CN105420367A - Molecular marker method of main effect QTL site qRR3 of rooting of cutting ratio of catalpa bungei - Google Patents
Molecular marker method of main effect QTL site qRR3 of rooting of cutting ratio of catalpa bungei Download PDFInfo
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- CN105420367A CN105420367A CN201510956104.4A CN201510956104A CN105420367A CN 105420367 A CN105420367 A CN 105420367A CN 201510956104 A CN201510956104 A CN 201510956104A CN 105420367 A CN105420367 A CN 105420367A
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Abstract
The invention provides a molecular marker method of a main effect QTL site qRR3 of a rooting of cutting ratio of catalpa bungei and belongs to the field of molecular genetics. 70 pairs of SSR molecular markers are used for determining genotypes of 87 clone materials of the catalpa bungei, the rooting of cutting ratio is combined for performing whole genome association study, and the main effect QTL site qRR3 of the rooting of cutting ratio of catalpa bungei is detected, 19.16% phenotypic variation is explained and CB202 of the SSR molecular marker is obviously related. The molecular marker method is favorable for solving the problem of slow breeding development of catalpa bungei in China, and is favorable for solving technical problems of high cost, long time, low stability and the like of the rooting of cutting ratio of catalpa bungei of the existing breeding technology, and the development of new species culturing and clone forestry developing in China are greatly accelerated.
Description
Technical field
The invention provides the molecule marking method of Chinese catalpa cutting plantation main effect QTL site qRR3, belong to molecular genetics field, be exclusively used in the Chinese catalpa new variety that directive breeding cuttage is easily taken root.
Background technology
Chinese catalpa (
catalpabungei) belong to Bignoniaceae (
bignoniaceae) Chinese catalpa genus (
catalpa) excellent Precious Timber Species.Along with industrial expansion and world population growth, timber demand and increase severely day, timber shortage becomes worldwide problem, and China is particularly outstanding.Development Developing Clonal Forestry becomes the effective way alleviating China's timber shortage problem, and cutting plantation is the bottleneck of restriction Developing Clonal Forestry development.Chinese catalpa is the seeds that cutting plantation is low, and cuttage root-taking difficulty limits the development of Chinese catalpa clones forestry.Therefore excavate the marker site relevant to cutting plantation to have great importance to the Chinese catalpa new variety that cultivation cuttage is easily taken root.
SSR molecular marker is one of current most widely used molecular marking technique, is widely used in the aspect such as structure of the assignment of genes gene mapping, QTL location, marker-assisted breeding and genetic linkage maps.(the Chen Yongxia such as Chen Yongxia, Zhang Xinquan, Ma Xiao, thank to literary composition just .SSR mark and the association analysis of Hemarthria compressa economical character. hubei agricultural science, 2011,50 (7): 1494-1498.) select 10 pairs of SSR primer pairs 44 Hemarthria compressa that portion's morphological differences is larger especially to scan, carry out the association analysis of proterties and SSR marker, wherein find 18 SSR marker and 8 economical character significant correlations, and filter out excellent germplasm, accelerate the breeding process of Hemarthria compressa.
Early-stage Study finds that between different Chinese catalpa clones, cuttage radication capability exists significant difference, to take root number and 3 indexs can be divided into rootability is poor, rootability is medium, rootability better and easily takes root 4 groups such as class (the horse tinkling of pieces of jades such as the longest root of individual plant is long according to rooting rate, individual plant, Wang Peng, Zhang Zhenyu, Li Linfang, Yang Rutong, Li Ya. the evaluation of catalpa epicormic branch cuttage rootability. northern gardening, 2014, (15): 72-77.).This result of study shows to utilize this group expansion association analysis, the QTL site that cutting plantation is relevant or molecule marker may be detected.Yet there are no the paper or patent of carrying out correlative study.
Summary of the invention
The object of the invention is to announce Chinese catalpa cutting plantation main effect QTL site and molecule marker thereof.
The object of the invention is to be achieved through the following technical solutions: utilize forward primer the sequence 5 '-CTTGGAGCGACGTTTCTTTC-3 ' of SSR molecular marker primer CB202 and reverse primer sequences 5 '-CCAAACATCATCGACAAACG-3 ' in conjunction with the genomic dna of round pcr amplification Chinese catalpa tender leaf, if the DNA fragmentation of 95bp can be amplified, then indicate the existence of Chinese catalpa cutting plantation main effect QTL site qRR3, utilizing the GLM program of TASSEL2.1 software to record the contribution rate of Chinese catalpa cutting plantation is 19.16%.
Beneficial effect: the present invention discloses main effect QTL site and the molecule marker thereof of a Chinese catalpa cutting plantation first, this mark will be conducive to the breeding cycle shortening Chinese catalpa, reduce breeding cost, the Chinese catalpa kind that directive breeding cutting plantation is high, and then accelerate the popularization of Chinese catalpa, final for playing an important role in the problem of high-quality timber shortage alleviating China.
Embodiment
Chinese catalpa cutting plantation statistical study: in early March, 2013 and in early March, 2014, is horizontally-arranged at equably in smooth husky bed after five of 87 Chinese catalpa clones years raw branches are cut into 30cm length, then covers the fine sand of 3 ~ 5cm, water permeable.Husky bed is irrigated weekly with the derosal turbid liquid sprinkling of 500 times.Peat and perlite are irrigated by putting into after the volume ratio mixing of 1:1 in seedling-growing container (specification is 15 × 15 × 20cm) and with the turbid liquid sprinkling of derosal of 500 times.When the spray height sprouted new in husky bed reaches 10 ~ 15cm, remove the top of spray, after one week, the heel of spray band is taken off, as cuttings.Blade in cuttings is cut together with petiole, only the leaf at surplus top, and cut half.The cuttings base portion handled well is dipped the alcohol disinfecting 30s of 75%, rinse well with clear water, then in the IBA of 3,000mg/L, dip 1min, cuttings is inserted seedling pan, each cave cup inserts 1, and the degree of depth is 1/2 ~ 2/3 of cutting length.Each clone cuttage 20 cuttings, repeat 3 times.Hide sun-proof with 85% shade net after cuttage is complete, automatic sprayer spraying feedwater.Irrigate with the turbid liquid sprinkling of derosal of 500 times weekly.Within after cuttage 70 days, add up each clone rooting rate, found that the rooting rate very different between Chinese catalpa different clones, rooting rate luffing is 10.06% ~ 95.80%.
Chinese catalpa population genetic variations is analyzed: utilize CTAB method to extract Chinese catalpa tender leaf genomic dna, then to increase 12 Chinese catalpa clones DNA with 277 pairs of SSR primer random PCRs, found that 70 pairs of primers successfully can amplify the clear and band that polymorphism is good of band, the operability of synthetic primer is 25.26%.The frequency of polymorphism of 70 pairs of SSR primers is between 40% ~ 100%, and average frequency of polymorphism is 87.17%, rich polymorphism (table 1).SSR primer is synthesized by Si Pujin bio tech ltd, Nanjing.PCR amplification system is that 10 μ L comprise 20ng genomic dna, the primer of the dNTPs of the MgCl2 of 2.5mM, 0.5mM, 20ng, 0.5UTaqDNA polysaccharase.PCR response procedures is: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 30 seconds, and 57 DEG C of annealing 45 seconds, 72 DEG C extend 60 seconds, circulates 32 times, last 72 DEG C of extensions 5 points of kinds.PCR reaction completes on Bole T100PCR instrument.Pcr amplification product detects: the polyacrylamide gel electrophoresis utilizing 8% concentration, carry out PCR primer detection, applied sample amount is 1.5 μ L, and electrophoretic buffer is 1 × TBE, and voltage is set to 220V, and electrophoresis is to glue least significant end run out of by tetrabromophenol sulfonphthalein band.Film silver staining method dyes: first use stationary liquid (deionized water, 10% ethanol, 1% acetic acid) to fix 10min, 1.5% silver nitrate solution soaks 10min again, after deionized water washs rapidly 2 times, develop the color nitrite ion (deionized water, 1.5% sodium hydroxide, 1% formaldehyde) 10min, finally use deionized water rinsing, put on film illuminator and take pictures.With binary recording SSR primer amplification result, the band same site with identical mobility is designated as 1, is designated as 0 without band, obtains the genotype data of 87 Chinese catalpa clones.Utilize Structure2.1 software to belong to group structure in conjunction with genotype data to Chinese catalpa to analyze, result shows that corresponding likelihood value lnP (D) continues with the increase of K value to increase, therefore with reference to (EvannoG such as Evanno, RegnautS, GoudetJ.Detectingthenumberofclustersofindividualsusingth esoftwareSTRUCTURE:asimulationstudy.Molecularecology, 2005,14 (8): 2611-2620.) true defining K value is carried out by △ K.There is peak value when K=7 in △ K, so 87 parts of Chinese catalpa materials can be divided into 7 subgroups.K=7 is substituted into Structure software to rerun, obtain the corresponding Q value of each material, as the concomitant variable of next step rooting effect and SSR marker association analysis, effectively can reduce the impact of group structure on association analysis.
, in the SSR site of 855 pair-wise combination, there is LD to a certain degree in Chinese catalpa linkage disequilibrium value: by 70 SSR marker, 486 Locus Analysis in Shoots, show 117.The LD that statistical probability (P < 0.01) is supported becomes loci 77,106, account for 65.42% of whole Sites Combination, mean value is 0.557, and the LD Sites Combination number be worth more than 0.4 is 42,523, account for 55.15% of total LD Sites Combination number, the number of sites wherein between 0.8-1 is maximum, has 29,667, between above all explanations site, overall LD level is higher.
The association analysis of Chinese catalpa cutting plantation: utilize the GLM program of TASSEL2.1 software with Q value corresponding to 87 clones for concomitant variable, 70 SSR marker and rooting rate are carried out linear regression analysis, result detects the QTL of 6 cutting plantation, respectively called after qRR1 ~ qRR6.QTL site qRR3 is closely linked is labeled as CB202, and the phenotypic variation rate of explanation is 19.16%.The forward primer sequence of CB202 is: 5 '-CTTGGAGCGACGTTTCTTTC-3 ', and reverse primer sequences is: 5 '-CCAAACATCATCGACAAACG-3 ', and amplified fragments size is 95bp.
The amplified fragments polymorphism of table 170 pair SSR primer
SEQUENCELISTING
<110> Institute of Botany
The molecule marking method of <120> Chinese catalpa cutting plantation main effect QTL site qRR3
<130> specification sheets
<160>2
<170>PatentInversion3.1
<210>1
<211>20
<212>DNA
<213> artificial sequence
<220>
<221>CB202 forward primer sequence
<222>(1)..(20)
<223>
<400>1
cttggagcgacgtttctttc20
<210>2
<211>20
<212>DNA
<213> artificial sequence
<220>
<221>CB202 reverse primer sequences
<222>(1)..(20)
<223>
<400>2
ccaaacatcatcgacaaacg20
Claims (1)
1. the molecule marking method of Chinese catalpa cutting plantation main effect QTL site qRR3, it is characterized in that: utilize forward primer the sequence 5 '-CTTGGAGCGACGTTTCTTTC-3 ' of SSR molecular marker primer CB202 and reverse primer sequences 5 '-CCAAACATCATCGACAAACG-3 ' in conjunction with the genomic dna of round pcr amplification Chinese catalpa tender leaf, if the DNA fragmentation of 95bp can be amplified, then indicate the existence of Chinese catalpa cutting plantation main effect QTL site qRR3, utilizing the GLM program of TASSEL2.1 software to record the contribution rate of Chinese catalpa cutting plantation is 19.16%.
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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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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 (5)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 《中国博士学位论文全文数据库 农业科技辑》 * |
| 《北方园艺》 * |
| 《植物资源与环境学报》 * |
| 《江苏农业学报》 * |
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