CN103642913A - Method for constructing boehmeria nivea core idioplasm by using EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) molecular marker - Google Patents
Method for constructing boehmeria nivea core idioplasm by using EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) molecular marker Download PDFInfo
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Abstract
The invention discloses a method for constructing boehmeria nivea core idioplasm by using an EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) molecular marker. The method comprises the steps of firstly extracting DNA (Deoxyribonucleic Acid) of boehmeria nivea idioplasm resources, then, carrying out PCR (Polymerase Chain Reaction) amplification through EST-SSR primers, and recording the banding pattern of each idioplasm resource according to amplification bands; analyzing the obtained banding patterns, and constructing the boehmeria nivea core idioplasm by using heuristic search and the principle of the DNA amplified banding pattern maximization of the idioplasm resources. According to the method, the forming of the EST-SSR primers and the experimental operating processes are simple, the operation is easy, the amplified banding patterns are clear and stable, and the repeatability is good. Compared with field-property core idioplasm construction, the molecular marker based boehmeria nivea core idioplasm construction has the characteristics of time saving, labor saving, good stability and the like. According to the core idioplasm constructed based on the molecular marker, the maximization variety of the DNA amplified banding patterns is guaranteed, the representativeness is very good, and the core idioplasm has important significance in preservation, evaluation and utilization of the idioplasm of boehmeria nivea.
Description
Technical field
The invention belongs to plant germplasm resource evaluation technique field, be specifically related to a kind of method that the EST-SSR of utilization molecule marker builds ramie Core Germplasms.
Background technology
Ramie (Boehmeria nivea L.Gaud), is again " China's grass ", originates from China, is the perennial root herbaceous plant of Urticaceae Section of Genus Boehmeria, is the natural textile raw material [1] of distinct Chinese characteristics.Except traditional fiber is worth, the multifunctional usages such as the pharmaceutical use of ramie, feed purposes, soil conservation, industrial raw material, bioenergy have started to receive publicity
[2-5].Up to now, China is collection, preserves the maximum country of Section of Genus Boehmeria germ plasm resource, and only ramie garden, national germplasm Changsha just has more than 2000 part of material, and cultivation resource wherein is extensively utilized by breed and production
[6].Plant genetic resources is the basis of breeding of new variety, the collection of germ plasm resource, preservation, evaluate and utilize extremely important, yet huge germ plasm resource is unfavorable for preservation and the utilization of elite germplasm.For solving this difficult problem, Frankel etc.
[7]and Brown
[8]the concept of Core Germplasms has been proposed.Core Germplasms refers to a part of selecting whole germ plasm resource with certain method, repeats farthest to represent the diversity of whole germ plasm resource with minimum resource quantity and heredity.At present, the structure of Core Germplasms not only successful application at wheat
[9], corn
[10], paddy rice
[11]on annual crop, in plum blossom
[12],, coffee
[13], peach
[14], tea tree
[15], white birch
[16]deng also having obtained success on perennial crop.This seminar has carried out research for many years to ramie Core Germplasms construction process, and in 2010, has delivered international first piece of report about ramie Core Germplasms structure aspect
[17].But the method for the structure ramie Core Germplasms that we deliver, mainly based on Agronomic characteristic.The method that adopts field investigation kind economical character, the time is long, and expense is high; And ramie is perennial root perennial plant, and life cycle is long, the morphological differences proterties between germ plasm resource is few.Therefore, utilize Agronomic characteristic to build ramie Core Germplasms and have a lot of deficiencies.
Molecule marker is directly on DNA level, genome to be carried out to polymorphic detection, the difference of the polymorphism main manifestations nucleotide sequence of DNA level, for the difference of the nucleotide sequence of any type all useful molecules labeling technique it is detected.Simple repeated sequence (Simple Sequence Repeat, SSR), claims again microsatellite sequence (Microsatellite), be at present application relatively extensively, development rapidly, the technical molecule marker of PCR-based.SSR mark presents following advantage
[18]: (1) SSR distributes extensively in eukaryotic gene group, rich polymorphism; (2) when its product carries out sequencing gel electrophoretic separation, single base resolving power is high, codominant marker's genetic information amount is large; (3) be Mendelian inheritance, have good stability, DNA consumption is few, and technical requirements is low, with low cost, and the repeatability of pcr amplification is high.Therefore, utilize SSR mark to build ramie Core Germplasms and can make up the low and easy deficiency affected by environment of Agronomic characteristic polymorphism.
At present, not yet there is the method for utilizing SSR molecule marker to build ramie Core Germplasms.
Summary of the invention
The object of the invention is intended to found a kind of method that the EST-SSR of utilization molecule marker builds ramie Core Germplasms, and the method can be set up the Core Germplasms of 108 ramee varieties efficiently, reliably, all significant to the preservation of Ramie Germplasms, evaluation, utilization.
Utilize EST-SSR molecule marker to build a method for ramie Core Germplasms, comprise the following steps:
(1) extract each sample Ramie Germplasms DNA;
(2) design ramie EST-SSR primer, carries out the Genomic PCR amplification of ramie;
(3) record the DNA cloning banding pattern of every pair of primer of each sample;
(4) according to DNA cloning banding pattern, build ramie Core Germplasms;
21 pairs of described ramie EST-SSR primer sequences are as follows:
PCR reaction system: 20 μ L,
| Composing system | Final concentration |
| Mg 2+ | 2.0mmol/L |
| Taq?Buffer | 1× |
| dNTP?Mix | 200μmol/L |
| Taq?Enzyme | 1U |
| Primers | 0.25μmol/L |
| DNA | 90ng |
In system, surplus is water.
Pcr amplification program: 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 55 ℃ of renaturation 50sec, 72 ℃ are extended 1min, the rear 72 ℃ of extension 10min of 29 circulations.
Step (4) is that each banding pattern is carried out to assignment numbering, then according to heuristic search and banding pattern maximization principle, builds ramie Core Germplasms.
Described method can build the Core Germplasms of 108 ramee varieties.
Prior art adopts the method for field investigation varietal character, and the time is long, and expense is high; And ramie is perennial root perennial plant, and life cycle is long, the morphological differences proterties between germ plasm resource is few; Therefore, utilize field proterties to build ramie Core Germplasms and have a lot of deficiencies.The present invention utilizes EST-SSR mark to build ramie Core Germplasms can make up the low and easy deficiency affected by environment of field proterties polymorphism, consuming time short, expense is low, overcome the various shortcoming of the method for in the past utilizing the ramie Core Germplasms that Agronomic characteristic builds, and the Core Germplasms building is also more reliable.
Embodiment
Below in conjunction with embodiment, be intended to further illustrate the present invention, and unrestricted the present invention.
Embodiment 1
1 materials and methods
1.1 material
108 parts of Ramie Germplasms resources.Above material is all planted in Ramie Germplasms garden, national Changsha.
1.2 method
1.2.1 the extraction of genomic dna
In ramie garden, national germplasm Changsha, get the tender shoots newly sending of 108 parts of Ramie Germplasms (table 1) plant, utilize a day root test kit to extract DNA.DNA after extraction passes through after electrophoresis detection concentration, calculation sample DNA concentration, and be diluted to desired concn.
1.2.2 design of primers is with synthetic
Utilize SSRhunter in conjunction with manual search, to find the ramie est sequence that contains SSR, the standard of search is: multiplicity >=16 of mononucleotide, multiplicity >=6 of dinucleotides, multiplicity >=4 of trinucleotide, multiplicity >=3 of tetranucleotide, multiplicity >=3 of pentanucleotide.Multiplicity >=2 of Hexanucleotide.For further utilizing ramie EST to set up SSR mark, in the other adjacent sequence of rejecting SSR, be less than after the EST of 20bp, to software Primer5 design ramie SSR primer for remaining EST-SSR.The SSR primer designing is synthetic in Nanjing Jin Site company.
1.2.3SSR-PCR analyze
PCR reaction system: 20 μ L reaction compositions are in Table 2
Table 2PCR reaction composition
| Composing system | Final concentration |
| Mg 2+ | 2.0mmol/L |
| Taq?Buffer | 1× |
| dNTP?Mix | 200μmol/L?each |
| Taq?Enzyme | 1U |
| Primers | 0.25μmol/L?each |
| DNA | 90ng |
Pcr amplification program: 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 55 ℃ of renaturation 50sec, 72 ℃ are extended 1min, the rear 72 ℃ of extension 10min of 29 circulations.Pcr amplification carries out (MJ Research.Inc.) on PTC-200 amplification instrument.
1.2.4PCR amplified production detects
Amplified production carries out 8% polyacrylamide gel electrophoresis, and the method for employing Zhang Jun [19] is carried out silver and dyed.Record banding pattern.
1.2.5SSR the genetic diversity in site
Utilize the genetic diversity in popgene32 computed in software SSR site.
1.2.6 Core Germplasms builds
With filtering out the SSR primer that polymorphism is good, the total DNA sample of the Ramie genome of each germ plasm resource is carried out to pcr amplification.
The different banding patterns of each primer are carried out to assignment.
Adopt heuristic search and germ plasm resource banding pattern maximization principle to build ramie Core Germplasms.
2 results and analysis
The feature of 2.1 ramie EST-SSR primers
320 est sequences downloading from NCBI, have 70 and contain SSR sequence, wherein 6 contain 2 above SSR sequences.The EST-SSR abundant species of ramie, mononucleotide to Hexanucleotide repeats to detect.But the frequency that all types of EST-SSR occurs differs widely.Designed altogether 76 pairs of SSR primers, the advantage repeating unit of designed ramie EST-SSR primer is trinucleotide, mononucleotide and dinucleotides. wherein the shared ratio of trinucleotide primitive is the highest, is 46.1%; Single base primitive proportion takes second place, and is 19.7%; Double alkali yl primitive proportion reaches 15.8%.The shared ratio of four bases, five bases and hexabasic base is lower, between 3.9%-7.9%.
The screening of 2.2 ramie EST-SSR primers
Utilize 76 pairs of EST-SSR primers, first 12 parts of Ramie Germplasms DNA increased, filter out 21 pairs of bands clearly primer for the DNA cloning of 108 parts of germplasms.
The feature of 2.321 pairs of EST-SSR primers
The genetic diversity of table 3SSR primer
| Locus | NA | Obs_Het | Exp_Het |
| b50 | 2 | 0.23 | 0.40 |
| b35 | 3 | 0.72 | 0.53 |
| b38 | 2 | 0.48 | 0.39 |
| b40 | 3 | 0.37 | 0.45 |
| b43 | 2 | 0.40 | 0.32 |
| c03 | 2 | 0.46 | 0.50 |
| c07 | 2 | 0.93 | 0.50 |
| b27 | 2 | 0.35 | 0.45 |
| b28 | 2 | 0.29 | 0.41 |
| b11 | 2 | 0.34 | 0.50 |
| b16 | 2 | 0.46 | 0.46 |
| b24 | 2 | 0.34 | 0.47 |
| b34 | 2 | 0.28 | 0.40 |
| b64 | 2 | 0.24 | 0.21 |
| b65 | 3 | 0.37 | 0.42 |
| c17 | 2 | 0.58 | 0.41 |
| c18 | 3 | 0.67 | 0.64 |
| b53 | 3 | 0.65 | 0.60 |
| b56 | 3 | 0.59 | 0.61 |
| c10 | 3 | 0.51 | 0.64 |
| b57 | 2 | 0.59 | 0.50 |
Utilize popgene32 software, investigated the genetic diversity of 21 pairs of primers.Result shows (table 3), and in 21 pairs of SSR primers, the number of sites of every pair of primer amplification is between 2-3, and wherein 14 pairs of primer amplifications go out 2 sites, and 7 pairs of primer amplifications go out 3 sites.Observation heterozygosity (the H of 21 pairs of primers
o) between 0.23 to 0.93, expectation heterozygosity (H
e) between 0.21 to 0.64.
The structure of 2.4 Core Germplasms
Adopt heuristic search and germ plasm resource banding pattern maximization principle, built 22 parts of Core Germplasms that Ramie Germplasms forms.
2.5 Core Germplasms Genetic diversity evaluations
21 pairs of SSR primers DNA cloning banding pattern number, the shanon exponential sum Nei index in Core Germplasms and former germplasm sees the following form.Result shows, the Core Germplasms DNA cloning banding pattern retention rate 100% of structure, and Core Germplasms has retained all banding patterns of former germplasm.
Calculated respectively the ratio of the shanon exponential sum nei index of Core Germplasms and original germplasm, result shows, in 21 pairs of SSR primers, the shannon index of 3 pairs of SSR primer Core Germplasms is lower than original germplasm, account for 14% of total primer, but difference does not all reach conspicuous level (p<0.05).The nei index of 4 pairs of SSR primer Core Germplasms, lower than original germplasm, accounts for 19% of total primer, and difference does not all reach conspicuous level (p<0.05) yet.Show that the Core Germplasms building has represented the genetic diversity of original germplasm well.
The genetic diversity of table 5 Core Germplasms and former germplasm
3 discuss
Ramie Germplasms resource is the important substance basis of breeding, scientific research, teaching and production, adopts over the years field planting to concentrate and preserves, and this preserving type is difficult to cut off the propagation of disease between each germplasm, can not effectively withstand natural calamities.As the national germplasm ramie garden ramie bacterial wilt of 1963 evil, resource all infects, and finally burns and again builds garden; The Yuanjiang flood disaster of 1996, in garden, more than 1000 part of germplasm lost
[20].Therefore, germplasm is backed up, the risk that germplasm can be preserved drops to minimum.But because ramie resource quantity is large, and can not seminal propagation preserve, can only adopt vegetative propagation to preserve, backup will strengthen retain costs greatly.Core Germplasms is with the minimum diversity that repeats farthest to represent whole germ plasm resource, and Core Germplasms is backed up, and the risk that germplasm can be preserved drops to minimum, and has reduced retain costs.Therefore, structure ramie Core Germplasms is significant.
As the special crop of China, ramie is playing the part of the role of important fibre crops always.In recent years, the purposes of ramie is constantly expanded, and the multifunctional usages such as the pharmaceutical use of ramie, feed purposes, soil conservation, industrial raw material, bioenergy have started to receive publicity.For example, Ministry of Water Resources is asserted southern hills area soil conservation crop ramie
[5]; Ramie leaf has entered the pilot scale stage as animal-feed
[4]; The pharmaceutical use research of ramie leaf extract has obtained important achievement
[2].For genetic improvement and the assignment of genes gene mapping of these new purposes ramies, indispensable as the effect of the germ plasm resource on scientific research basis.But Ramie Germplasms quantity is large, large to the screening operation amount of specific end use.Core Germplasms can represent the genetic diversity of whole germ plasm resource, can improve determination rates.For example, Holbrook
[21]leaf spot resistance to Peanut Core Collection identifies, than using whole Idioplasm identification, efficiency is doubled.Jiang Huifang etc.
[22]set up after Peanut Core Collection, its flavus is infected and produces malicious resistance and analyze, therefrom excavate out aspergillus flavus infection resistance and produce each 8 parts of seed culture of viruses matter, compare with analyzing all peanut resources, improved efficiency.Therefore, build ramie Core Germplasms, can take a firm foundation for the research of the new purposes of ramie.
2010, the Core Germplasms that our seminar utilizes 18 field proterties to carry out 790 portions of ramies built.But because the acquisition of Agronomic characteristic is wasted time and energy, and polymorphism proterties is few, therefore, utilize Agronomic characteristic to there is significant limitation.This research and utilization EST-SSR molecule marker builds ramie Core Germplasms and has more advantage.First, SSR molecule marker polymorphism is high, and every pair of SSR primer can produce 2-9 kind banding pattern; Secondly, SSR labeling technique test operation is simple, and acquisition data are not subject to seasonal restrictions; Finally, easily, repeatability is high for the band statistics of SSR labeling technique amplification.Therefore, utilize EST-SSR labeling technique to build ramie Core Germplasms time saving and energy saving, reliable results.
In the present invention, we have adopted heuristic search to carry out the structure of ramie Core Germplasms.Heuristic search is to state space search method.State space search is that problem solving process is shown as to the process of finding this path from original state to dbjective state.In fact solving of problem be exactly to find the paths can be from starting to result.Heuristic search is exactly that search in state space is assessed the position of each search, obtains best position, then searches for until target from this position.Therefore it is unique, utilizing the Core Germplasms that heuristic search obtains.
After obtain 22 parts of ramie Core Germplasms are evaluated, show, this Core Germplasms has retained all banding patterns of 108 parts of Ramie Germplasms resources; Calculate respectively the shannon index of Core Germplasms and original germplasm and the ratio of nei index is also found, in Core Germplasms, only have the shannon index of 14% and 19% SSR primer and nei index lower than original germplasm, but difference does not all reach conspicuous level (p<0.05).Show that Core Germplasms has represented the genetic diversity of original germplasm well.
5. conclusion
The present invention is the structure for ramie Core Germplasms EST-SSR molecular marking technique first.Result shows, EST-SSR primer form and experimental implementation process simple, easy to operate, the banding pattern that increases is steady and audible, reproducible.Based on molecule marker, build Core Germplasms, with respect to Agronomic characteristic, build Core Germplasms, there is the features such as time saving and energy saving, good stability.The Core Germplasms building based on molecule marker, has guaranteed the maximization of DNA cloning banding pattern, can represent well the genetic diversity of original germplasm, representative strong.All significant to the preservation of Ramie Germplasms resource, evaluation and utilization.
Reference
1. Lu Haoran. Chinese crudefiber crop cultivation. Beijing: agriculture press, 1992
2 hide consolidation, Zhao Lining, Chen Jianhua, Li Yujun. ramie medicinal health value and prospect exploitation simple analysis thereof. and Chinese numb industry, 2002,24:27-29.
3. explain the spring bright, Chen Jianrong, Wang Yanzhou. ramie molecular breeding and feed ramie progress. Chinese numb industry science, 2007,29:389-392.
4. bear is peaceful. present situation and policy and suggestion that China's crudefiber crop is produced. and Chinese numb industry science, 2010,32:301-304.
5. Tu Xiaoning, Chen Shuchun. the effective soil conservation plant-ramie ploughing in southern slope. international sea-buckthorn research and development, 2007,5:45-48.
6. bear is peaceful. crudefiber crop thremmatology. and Beijing: Chinese agriculture science and technology press, 2008.pp46 – 50.
7.Frankel?O?H,Brown?A?H?D.Current?Plant?Genetic?Resources-a?critical?appraisal?In:genetics:New?Frontiers?Vol?IV.New?Delhi,India:Oxford?and?IBH?Publishing,1984.pp1–11.
8.Brown?A?H?D.Core?collection:a?practical?approach?to?genetic?resources?management.Genome,1989,31:818–824.
9.Hao?C?Y,Zhang?X?Y,Wang?L?F,Dong?Y?S,Shang?X?W,Jia?J?Z._Genetic?diversity?and?core?collection?evaluations?in?common?wheat?germplasm?from?the?northwestern?spring?wheat?region?in?china.Mol?Breed,2006,17:69–77.
10. Yao Qi human relations, Fang Ping, Pan Guangtang. based on SSR mark, build the method for Southwest Maize local variety Core Germplasms. Agricultural University Of Hunan's journal natural science edition, 2009,35:225 – 228
11. are loud and clear, Li Zichao, Cao Yongsheng, Qiu Zongen, Yu Ping, Wang Xiangkun. on phenotypic level, check the parameter comparison of Rice Core Germplasm. and Acta Agronomica Sinica, 2003,29:252 – 257.
12.Ming army, Zhang Qixiang, Lan Yanping. plum blossom variety source Core Germplasms builds. Beijing Forestry University's journal, 2005,27:65 – 69.
13Prakash?N?S,Combes?M?C,Dussert?S,Naveen?S,Lashermes?P.Analysis?of?genetic?diversity?in?Indian?robusta?coffee?genepool(Coffea?canephora)in?comparison?with?a?representative?core?collection?using?SSRs?and?AFLPs.Genet?Resour?Crop?Evol,2005,52:333–343.
14. Li Yinxia, An Lijun, Jiang Quan. structure and the evaluation of peach kind Core Germplasms. China Agricultural University's journal, 2007,12 (5): 22 – 28.
15. Wang Xin are super, Liu Zhen, Yao Ming wise man. Chinese tea tree primary core collection Sampling Strategy research. and tea science, 2009,29 (2): 159 – 167.
16. Wei Zhi are firm, Gao Yuchi, Yang Chuanping. the methods of sampling that white birch Core Germplasms builds. and Journal of northeast Forestry university, 2009,37 (7): 1 – 4.
17. Luan Ming are precious, Chen Jianhua, Xu Ying, Wang Xiaofei, Sun Zhimin. ramie Core Germplasms construction process. and Acta Agronomica Sinica, 2010,36:2099-2106.
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Claims (6)
1. utilize EST-SSR molecule marker to build a method for ramie Core Germplasms, it is characterized in that, comprise the following steps:
(1) extract each sample Ramie Germplasms DNA;
(2) utilize 21 pairs of ramie EST-SSR primers of design, the genomic dna of amplification ramie;
(3) record the DNA cloning banding pattern of every pair of primer of each sample;
(4) according to DNA cloning banding pattern, build ramie Core Germplasms;
21 pairs of described ramie EST-SSR primer sequences are as follows:
21 pairs of SSR primer sequences
Primer name
2. the method for utilizing EST-SSR molecule marker to build ramie Core Germplasms according to claim 1, is characterized in that,
PCR reaction system: 20 μ L,
In system, surplus is water.
3. the method for utilizing EST-SSR molecule marker to build ramie Core Germplasms according to claim 1 and 2, is characterized in that,
Pcr amplification program: 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 55 ℃ of renaturation 50sec, 72 ℃ are extended 1min, the rear 72 ℃ of extension 10min of 29 circulations.
4. the method for utilizing EST-SSR molecule marker to build ramie Core Germplasms according to claim 1, is characterized in that,
Step (4) is that each banding pattern is carried out to assignment numbering, then according to heuristic search and banding pattern maximization principle, builds ramie Core Germplasms.
5. the method for utilizing EST-SSR molecule marker to build ramie Core Germplasms according to claim 1, is characterized in that,
Described method can build the Core Germplasms of 108 ramee varieties.
6. utilize EST-SSR molecule marker to build ramie Core Germplasms primer used, it is characterized in that,
21 pairs of SSR primer sequences
Primer name
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104212897A (en) * | 2014-09-05 | 2014-12-17 | 中国农业科学院麻类研究所 | Method for large-scale development of ramie genome SSR (simple sequence repeat) markers and primers developed by method |
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| CN106011135B (en) * | 2016-06-29 | 2019-03-12 | 中国农业科学院麻类研究所 | With the associated SSR marker of ramie stem thickness and its application |
| CN108280327A (en) * | 2018-02-09 | 2018-07-13 | 北京科技大学 | A kind of multifarious warehouse-out method of raising sample database sample |
| CN108280327B (en) * | 2018-02-09 | 2020-12-29 | 北京科技大学 | Ex-warehouse method for improving sample diversity of sample library |
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