CN105368964B - The molecule labelling method of the indefinite radical major gene loci qRN4 of Chinese catalpa cuttage - Google Patents
The molecule labelling method of the indefinite radical major gene loci qRN4 of Chinese catalpa cuttage Download PDFInfo
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- CN105368964B CN105368964B CN201510956023.4A CN201510956023A CN105368964B CN 105368964 B CN105368964 B CN 105368964B CN 201510956023 A CN201510956023 A CN 201510956023A CN 105368964 B CN105368964 B CN 105368964B
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Abstract
The present invention provides the molecule labelling methods of the indefinite radical major gene loci qRN4 of Chinese catalpa cuttage, belong to molecular genetics field.Determine that 87 parts of clonal genotype of Chinese catalpa combine the indefinite radical of its cuttage to carry out full-length genome and are associated with linkage analysis using 70 pairs of SSR molecular markers, detect the indefinite radical major gene loci qRN4 of Chinese catalpa cuttage, explain that 10.72% phenotypic variation, SSR molecular marker CB250 are extremely significantly correlated therewith.The present invention, which not only helps, solves the problems, such as that China's Chinese catalpa Advances in Breeding is slow, and help to solve existing breeding technique to improving the technical barriers such as the indefinite radical of Chinese catalpa cuttage is of high cost, the time is long, stability is low, China's Chinese catalpa rearing new variety will be accelerated and developed with Developing Clonal Forestry.
Description
Technical field
The present invention provides the molecule labelling methods of the indefinite radical major gene loci qRN4 of Chinese catalpa cuttage, belong to molecule something lost
Field is passed, the high Chinese catalpa new varieties of directive breeding cuttage radication capability are exclusively used in.
Background technology
Chinese catalpa (Catalpa bungei) belong to Bignoniaceae (Bignoniaceae) Chinese catalpa category (Catalpa) excellent precious use
Wood species.Increase severely with the development of industry with the growth of world population, timber demand and day, timber shortage has become worldwide ask
Topic, China are especially prominent.Developing Developing Clonal Forestry becomes the effective way for alleviating China is short of timber problem, and cutting plantation
It is the bottleneck for restricting Developing Clonal Forestry development.Chinese catalpa is that cutting plantation is low and several few seeds that take root, cuttage root-taking it is difficult and
The weak development for limiting Chinese catalpa clones forestry of cuttage seeding growing way.Therefore it excavates and the relevant marker site pair of the indefinite radical of cuttage
Fast-growing Chinese catalpa new varieties are cultivated to have great importance.
SSR molecular marker is one of current most widely used molecular marking technique, be widely used in the assignment of genes gene mapping,
QTL positioning, marker-assisted breeding and genetic linkage maps structure etc..Chen Yongxia etc. (Chen Yongxia, Zhang Xinquan, Ma Xiao,
Thank literary just SSR markers and Hemarthria compressa economical character association analysis hubei agricultural sciences, 2011,50 (7):1494-
1498.) selecting 10 pairs of SSR primer pairs 44, especially morphological differences larger Hemarthria compressa in portion's is scanned, and carries out character and SSR
The association analysis of label wherein finding 18 SSR markers and 8 economical characters are significantly correlated, and filters out excellent germplasm, accelerates
The breeding process of Hemarthria compressa.
There are significant differences for cuttage radication capability between early-stage study finds different Chinese catalpa clones, according to rooting rate, single plant
3 indexs of number and single plant longest root long etc. of taking root can be classified as rootability is poor, rootability is medium, rootability compared with
Good and easily take root 4 groups such as class (horse tinkling of pieces of jades, Wang Peng, Zhang Zhenyu, Li Linfang, Yang Rutong, Li Ya catalpa sprays
The north the evaluation gardening of cuttage radication capability, 2014, (15): 72-77.).The result of study shows asexual using Chinese catalpa
It is group expansion association analysis, may detect that the relevant major gene resistance of the indefinite radical of cuttage or molecular labeling.It yet there are no
Carry out the paper or patent of correlative study.
Invention content
The purpose of the present invention is announce the indefinite radical major gene loci of Chinese catalpa cuttage and its molecular labeling.
The purpose of the present invention is what is be achieved through the following technical solutions：Forward direction using SSR molecular marker primer CB250 is drawn
5 '-CCTCGTTGTAGGGCAGGATA-3 ' of object sequence and reverse primer sequences 5 '-GAATACGCAAGCACGTTTGA-3 ' are combined
The genomic DNA of round pcr amplification Chinese catalpa tender leaf shows Chinese catalpa cuttage not if the DNA fragmentation of 190 bp can be amplified
The presence for determining radical major gene loci qRN4 is measured using the GLM programs of TASSEL2.1 softwares to the indefinite radical of Chinese catalpa cuttage
Contribution rate be 10.72%.
Advantageous effect：The present invention discloses the major gene loci and its molecule mark of an indefinite radical of Chinese catalpa cuttage for the first time
Note, the label are beneficial to shorten the breeding cycle of Chinese catalpa, reduce breeding cost, the high Chinese catalpa of directive breeding cuttage radication capability
Kind, and then accelerate the popularization of Chinese catalpa, final is to play an important role in the problem of high-quality timber shortage for alleviating China.
Specific implementation mode
The indefinite radical statistical analysis of Chinese catalpa cuttage：In early March, 2013 and in early March, 2014,87 Chinese catalpas are asexual
5 years raw branches of system, which are cut into after 30cm long, to be equably horizontally-arranged in smooth sand bed, is then covered the fine sand of 3~5cm, is irrigated
Water.Weekly sand bed is irrigated with 500 times of carbendazim turbid sprinkling.Peat and perlite are pressed 1:It is put into after 1 volume ratio mixing
Seedling-growing container（Specification is 15 × 15 × 20 cm）In and with 500 times of carbendazim turbid sprinkling irrigate.It is tender when what is newly sprouted in sand bed
When branch height reaches 10~15cm, remove the top of spray, after a week, the heel of spray band is removed, as cuttings.It will be in cuttings
Blade is cut together with petiole, only the leaf at surplus top, and is cut half.The cuttings base portion handled well is dipped
75% alcohol disinfecting 30s, is rinsed well with clear water, then dips 1min in the IBA of 3,000 mg/L, and cuttings insertion is educated
Seed plate, each cave cup insert 1, and depth is the 1/2~2/3 of cutting length.Each clone 20 cuttings of cuttage, are repeated 3 times.Skewer
Sun-proof, automatic sprayer spraying water supply is covered after having inserted with 85% shading net.It is sprayed with 500 times of carbendazim turbid weekly
It irrigates.The indefinite radical of each clone is counted within 70 days after cuttage, as a result, it has been found that the indefinite radical difference pole between Chinese catalpa different clones
Greatly, indefinite radical luffing be 1.0/plant~14.2/plant.
Chinese catalpa population genetic variations are analyzed：Chinese catalpa tender leaf genomic DNA is extracted using CTAB methods, is then drawn with 277 couples of SSR
Object random PCR expands 12 Chinese catalpa clones DNA, as a result, it has been found that can successfully to amplify band clear and polymorphic for 70 pairs of primers
Property good band, the availability of synthetic primer is 25.26%.The frequency of polymorphism of 70 pairs of SSR primers is average between 40%~100%
Frequency of polymorphism be 87.17%, rich polymorphism（Table 1）.SSR primers are synthesized by the Nanjing bio tech ltd Si Pujin.
PCR amplification system be 10 μ L include 20 ng genomic DNAs, the dNTPs of the MgCl2 of 2.5mM, 0.5mM, the primer of 20 ng,
0.5U Taq DNA polymerases.PCR response procedures are：94 DEG C of pre-degenerations 5 minutes, 94 DEG C are denaturalized 30 seconds, 57 DEG C of annealing 45
Second, 72 DEG C extend 60 seconds, recycle 32 times, 5 points of kinds of last 72 DEG C of extensions.PCR reactions are completed on Bole's T100PCR instrument.
Pcr amplification product detects：Using the polyacrylamide gel electrophoresis of 8% concentration, PCR product detection is carried out, applied sample amount is 1.5 μ
L, electrophoretic buffer are 1 × TBE, and voltage is set as 220 V, and electrophoresis is until bromophenol blue band runs out of glue least significant end.Film silver staining
Method dyes：First use fixer（Deionized water, 10% ethyl alcohol, 1% acetic acid）10 min are fixed, then 1.5% silver nitrate solution impregnates 10
Min, after deionized water washs rapidly 2 times, developing solution（Deionized water, 1.5% sodium hydroxide, 1% formaldehyde）Develop the color 10 min, finally
It is rinsed with deionized water, is put on film illuminator and takes pictures.With binary recording SSR primer amplifications as a result, having on same site
There is the band of identical mobility to be denoted as 1, no band is denoted as 0, obtains the genotype data of 87 Chinese catalpa clones.It utilizes
2.1 software combination genotype datas of Structure belong to group structure to Chinese catalpa and analyze, the results showed that corresponding likelihood value lnP
(D) the lasting increase with the increase of K values, therefore with reference to (Evanno G, Regnaut S, the Goudet J. such as Evanno
Detecting the number of clusters of individuals using the software STRUCTURE:
a simulation study. Molecular ecology, 2005, 14(8):2611-2620.) K determined by △ K
Value.There is peak value in K=7 in △ K, so 87 parts of Chinese catalpa materials can be divided into 7 subgroups.K=7 is substituted into Structure softwares
It reruns, the correspondence Q values for obtaining each material can as the covariant of next step rooting effect and SSR marker association analysis
Effectively to reduce influence of the group structure to association analysis.
Chinese catalpa linkage disequilibrium value：By to 70 SSR markers, 486 Locus Analysis in Shoots, show 117,855 at
To in the sites SSR of combination, there are a degree of LD.Statistical probability（P ＜ 0.01）The LD of support is at loci 77,106
It is a, the 65.42% of whole Sites Combinations, average value 0.557 are accounted for, value is 42,523 in 0.4 or more LD Sites Combination numbers,
The 55.15% of total LD Sites Combinations number is accounted for, wherein the number of sites between 0.8-1 is most, there are 29,667, the above description shows that
Entirety LD levels are higher between site.
The association analysis of Chinese catalpa cutting plantation：It is corresponding with 87 clones using the GLM programs of TASSEL2.1 softwares
Q values are covariant, and 70 SSR markers and the indefinite radical of cuttage are carried out linear regression analysis, as a result detect that 4 cuttages are indefinite
The QTL of radical, is respectively designated as qRN1 ~ qRN4.The label of QTL site qRN4 close linkages is the phenotypic variation of explanation
Rate is 10.72%.The forward primer sequence of CB250 is：5 '-CCTCGTTGTAGGGCAGGATA-3 ', reverse primer sequences are：
5 '-GAATACGCAAGCACGTTTGA-3 ', amplified fragments size are 190 bp.
The amplified fragments polymorphism of 1 70 pairs of SSR primers of table
SEQUENCE LISTING
<110>Institute of Botany
<120>The molecule labelling method of the indefinite radical major gene loci qRN4 of Chinese catalpa cuttage
<130>Specification
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221>CB250 forward primer sequences
<222> (1)..(20)
<223>
<400> 1
cctcgttgta gggcaggata 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221>CB250 reverse primer sequences
<222> (1)..(20)
<223>
<400> 2
gaatacgcaa gcacgtttga 20
Claims (2)
1. a kind of molecular labeling primer CB250 for the indefinite radical major gene loci detection of Chinese catalpa cuttage, it is characterised in that：
The forward primer sequence of the molecular labeling primer CB250 is 5 '-CCTCGTTGTAGGGCAGGATA-3 ', reverse primer sequences
For 5 '-GAATACGCAAGCACGTTTGA-3 '.
2. the molecule of the radical major gene loci qRN4s indefinite to Chinese catalpa cuttage of molecular labeling primer CB250 described in claim 1
Labeling method, it is characterised in that：The genome of Chinese catalpa tender leaf is expanded by the molecular labeling primer CB250 combination round pcrs
DNA indicates depositing for the indefinite radical major gene loci qRN4 of Chinese catalpa cuttage if the DNA fragmentation of 190bp can be amplified
.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101243752A (en) * | 2008-03-20 | 2008-08-20 | 中国林业科学研究院林业研究所 | Method for forcing germination epicormic branch cuttage of Catalpa bungei root segment |
| CN103621306A (en) * | 2013-12-19 | 2014-03-12 | 江苏省中国科学院植物研究所 | Method for increasing tender bud cutting and rooting rate of catalpa bungei in mode of removing terminal buds of cutting slips |
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2015
- 2015-12-21 CN CN201510956023.4A patent/CN105368964B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101243752A (en) * | 2008-03-20 | 2008-08-20 | 中国林业科学研究院林业研究所 | Method for forcing germination epicormic branch cuttage of Catalpa bungei root segment |
| CN103621306A (en) * | 2013-12-19 | 2014-03-12 | 江苏省中国科学院植物研究所 | Method for increasing tender bud cutting and rooting rate of catalpa bungei in mode of removing terminal buds of cutting slips |
Non-Patent Citations (6)
| Title |
|---|
| 中国楸树（catalpa bungei C.A.Mey）种质资源遗传多样性的ISSR分析;石欣等;《江苏农业学报》;20111231;第27卷(第3期);634-639 * |
| 桉树遗传图谱的EST-CAPS标记整合及生长和扦插性状的QTL定位;于晓丽;《中国优秀硕士学位论文全文数据库 农业科技辑》;20120115(第1期);D049-210 * |
| 桑树绿枝扦插高效生根的转录组测序分析及相关基因的验证;聂浩;《中国优秀硕士学位论文全文数据库 农业科技辑》;20140815(第8期);D051-30 * |
| 梓属植物嫩枝扦插生根能力的评价;马玲玲等;《北方园艺》;20140815(第15期);72-77 * |
| 楸树不同无性系枝条萌芽力及催芽条件初探;杨如同等;《植物资源与环境学报》;20141231;第23卷(第1期);104-106 * |
| 楸树优良无性系2-8苗期生理变化与基因表达对干旱胁迫的响应;麻文俊;《中国博士学位论文全文数据库 农业科技辑》;20140315(第3期);D049-59 * |
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