Analysis of SSR loci and development of SSR primers in Eucalyptus
Introduction
Simple sequence repeats(SSRs),or microsatellites,are arrays of short motifs 1–6 base pairs long.They are abundantly and uniformly distributed throughoutthe eukaryotic genome and inherited codominantly.They can provide multiple allelic,highly informative content,with good reproducibility and are easy to manipulate(Tautz 1989;Powell et al.1996;Gupta and Varshney 2000).The number of repetitions in SSRs varies greatly,and they are rich in polymorphisms.Because they can be assayed relatively quickly with low technical dif fi culty and cost,SSR markers have found wide application in genetic analyses of Eucalyptus species and genotypes,including for fi ngerprinting,genetic mapping,genetic diversity assessment,genetic structure analyses and molecular marker assisted selection(MAS)(Bradbury et al.2013;Zhang et al.2013;Liu and Xie 2012;Maria et al.2011;Zhou 2011;Kengavanar et al.2011;Shanmugapriya et al.2011;He 2010;Wang 2009).
SSR markers are generally classi fi ed into two categories:genomic SSRs(gSSR)and expressed sequence tag SSRs,(EST-SSRs or eSSRs).Genomic SSRs are based on the genome sequences,and EST-SSRs exist in the expressed gene sequences(Xu et al.2014;Zhang et al.2011).EST-SSRs have a higher transferability among closely related species(Varshney et al.2005),and the methods for their development are relatively simple and of lower cost in comparison to genomic SSRs.The latter come from the noncoding sequences in the genome,and they have higher degrees of polymorphism.With the dis coveriesofcontrolfunctions ofsome ‘noncoding sequences’,gSSRs have signi fi cant potential to be linked to genomic sequences in fl uencing phenotypic traits of importance.
在电子对抗阶段，因为借助于软件环境，准备阶段的工作量不是很大。在实施过程中，由5人组队，组建在同一市场环境下竞争的企业，分别担任公司的总经理、人力资源总监、财务总监、销售总监、研发总监；比赛正式开始前两天进行两期的指引培训，使参赛选手熟悉流程；之后进入正式比赛，根据主持人的指示依次完成每一步的决策；每月为一期，进行2-4轮后最终确定比赛结果。
At present,the public database GenBank(http://www.ncbi.nlm.nih.gov/genbank/)has published a large number of Eucalyptus genomes,EST sequences of Eucalyptus,and the complete genome sequence of Eucalyptus grandis(http://www.phytozome.net/cgi-bin/gbrowse/eucalyptus/)(Myburg et al.2014;DOE 2008).This database provides direct access to genome sequences and EST sequences that can be used to develop Eucalyptus SSR markers.Over the past 20 years,many articles about development of SSR markers of Eucalyptus have been published worldwide.For example,Steane et al.(2001)developed 12 E.globulus microsatellite loci for fi ngerprinting and future studies in genome mapping,gene fl ow and genetic diversity;Wang(2009)developed 185 pairs of EST-SSR primers to construct genetic maps in Eucalyptus;He(2010)developed 206 EST-SSR markers and used 90 genomic-SSR markers to evaluate genetic variation among 20 different genotypes of Eucalyptus;Zhou(2011)developed 295 EST-SSR markers based on pool-cloning sequencing of PCR products;and Kengavanar et al.(2011)developed 179 orthologous genic SSR markers in E.camaldulensis.
In contrast,comprehensive studies about developing genomic SSRs and EST SSRs have rarely been published.Yang et al.(2011)developed 37 pairs of SSR primers based on an EST library and 95 pairs based on a genomic library;Zhang et al.(2011)explored genetic differences between genomic SSRs and EST SSRs in 15 species of Poplar with 48 pairs of genomic SSR primers and 48 pairs of EST SSR primers;Ding et al.(2015)explored genetic differences between genomic SSR and EST SSR in 12 species of Stylosanthes using 20 pairs of genomic SSR primers and 20 pairs of EST SSR primers;and Wen et al.(2010)developed 20 genomic SSRs and 36 EST SSRs to analyze the genetic diversity among 45 Jatropha curcas accessions.
The research reported here was aimed at developing genomic SSRs and EST SSRs of Eucalyptus by detecting and analyzing SSR and EST sequences in the genomes of Eucalyptus species and screening the effectiveness of primers.PhyML3.0 software was then used to construct a maximum likelihood phylogentic tree for six accessions of eucalypts,represented by fi ve species of the genus Eucalyptus and one of the genus Corymbia.The results will provide resources and theoretical foundations for evaluating the genetic diversity and phylogenetics of eucalypts by SSR markers.
2.2 两组患者裸眼视力比较 治疗前，两组患者裸眼视力比较，差异无统计学意义(P>0.05)；治疗后，两组患者的裸眼视力高于本组治疗前(P<0.05)，且B组高于A组(P<0.05)。见表2。
Statistics on the 45,257 DNA sequences from Eucalyptus downloaded from GenBank are presented in Table 2.The genomic sequences included 13,373 from E.camaldulensis,1206 from E.globulus,5751 from E.grandis,7803 from E.gunnii,265 from E.nitens and 293 from E.urophylla.The EST sequences included 588 from E.globulus and 16,008 from E.grandis.After removing redundancies,14,121 contigs were obtained and 1785 effective sequences detected that contained SSRs,accounting for 12.6% of the total contigs.The effective Eucalyptus sequences obtained consisted of820 EST sequencesand 965 genome sequences.
Materials and methods
Plant material and DNA isolation
DNA samples were obtained from fi ve species of Eucalyptus subgenus Symphyomyrtus and one species of Corymbia(Table 1).The taxonomy of samples was based on the classi fi cations of Pryor and Johnson(1971)and Hill and Johnson(1995).
TotalgenomicDNAwasextractedfromfreshleaveswith DNeasy Plant Mini Kit(QIAGEN,Germany).The quantity of DNA was checked on 1.5%agrose gels and determined using a Nanodrop nucleic acid-protein analyzer.The DNA templates constituted a genomic DNA pool,obtained by combining the DNA from the fi ve accessions of Eucalyptus species and one Corymbia species.
SSR mining and primer design
A total of 45,557 sequences of Eucalyptus were obtained from GenBank(http://www.ncbi.nlm.nih.gov/genbank/),including 28,691 genome sequences and 16,566 EST sequences of Eucalyptus.These sequences were checked to remove redundancies,and then assembled and clustered using DNAStar 7.1 software(http://www.dnastar.com/).Next,the sequences were searched for the presence of SSR repeats using SSRHunter 1.03 software(http://en.bio-soft.net/dna/SSRHunter.html).For SSR identi fi cation,a criterion of a minimum length of 18 bases was adopted.
PCR primer pairs fl anking the SSR repeats were designed using softwarePrimer5.0 (http://www.pre mierbiosoft.com/).For designing PCR primers,the length ranged from 18-to 25-mer,and the optimum annealing temperature ranged from 50 to 60°C,the optimum GC content was 40–60%,and the rest of the parameters were set at default values.And these primers were checked with Oligo 6.0 software(http://oligo.net/).The primers were then synthesized by Invitrogen Trading(Shanghai).
SSR analysis
The SSRs were classi fi ed based on the length of the SSR motifs in their sequences.The different repeats of the SSR motifs and the frequencies of SSR motifs were used to analyse the characteristics of the SSRs.The types of SSR motifs were analysed to evaluate the speci fi city of SSRs in Eucalyptus.
SSR screening
To obtain SSR-PCR products with higher speci fi cities and more stable rates,the SSR-PCR conditions for primer screening were optimized by adjusting Mg2+concentration and the annealing temperature(Tm).The PCR system and PCR program used followed methodologies described by Li et al.(2010):Mg2+concentration was set to 1.5,2.0,2.5 mmol L-1,and annealing temperature(Tm)were set to 50,56 and 60°C.After the optimum Mg2+concentration and annealing temperature were determined,effective primers that yielded good PCR results were reserved at each round,and ineffective primers that did not yield ideal products were discarded.
Previous research has shown that the formation of SSR loci might be associated with DNA replication slippage,alternation of nucleic acids and unbalanced recombination(Tóth et al.2000;Ma et al.2015).The combinations of CA,GA,and GT in SSR repeat motifs could affect DNA recombination by impacting the DNA structure(Biet et al.1999).Therefore,the composition of nucleotides in SSRs can affect the activities of life,and hence analyses of the structure of motifs and distributions of SSRs can be of great importance.In this current study,the repeat motifs of AG/TC and GA/CT accounted for the vast majority(94.3%)of dinucleotide repeat motifs,and CCG/CGG repeat motifs were the most common(16.4%)trinucleotide repeat motifs.These results are in perfect agreement with the results of other studies in Eucalyptus(Zhou 2011;Yasodha et al.2008).
Table 1 Information on Eucalyptus and Corymbia species used in this study
All sampled trees were in Zhanjiang,Guangdong
Code Species Genus Subgenus Section Location of trees 1 E.tereticornis Eucalyptus Symphyomyrtus Exsertaria South China Experimental Nursery 2 E.grandis Eucalyptus Symphyomyrtus Transversaria South China Experimental Nursery 3 E.urophylla Eucalyptus Symphyomyrtus Transversaria Leizhou trial forest 4 E.camaldulensis Eucalyptus Symphyomyrtus Exsertaria Leizhou trial forest 5 C.citriodora Corymbia Ochraria Leizhou trial forest 6 E.pellita Eucalyptus Symphyomyrtus Transversaria South China Experimental Nursery,Zhanjiang,Guangdong
Statistical analyses
Data for length of SSRs,the numbers of repeats,the frequencies of SSRs,and the SSRs types,were collated and statistics analyzed using Excel 2010 software(Microsoft,WA,USA).
源流各级水行政主管部门要在当地政府的大力支持和帮助下，成立水事务管理局，对当地的水行政事务实行一体化管理。水务一体化管理，是实现水资源优化管理和可持续利用的前提，为水资源的合理开发、高效利用和有效保护提供了体制保证。水务管理部门作为政府水行政工作的职能部门，要突破部门利益的束缚，从政府的角度而不是部门的角度实现对区域内一切水事行为的集中统一管理。水务管理部门要实行政企分开、政事分开，不直接进行水的经营和水企业的管理，而是通过政策法规的制定对水企业进行监督管理。
Phylogenetic analyses
Phylogenetic analyses to examine the genetic relationships of the fi ve Eucalyptus and one Corymbia species were conducted using maximum likelihood and 1000 times bootstrapping was used to statistically support the groups using PhyML 3.0 software.The SSR-PCR sequences of six species were presented using the tree- fi gure drawing tool FigTree version 3.0 from the output fi le of PhyML 3.0.Information on the morphological features of Eucalyptus in China is from Qi(2002)and was compared with the results of phylogenetic analyses.
Results
accounted for only 3.2 and 2.8%,respectively.These results agreed closely with those of He(2010),Li(2010)and Zhou(2011),who also examined frequencies of SSRs in EST sequences of Eucalyptus.
纵观陈凯歌导演“以士为影”的创作历程，我们会发现，对人文精神的追求是其不变的内核。不管是早期农村题材、影像美学、寓言手法，还是后期城市题材，创作者从未停止过对人们存在的思考以及对我们所处世界的探寻。
The frequencies of different nucleotide repeat motifs in the SSRs contained within the Eucalyptus DNA sequences obtained from the GenBank database are shown in Fig.1.Frequencies of motifs varied greatly;dinucleotide SSRs were the most common(44.5% of total),followed by trinucleotide SSRs(32.9%).The proportion of tetranucleotide and pentanucleotide SSRs were relatively low(3.2 and 2.8% of total,respectively).
In the analysis conducted with SSRHunter software,435 SSR repeat motifs were found among the 2292 SSRs obtained.Of these SSR repeat motifs,12 were dinucleotides,50 were trinucleotides,51 were tetranucleotides,54 were pentanucleotides and 268 were hexanucleotides.
Among the repeat motifs,CT/GA,TC/AG repeat motifs were the most common of the di-nucleotide types,occurring in 21.3 and 20.7% of SSRs,respectively.The same repeat motifs also accounted for 94.3% of all the dinucleotide repeat motifs obtained.In contrast,CG/GC repeat motifs accounted for only 0.4% of the dinucleotide repeat motifs.The CGG/GCC,CCG/CGG repeat motifs were the highest frequency trinucleotide repeat motifs,and occurred in 4.3%,3.9% of the SSRs,respectively.
Table 2 Statistics for SSRs generated from Eucalyptus species
EST sequences Genome sequences E.urophylla Total number of sequences downloaded Total E.grandis E.globulus E.grandis E.globulus E.camaldulensis E.nitens E.gunnii 16,008 558 5751 1206 13,373 265 7803 293 45,257 Contigs 7719 523 1824 266 153 33 3516 87 14,121 Total no.of detected SSRs 796 24 630 51 47 3 181 53 1785 Dinucleotides 286 10 506 28 48 3 80 60 1021 Trinucleotides 405 7 244 31 7 0 58 3 755 Tetranucleotides 30 1 32 1 0 0 8 1 73 Pentanucleotides 25 0 30 3 1 0 6 0 65 Hexanucleotides 199 7 119 4 5 0 43 1 378
Among the genome sequences and EST sequences of Eucalyptus,the repeat numbers varied from 3 to 50.Repeat numbers of 3–9 were predominant in the SSRs,totalling 1382 or 60.3% of the total SSRs.Repeat numbers of 10–20 accounted for 768 or 33.5% of the total number of SSRs,repeat numbers of 21–30 accounted for 130 or 5.7% of SSRs,whilst repeat number of 31–40 and more than 40 were accounted for just 10(0.4%)and 2(0.1%)of the SSRs,respectively(Fig.2).From the SSR repeat motifs analyzed,532 or 52.1% of the di-nucleotide repeat motifs occurred between repeat numbers of 10–15.Of the other repeat motifs,most were concentrated in among the 3–9 motif repeats,including 691 or 91.5% of trinucleotide repeat motifs,72 or 98.6%tetranucleotide repeat motifs,65 or 100% of pentanucleotide repeat motifs and 376 or 99.5% of hexanucleotide repeat motifs.
Fig.1 Frequencies of different nucleotide repeat motifs in the SSRs
The DNA templates were from a mixed genomic DNA pool obtained by combining the DNA from the fi ve Eucalyptus and one Corymbia species sampled(see Table 1).After nine rounds of screening by optimizing the PCR conditions,340 pairs of primers successfully ampli fi ed the target fragments with a success ratio up to 86.1%;136 pairs of effective primers were screened from 150 pairs of eSSR primers,with a success ratio of 90.7%;204 pairs of effective primers were screened from 245 pairs of gSSR primers,with a success ratio of 83.3%.Some of the results from screening partial eSSR and gSSR primers are shown in Figs.3 and 4 respectively.
Fig.2 Frequency distributions of SSR repeats
The number of SSRs in repeat motifs of different nucleotide lengths seemed to be negatively correlated with the length of the repeat motifs;the SSRs with longer lengths of nucleotide repeat motifs had lower frequencies ofnucleotide repeatmotifs in Eucalyptus genome sequences.However,the frequencies of tri-nucleotide repeat motifs were higher than other nucleotide repeat motifs in EST sequences of Eucalyptus.
In Table 4,the 970 SSRs found in the Eucalyptus EST sequences accounted ig for 42.3% of the total SSRs,and 1322SSRswerefoundintheEucalyptusgenome sequences,accounting for 57.7% of the total SSRs.The number of dinucleotide,tetranucleotide and pentanucleotide repeat motifs found in the Eucalyptus genome sequencesexceededthenumberfoundintheEST sequences of Eucalyptus.In contrast,more tri-and hexanucleotide repeat motifs were found in Eucalyptus EST sequences than in Eucalyptus genome sequences.Dinucleotide repeat motifs were predominant in Eucalyptus genome sequences,accounting for 54.8% of total SSRs,and the frequency of trinucleotide repeat motifs was highestamong the EST sequences ofEucalyptus,accounting for 42.5% of the total SSRs.
Table 3 Information on SSR motifs in Eucalyptus
Types of repeat No. Maximum repetitions Average repetition Average no.bp Major repetition motifs Dinucleotide 1021 48 15.02 30 AG/TC,CT/GA,AC/TG,CA/GT,AT/TA,CG/GC Trinucleotide 755 29 7.51 23 CCG/GGC,CGG/GCC,CGC/GCG,CTT/GAA,CTC/GAG,AGG/TCC,AAG/TTC,CAG/GTC Tetranucleotide 73 16 5.72 23 CGAG/GCTC,TTCT/AAGA,CTCC/GAGG,GCGA/CGCT,TCCT/AGGA Pentanucleotide 65 6 4.31 22 GAGGA,AGCCG,TCGGT,TCTCC,TTATT,TTTTC Hexanucleotide 378 16 3.24 19 ATTTTT,CGCCGT,CGGCGA,CGGCTC Total 2292 23
Table 4 Characterization of SSRs in Eucalyptus genome sequences and EST sequences of Eucalyptus
Type EST sequences Genome sequences No.of repeats No.(%) Maximum repetition Mean repetition No.(%) Maximum repetition Mean repetition Dinucleotide 296(30.52) 20 11.89 725(54.84) 48 16.278 12 CT/GA 143 345 TC/AG 171 304 AC/TG 0 49 Trinucleotide 412(42.47) 13 7.21 343(25.94) 29 7.9525 50 CCG/GGC 25 65 CGG/GCC 40 59 GAA/CTT 30 43 Tetranucleotide 31(3.20) 8 5.185 42(4.33) 16 6.083 51 TTCT/AAGA 5 4 CGAG/GCTC 10 0 CTCC/GAGG 4 1 Pentanucleotide 25(2.58) 6 4.24 40(3.02) 6 4.333 54 TCTCC/AGAGG 2 1 AGCCG/TCGGC 2 1 Hexanucleotide 206(21.24) 9 3.245 172(13.01) 16 3.238 268 CGCCGT 3 2 CGGCGA 2 4 ATTTTT 1 5 Total 970 1322 435
SSR primer design
After primer design and veri fi cation,a total of 395 SSR primers were synthesized for use in subsequent analyses.These included 150 pairs of EST-SSR(eSSR)primers,of which nine pairs were designed using EST sequences of E.globulus,and 141 pairs were designed using EST sequences of E.grandis.The 245 pairs of genomic-SSR(gSSR)designed included 168 pairs,which were designed using genome sequences of E.grandis,13 pairs designed using genome sequences of E.globulus,17 pairs designed using genome sequences of E.camaldulensis,36 pairs designed using genome sequences of E.gunnii,and 11 pairs designed using genome sequences of E.urophylla.The length of primers varied from 18 to 22 bp,and the length of target fragments varied from 200 to 500 bp.Selection details for these SSR primers are presented in Table 5.
稻鸭共育是以水田为基础，在水稻田进行家鸭野养的一项生态农业技术，具有除草吃虫、增肥控蘖、提高品质、增加效益、减少污染等优点，是目前较为理想的优质高效技术之一。同时，稻鸭共育技术的实施及示范减少了农药和化肥用量，保护了环境，提高了农民的收入，也满足消费者对绿色食品、无公害农产品的需求。为有效控制水稻虫害，提高水稻害虫的控制水平，减少农药在稻米和环境中的残留，保障稻米质量安全，于2007～2011年推广实施稻鸭共育技术，取得了显著的社会、经济和生态效益。
Screening for effective SSR primers of Eucalyptus
The characterization of SSRs in genome sequences and EST sequences of Eucalyptus are presented in Table 3.Dinucleotide repeat motifs occurred at higher frequency in the genome sequences(48 repeat motifs),than in EST sequences(20 repeat motifs)of Eucalyptus.In contrast,the frequency of trinucleotide repeat motifs was higher in the Eucalyptus genome sequences(29 repeat motifs)than in the EST sequences(13 repeat motifs),as were the frequencies of both tetranucleotide and hexanucleotide repeat motifs.
Results from primer screening are presented in Table 6.From among the nine rounds of primer screening,an annealing temperature of 56°C with 2.0 or 2.5 mmol L-1 Mg2+of provided the highest success ratios.The maximum eSSR primer success ratio was 56.0%,achieved with an annealing temperature of 56°C and 2.5 mmol L-1Mg2+,while the maximum gSSR primer success ratio was 51.4%,which was achieved at the same annealing temperature but with 2.0 mmol L-1Mg2+.
Phylogenetic analysis using SSR-PCR sequences
To assess the genetic relationships of the six accessions( fi ve Eucalyptus and one Corymbia species),data from fi ve pairs of effective SSR primers that had good stability,strong signals and high polymorphisms were analysed.This analysis generated a maximum likelihood phylogenetic tree based on fi ve combined ampliconic sequences of the six species;see Fig.5.
The results of combined-ML phylogenetic analyses revealed that C.citriodora had a greater genetic distance from the other fi ve species,consistent with morphological taxonomy.Based on this analysis,the kinship of E.camaldulensis and E.pellita is somewhat closer,while E.tereticornis and E.urophylla had the closest genetic relationship of all the species examined.These results differed somewhat from what was expected based on the taxonomy of these species described by both Pryor and Johnson(1971)and Hill and Johnson(1995).
Further analyses using the six pairs of SSR primers in the fi ve Eucalyptus species(the primer of gSSR-GU023 had no ampliconic sequence in C.citriodora)examined the genetic relationships among only species of subgenus Symphyomyrtus(Fig.6).The combined ML phylogenetic tree of these species shows that E.pellita,E.camaldulensis and E.grandis had a relatively close genetic relationship,while the shortest genetic distance with this group of fi ve species was between E.tereticornis and E.urophylla.This latter result concurs with the combined ML phylogenetic tree developed from data using the fi ve pairs of SSR primers for the six eucalypt species(Fig.5).
Table 5 Basic information for fi ve pairs of SSR primers
Code Forward primer sequence/Reverse primer sequence(5′–3′) Length of target fragment(bp) SSR type eSSR-GR046 AAACAACCCGAACGAAAGAA/GGAGGTCCTACTACACCCACAA 170 (GGACCG)4 eSSR-GR124 ATTGATTCACGGACACGG/GAACGCAATCCCACCTAA 279 (GTT)8 eSSR-GR127 CGCTGTCCATCGTGTATTTG/TGACGCTGTCGGTGAAACT 371 (GCAGCG)3 gSSR-CA013 AGATGGGACGGACGAGGAT/GGTGGTGCTGGGAAGAAT 121 (CT)10 gSSR-GU023 GAGCCTGTTACAAATGGA/TCACAGCAGTCGGTCTTT 316 (AGAAAA)3
Discussion
Characterization of SSRs in Eucalyptus
EST-SSR primers come from the expressed gene conservative region(Ding et al.2015).Numerous studies have shown that polymorphisms in EST-SSR primers are lower than in genomic SSR primers(Qi et al.2009),that EST sequences tend to be more conservative than genome sequences,and that EST-SSR primers are better than genomic SSR primers in transferability across species(Qi et al.2009;Yang et al.2011;Xu et al.2014;Zhang et al.2011).In this current study,trinucleotide repeat motifs(42.5%)were more frequent than the other nucleotide repeat motifs in EST sequences of Eucalyptus,while in genomic sequences dinucleotide repeat motifs were more frequent(54.8%).This result concurs with fi ndings of Li(2010)on the content of microsatellites in EST sequences of Eucalyptus.Trinucleotide repeat motifsis where excessive enrichment might occur because the genetic code only allows triplet repeats to have mutations.
[3] Jerel A. Rosati, The Carter administration’s quest for global community: beliefs and their impact on behavior, South Carolina: University of South Carolina Press, 1987, p. 16.
Dinucleotide repeat motifs are generally the most common types of repeat motifs in dicotyledons,and trinucleotide repeat motifs are most common in graminaceous plants(Biet et al.1999).In this current study,dinucleotide and trinucleotide repeat motifs were found to be the most frequent in Eucalyptus, accounting for 44.4 and 32.8%,respectively,ofthe totalrepeatmotifs.The tetranucleotide and pentanucleotide repeat motifs
Characterization of SSRs in Eucalyptus genome sequences and EST sequences of Eucalyptus
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Table 6 Results of primer screening
Screening turns Mg2+(mmol L-1) Temperature°C Successful ampli fi cation(pairs) Success ratio in ampli fi cation(%)Genomic-SSR EST-SSR Genomic-SSR EST-SSR 1 2.0 56 126 22 51.4 14.7 2 50 8 2 3.3 1.3 3 60 22 4 9.0 2.7 4 1.5 56 2 12 0.8 8.0 5 50 6 10 2.4 6.7 6 60 25 1 10.2 0.7 7 2.5 56 6 84 2.4 56.0 8 50 5 1 2.0 0.7 9 60 4 0 1.6 0 Total 204 136 83.3 90.7
Fig.3 Fast screening result of partial eSSR primers.Lanes 1,2,3,4,7,8,10,11,12,14,15,16,18,20 lane have bands that indicate successfully screens of primers
Fig.4 Fast screening result of partial gSSR primers.Lanes 2,5,6,9,11,13,15,17,18,20 have bands that indicate successful screens of primers
Fig.5 Combined ML phylogenetic analysis of Eucalyptus(including 1 Corymbia species)using 5 combined sequences.Numbers on branch points represent the percentage of 1000 bootstraps by heuristic searching
Fig.6 Combined ML phylogenetic analysis of the fi ve Eucalyptus species using the six combined sequences.Numbers on branch points represent the percentage of 1000 bootstraps by heuristic searching
PCR was performed in a volume of 20 μL,containing genomic DNA(about 100 ng),0.1 μM of each primer,Taq DNA polymerase(2 U),0.2 mM of each dNTP,MgCl2(1.5,2.0 or 2.5 mM).The PCR pro fi le consisted of denaturing the template DNA at 94°C for 4 min.,followed by 35 cycles,each at 94 °C for 30 s,50,56 or 60 °C for 30 s,and 72 °C for 1 min,followed by 72 °C for 10 min.The PCR products were separated by electrophoresis in 1.5%agarose.
Among 14,141 contigs assembled from 45,257 genome and EST sequences of Eucalyptus,1785 SSRs(12.6%)were detected.This frequency is consistent with results obtained by Ellis and Burke(2007)and Yasodha et al.(2008)who obtained frequencies of 12.3 and 12.9%respectively.However,He(2010)reported a somewhat higher frequency of SSRs,25.3%,in EST sequences of Eucalyptus,and Zhou(2011)reported a frequency of SSRs of 21.7%in 36,029 unigenes assembled from EST sequences of Eucalyptus.Similarly,Rabello et al.(2005)and Ceresini et al.(2005)reported frequencies of SSRs in Eucalyptus of 25.5 and 25.6%,respectively.
Analyses of the data on the maximum repetition of SSRs in this study revealed that the repetition of di-,tri-,tetraand hexanucleotide repeat motifs was greater in genome sequences than in EST sequences of Eucalyptus.Pentanucleotide repeat motifs were an exception,as the repetitions of these in EST sequences equaled that in genomic sequences.The average repetitions of di-,tri-,tetra-and pentanucleotide repeat motifs in genomic sequences were higher than in EST sequences of Eucalyptus,but the average repetitions of hexanucleotide repeat motifs in genomic sequences was equal to that in EST sequences of Eucalyptus.These results clearly indicate that the length and polymorphism of SSRs in genomic sequences were superior to those in EST sequences of Eucalyptus.
In molecular genetics,development and utilization of molecular markers to diagnose and detect DNA polymorphisms and analyze genetic diversity have valuable applications in understanding genetic relationships,accelerating breeding and facilitating genetic improvement(Zhou 2011).SSR molecular marker technology is based on PCR,and for this,the operability of the assembly sequences must be considered in the design of primers(Yang et al.2011).The research of Temnykh et al.(2001)showed that when the SSRs of lengths greater than or equal to 20 bp tend to have a higher degree polymorphism,SSRs of lengths between 12 and 20 bp tend to have a medium degree of polymorphism,and SSRs of lengths less than 12 bp tend to have extremely low degree of polymorphism.To ensure a high degree of polymorphism in SSR primers in this current study,selection criteria for SSRs included a minimum length of 18 bp.The lengths of the 395 SSR primers used in this current study were 18–22 bp,and the lengths of their target sequences were 200–500 bp.Those lengths can guarantee the quantity of target sequences and the fi delity of SSR primers,both sides of SSR loci must have a certain length of sequences,and it would be more convenient to select the parameters of SSR primers,as an appropriate primer length,GC content,annealing temperature,and target sequence length(Temnykh et al.2001;Picoult-Newberg et al.1999).
The screening of SSR primers
To evaluate the effectiveness of SSR primers of Eucalyptus,we screened 150 pairs of eSSR primers and 245 pairs of gSSR primers.After nine rounds of screening to optimize PCR conditions,340 pairs of primers were screened and found to be effective primers.Among the 340 pairs of primers,136 pairs were eSSR primers,and 204 pairs were gSSR primers;the overall success ratio was 86.1%.This result demonstrated that the SSR primers had good transferability and that one SSR marker could probably be shared among species with a close genetic relationship.This conclusion is agrees with results reported by Cupertino et al.(2011)who found that genomic SSR and EST SSR had no signi fi cant differences among 112 hybrid taxa of Eucalyptus.Ellis and Burke(2007)reported that the length of SSRs and their stability varied between different species and between different SSR loci within the one species.Ellis showed that gSSRs had higher polymorphism than eSSRs,and the transferability of gSSRs was worse than eSSRs.In this current study,the success ratio of screening for eSSRs(90.7%)was somewhat higher than thatforgSSRs (83.3%);a resultthatmightbe attributable to the EST sequences accounting for 57.7% of the genome sequences in this study.
Conclusion
By detecting the SSRs in both genome and EST sequences of Eucalyptus,this study obtained 970 SSRs in EST sequences and 1322 SSRs in genome sequences.The software Primer 5.0 designed 150 pairs of eSSR primers and 245 pairs of gSSR primers.PCR reaction used a pool of genomic DNA,obtained from six Eucalyptus species,as a DNA template.PCR conditions were optimized and used to obtain 136 pairs of eSSR primers and 204 pairs of gSSR primers from the screening of 395 pairs of SSR primers,providing screening success ratios of 90.7 and 83.3%,respectively.The 340 pairs of SSR primers developed in the study along with insights on SSRs provide important resources for future studies on genetic diversity,phylogenetics and other genetic aspects in Eucalyptus.
The classi fi cation of Eucalyptus by Pryor and Johnson(1971)and Hill and Johnson(1995)acknowledges C.citriodora as belonging to a separate genus,Corymbia,and that E.tereticornis,E.grandis,E.urophylla,E.camaldulensis and E.pellita belonged to the genus Eucalyptus.Their classi fi cations also place E.tereticornis and E.camaldulensis into the subgeneric taxon known as section Exsertaria,whilst E.grandis,E.urophylla and E.pellita are placed into section Latoangulatae.Within these sections,they placed E.grandis into series Salignae,and E.urophylla and E.pellita into series Resiniferinae.In the combined ML phylogenetic analyses in the current study,the results at the genus level were consistent with thothe classi fi cations of Pryor and Johnson(1971)and Hill and Johnson(1995).
However,the genetic relationships among the fi ve Eucalyptus revealed by analyses conducted in this current study differed somewhat from those suggested by Pryor and Johnson(1971)and Hill and Johnson(1995).The phylogenetic trees obtained in this study indicate that E.camaldulensis and E.grandis have a close genetic relationship even though traditional taxonomy placed them in different subgeneric sections,Exsertaria and Latoangulatae respectively.And the same status applied to E.tereticornis and E.urophylla.
从灌浆资料统计结果来看，各次序孔的单位注入量和透水率有依次递减的规律。水泥注入总量和单位注入量依次减少，透水率也依次减少。从各个灌浆孔的单位吸水率区间来看，符合灌浆施工的一般规律。从主副坝30个检查孔（主坝 15个，副坝15个）的122段压注水试验成果看，坝土渗透系数 6.84×10-6cm/s～6.47×10-7cm/s， 基岩透水率 2.86～8.81 Lu，均满足设计要求，符合规范规定。
在扫描区域布设标靶，采用无棱镜全站仪测量标靶具体坐标，将扫描数据坐标转换至工程独立坐标中。为了提高扫描数据坐标转换精度，应在测区周围均匀布设四个及以上的反射片标靶。这些反射片标靶应避免处于同一面上或同一条线中。为了防止影响地面三维激光扫描仪扫描及后期拼接精度，反射片标靶的布设不能太远，一般距离保持在30 m左右为宜。
In the analysis of the morphological features of six eucalypt species,the features of young leaves and mature leaves of E.pellita were similar to those of E.camaldulensis:3–4 pairs of young leaves opposite,ovoid to lanceolate,and mature leaves alternate,lanceolate.Species in section Latoangulatae have mature leaves,upper and lower sides concolorous;juvenile leaves on seedlings are sessile.Zhang et al.(2010)analyzed the genetic diversity of four species of Eucalyptus and illustrated that genetic similarity of E.camaldulensis and E.grandis was greater,in agreement with the phylogenetic trees in the present study.Thus,the reliability of classi fi cation schemes based on foliar features or petiole features need further study.
Acknowledgements This research was funded by the Special Fund for Forestry Scienti fi c Research in the Public Interest(201504204).The authors are indebted to numerous persons who provided help for this study,in particular Roger Arnold,Luo Jianzhong,Wu Zhihua,and the forestry administration of Leizhou and South China Experimental Nursery who provided Eucalyptus samples for this study.
3.异议审查的回应机制。如果说赋予公民异议审查申请的权利，但没有规定审查机关的回应义务，那么异议审查机制就只能停留在纸上，成为有异议不回答的虚置性制度。为此，要建立异议审查回应机制。明确审查机关对地方规范性文件异议的具体回应，包括合法性和合目的性的答复。对于减少政府自身职责、剥夺公民平等权和发展权内容的地方规范性文件，审查机构经同级人民政府批准可以撤销或者纠正、废止。
目前临床对于肩袖分层撕裂，无论是行双排固定或缝线桥固定，仍以全层缝合为主，可获得较好的疗效［6,13-15］。Park 等［15］纳入的 36 例肩袖撕裂患者中，肩袖分层撕裂的发生率为58.3%，手术均使用双排锚钉全层缝合修复肩袖分层撕裂，患者术后疼痛和功能均获得明显改善，且全层缝合能够减少双侧结构之间的剪切力，促进肩袖更好的愈合。
早在上世纪中期，人工智能技术就已诞生。截至目前，人工智能手段已经逐步渗透于各个现有的领域中。从本质上讲，人工智能技术最关键的特征在于模仿人脑固有的某种工作机制，从而实现了决策制定、精确操控以及其他相关事项。在人工智能的范围内，深度学习技术应当属于很关键的技术分支，其指的是借助智能化手段来仿照人脑机能，从而集合了多层次的神经元。具体在涉及到处理各类有关信息时，运用人工智能手段可以保证顺利连接各个层次的神经突触，据此实现了全方位的信息流动。
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http://crea tivecommons.org/licenses/by/4.0/),which permits unrestricted use,distribution,and reproduction in any medium,provided you give appropriate credit to the original author(s)and the source,provide a link to the Creative Commons license,and indicate if changes were made.
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