CN105420367B - The molecule labelling method of Chinese catalpa cutting plantation main effect QTL site qRR3 - Google Patents
The molecule labelling method of Chinese catalpa cutting plantation main effect QTL site qRR3 Download PDFInfo
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Abstract
The present invention provides the molecule labelling methods of Chinese catalpa cutting plantation main effect QTL site qRR3, belong to molecular genetics field.Determine that the genotype of 87 parts of clonal materials of Chinese catalpa combines its cutting plantation to carry out full-length genome association linkage analysis using 70 pairs of SSR molecular markers, detect Chinese catalpa cutting plantation main effect QTL site qRR3, explain 19.16% phenotypic variation, SSR molecular marker CB202 extremely significant correlation 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 the technical problems such as Chinese catalpa cutting plantation is at high cost, the time is long, stability is low are improved, it greatly speeds up China's Chinese catalpa rearing new variety and Developing Clonal Forestry develops.
Description
Technical field
The present invention provides the molecule labelling methods of Chinese catalpa cutting plantation main effect QTL site qRR3, belong to molecular genetic
Field is exclusively used in the Chinese catalpa new varieties that directive breeding cuttage is easily taken root.
Background technique
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 the low tree species of cutting plantation, cuttage root-taking difficulty limit Chinese catalpa without
The development of property system forestry.Therefore the Chinese catalpa new varieties that marker site relevant to cutting plantation easily takes root to cultivation cuttage are excavated
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 building of genetic linkage maps etc..Chen Yongxia etc. (Chen Yongxia, Zhang Xinquan, Ma Xiao,
Thank literary just SSR marker and Hemarthria compressa economical character association analysis hubei agricultural science, 2011,50 (7): 1494-
1498.) selecting 10 pairs of SSR primer pairs 44, especially morphological differences biggish 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 significant related, 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 such as several and single plant longest root long 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 spray
The north the evaluation gardening of cuttage radication capability, 2014, (15): 72-77.).The result of study shows to open using this group
Association analysis is opened up, may detect that the relevant QTL site of cutting plantation or molecular labeling.It yet there are no development correlative study
Paper or patent.
Summary of the invention
The purpose of the present invention is announce Chinese catalpa cutting plantation main effect QTL site and its molecular labeling.
The purpose of the present invention is what is be achieved through the following technical solutions: the forward direction using SSR molecular marker primer CB202 is drawn
Object sequence 5 '-CTTGGAGCGACGTTTCTTTC-3 ' and reverse primer sequences 5 '-CCAAACATCATCGACAAACG-3 ' are combined
Round pcr expands the genomic DNA of Chinese catalpa tender leaf, if the DNA fragmentation of 95 bp can be amplified, indicates that Chinese catalpa cuttage is raw
The presence of root rate main effect QTL site qRR3, the tribute to Chinese catalpa cutting plantation is measured using the GLM program of TASSEL2.1 software
Offering rate is 19.16%.
The utility model has the advantages that the present invention discloses the main effect QTL site and its molecular labeling of a Chinese catalpa cutting plantation for the first time,
The label be beneficial to shorten Chinese catalpa breeding cycle, reduce breeding cost, the high Chinese catalpa kind of directive breeding cutting plantation,
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 embodiment
Chinese catalpa cutting plantation statistical analysis: in early March, 2013 and in early March, 2014, by 87 Chinese catalpa clones
5 years raw branches be cut into after 30cm long and be equably horizontally-arranged in smooth sand bed, then cover the fine sand of 3~5cm, sprinkle profoundly water.
Sand bed is irrigated with 500 times of carbendazim turbid sprinkling weekly.Nursery is put into after peat and perlite are mixed by the volume ratio of 1:1
It is irrigated in basin (specification is 15 × 15 × 20 cm) and with the sprinkling of 500 times of carbendazim turbid.When the spray newly sprouted in sand bed is high
When degree reaches 10~15cm, remove the top of spray, after a week, the heel of spray band is removed, as cuttings.By the blade in cuttings
It 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, rinsed well with clear water, then dip 1min in the IBA of 3,000 mg/L, cuttings is inserted into nursery
Disk, each cave cup insert 1, and depth is the 1/2~2/3 of cutting length.20 cuttings of each clone cuttage, are repeated 3 times.Cuttage
Sun-proof with the covering of 85% shading net after complete, automatic sprayer is sprayed water supply.It is poured with 500 times of carbendazim turbid sprinkling weekly
Thoroughly.Each clone rooting rate is counted within 70 days after cuttage, as a result, it has been found that the rooting rate very different between Chinese catalpa different clones, takes root
Rate luffing is 10.06%~95.80%.
The analysis of Chinese catalpa population genetic variations: Chinese catalpa tender leaf genomic DNA is extracted using CTAB method, 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 primer is synthesized by Nanjing Si Pujin Biotechnology Co., Ltd.
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 polymerase.PCR response procedures are as follows: 94 DEG C initial denaturation 5 minutes, 94 DEG C be denaturalized 30 seconds, 57 DEG C annealing 45
Second, 72 DEG C extend 60 seconds, recycle 32 times, 5 points of kinds of last 72 DEG C of extensions.PCR reaction is completed on Bole's T100PCR instrument.
Pcr amplification product detection: 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 dyeing: first fixing 10 min with fixer (deionized water, 10% ethyl alcohol, 1% acetic acid), 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) 10 min of colour developing, finally
It is rinsed with deionized water, is put on film illuminator and takes pictures.With binary recording SSR primer amplification 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 data of Structure belongs to group structure to Chinese catalpa and analyzes, the results showed that corresponding likelihood value lnP
(D) the lasting increase with the increase of K value, therefore referring 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 software
It reruns, the correspondence Q value for obtaining each material can as the covariant of next step rooting effect and SSR marker association analysis
Influence of the group structure to association analysis is effectively reduced.
Chinese catalpa linkage disequilibrium value: by 70 SSR markers, 486 Locus Analysis in Shoots, show 117,855 at
To in the combined site SSR, there are a degree of LD.The LD that statistical probability (P < 0.01) is supported 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 number,
The 55.15% of total LD Sites Combination 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 level is higher between site.
The association analysis of Chinese catalpa cutting plantation: corresponding with 87 clones using the GLM program of TASSEL2.1 software
Q value is covariant, and 70 SSR markers and rooting rate are carried out linear regression analysis, as a result detect 6 cutting plantations
QTL is respectively designated as qRR1 ~ qRR6.The label of QTL site qRR3 close linkage be the phenotypic variation rate explained as
19.16%.The forward primer sequence of CB202 is: 5 '-CTTGGAGCGACGTTTCTTTC-3 ', reverse primer sequences are as follows: 5 '-
CCAAACATCATCGACAAACG-3 ', amplified fragments size are 95 bp.
The amplified fragments polymorphism of 1 70 pairs of SSR primers of table
SEQUENCE LISTING
<110>Institute of Botany
<120>molecule labelling method of Chinese catalpa cutting plantation main effect QTL site qRR3
<130>specification
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<220>
<221>CB202 forward primer sequence
<222> (1)..(20)
<223>
<400> 1
cttggagcga cgtttctttc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<220>
<221>CB202 reverse primer sequences
<222> (1)..(20)
<223>
<400> 2
ccaaacatca tcgacaaacg 20
Claims (2)
1. a kind of molecular labeling primer CB202 for the detection of Chinese catalpa cutting plantation major gene loci, it is characterised in that: institute
The forward primer sequence for stating molecular labeling primer CB202 is 5 '-CTTGGAGCGACGTTTCTTTC-3 ', and reverse primer sequences are
5’-CCAAACATCATCGACAAACG-3’。
2. molecular labeling primer CB202 described in claim 1 is to the molecule mark of Chinese catalpa cutting plantation major gene loci qRR3
Note method, it is characterised in that: the genome of Chinese catalpa tender leaf is expanded by the molecular labeling primer CB202 combination round pcr
DNA indicates the presence of Chinese catalpa cutting plantation major gene loci qRR3 if the DNA fragmentation of 95bp 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 CN201510956104.4A patent/CN105420367B/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 |
|---|
| 于晓丽.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.《中国优秀硕士学位论文全文数据库 农业科技辑》.2012,(第1期), * |
| 杨如同等.楸树不同无性系枝条萌芽力及催芽条件初探.《植物资源与环境学报》.2014,第23卷(第1期), * |
| 石欣等.中国楸树（catalpa bungei C.A.Mey）种质资源遗传多样性的ISSR分析.《江苏农业学报》.2011,第27卷(第3期), * |
| 聂浩.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.《中国优秀硕士学位论文全文数据库 农业科技辑》.2014,(第8期), * |
| 马玲玲等.梓属植物嫩枝扦插生根能力的评价.《北方园艺》.2014,(第15期), * |
| 麻文俊.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.《中国博士学位论文全文数据库 农业科技辑》.2014,(第3期), * |
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