ~ 40 DEG C of 2 ~ 3h of enzymolysis（It is preferred that the min of 2 h 40 are digested at 37 DEG C）；

（4）Meristernatic zone is then taken out, embathes 10 ~ 20 min in distilled water；Meristernatic zone is placed in fixer（Now with existing With absolute methanol：Glacial acetic acid=3:1）, 4 DEG C are fixed 1 h；

（5）Meristernatic zone is taken, is placed on precooling cleaning slide, local cells suspension is made；Then fixer, which is added dropwise, makes suspension It is scattered in whole slide, naturally dry；

（6）Use Giemsa stain（1:30 dilutions）15 min are dyed, then running water rinses, naturally dry；

Under fluorescence microscope, high quality stains body sample is selected, and marks target chromosome region, is recorded per group chromosome Corresponding coordinate position；

It should be noted that before carrying out follow-up hybridization mark, above-mentioned prepared chromosome specimen need to be pre-processed, specifically pressed Following steps are handled：

（1）Slide is soaked in 1 ~ 2 min in 45% acetic acid, takes off Giemsa stain color；

（2）Used at 37 DEG C and contain 100 μ g mL^-1RNase A 2 × SSC processing slides 1 h；Developed a film 3 times with 2 × SSC again, every time 5 min；

（3）Sample is dehydrated step by step using the ethanol of the 70% of precooling, 85% and 100%；

（4）With the min of 1% pepsin film-making mark zone 30（37℃）, 1 × PBS washes 2 times；

（5）Then the min of slide marker area 10 is handled with 1% paraformaldehyde, 2 × SSC is washed 2 times；

（6）Add 70% deionized formamide in slide marker area, be capped piece, 2 min are denatured at 70 DEG C；

（7）Film-making after denaturation is sequentially placed into 70%, 85% and 100% ethanol of -20 DEG C of precoolings and be dehydrated step by step, it is then natural Dry；

（Two）Prepare BAC probes

To the BAC mark groups for being used to distinguish 13 pairs of sesame chromosome（17 BAC probes）, carry out red fluorescence mark and it is green Color fluorescence labeling；Specifically：

First, BAC libraries are built, and extract BAC DNA；

Secondly, BAC probe marks are carried out using random priming；From red fluorescence element（Rhodamine）Or green fluorescein （Fluorescein）When carrying out fluorescence labeling to BAC DNA fragmentation, 5 μ L mark reaction system design is as follows：

BAC DNA fragmentation, 300 ~ 1000 ng；

DATP, dGTP, dCTP, each 0.1 mM；

DTTP, 0.065 mM；

Tetramethyl-Rhodamine-5-dUTP（Or Fluorescein-12-dUTP）Fluorescein, 0.035 mM；

Random Primer pd (N) 6,100 ng；

Klenow Fragment, 2.5 U；

10 × Klenow Buffer buffer solutions, 0.5 μ L；

Ultra-pure water adds to the μ L of cumulative volume 5；

Reaction condition：The h of lucifuge water-bath 20 at 37 DEG C；5 min at 65 DEG C again；Directly use or -20 DEG C save backup；

（Three）FISH

Using BAC-FISH continuous hybrid technologies, step is utilized（Two）BAC probes after middle mark are to step（One）In dyeing Body sample is marked, specifically：

Take the μ L of BAC probes 8 with red fluorescence mark or green fluorescent label（If double-colored in situ hybridization, two kinds of probes Each 4 μ L）, after processing（Specifically：95 DEG C of denaturation 10 min, 0 DEG C of 10 min of processing）, it is added dropwise to by pre-processing slide Mark zone, it is capped piece, mounting, lucifuge hybridization（Specifically, lucifuge hybridizes 20 h at 37 DEG C）；

3 times are embathed with 2 × SSC containing 0.1% SDS, every time 5 min；After distilled water flushing, lucifuge is dried；

4 μ L are added dropwise in slide marker area and contain 4 μ gmL^-1DAPI Vectashield H1000, it is capped piece, fluorescence microscopy Under mirror, hybridization signal is observed, and IMAQ is carried out under cold light source CCD, and image is handled；

When repeat FISH, after above-mentioned FISH hybridization, sample cover plate is thrown off, with containing 0.2% Tween20's 2 × SSC embathes 10 min, after distilled water flushing, naturally dry, is used further to next hybridization；

（Four）Differential staining body

To step（Three）In last BAC-FIFH results of hybridization picture carry out confluence analysis, 13 sesame chromosomes are numbered And make a distinction；

During specific differentiation, with reference to following 13 genome idiograms of sesame（Based on the positioning of BAC marks and sesame chromosome Caryogram structure obtains）Make a distinction：

When being made a distinction to sesame 13 to chromosome, respectively there are 2 specific B AC marks on Chr.1, Chr.2, Chr.4 and Chr.7 Remember respectively there is 1 specific B AC mark on remaining 9 pairs of chromosome；Furthermore：

53G and 45G probes mark the galianconism and long-armed area in No. 1 chromosome respectively；

87R, 48G probe are marked in the long-armed area of No. 2 chromosome；

25G probes are marked in the galianconism area of No. 3 chromosome；

39G, 15R probe are marked in the galianconism area of No. 4 chromosome；

42R probes are marked in the long-armed area of No. 5 chromosome；

102R probes are marked in the galianconism area of No. 6 chromosome；

76R, 21R probe are marked in the long-armed area of No. 7 chromosome；

57G probes are marked in the long-armed area of No. 8 chromosome；

3R probes mark the long-armed nearly centric region in No. 9 chromosome；

31G probes are marked in the galianconism area of No. 10 chromosome；

4G probes are marked in the long-armed area of No. 11 chromosome；

68R probes are marked in the long-armed area of No. 12 chromosome；

54R probes are marked in the long-armed area of No. 13 chromosome.

Compared with existing sesame chromosome differentiating method, important technical advantage of the present invention may be summarized to be：

（1）By screening, can be used for distinguishing BAC mark of the sesame 13 to chromosome present invention obtains one group；And utilize BAC When probe hybridizes, have hybridization signal stable（Results of hybridization is reliable and stable）, high specificity the advantages that, not by chromosome morphology, The factors such as size are influenceed, and the in situ hybridization and chromosome that can be completely used for sesame chromosome are distinguished；

（2）The present invention is first integrated BAC sequence informations and designation of chromosome position, chromosome to information such as length Correspondingly, and sesame karyotype characteristic pattern is constructed, tentatively completes karyological character analysis, to complete sesame in next step The integration of cytogenetics collection of illustrative plates, genetic map and the fine figure of Sesame group provides possibility；

（3）The present invention has carried out specific B AC marks to 3 sat chromosomes of sesame, is ground for follow-up sesame karyological character characteristic Study carefully and provide data support.

In general, 17 BAC marks provided herein are single specific site on chromosome, have site Specificity and uniqueness；It can be accurately identified using differentiating method provided herein and determine each bar chromosome of sesame, can The research such as check and correction is finely schemed for sesame high-density cells genetic map construction, Sesame group, there is the theoretical meaning of important scientific research Justice and scientific research value, it can be studied for follow-up sesame genomics, sesame breeding provides theories integration and establishes application foundation.

Brief description of the drawings

Fig. 1 is the BAC hybridization regions component of sesame normal chromosomal, wherein：

A, 102R, 45S rDNA (G) probe results of hybridization；

B, 87R, 53G probe results of hybridization；

C, 42R, 45G probe results of hybridization；

D, 76R, 31G probe results of hybridization；

E, 15R, 48G probe results of hybridization；

F, 3R, 25G probe results of hybridization；

G, 21R, 57G probe results of hybridization；

H, 39G probe results of hybridization；

I, it is a, b, c, d, e, f, g and h integrated results；

J, for the sesame chromosome karyotype analysis with 15 BAC marks and 45S rDNA marks；

" G " represents that the probe is green fluorescent label in probe title, and " R " represents that the probe marks for red fluorescence；Figure acceptance of the bid Chi is 5 μm；

Fig. 2 is that result is distinguished in the BAC hybridization of sesame sat chromosome；Wherein：

A, 102R, 45S rDNA (G) probe results of hybridization；

B, 64R, 4G probe results of hybridization；

C, 61R, 18G probe results of hybridization；

D, 51R, 2G probe results of hybridization；

E, 54R, 43G probe results of hybridization；

F, 68R, 100G probe results of hybridization；

G, it is a, b, c, d, e and f integrated results；

H, for the sesame chromosome karyotype analysis with 11 BAC and 45S rDNA marks；Scale is 5 μm in figure；

Fig. 3 is sesame chromosome BAC marking mode figures, wherein：Hollow posts are chromosome；RED sector is satellite DNA position （Chr.11, Chr.12, Chr.13 topmost portion）；Black line is BAC positions in post；Left ordinate scale represents that chromosome is relative Length（Relative chromosome length）, right ordinate scale expression DNA sequence sequence length （Chromosome length）；

Fig. 4 is the DNA the selection results of the BAC positioned at No. 2 and No. 4 chromosome, wherein：

A, 24R, 48G probe results of hybridization；

B, 357R, 316G probe results of hybridization；

C, 305R, 337G probe results of hybridization；

D, 15R, 364G probe results of hybridization；

E, 58R, 291G probe results of hybridization；

F, 459G probe results of hybridization；

G, 487G probe results of hybridization；

H, 660G probe results of hybridization

Scale is 5 μm in figure；

Fig. 5 is the DNA station-keeping mode figures of the BAC positioned at No. 2 and No. 4 chromosome；Wherein：Hollow posts represent No. 2 or No. 4 dyes of sesame Colour solid；Black line is BAC positions in post；The BAC probes to be determined for positioning of red display（BAC probes 364G to be determined It is located at No. 2 chromosomes with 337G and known mark 48G；BAC probes 316G, 357R, 487G, 58R and 291G to be determined with it is known Mark 15R is located at No. 4 chromosomes）；Lower right corner hollow posts represent 1% length of total chromosome.

Embodiment

The technical scheme of the application is explained further with reference to embodiment as follows.Before specifically embodiment is introduced, just Part biological material and related experiment background are described as follows in following embodiments.

Biomaterial：

Henan sesame No. 11, a kind of conventional sesame variety of commercialization；Involved BAC clones come from In Henan Agriculture in embodiment The BAC storehouses of academy of sciences's sesame research center structure, are stored in -80 DEG C；Specific construction method refers to existing patent（Application number 2014103220094）Or other prior art literatures；

45S rDNA probes synthetic works are completed and provided by Shanghai life work；

Experiment reagent：

BAC-FISH hybridization related reagents are purchased from German Roche companies；

Hybridization dyestuff and portion of reagent come from Shanghai Sheng Gong Reagent Companies；

Other reagents are conventional analysis pure reagent, no longer independent explanation；

Embodiment 1

The present embodiment is mainly introduced to be used to distinguish what sesame 13 marked 17 BAC of chromosome for described herein Screening process.Detailed process is described as follows.

（1）It is prepared by sesame chromosome specimen

（A）Smear method prepares sesame chromosome specimen

Wall Low Osmotic Method is removed in reference（Chen Ruiyang etc., prepared by plant chromosome sample removes wall, Low Osmotic Method and its in cytogenetics Meaning, 1982）And improved, sesame chromosome specimen is prepared, is comprised the following steps that：

（1）No. 11 healthy seeds of Henan sesame are taken, is sowed at and is covered with the culture dish of wet filter paper, 21 DEG C of long roots of light culture；

（2）The long tips of a root of 1.2 ~ 1.8cm are taken, 4 ~ 5mm tip of a root top of supply line is cut, is placed in glass bottle, adds 0.002M 8- In oxyquinoline, 21 DEG C of lucifuges pre-process 1h, and the transfer tip of a root is into fixer（It is now with the current, absolute methanol：Glacial acetic acid=3:1）, 4 DEG C of fixed 1h；

（3）After distilled water embathes 2-3 times, 1mm or so places Meristernatic zone is cut, then every 10 Meristernatic zone add 20 μ L Mixed enzyme solution（2.5% cellulase and 2.5% pectase）, the min of 2 h 40 are digested at 37 DEG C.

（4）Meristernatic zone is then taken out, embathes 15 min or so in distilled water；Meristernatic zone is placed in fixer （It is now with the current, absolute methanol：Glacial acetic acid=3:1）, 4 DEG C are fixed 1 h；

（5）Meristernatic zone is taken, is placed on precooling cleaning slide, local cells suspension is made；Then fixer, which is added dropwise, makes suspension It is scattered in whole slide, naturally dry；

（6）Use Giemsa stain（1:30 dilutions）15 min are dyed, then running water rinses, naturally dry.

Fluorescence microscope（Nikon 80i）Under, high quality stains body sample is selected, and mark target chromosome location Domain, record coordinate position corresponding to per group chromosome.

（B）Chromosome specimen processing

Before carrying out follow-up hybridization mark, above-mentioned prepared chromosome specimen need to be pre-processed, specifically carried out as follows Processing：

（1）Slide is soaked in 1 ~ 2 min in 45% acetic acid, takes off Giemsa stain color；

（2）Used at 37 DEG C and contain 100 μ g mL^-1RNase A 2 × SSC processing slides 1 h；Developed a film 3 times with 2 × SSC again, every time 5 min；

（3）Sample is dehydrated step by step using the ethanol of the 70% of precooling, 85% and 100%；

（4）With the min of 1% pepsin film-making mark zone 30（37℃）, 1 × PBS washes 2 times；

（5）Then the min of slide marker area 10 is handled with 1% paraformaldehyde, 2 × SSC is washed 2 times；

（6）Add 70% deionized formamide in slide marker area, be capped piece, 2 min are denatured at 70 DEG C；

（7）Film-making after denaturation is sequentially placed into 70%, 85% and 100% ethanol of -20 DEG C of precoolings and be dehydrated step by step, it is then natural Dry.

（2）Prepare BAC probes

（A）Extract B AC DNAs

With reference to Zhang et al., (2002) method（Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research）, based on support C opyControl^TM pCC1BAC^TM(Hind III Cloning-Ready) Vector specifications, build No. 11 Genomic BAC libraries of Henan sesame；This article Storehouse includes 57600 clones, is divided in 384 orifice plates, is preserved at -80 DEG C；

Random 100 BAC clones of picking, are inoculated into the LB fluid nutrient mediums that 5 mL contain 12.5 μ g/mL chloramphenicol, 37 DEG C Under, the h of 240r/min rotating speeds concussion and cultivate 12；

Extract DNA（Use qiagen plasmid extractant box）, detect BAC plasmid DNA concentrations（Use nucleic acids instrument ND2000（Thermo Electron, the U.S.））；

The BAC DNAs extracted are carried out at 37 DEG CNotThe h of I digestion 4；

Then carry out 1% agarose PFGE, 5 ~ 15 s, 120 °, 6V/cm, 4 DEG C of h of electrophoresis 18；According to DNA MARKER, the size of the DNA Insert Fragments of detection BAC clones.

（B）Label probe

Take the 20 above extracted BAC of μ L DNA, 0.1MPa, interrupt processing 5min at random under the conditions of 120 DEG C；Then using with Machine primer method carries out BAC probe marks（Specific steps are with reference to Fluorescein High Prime methods（Roche, Germany）Enter OK）；Specifically：

From red fluorescence element（Rhodamine）Or green fluorescein（Fluorescein）Fluorescence is carried out to BAC DNA fragmentation During mark, 5 μ L mark reaction system design is as follows：

The DNA fragmentation of BAC plasmids, 300 ~ 1000 ng；

DATP, dGTP, dCTP, each 0.1 mM；

DTTP, 0.065 mM；

Tetramethyl-Rhodamine-5-dUTP（Or Fluorescein-12-dUTP）Fluorescein, 0.035 mM；

Random Primer pd (N) 6,100 ng；

Klenow Fragment, 2.5 U；

10 × Klenow Buffer buffer solutions, 0.5 μ L；

Ultra-pure water adds to the μ L of cumulative volume 5；

Reaction condition：The h of lucifuge water-bath 20 at 37 DEG C；5 min at 65 DEG C again；Directly use or -20 DEG C save backup.

Before hybridization mark, hybridization buffer is used（Containing 50% deionized formamide, 10% dextran sulfate, 0.5 μ g μ L^-1Salmon Milt DNA, 1 × SSC buffer solutions）10 × dilution is carried out to marked product, produces crossing work liquid.

On the other hand, with reference to Chen Chengbin etc.（2013）（A kind of FISH sides of 45SrDNA on plant chromosome Method）The sequent synthesis 45S rDNA probes of announcement, in 5' ends mark green fluorescence group FAM marks.

（3）FISH

Using BAC-FISH continuous hybrid technologies, the BAC probes after being marked using step 2 kind are to the chromosome specimen in step 1 It is marked, specifically：

Take the μ L of BAC probes 8 with red fluorescence mark or green fluorescent label（If double-colored in situ hybridization, two kinds of probes Each 4 μ L）, after processing（Specifically：95 DEG C of denaturation 10 min, 0 DEG C of 10 min of processing）, it is added dropwise to by pre-processing slide Mark zone, piece is capped, mounting, lucifuge hybridizes 20 h at 37 DEG C；

3 times are embathed with 2 × SSC containing 0.1% SDS, every time 5 min；After distilled water flushing, lucifuge is dried；

4 μ L are added dropwise in slide marker area and contain 4 μ gmL^-1DAPI Vectashield H1000, it is capped piece, fluorescence microscopy Mirror（Nikon 80i）Under, hybridization signal is observed, and IMAQ is carried out under cold light source CCD, using the softwares of Spot Rtke 4.1 Image synthesis is carried out, and utilizes the softwares of Photoshop 7.0（Adobe companies, the U.S.）Image is adjusted；

Such as carry out repeating FISH, after above-mentioned FISH hybridization, sample cover plate is thrown off, with containing 0.2% Tween20's 2 × SSC embathes 10 min, after distilled water flushing, naturally dry, is used further to next hybridization.

（4）Sesame chromosome is distinguished

On above-mentioned experiment basis, finally preliminary screening goes out the up to a hundred BAC with stable specific cross signal from BAC storehouses Probe；

Further, carry out sesame chromosomal region go-on-go according to existing achievement in research to test, and finally determine proper probes the most, specifically For：

（1）Chromosomal hybridation is carried out using 45S rDNA probes（Fig. 1 a）, 13 pairs of sesame chromosomes are divided into 2 groups：Group I includes 10 pairs of chromosomes, it is normal chromosomal；Group II includes 3 pairs of chromosomes, is marked with 45S rDNA, that is, is with satellite Chromosome；

（2）From 20 specific B AC probes, using repeated crossing technology, carry out continuously repeating hybridization on a slide, To which specific B AC probes are located on 10 pairs of chromosomes of Group I；As a result showing, the slide is total to successful cross 8 times, into Work(located 15 BAC probes（Fig. 1 a-i）；It is superimposed by 8 hybridization pictures, 10 pairs of chromosomes of Group I is distinguished one by one （Fig. 1）；

（3）From other more than 20 specific Bs AC probes, one group is labeled as with red, green 2 BAC every time, it is miscellaneous using being repeated several times Friendship technology, carry out continuously repeating hybridization on another slide, to position specific B AC on the chromosome of the 3 of Group II pair Probe；As a result show, the slide is total to successful cross 6 times, successfully located 11 BAC probes（Fig. 2 a-g）.Pass through 6 hybridization Picture is superimposed, and 3 pairs of chromosomes of Group II are distinguished one by one（Fig. 2）；

（4）2 groups of results of hybridization are integrated by more than, it is determined that distinguish sesame 13 and 17 BAC of chromosome are marked（Referring to preceding State the BAC mark group form datas of " content of the invention " part）；In sesame 13 on chromosome, Chr.1, Chr.2, Chr.4 and Respectively there are 2 specific B AC marks on Chr.7, respectively there is 1 specific B AC mark on remaining 9 pairs of chromosome.

According to 17 BAC probes hybridization signals relative position on chromosome and chromosome image, sesame 13 is built Genome idiogram（Fig. 3）, the relative position of each bar chromosome numbers and specific B AC on chromosome is specify that, i.e.,：

53G and 45G probes mark the galianconism and long-armed area in No. 1 chromosome respectively；

87R, 48G probe are marked in the long-armed area of No. 2 chromosome；

25G probes are marked in the galianconism area of No. 3 chromosome；

39G, 15R probe are marked in the galianconism area of No. 4 chromosome；

42R probes are marked in the long-armed area of No. 5 chromosome；

102R probes are marked in the galianconism area of No. 6 chromosome；

76R, 21R probe are marked in the long-armed area of No. 7 chromosome；

57G probes are marked in the long-armed area of No. 8 chromosome；

3R probes mark the long-armed nearly centric region in No. 9 chromosome；

31G probes are marked in the galianconism area of No. 10 chromosome；

4G probes are marked in the long-armed area of No. 11 chromosome；

68R probes are marked in the long-armed area of No. 12 chromosome；

54R probes are marked in the long-armed area of No. 13 chromosome.

Double end sequencings are carried out to the DNA of above-mentioned 17 BAC probes, can further obtain double end sequences, finally Sequence information is as shown in sequence table SEQ ID NO.1 ~ 34.

Embodiment 2

By examine above-described embodiment 1 final determination BAC mark groups accuracy and specificity, while also dive to illustrate the invention In purposes, further, still by taking No. 11 seeds of Henan sesame as an example, inventor is filtered out positioned at No. 2 dyes from 14 unknown BAC clones The BAC clones of colour solid and No. 4 chromosome, and determined relative positions of the BAC clones DNA to be determined on respective chromosome, phase Experiment is closed to be briefly discussed below.

（1）Sesame chromosome specimen is prepared, related preparation process reference implementation example 1 is carried out.

（2）BAC probes are prepared, when extracting BAC DNAs, from the individual Henan sesames 11 up to a hundred for having identified specific hybridization signal In number BAC clone, 14 BAC clones are selected at random（Clone's numbering refers to following table）, therefrom filter out positioned at No. 2 chromosomes and 4 The BAC clones of number chromosome.

Positioned at the BAC colony screening results of No. 2, No. 4 chromosome：

。

On the basis of embodiment 1, the specific B AC clones of the molecular labeling as No. 2 chromosomes and No. 4 chromosome are picked out 3L8（Probe is labeled as 48G）And 119C1（Probe is labeled as 15R）, carry out cross experiment.

Related plasmids DNA is extracted and probe mark, the description of reference implementation example 1 are operated.

（3）FISH, by 14 probes to be determined and 2 known probes（48G, 15R）, according to red, green mark Any combination of two is carried out, carries out FISH（Relevant treatment reference implementation example 1）, it is continuously glimmering finally to successfully complete 8 wheels Light in situ hybridization.

As a result show：In 14 probes to be determined, in addition to there is not hybridization signal in 3 probes, other 11 wholes Navigate on chromosome（Fig. 4）；Wherein：

There are BAC probes 364G and 337G on No. 2 chromosomes；

There are BAC probes 316G, 357R, 487G, 58R and 291G on No. 4 chromosomes；

Other 4 BAC probes, i.e. 24R, 459G, 305R and 660G are co-located on another item chromosome；

With reference to karyotyping, it further determined that and be positioned at the relative position of No. 2 and 7 BAC on No. 4 chromosomes（Fig. 5）.

On the one hand this result further demonstrates the specificity and accuracy that the application screens gained BAC marks, another Aspect shows that the BAC that acquisition is screened using the application is marked, and can be further used for screening positioned at the BAC's of specific chromosome DNA fragmentation, and these the selection results can be provided for the analysis of further sesame genomics and genomics using support.

SEQUENCE LISTING

<110>Henan Academy of Agricultural Sciences's sesame research center

<120>One group is used to distinguish BAC mark of the sesame 13 to chromosome

<130> none

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 1

gggtaaccaa aggactttcc agtccgacgt tgtaaaacga cggccagtga attgtaatac 60

gactcactat agggc 75

<210> 2

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 2

cacccaatac tcaaggctaa aaattgtctg tagtatggtt tgctttatgt atattatact 60

agaagtgtta acatt 75

<210> 3

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 3

ccacgaagcc cagggatttc caagtcacga cgttgtaaaa cgacggccag tgaattgtaa 60

tacgactcac tatag 75

<210> 4

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 4

tcttttccct caggctttaa cgggcagtag tcttgtttca atgatcaaaa ttactacctt 60

tgggtctaca gtcca 75

<210> 5

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 5

ggcgttcccg gtgattccag tcacgacgtt gtaaacgacg gccagtgaat tgtaatacga 60

ctcactatag ggcga 75

<210> 6

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 6

ccaaaatatt caagcttctc cttgctctgc cataaccctt tgtcgagcct cagctgctgc 60

cctctccaca gcagc 75

<210> 7

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 7

accttgccaa ggcttttcca gtcacgacgt tgtaaaacga cggccagtga attgtaatac 60

gactcactat agggc 75

<210> 8

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 8

cacaaattcc agcttttgtg ttgtgttgat tgagaagaca aaggtgaagg cttttgttat 60

ctcaaaggcc attct 75

<210> 9

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 9

aggcctcgcc aaagagttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat 60

acgactcact atagg 75

<210> 10

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 10

ctaattactt aaacttatga gaggtggtga tttagtgtat ttttgctgtt tccatgaatc 60

atttttgttg cagag 75

<210> 11

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 11

agccctccca aagtattcag gtcacgacgt tgtaaacgac ggccagtgaa ttgtaatacg 60

actcactata gggcg 75

<210> 12

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 12

gtcaaaaaat ccagctggac tatgggtgaa agagtcacgt atcgtcaagg gtgatgggaa 60

catacggcta ctgtg 75

<210> 13

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 13

tctctaccca ggggttttcc aagtcacgac gttgtaaacg acggccagtg aattgtaata 60

cgactcacta taggg 75

<210> 14

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 14

tctttctacc aagcttgtgt cggtatttat gtgcattcca tcggtggaac gtaaaaatgt 60

cagttgatag ttctg 75

<210> 15

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 15

ccttagccca gggaattcca gtcacgacgt tgtaaacgac ggccagtgaa ttgtaatacg 60

actcactata gggcg 75

<210> 16

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 16

ccaggaaatt caggcttggg gcgtgcttga cgtcagccaa atcctggaaa attcttctga 60

aaatgcagtt gtgga 75

<210> 17

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 17

agctagagag aaggggtttc cagtcacgac gttgtaaacg acggccagtg aattgtaata 60

cgactcacta taggg 75

<210> 18

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 18

gaattttact cagctttctg agtaccaaac caaataatta agaatgttgg gagccctaat 60

aatatagtac cttcg 75

<210> 19

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 19

cgcgaaaccc gaagcttttc cagtcacgac gttgtaaacg acggccagtg aattgtaata 60

cgactcacta taggg 75

<210> 20

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 20

caatgatatc caggctttgg tcatgcatgg agctcaagct caagttgggg gctcaatgtg 60

cctattgttt gtcgc 75

<210> 21

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 21

cgccttaacc gaaaggattc cagtcacgac gttgtaaaac gacggccagt gaattgtaat 60

acgactcact atagg 75

<210> 22

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 22

aacatttcca tcaaggcctt gtgatctttt ttttttccat ttccttttgc atttcttgac 60

ttcactctga aaatc 75

<210> 23

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 23

acgctcacaa gagactttcc agtcacgacg ttgtaaacga cggccagtga attgtaatac 60

gactcactat agggc 75

<210> 24

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 24

cccttttcct caaggcttgc tcatcataca gacctccatc atatctttgc cctctgctca 60

aacgacgcag caagc 75

<210> 25

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 25

ccgctcatgc aaggggtttt ccagtcacgg acgttgtaaa acgacggcca gtgaattgta 60

atacgactca ctata 75

<210> 26

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 26

aatataacaa gccttcccgg cccggtttga caaaaatgag gatcccaaat tgcatgagca 60

ataggtctta catgt 75

<210> 27

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 27

cctcggccag cggatttcca agtcacgacg ttgtaaacga cggccagtga attgtaatac 60

gactcactat agggc 75

<210> 28

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 28

ccattcaaca tcaagcttcg atttgtgagg acaaagcata ccaccagtga tggagaaccc 60

gagcagactt caaat 75

<210> 29

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 29

ggttgacaca agagggttcc caatcacgac gtttgtaaaa cgacggccag tgaattgtaa 60

tacgactcac tatag 75

<210> 30

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 30

cccttaatta agctcggatt aagggtgatg taattgcctt tcttggtaaa attcaatgct 60

cttaaaaatg tgaac 75

<210> 31

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 31

accataacca aggggattcc cagtcacgac gttgtaagcg acggccagtg aattgtaata 60

cgactcacta taggg 75

<210> 32

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 32

ccttaatcat caagcttgtt tctcaattat tacttaactc cattatatag tcttaactat 60

tatttttttt actgt 75

<210> 33

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 33

ccacgaagcc cagggatttc caagtcacga cgttgtaaaa cgacggccag tgaattgtaa 60

tacgactcac tatag 75

<210> 34

<211> 75

<212> DNA

<213> Sesamum indicum

<400> 34

tcttttccct caggctttaa cgggcagtag tcttgtttca atgatcaaaa ttactacctt 60

tgggtctaca gtcca 75

Claims (6)

1. one group is used to distinguish BAC mark of the sesame 13 to chromosome, it is characterised in that the BAC group echos include 17 BAC altogether Mark, shown in BAC mark essential information tables specific as follows, the double terminal base sequence information of BAC are specifically such as SEQ ID NO.1 ~ 34 It is shown；

It is to be understood that " G " represents that the probe is green fluorescent label in probe title, " R " represents that the probe is glimmering for red Signal.

2. using described in claim 1 be used for distinguish sesame 13 to the differentiation sesame 13 of the BAC mark groups of chromosome to chromosome Differentiating method, it is characterised in that specifically include following operating procedure：

（One）It is prepared by sesame chromosome specimen

Using the sesame tip of a root as sample, root tip chromosomes piece sample is prepared；

（Two）Prepare BAC probes

To the BAC mark groups for being used to distinguish 13 pairs of sesame chromosome, red fluorescence mark and green fluorescent label are carried out；

（Three）FISH

Utilize step（Two）BAC probes after middle mark are to step（One）In chromosome specimen be marked；

（Four）Differential staining body

To step（Three）In last BAC-FIFH results of hybridization picture carry out confluence analysis, 13 sesame chromosomes are numbered And make a distinction；During specific differentiation, made a distinction with reference to following 13 genome idiograms of sesame：

When being made a distinction to sesame 13 to chromosome, respectively there are 2 specific B AC marks on Chr.1, Chr.2, Chr.4 and Chr.7 Remember respectively there is 1 specific B AC mark on remaining 9 pairs of chromosome；

53G and 45G probes mark the galianconism and long-armed area in No. 1 chromosome respectively；

87R, 48G probe are marked in the long-armed area of No. 2 chromosome；

25G probes are marked in the galianconism area of No. 3 chromosome；

39G, 15R probe are marked in the galianconism area of No. 4 chromosome；

42R probes are marked in the long-armed area of No. 5 chromosome；

102R probes are marked in the galianconism area of No. 6 chromosome；

76R, 21R probe are marked in the long-armed area of No. 7 chromosome；

57G probes are marked in the long-armed area of No. 8 chromosome；

3R probes mark the long-armed nearly centric region in No. 9 chromosome；

31G probes are marked in the galianconism area of No. 10 chromosome；

4G probes are marked in the long-armed area of No. 11 chromosome；

68R probes are marked in the long-armed area of No. 12 chromosome；

54R probes are marked in the long-armed area of No. 13 chromosome.

3. utilize distinguish differentiation sesame 13 of the sesame 13 to the BAC mark groups of chromosome to chromosome as claimed in claim 2 Differentiating method, it is characterised in that step（One）In, using 1.2 ~ 1.8cm tips of a root of No. 11 seeds of Henan sesame as sample, prepare sesame Chromosome specimen.

4. utilize distinguish differentiation sesame 13 of the sesame 13 to the BAC mark groups of chromosome to chromosome as claimed in claim 3 Differentiating method, it is characterised in that when preparing sesame chromosome specimen, with reference to going wall Low Osmotic Method to be prepared, or going wall low Ooze and improve on the basis of method, specifically prepared as follows：

（1）Healthy sesame seed is taken, is sowed at and is covered with the culture dish of wet filter paper, 21 DEG C of long roots of light culture；

（2）The long tips of a root of 1.2 ~ 1.8cm are taken, 4 ~ 5mm tip of a root top of supply line is cut, is placed in glass bottle, adds 0.002M 8- In oxyquinoline, 21 DEG C of lucifuges pre-process 1h, and the transfer tip of a root is into fixer, 4 DEG C of fixed 1h；

（3）After distilled water embathes 2 ~ 3 times, 1mm or so places Meristernatic zone is cut, then every 10 Meristernatic zone add 20 μ L Mixed enzyme solution, 35 ~ 40 DEG C of 2 ~ 3h of enzymolysis；

（4）Meristernatic zone is then taken out, embathes 10 ~ 20 min in distilled water；Meristernatic zone is placed in fixer, 4 DEG C solid Fixed 1 h；

（5）Meristernatic zone is taken, is placed on precooling cleaning slide, local cells suspension is made；Then fixer, which is added dropwise, makes suspension It is scattered in whole slide, naturally dry；

（6）15 min are dyed with Giemsa stain, then running water rinses, naturally dry；

Under fluorescence microscope, high quality stains body sample is selected, and marks target chromosome region, is recorded per group chromosome Corresponding coordinate position.

5. utilize distinguish differentiation sesame 13 of the sesame 13 to the BAC mark groups of chromosome to chromosome as claimed in claim 2 Differentiating method, it is characterised in that step（Two）In, BAC probe marks are carried out using random priming.

6. utilize distinguish differentiation sesame 13 of the sesame 13 to the BAC mark groups of chromosome to chromosome as claimed in claim 2 Differentiating method, it is characterised in that step（Three）In, during FISH, using BAC-FISH continuous hybrid technologies.