CN102174510A - Method for constructing lichi core collection by using EST-SSR (Expresses Sequence Tag-Simple Sequence Repeat) molecular marker - Google Patents
Method for constructing lichi core collection by using EST-SSR (Expresses Sequence Tag-Simple Sequence Repeat) molecular marker Download PDFInfo
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Abstract
The invention discloses a method for constructing lichi core collection, which comprises the following steps: selecting tender lichi leaves, and extracting genome DNAs according to varieties to obtain genome DNA samples of lichi; searching EST-SSR (Expresses Sequence Tag-Simple Sequence Repeat) of the lichi according to an EST sequence of the lichi, and designing an EST-SSR primer; carrying out PCR (Polymerase Chain Reaction) by using the EST-SSR primer to amplify the genome DNAs; carrying out electrophoretic analysis on PCR products to obtain existence data of alleles of the PCR products; and analyzing the obtained data to construct the core collection. In the method disclosed by the invention, the processes of EST-SSR-marked primer formation and experiment operation are simple and are easy to operate, an amplification band is clear, results are stable, and the method is suitable for analyzing genetic diversity among lichi resources, the core collection constructed on the basis almost represents all the genetic diversity of the original germplasm and has better representativeness, and the method has realistic significances for lichi germplasm management, new germplasm collection and germplasm reproduction and renewal and has great importance for promoting the exploration in the utilization of the lichi germplasm resources, so that the utilization efficiency of the lichi germplasm resources is improved.
Description
Technical field
The present invention relates to the construction process of lichee Core Germplasms.
Background technology
Lichee (
Litchi chinensisSonn.) be Sapindaceae (
Sapindaceae) the lichee genus (
Litchi) plant, originate in south China, it is important fruit tree in many tropical and subtropical countries, also be the most ancient one of the cultivating apple trees in the world, be distributed in the torrid zone and the semi-tropical wide area in east, the western hemisphere, China's lichee mainly be distributed in 18 °～24 ° 30 of north latitude ' the tropical and subtropical zone area, main cultivation province is Guangdong, Guangxi, Fujian, Hainan, Taiwan, Yunnan, in addition, in Sichuan, also there is a small amount of plantation (Zhang Zhanwei etc., 1997) in some special miniclimate districts in Guizhou, Zhejiang.The lichee germ plasm resource of various places had been carried out statistics in Wu Shuxian etc. (1996) years and thought that the lichee germplasm that China has found reaches 222 parts, has now increased to more than 300 part (Chen Jiezhen etc. 2003).Wherein, Guangdong lichee cultivated area maximum, variety source is abundant, and Cultivar is also more, and at present, it mainly is to utilize the germ plasm resource garden that China's lichee germ plasm resource is preserved.More than 130 part of germplasms such as wild, the semi-wild on ground such as comprising Guangdong, Guangxi, Fujian, Sichuan, Yunnan and Cultivar have been preserved in the lichee garden of setting up in 1988, Guangzhou up to the present, are maximum in the world lichee germplasm gene pools.
The existing resource preserving type of lichee is single, and various places are almost only adopted and set up the resource garden and carry out field planting and preserve, and resource repeat that to preserve phenomenon more serious; Its protection also is nowhere near, and the genetic diversity of lichee suffers serious destruction (Wang Yan fine jade 2008).In addition, because lichee is the perennial woody plant, the tree body is big, preserves as resource, and floor space is big, along with the increase of genetic resources quantity is in recent years also given preservation, the research of germ plasm resource and utilized and brought very big difficulty.
It promptly is to repeat to represent to greatest extent the genetic diversity of these species with the resource umber of minimum and heredity that Frankel (1984) proposes Core Germplasms (Core collection) the earliest.The structure of Core Germplasms can effectively solve the preservation that repeats of resource, and the difficulty of bringing for the breeding work person greatly owing to resource quantity also can solve thereupon.
Making up of traditional Core Germplasms with agronomy and morphological data, but these phenotypic datas can accurately not react genetic affinity (the Gu et al of colony's inherent heritable variation and storeroom, 2005), compare with phenotypic data, the dna molecular marker data can directly reflect the variation on the dna sequence dna level, are the validity feature data (Li Yinxia etc. 2007) that make up Core Germplasms.
At present, Core Germplasms has become the focus of international genetic resources research, and being used widely on many annual crops such as paddy rice, (Chandra 2002; Amalraj 2006; Juneja 2006; Ronfort 2006; Upadhyaya 2007), on garden crop flowers turmeric (Raamsdonk 2000), plum blossom (bright army 2005) and wax plum (Zhao Bing 2007) etc., report is arranged also, on fruit tree, only on apple (Hokanson 1998), fruit plum (Gao Zhihong etc. 2008), shaddock class (Liu Yong 2006) and peach (Li Yinxia etc. 2007), a small amount of report is arranged.
Present stage, the construction process of shortage lichee Core Germplasms and molecular fingerprint.
Summary of the invention
The object of the present invention is to provide the construction process of lichee Core Germplasms and molecular fingerprint.
The technical solution used in the present invention is:
The construction process of lichee Core Germplasms may further comprise the steps:
1) chooses the tender leaf of lichee, extract its genomic dna, obtain the genome DNA sample of lichee by kind;
2) according to the est sequence of lichee, the EST-SSR of search lichee designs the EST-SSR primer;
3) use the EST-SSR primer to carry out PCR, the genomic dna of amplification lichee;
4) the PCR product is carried out electrophoretic analysis, obtain its allelic data that exist;
5) data that obtain are carried out analyzing and processing, make up Core Germplasms.
Preferably, the amplification program of PCR is: 94 ℃, and 4min; 94 ℃, 1min; 50 ℃, 1min; 72 ℃, 2min; 35 circulations; 72 ℃, 10min; 4 ℃, finish amplification.
In the inventive method, the primer formation and the experimental implementation process of EST-SSR mark are simple, easy to operate, the amplification bands of a spectrum are clear, the result is stable, be suitable for analyzing the genetic diversity between the lichee resource, the Core Germplasms of Gou Jianing has almost been represented the whole genetic diversity of former germplasm on this basis, good representativeness is arranged, the management of lichee germplasm, new germ plasm collection and germplasm breeding renewal had realistic meaning, the more important thing is to have promoted going deep into of lichee germ plasm resource utilization, thus the utilising efficiency of raising lichee germ plasm resource.
Description of drawings
Fig. 1 extracts the allelotrope number that different sample numbers keep under three kinds of similarity factors.
Fig. 2 is the Core Germplasms of structure under three kinds of coefficients and the principal dimension figure of former germplasm.
Fig. 3 is the polymorphism classification of type sketch of 9 EST-SSR marks.
Fig. 4～6 are to use EST-SSR mark EST-21, EST-29, EST-30 96 parts of lichee germ plasm resources to be carried out the electrophoretic analysis figure of PCR product respectively.
Embodiment
Below in conjunction with embodiment, further specify the present invention.
1 materials and methods
1.1 vegetable material:
96 lichee kinds that this experiment is adopted all take from academy of agricultural sciences, Guangdong Province fruit tree research national lichee resource garden, during sampling, select the shoot growth stalwartness, and blade does not have the tender leaf of disease and pest, put into freshness protection package, hold with ice chest then, take back the laboratory after having got rapidly, the Ultralow Temperature Freezer that is placed on-80 ℃ is in store for.
1.2 method
1.2.1 the extraction of DNA
Adopt improved SDS method (Chen Liang etc., 1997) (Chen Liang, Chen Daming, Gao Qikang etc. the tea tree genomic dna is purified and is identified, tea science, 1997,17 (2): 172-176) extract genomic dna.Measure it at 230nm with PERKIN ELMER PTP6 type ultraviolet spectrophotometer, 260nm, the ultra-violet absorption spectrum at 280nm place is according to OD
260Value is calculated the concentration of DNA in the sample, and judges quality and the concentration of DNA in conjunction with 0.8% agarose gel electrophoresis.For the convenience and the unification of final experiment, the DNA of every kind of sample is diluted to about 10ng/ μ L ,-20 ℃ of preservations are standby.
1.2.2 determining of primer design and annealing temperature
Est sequence from lichee cDNA library order-checking gained, use SSRIT (simple sequence repeat identification tool) software online (http://www.gramene.org/db/markers/ssrtool) search EST-SSR, use primer-design software Primer Premier 5.0 design primers, synthetic by Shanghai biotechnology Services Co., Ltd.With reference to the Tm value of different primers, adopt grads PCR to determine corresponding annealing temperature.Select for use the genomic dna of 10 kinds to carry out the polymorphism screening, select the core primers of the primer of polymorphism height, favorable reproducibility as later on whole 96 parts of resources amplifications to expanding the primer that the purpose band.
1.2.3 pcr amplification and electrophoresis detection
25 μ l reaction system: ddH are adopted in this experiment
2O 17.5 μ l, Buffer (contains Mg
2+) 2.5 μ l, dNTPs 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l, each 1 μ l of primer, the amplified reaction program of the lichee SSR-PCR that template DNA 2 μ l. optimize: 94 ℃, 4min; 94 ℃, 1min; 50 ℃, 1min; 72 ℃, 2min; 35cycles; 72 ℃, 10min; 4 ℃, finish amplification.The PCR product detects on 2.5% agarose gel electrophoresis earlier, and then on 8% polyacrylamide gel electrophoresis, silver dyes detection.Take pictures with digital camera at last, preserve.
1.2.4 the statistics of data, processing and analysis
The statistics of data: SSR is a kind of codominant marker, the position and the frequency of bands of a spectrum occur according to its amplified production on running gel, and the allelotrope that the differing materials site is existed is designated as 1, non-existently is designated as 0.
The processing of data and analysis: the equal input computer of the data of all records, utilize NTSYS 2.10e biological software to carry out the similarity data analysis then, similarity factor adopts Dice, SM and Jaccard coefficient to carry out the UPGMA cluster.
1.2.5 the structure of Core Germplasms
Utilize similarity factor Dice, SM and Jaccard estimation genetic distance, carry out the UPGMA cluster analysis with NTSYSpc-2.10e software, repeatedly clustering method (Hu J according to (2000) such as Hu, Zhu J, Xu H M. Methods of constructing core collections by stepwise clustering with three sampling strategies based
1.2.6 the evaluation of Core Germplasms and affirmation
Calculate allelotrope number (A), the various degree of Nei ' s gene (He), shannon information index (Ho) by POPGENE 1.32, carry out t check and calculate Core Germplasms and confirm its representativeness with SPSS 13.0 softwares, adopt cylindrical coordinates axle analytical method to confirm Core Germplasms then with respect to the retention rate of former germplasm.
2 results and analysis
2.1 the characteristics of EST-SSR and polymorphism
From 3391 lichee est sequences, filter out 305 and contain SSR, ratio is 8.99%, is 1.34% and Chen Haimei (2005) obtains ratio on wheat, illustrates in the est sequence of lichee to contain abundant SSR.It is right to have designed 100 pairs of EST-SSR primers altogether according to above SSR sequence, there are 62 pairs amplified production is arranged on lichee, there have the 30 pairs of primers can 96 to expand on the lichee samples to be clear, the band that polymorphism is high, expand altogether and 284 bands, the amplified band of different primers is at 3～18, and average 9.47, wherein have 282 to be the polymorphism band, polymorphic rate is up to 99.30 %.
2.2 analysis of genetic diversity
With the EST-SSR mark 96 parts of lichee resources are increased, its genetic diversity sees Table 1. and finds out from table, the various degree scope 0.186～0.396 of Nei ' s gene of every pair of primer, shannon information index scope 0.318～0.558 illustrates that thus 96 parts of lichee resources have abundant genetic diversity.
The genetic diversity of the following 96 parts of lichee resources of table 1 EST-SRR mark
2.3
Determining of Core Germplasms sampling ratio
This research is carried out cluster with three kinds of similarity factor Dice, SM and Jaccard, adopts the site precedence method to sample then, and the allelotrope number is seen Fig. 1 with the variation of sample number.When extracting 49 samples, the Core Germplasms that DICE coefficient and J coefficient make up has down been lost an allelotrope, and does not lose under the SM coefficient.When the sample number that extracts during less than 19, the allelotrope numbers that three kinds of coefficients keep down obviously reduce, when sample number greater than the time, three kinds of following allelotrope numbers that keep of coefficient reach unanimity, when sample number is 24, lost 3 allelotrope under the SM coefficient, and DICE coefficient and J coefficient 6 have been lost.And according to the maintenance situation of heritable variation amount and allelic variation, the Core Germplasms number is that the 20%-30% of original seed prime number is considered to have representative preferably (Yonezawa 1995) (Yonezawa K, Nomura T, Morishima H. Sampling strategies for use in stratified germplasm collections[A]. In:Hodgkin T, eds.Core Collection of Plant Genetic Resources[C] .Chichester:John Wiley; Sons, 1995,35-53).Take all factors into consideration, it is Core Germplasms that present method adopts 29 samples (ratio 30.2%), lose 3 allelotrope under the SM coefficient, lose 4 allelotrope under DICE coefficient and the J coefficient, the allelotrope number of losing is less, reaches with the minimal sample number to represent the multifarious purpose of former blastogenesis to greatest extent.
Detect and affirmation 2.4 Core Germplasms is representative
The Core Germplasms of three kinds of following structures of similarity factor and the genetic diversity of former germplasm relatively see Table 2, and it has been carried out the t check, as seen from Table 2, the allelotrope number of three kinds of following Core Germplasms that make up of coefficient, the retention rate of various degree of Nei ' s gene and shannon information index is all up to more than 90%, and following three parameters of DICE and J coefficient have all surpassed 99%, as seen these three kinds of Core Germplasms can well be represented former germplasm, CoreSM loses 3 allelotrope altogether in addition, retention rate is 98.9%, the t check shows in 30 pairs of primers, the various degree of Nei ' s gene of EST-12 and EST-26 and shannon information index and former germplasm have significant difference, so CoreSM has kept the genetic diversity of former germplasm 93.3%.CoreDICE has lost 4 allelotrope altogether, retention rate is 98.6%, t checks demonstration, there is not significant difference with former germplasm on the various degree of Nei ' s gene, kept the whole genetic diversity of former germplasm, and shannon information index has kept the hereditary various of former germplasm 97.7%, CoreJ is the same with CoreDICE, therefore three kinds of Core Germplasms can both well be represented the genetic diversity of former germplasm, and the representativeness of CoreDICE and CoreJ is better than CoreSM.In addition, we adopt principal coordinates method that the representativeness of three kinds of Core Germplasms is confirmed to see Fig. 2 again.As seen from the figure, Core Germplasms spreads all over whole coordinate diagram, has guaranteed the representativeness of Core Germplasms.
Three kinds of similarity factors of table 2 make up Core Germplasms and former blastogenesis diversity down relatively
2.5 the checking of determining to reach molecular fingerprint of Core Germplasms
By analyzing, present method has obtained the Core Germplasms of being made up of 29 parts of lichee resources that made up by DICE and J coefficient, and with 9 clear and legible EST-SSR mark EST1 of polymorphism type difference, EST8, EST9, EST16, EST21, EST24, EST25, EST28, EST30(uses A respectively, B, C, D, E, F, G, H, I represents) for basic, the polymorphism classification of type of each mark (is used a respectively, b, c, d, e, f, g, h, i, j, k, l, m, expressions such as n) (see figure 3), the Core Germplasms structure molecular fingerprint that these 29 portions of lichee are formed is verified its diversity and representativeness, found that between these 29 parts of materials tangible difference is arranged, the molecular fingerprint of each part all is unique, illustrates that these 29 parts of Core Germplasms have fully represented the genetic diversity (seeing Table 3) of former germplasm colony (96 parts of germplasms).
The molecular fingerprint of 29 portions of primary election lichee of table 3 Core Germplasms
After using EST-SSR mark EST-21, EST-29, EST-30 that 96 parts of lichee germ plasm resources are carried out pcr amplification, the PCR primer is carried out electrophoretic analysis, shown in Fig. 4～6, the marker among the figure is pBR322 DNA/MspI to its electrophoresis result respectively.
3 discuss
3.1 three kinds of genetic distances are to making up the influence of lichee Core Germplasms
Will analyze mass data in the structure of Core Germplasms, wherein most important cluster analysis can effectively be sorted out the material of similar proterties. and genetic distance directly influences cluster result, and then influence the selection of core material.From document (Hokanson S C, Lamboy W F, Szewc-McFadden A K, McFerson J R.Microsatellite (SSR) variation in a collection of Malus (apple) speciesand hybrids. Euphytica, 2001,18:281-294.; Lima M L A, Garcia A A F, Oliveira K M, Matsuoka S, Arizono H, deSouza Jr C L, de Souza A P. Analysis of genetic similarity detected byAFLP and coefficient of parentage among genotypes of sugar cane (Saccharum spp.). Theoretical and Applied Genetics, 2002,104:30-38.; Yuan Z H, Chen X S, He T M, Feng J R, Feng T, Zhang C Y.Pop μ lation genetic structure in apricot (Prunus armeniaca L.) c μ ltivars revealed by fluorescent-AFLP markers in southern Xinjiang, China. Journal of Genetics and Genomics, 2007,34 (11): learn that 1037-1047.) genetic distance SM, DICE and J are three kinds of the most frequently used methods of cluster.This research can both well be represented the hereditary various of former germplasm with the Core Germplasms that three kinds of genetic distances make up, and principal dimension figure shows that the Core Germplasms that three kinds of genetic distances make up spreads all over whole coordinate diagram, has further confirmed their representativeness.And the situation that CoreDICE and CoreJ make up under three kinds of genetic distances is duplicate, when choosing 29 samples, the allelic retention rate of three kinds of following Core Germplasms that make up of genetic distance is almost as broad as long to be respectively 98.9% and 98.6%, and on various degree of Nei ' s gene and shannon information index, CoreDICE and CoreJ are higher than CoreSM, illustrate that representativeness is better the relative CoreSM with CoreJ of CoreDICE, this (2009) (Zhang Chunyu such as Zhang Chunyu that just in time coincide, Chen Xuesen, Zhang Yanmin etc. adopt molecule marker to make up the method for Xinjiang codlin Core Germplasms. Scientia Agricultura Sinica [J], 2009,42 (2): 597-604) think for the codominance molecule marker, as RFLP and SSR, utilize Nei ﹠amp; Li and Jaccard similarity factor are ideal comparatively, because two materials all do not have a certain banding pattern in a certain position, for all not having this specific allelic variation, and do not mean that they have identical allelic variation in this position.
Exploitation and Core Germplasms that the inventive method applies to the EST-SSR molecule marker lichee molecule marker first make up.The result shows, the primer formation and the experimental implementation process of EST-SSR mark are simple, easy to operate, the amplification bands of a spectrum are clear, the result is stable, be suitable for analyzing the genetic diversity between the lichee resource, the Core Germplasms of Gou Jianing has almost been represented the whole genetic diversity of former germplasm on this basis, good representativeness is arranged, the management of lichee germplasm, new germ plasm collection and germplasm breeding renewal had realistic meaning, the more important thing is to have promoted going deep into of lichee germ plasm resource utilization, thus the utilising efficiency of raising lichee germ plasm resource.
Subordinate list: 30 pairs of EST-SSR primer sequences that use in the inventive method
＜110〉Fruit Tree Inst., Guangdong prov. Academy of Agricultural Sciences
＜120〉utilize the EST-SSR molecule marker to make up the method for lichee Core Germplasms
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Claims (3)
1. the construction process of lichee Core Germplasms may further comprise the steps:
1) chooses the tender leaf of lichee, extract its genomic dna, obtain the genome DNA sample of lichee by kind;
2) according to the est sequence of lichee, the EST-SSR of search lichee designs the EST-SSR primer;
3) use the EST-SSR primer to carry out PCR, the genomic dna of amplification lichee;
4) the PCR product is carried out electrophoretic analysis, obtain its allelic data that exist;
5) data that obtain are carried out analyzing and processing, make up Core Germplasms.
2. the construction process of lichee Core Germplasms according to claim 1 is characterized in that: the amplification program of PCR is: 94 ℃, and 4min; 94 ℃, 1min; 50 ℃, 1min; 72 ℃, 2min; 35 circulations; 72 ℃, 10min; 4 ℃, finish amplification.
3. the construction process of lichee Core Germplasms according to claim 1 is characterized in that: the EST-SSR primer that uses is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:60.
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