CN103232996B - Chrysanthemum-branching-trait-related molecular marker acquisition method - Google Patents
Chrysanthemum-branching-trait-related molecular marker acquisition method Download PDFInfo
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Abstract
The invention relates to a chrysanthemum-branching-trait-related molecular marker acquisition method, and belongs to the technical field of biology. The method comprises the steps that: a, a testing material and phenotypic data thereof are obtained; b, a chrysanthemum linkage map is established; c, chrysanthemum branching trait QTL analysis is carried out according to the phenotypic data and a molecular genetic map; and d, chrysanthemum-branching-trait-related molecular markers are determined. According to the invention, 'QX-145' (P1) is adopted as a female parent, NanNongYinShan is adopted as a male parent (P2), and 92 F1 segregating populations obtained by hybridization are adopted as the testing material; and a plurality of molecular markers substantially related to chrysanthemum branching traits are obtained. The molecular markers can be used in chrysanthemum branching trait good gene fine positioning and cloning, such that selection efficiency can be greatly improved, and breeding process can be accelerated.
Description
Technical field
The preparation method that the invention discloses the associated molecule marker of a kind of chrysanthemum branch proterties, belongs to biological technical field, for the location of chrysanthemum branch proterties excellent genes and the cultivation of clone and chrysanthemum new variety.
Background technology
Chrysanthemum is one of the famous-brand and high-quality and world's four large cut-flowers of China's ten great traditions, in the production of flowers and plants and afforestation, occupies critical role.In recent years, more about fancy points genetic researches such as chrysanthemum plant type, floral organs, promote to a certain extent chrysanthemum cross-breeding process.But along with deepening continuously of breeding work, traditional cross breeding method is because of consuming time longer, and cannot orderly improvement specific trait, in breeding, there is certain limitation.Molecular marker assisted selection breeding provides new Research Thinking for chrysanthemum breeding work.
Along with the development of molecular marking technique, Genetic linkage map builds with Quantitative Trait Genes location (quantitative trait loci, QTL) research and in many ornamental plants such as Chinese rose, cuckoo, lily, carnation, launches successively.At present, also there is relevant report in the molecule marker site relevant about chrysanthemum fancy points, but the preparation method of the associated molecule marker of chrysanthemum branch proterties there is not yet report.
The quality of cut-flower is mainly determined by proterties such as flower type, florescence and plant types, and branch proterties is chrysanthemum fancy points---the important composition factor of plant type is also one of major objective proterties of Dendranthema morifolium Varieties improvement.Further understand the genetic mechanism of Cut Flower Chrysanthemum Morifolium branch proterties, and obtain the main effect QTL of controlling branch proterties, the molecular mark that can be chrysanthemum branch proterties creates conditions.The present invention is by the molecule marker being associated with chrysanthemum branch proterties obtaining, for accurate location and the clone of chrysanthemum breeding of new variety, branch trait related gene have established theoretical basis.
Reference:
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Summary of the invention
For the accurate location of current chrysanthemum branch proterties Fineness gene and clone's research blank this present situation almost at home and abroad, the object of the invention is: filter out closely associated molecule marker of one or more and chrysanthemum branch character gene, set up chrysanthemum branch proterties molecular marker assisted selection system, thereby improve the efficiency of selection of the good branch proterties of chrysanthemum, for chrysanthemum breeding of new variety lays the foundation.
Object of the present invention can be achieved through the following technical solutions:
A preparation method for the associated molecule marker of chrysanthemum branch proterties, comprises the following steps:
The acquisition of a test materials and phenotypic data thereof: " Chinese Chrysanthemum germ plasm resource is preserved the Dendranthema morifolium Varieties of " center " in order to be stored in Agricultural University Of Nanjing for examination material.First Year selects two Dendranthema morifolium Varieties of branch proterties obvious difference to carry out artificial hybridization, obtains F
1cenospecies, making an inventory and broadcast in menophania in March next year cave, is colonizated in that " Chinese Chrysanthemum germ plasm resource is preserved " center ", the same land for growing field crops of Routine Management together with parent's cuttage seeding after mid or late April individual plant label.Respectively at Second Year and initial bloom stage investigation in the 3rd year autumn parent and F
1the branch proterties of colony plant, individual plant repeats 3 times, and calculates the mean value of proterties investigation in 2 years.Utilize Microsoft Excel2007 software and SPSS15.0 software to carry out respectively basic descriptive statistical analysis to branch proterties at the phenotypic data in two times.
B chrysanthemum linkage map builds:
1) adopt CTAB micromethod to extract parent and F thereof
1filial generation genomic dna;
2) utilize hybrid strain to carry out polymorphism screening to SRAP and SSR combination of primers, the polymorphism primer combination filtering out is increased for the polymorphism of mapping population and added up the polymorphic bands data after amplification;
For codominant marker, according to polymorphism mark electrophoretic image bands of a spectrum, represent respectively parent's banding pattern with a, b, represent heterozygosis banding pattern with h, represent shortage of data with u.For dominant marker, if female parent has band, male parent, without band, has band to be designated as d, is designated as b without band; If male parent has band, maternal without band, there is band to be designated as c, be designated as a without band, represent shortage of data with u.Be referred to as the title of pleomorphism site with primer name, if same primer amplification goes out multiple pleomorphism sites, after primer or combination of primers title according to molecular weight from big to small for order mark Arabic numerals as suffix to show difference.
Experiment use Lambda DNA, Taq archaeal dna polymerase, dNTPs and 100bp DNA marker(100,300,500,700,1000,1500,2000bp) purchased from precious biotechnology (Dalian) company limited, SRAP and EST ?SSR primer synthesized by Shanghai Jierui Biology Engineering Co., Ltd.
SRAP ?PCR reaction system and response procedures: SRAP ?PCR reaction mixture cumulative volume be 10 μ l, comprising 10 × PCR buffer, 3mM Mg
2+, 200 μ M dNTP, 0.5U Taq archaeal dna polymerase, 10 μ M SRAP primer and 25ng template DNAs.SRAP ?PCR response procedures: 94 DEG C/5min of denaturation; 94 DEG C/1min of sex change, 35 DEG C/1min of annealing renaturation, extends 72 DEG C/1min, 5 circulations; 94 DEG C/1min of sex change, 50 DEG C/1min of annealing renaturation, extends 72 DEG C/1min, 35 circulations; Extend 72 DEG C/7min; Finish reaction, 10 DEG C of preservations.SRAP ?PCR product adopt 8% native polyacrylamide gel electrophoresis, the preservation of taking pictures after silver dyes.SSR ?PCR reaction system and response procedures: SSR ?PCR reaction mixture cumulative volume be 25 μ l, comprising 2.5 μ l10 × PCR buffer, 1.5 μ l25 μ M Mg
2+, 2 μ l2.5mM dNTP, 0.5U Taq archaeal dna polymerase, the each 2 μ l of SSR primer, template DNA 50ng.SSR ?PCR response procedures: amplification condition: 94 DEG C of denaturation 3min; 94 DEG C of sex change 40s, 56 DEG C of annealing 30s, 72 DEG C are extended 50s, 35 circulations; 72 DEG C are extended 5min, preserve amplified production through 6% denaturing polyacrylamide gel electrophoresis for 4 DEG C.
3) use JoinMap4.0 software, selecting ' CP makes graph model ' that LOD is set is 4.0, respectively linkage analysis is carried out in parents' separation site, obtains parents' molecule marker linkage group, then uses Mapchart2.2 Software on Drawing linkage map.
The QTL location of c chrysanthemum branch proterties: the phenotypic data that the molecular genetic linkage map having built based on step b integrating step a obtain, use Win QTL Cartographer v2.5 software and composite interval mapping method to carry out chrysanthemum branch proterties qtl analysis, and estimate the genetic parameter such as the additive effect of each QTL and the contribution rate to phenotypic variation thereof.Relevant operating parameter is as follows: Walk speed=2cM; Window size=10.00; Model=6.Getting LOD threshold value is 2.5, can determine that this place exists a remarkable QTL site in the time that LOD peak value is greater than 2.5, and fiducial interval is determined according to each 1 the LOD value that declines in the peak value both sides of LOD value.QTL name is also slightly changed in accordance with (1997) methods such as McCouch substantially: the sequence number of proterties english abbreviation title (initial caps)+environment (E1, E2)+linkage group.For example, " PbnE1Q1 " represents the quantitative character gene locus therefor about one-level branch amount (Pbn) that utilizes (E1) phenotypic data in 2011 to detect on linkage group Q1.
Determining of the associated molecule marker of d chrysanthemum branch proterties:
According to determining between the mark zone at the main effect QTL place of step c gained and the tight associated molecule marker of chrysanthemum branch proterties.
According between the mark zone at the main effect QTL place of step c gained, determine with chrysanthemum branch proterties closely associated molecule marker be: with the tight associated molecule marker of one-level branch amount be PbnE1N4, its corresponding primer pair is SSR338 and SSR63, primer pair SSR338 sequence is: SEQ ID NO.1/SEQ ID NO.2, and primer pair SSR63 sequence is: SEQ ID NO.3/SEQ ID NO.4; Same level branch amount, the associated molecule marker of branch height is Bh E1N11, its corresponding primer pair is SSR185 and Me3Em15, and primer pair SSR185 sequence is: SEQ ID NO.5/SEQ ID NO.6, and primer pair Me3Em15 sequence is: SEQ ID NO.7/SEQ ID NO.8; The molecule marker associated with one-level branch length is PblE1N15, its corresponding primer pair is a pair of arbitrarily in SSR341, Me4Em3, Me3Em8, Me20Em4 and Me21Em5, primer pair SSR341 sequence is SEQ ID NO.9/SEQ ID NO.10, primer pair Me4Em3 sequence is SEQ ID NO.11/SEQ ID NO.12, primer pair Me3Em8 sequence is SEQ ID NO.7/SEQ ID NO.13, primer pair Me20Em4 sequence is SEQ ID NO.14/SEQ ID NO.15, and primer pair Me21Em5 sequence is SEQ ID NO.16/SEQ ID NO.17.
What beneficial effect the present invention selected is with ' QX ?145 ' (P
1) be that female parent, ' Nan Nongyin mountain ' they are male parent (P
2) hybridization obtain 92 strain F
1segregating population is examination material, utilizes SRAP and SSR molecule marker to build parents' Genetic Linkage Map spectrum, obtain the main effect QTL of chrysanthemum branch proterties and with its tight associated molecule marker.Compared with current technology, its advantage is:
(1) SSR is labeled as codominant marker, and polymorphism is good, and repeatability is high, covers whole genome, has multiallelic characteristic, is to build the comparatively ideal molecule marker of genetic linkage maps.The positive anti-primer of SRAP mark is respectively for genomic intron and exon region design, amplification region complementation with SSR mark, can be used as SSR mark supplemental markers, effectively increase density and the genome fraction of coverage of collection of illustrative plates, its polymorphism and potency ratio (producing the efficiency/cost of polymorphism) are all very high.
(2) molecular mark, overcomes the difficulty that chrysanthemum branch proterties excellent genes type is identified.Range of choice is wider, and intensity is larger.The conventional herd breeding method cycle of chrysanthemum is long, time-consuming effort again.Build the genetic linkage maps of chrysanthemum by the present invention, for laying a good foundation the QTL location of chrysanthemum fancy points; The acquisition of the associated molecule marker of chrysanthemum branch proterties can realize improved seeds and select ahead of time, reduces workload, greatly improves the new genotypic efficiency of selection of chrysanthemum, thereby accelerates breeding process.
Brief description of the drawings
The Dendranthema morifolium Varieties of Fig. 1 based on SRAP and SSR test cross marker site ' QX ?145 ' Genetic linkage map
The Genetic linkage map of the Dendranthema morifolium Varieties ' Nan Nongyin mountain ' of Fig. 2 based on SRAP and SSR test cross marker site
The distribution of the QTL of the each branch proterties of Fig. 3 and chrysanthemum significant correlation connection on genetic map
Embodiment
Embodiment 1
(1) acquisition of test materials and phenotypic data thereof
For examination material, in order to be stored in Agricultural University Of Nanjing, " Chinese Chrysanthemum germ plasm resource is preserved Cut Flower Chrysanthemum Morifolium kind ' QX ?145 ' and ' the Nan Nongyin mountain ' of " center ".The part branch proterties obvious difference of two kinds.Autumn in 2010, so that ' QX ?145 ' is as maternal, and ' Nan Nongyin mountain ' carries out artificial hybridization for male parent.' the well-developed bud of QX ?145 ', emasculation in the time that ligulate flower just reveals look, with sulfuric acid paper bag bagging, simultaneously by the inflorescence bagging on male parent ' Nan Nongyin mountain ' to choose female parent.Stretch out and jag is acute angle and secretion when mucus until maternal column cap, collect the fresh pollen of bagging male parent, with writing brush to female parent pollinate, bagging, repeat pollination next day.In the time that bennet flavescence is withered, gather pollination inflorescence, threshing, obtains 92 F
1cenospecies, making an inventory and broadcast in menophania in March next year cave, is colonizated in that " Chinese Chrysanthemum germ plasm resource is preserved " center ", the same land for growing field crops of Routine Management together with parent's cuttage seeding after mid or late April individual plant label.
By 92 strain F
1seedling obtains clone in the mode of cuttage, respectively at autumn in 2011 and 2012 initial bloom stage investigation parent and F
1the branch proterties of colony plant, comprises one-level branch amount, branch height and 3 proterties of one-level branch length, and individual plant repeats 3 times, and calculates the mean value of proterties investigation in 2 years.The method (" Chinese Chrysanthemum ", 1993) that concrete statistical method gradually waits with reference to Li Hong.Utilize Microsoft Excel2007 software and SPSS15.0 software to carry out respectively basic descriptive statistical analysis (in table 1) to branch proterties at the phenotypic data in two times.
Table 1 chrysanthemum ' QX-145 ', ' Nan Nongyin mountain ' and F thereof
1the branch proterties of colony is in the descriptive data in 2011～2012 years
(2) chrysanthemum linkage map builds
1) extract parent and F thereof with reference to CTAB micromethod (Murray & Thompson, 1980)
1filial generation genomic dna, Lambda DNA agarose gel electrophoresis detects DNA concentration and quality, finally concentration is adjusted to 50ng μ L
?1;
2) utilize hybrid strain respectively from 375 couples of SRAP and 38 pairs of SSR combination of primers (Zhang et el., 2011; Wang et el., 2013, primer sequence is in table 2, table 3) in carry out polymorphism screening, the polymorphism primer combination (59 couples of SRAP and 38 pairs of SSR combination of primers) after screening increase for the polymorphism of mapping population and is added up the polymorphic bands data after increasing.
Table 2 carries out SRAP primer title and the sequence thereof of polymorphism analysis for chrysanthemum mapping population
Note: SRAP primer sequence comes from open source literature (Zhang et el., 2011),
afor at Li & Quiros(2001) primer sequence collected in literary composition.
Table 3 carries out SSR primer title and the sequence thereof of polymorphism analysis for chrysanthemum mapping population
Note: SSR primer sequence comes from open source literature (Wang et el., 2013).
For codominant marker, according to polymorphism mark electrophoretic image bands of a spectrum, represent respectively parent's banding pattern with a, b, represent heterozygosis banding pattern with h, represent shortage of data with u.For dominant marker, if female parent has band, male parent, without band, has band to be designated as d, is designated as b without band; If male parent has band, maternal without band, there is band to be designated as c, be designated as a without band, represent shortage of data with u.Be referred to as the title of pleomorphism site with primer name, if same primer amplification goes out multiple pleomorphism sites, after primer or combination of primers title according to molecular weight from big to small for order mark Arabic numerals as suffix to show difference.Separation case according to pleomorphism site in Parent, adds appropriate prefix so that the reference of when mapping: pleomorphism site only appear at hybridization maternal ' QX ?in 145 ', prefixing " Q "; Pleomorphism site only appears in parent sire of hybrid pigs ' Nan Nongyin mountain ', prefixing " N ".To being only present in the polymorphism mark site of one of parent (Testcross marker, test cross marker site), carry out chi square test according to 1:1 Mendelian segregation ratio in 0.05 conspicuous level.Last gained flag data is in table 4.
The compartment analysis of table 4 polymorphic molecular marker in chrysanthemum mapping population
SRAP ?PCR reaction system and response procedures: SRAP ?PCR reaction mixture cumulative volume be 10 μ l, comprising 10 × PCR buffer, 3mM Mg
2+, 200 μ M dNTPs, 0.5U Taq archaeal dna polymerase, 10 μ M SRAP primer and 25ng template DNAs.SRAP ?PCR response procedures: 94 ° of C/5min of denaturation; 94 ° of C/1min of sex change, 35 ° of C/1min of annealing renaturation, extend 72 ° of C/1min, 5 circulations; 94 ° of C/1min of sex change, 50 ° of C/1min of annealing renaturation, extend 72 ° of C/1min, 35 circulations; Extend 72 ° of C/7min; Finish reaction, 10 ° of C preserve.SRAP ?PCR product adopt 8% native polyacrylamide gel electrophoresis, the preservation of taking pictures after silver dyes.SSR ?PCR reaction system and response procedures: SSR ?PCR reaction mixture cumulative volume be 25 μ l, comprising 2.5 μ l10 × PCR buffer, 1.5 μ l25 μ M Mg
2+, 2 μ l2.5mM dNTP, 0.5U Taq archaeal dna polymerase, the each 2 μ l of SSR primer, template DNA 50ng.SSR ?PCR response procedures: amplification condition: 94 ° of C denaturation 3min; 94 ° of C sex change 40s, 56 ° of C annealing 30s, 72 ° of C extend 50s, 35 circulations; 72 ° of C extend 5min, and 4 ° of C preserve.Amplified production is through 6% denaturing polyacrylamide gel electrophoresis.
Lambda DNA, Taq archaeal dna polymerase, dNTPs and 100bp DNA marker(100,300,500,700,1000,1500, the 2000bp of experiment use) purchased from precious biotechnology (Dalian) company limited, SRAP and SSR primer are synthesized by Shanghai Jierui Biology Engineering Co., Ltd; The key instrument using in experiment have Eppendorf5810R type high speed freezing centrifuge, DYY ?6C type electrophoresis apparatus, PTC ?100
tMtype PCR instrument, JS ?380 type gel image analysers.
3) utilize 329 marker sites of gained, use JoinMap4.0 software, selecting ' CP makes graph model ' that LOD is set is 4.0, respectively linkage analysis is carried out in parents' separation site, obtain parents' molecule marker linkage group, then use Mapchart2.2 Software on Drawing linkage map.
' QX ?145 ' genetic map contain 109 marker sites, remaining 51 marker sites does not have chainly to genetic map, the mapping rate of marker site is 68.1%.' QX ?145 ' genetic map form (Fig. 1) by 25 linkage groups, mean chart spectrum distance is from being 13.4cM, expectation genome is 2159.5cM, the actual acquisition of the present invention ' QX ?145 ' genetic map cumulative length be 1456.6cM, its genome fraction of coverage is about 67.9%.
' Nan Nongyin mountain ' genetic map contains 125 marker sites, and remaining 44 marker sites does not have chainly to genetic map, and the mapping rate of marker site is 73.9%.The genetic map on ' Nan Nongyin mountain ' forms (Fig. 2) by 21 linkage groups, mean chart spectrum distance is from being 11.6cM, expectation genome is 1452.1cM, and the actual acquisition of the present invention ' Nan Nongyin mountain ' genetic map cumulative length is 1982.7cM, and its genome fraction of coverage is about 73%.
(3) QTL of chrysanthemum branch proterties location
The phenotypic data that the molecular genetic linkage map having built based on step (two) integrating step () obtain, use Win QTL Cartographer v2.5 software and composite interval mapping method to carry out the analysis of the each branch proterties of chrysanthemum QTL, and estimate the genetic parameter (in table 5) such as the additive effect of each QTL and the contribution rate to phenotypic variation thereof.Relevant operating parameter is as follows: Walk speed=2cM; Window size=10.00; Model=6.Getting LOD threshold value is 2.5, can determine that this place exists a remarkable QTL site in the time that LOD peak value is greater than 2.5, and fiducial interval is determined according to each 1 the LOD value that declines in the peak value both sides of LOD value.QTL name is also slightly changed in accordance with (1997) methods such as McCouch substantially: the sequence number of proterties english abbreviation title (initial caps)+environment (E1, E2)+linkage group.For example, " PbnE1Q1 " represents the quantitative character gene locus therefor about one-level branch amount (Pbn) that utilizes (E1) phenotypic data in 2011 to detect on linkage group Q1.
The each branch proterties of table 5 chrysanthemum is at 2011 and 2,012 two annual QTL positioning analysises
Note: * P<0.05; * P<0.01.
For one-level branch amount, the PbnE2N4 that the PbnE1N4 detecting for 2011 detected with 2012 is positioned at identical between the mark zone in ' Nan Nongyin mountain ' genetic map N4 linkage group (N ?SSR338 ?6_N ?SSR63 ?4), illustrates that it should be same QTL; Due to its can explain the contribution rate of one-level branch amount phenotypic variation be 14.66% ?15.52%.Therefore, PbnE1N4(PbnE2N4) should be less main effect QTL affected by environment.
For branch height, the Bh E1N11 detecting for 2011 and the Bh E2N11 detecting for 2012 all between N ?SSR185 ?4_N ?Me3Em15 ?1 mark zone, illustrate that it should be same QTL in N11 linkage group; It can explain the contribution rate of branch height indicator form variation be 10.74% ?18.37%, therefore Bh E1N11(Bh E2N11) should be less main effect QTL affected by environment.
For one-level branch length, the PblE1Q1 detecting for 2011 and the PblE2Q1 detecting for 2012 are all positioned between the Q ?Me3Em8 ?3_Q ?Me20Em4 ?2 and Q ?Me20Em4 ?2_Q ?Me21Em5 ?8* mark zone in Q1 linkage group, adjacent between two mark zones, should belong to same QTL site, illustrate that this site is subject to such environmental effects less; PblE1Q1(PblE2Q1) contribution rate in 2011 and two kinds of environment in 2012 is respectively 7.98% and 14.80%, illustrates that this QTL is main effect QTL; N ?Me4Em3 ?4_N ?SSR341 ?2 marked regions of the same N15 linkage group that is distributed in ' Nan Nongyin mountain ' genetic map of PblE1N15 and PblE2N15, illustrate that in two kinds of environment, detecting is identical QTL site, it is subject to the impact of environment also smaller, the contribution rate of this site in 2 years be 10.69% ?24.38%, be also main effect QTL.
(4) determining of the associated molecule marker of chrysanthemum branch proterties
According to determining between the mark zone, main effect QTL place of step (three) gained and the tight associated molecule marker (in table 5, Fig. 3) of chrysanthemum branch proterties.
For one-level branch amount, PbnE1N4(PbnE2N4) be positioned at identical between the mark zone in ' Nan Nongyin mountain ' genetic map N4 linkage group (N ?SSR338 ?6_N ?SSR63 ?4), mark SSR338(SSR338F:SEQ ID NO.1/SSR338R:SEQ ID NO.2) with SSR63(SSR63F:SEQ ID NO.3/SSR63R:SEQ ID NO.4) closely associated with one-level branch amount; Same level branch amount, the associated molecule marker of branch height is SSR185(SSR185F:SEQ ID NO.5/SSR185R:SEQ ID NO.6) and Me3Em15(Me3:SEQ ID NO.7/Em15:SEQ ID NO.8); The associated molecule marker of one-level branch length is SSR341(SSR341F:SEQ ID NO.9/SSR341R:SEQ ID NO.10), Me4Em3(Me4:SEQ ID NO.11/Em3:SEQ ID NO.12), Me3Em8(Me3:SEQ ID NO.7/Em8:SEQ ID NO.13), Me20Em4(Me20:SEQ ID NO.14/Em4:SEQ ID NO.15) and Me21Em5(Me21:SEQ ID NO.16/Em5:SEQ ID NO.17).
Claims (4)
1. a preparation method for the associated molecule marker of chrysanthemum branch proterties, is characterized in that comprising the following steps:
The acquisition of a, test materials and phenotypic data thereof: First Year selects two Dendranthema morifolium Varieties ' QX-145 ' of branch proterties obvious difference to carry out artificial hybridization for female parent, ' Nan Nongyin mountain ' for male parent, obtains F
1cenospecies, make an inventory and broadcast in menophania in March next year cave, together with the field planting after mid or late April individual plant label of parent's cuttage seeding, the same land for growing field crops of Routine Management, respectively at Second Year and the 3rd year autumn initial bloom stage investigation parent and F
1the branch proterties of colony plant, individual plant repeats 3 times, and calculate the mean value of proterties investigation in 2 years, utilize Microsoft Excel 2003 softwares and SPSS 15.0 softwares to carry out respectively basic descriptive statistical analysis to branch proterties at the phenotypic data in two times;
B, chrysanthemum linkage map build:
1) adopt CTAB micromethod to extract parent and F thereof
1filial generation genomic dna;
2) utilize hybrid strain to carry out polymorphism screening to SRAP and SSR combination of primers, the polymorphism primer combination filtering out is increased for the polymorphism of mapping population and added up the polymorphic bands data after amplification;
3) use JoinMap4.0 software, selecting ' CP makes graph model ' that LOD is set is 4.0, respectively linkage analysis is carried out in parents' separation site, obtains parents' molecule marker linkage group, then uses Mapchart2.2 Software on Drawing linkage map;
The QTL location of c, chrysanthemum branch proterties: in conjunction with phenotypic data and molecular genetic linkage map, use Win QTL Cartographer v2.5 software and composite interval mapping method to carry out the qtl analysis of chrysanthemum branch proterties, and estimate genetic parameter, the additive effect that described genetic parameter comprises each QTL and the contribution rate to phenotypic variation thereof;
Determining of the associated molecule marker of d, chrysanthemum branch proterties: according to determining between the mark zone at the main effect QTL place of step c gained and closely associated molecule marker of chrysanthemum branch proterties: with the tight associated molecule marker of one-level branch amount be PbnE1N4, its corresponding primer pair is SSR338 and SSR63, primer pair SSR338 sequence is: SEQ ID NO.1 and SEQ ID NO.2, and primer pair SSR63 sequence is: SEQ ID NO.3 and SEQ ID NO.4; Same level branch amount, the associated molecule marker of branch height is Bh E1N11, its corresponding primer pair is SSR185 and Me3Em15, and primer pair SSR185 sequence is: SEQ ID NO.5 and SEQ ID NO.6, and primer pair Me3Em15 sequence is: SEQ ID NO.7 and SEQ ID NO.8; The molecule marker associated with one-level branch length is PblE1N15, its corresponding primer pair is a pair of arbitrarily in SSR341, Me4Em3, Me3Em8, Me20Em4 and Me21Em5, primer pair SSR341 sequence is SEQ ID NO.9 and SEQ ID NO.10, primer pair Me4Em3 sequence is SEQ ID NO.11 and SEQ ID NO.12, primer pair Me3Em8 sequence is SEQ ID NO.7 and SEQ ID NO.13, primer pair Me20Em4 sequence is SEQ ID NO.14 and SEQ ID NO.15, and primer pair Me21Em5 sequence is SEQ ID NO.16 and SEQ ID NO.17.
2. the preparation method of the associated molecule marker of a kind of chrysanthemum branch proterties according to claim 1, is characterized in that the molecule marker described in step b is that dominant marker SRAP and codominant marker SSR combine.
3. the preparation method of the associated molecule marker of chrysanthemum branch proterties according to claim 1, it is characterized in that the polymorphic bands data method after described statistics amplification is: for codominant marker, according to polymorphism mark electrophoretic image bands of a spectrum, represent respectively parent's banding pattern with a, b, represent heterozygosis banding pattern with h, represent shortage of data with u; For dominant marker, if female parent has band, male parent, without band, has band to be designated as d, is designated as b without band; If male parent has band, maternal without band, there is band to be designated as c, be designated as a without band, represent shortage of data with u; Be referred to as the title of pleomorphism site with primer name, if same primer amplification goes out multiple pleomorphism sites, after primer or combination of primers title according to molecular weight from big to small for order mark Arabic numerals as suffix to show difference, separation case according to pleomorphism site in Parent, prefixing is so that the reference of when mapping: pleomorphism site only appears in hybridization maternal ' QX-145 ', prefixing " Q "; Pleomorphism site only appears in parent sire of hybrid pigs ' Nan Nongyin mountain ', and prefixing " N ", to being only present in one of parent's polymorphism mark site, is carried out chi square test according to 1:1 Mendelian segregation ratio in 0.05 conspicuous level.
4. the preparation method of the associated molecule marker of chrysanthemum branch proterties according to claim 1, it is characterized in that the QTL location of described step chrysanthemum branch proterties: the phenotypic data that the chrysanthemum linkage map having built based on step b integrating step a obtain, use Win QTL Cartographer v2.5 software and composite interval mapping method to carry out chrysanthemum branch proterties qtl analysis, and estimate the additive effect of each QTL and the contribution rate genetic parameter to phenotypic variation thereof, relevant operating parameter is as follows: Walk speed=2cM, Window size=10.00, Model=6, getting LOD threshold value is 2.5, in the time that being greater than 2.5, LOD peak value can determine that this place exists a remarkable QTL site, fiducial interval is determined according to each 1 the LOD value that declines in the peak value both sides of LOD value.
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