Transcriptome resources and genome-wide marker development for Japanese larch (Larix kaempferi)

Transcriptome resources and genome-wide marker development for Japanese larch (Larix kaempferi )

Wanfeng LI 1,Suying HAN 2,Liwang QI 1,Shougong ZHANG (?)1

1State Key Laboratory of Tree Genetics and Breeding,Research Institute of Forestry,Chinese Academy of Forestry,Beijing 100091,China

2State Key Laboratory of Tree Genetics and Breeding,Research Institute of Forest Ecology,Environment and Protection,

Chinese Academy of Forestry,Beijing 100091,China

Abstract While the differential responses of trees to changes in climatic and environmental conditions have been demonstrated as they age,the underlying mechanisms and age control of tree growth and development are complex and poorly understood particularly at a molecular level.In this paper,we present a transcriptome analysis of Larix kaempferi ,a deciduous conifer that is widely-grown in the northern hemisphere and of signi ?cant ecological and economic https://www.wendangku.net/doc/8c5312929.html,ing high-throughput RNA sequen-cing,we obtained about 26million reads from the stems of 1-,2-,5-,10-,25-and 50-year-old L.kaempferi https://www.wendangku.net/doc/8c5312929.html,bining these with the published Roche 454sequen-cing reads and the expressed sequence tags (both mainly from Larix embryogenic cell cultures),we assembled 26670549reads into 146786transcripts,of which we annotated 79182to support investigations of the molecular basis of tree aging and adaption,somatic embryogenesis and wood https://www.wendangku.net/doc/8c5312929.html,ing these sequences we also identi ?ed many single-nucleotide polymorphisms,simple sequence repeats,and insertion and deletion markers to assist breeding and genetic diversity studies of Larix .Keywords Larix ,transcriptome,age,wood formation,somatic embryogenesis,molecular marker

1Introduction

Age is an important developmental cue for trees,and great changes occur as they age.For example,young trees reactivate earlier than old trees with increasing temperature in the spring,thus having a longer period of growth,which indicates that meristem activity is affected by age [1–4].Notably,cambium cell division in locally-heated portions of the Cryptomeria japonica stem during the dormant

period restarts earlier in younger than in older trees [3],indicating that age affects the activity of the lateral meristem in response to temperature signals.The regular division of meristem cells in shoot and stem contributes to the continued growth in height and girth of a tree for many years,and this involves orderly cell-cycle progression and the expression of cell-cycle genes.The sensitivity of cambium to auxin,which is important in the control of cambium activity and wood formation [5–11],declines as trees age [12–14].

In addition to the differential responses to environmental and hormonal stimuli,the underlying mechanisms for transition from vegetative growth to ?owering occurs as trees age are still poorly-de ?ned.Given the age-control of meristem activity and identity,the expression of implicated genes appears to be regulated by age.However,limited information is available on the molecular basis of tree aging,especially the regulatory mechanisms of meristem activity.

Investigations of the molecular mechanisms of tree aging have identi ?ed many age-related genes [15–22].Some of these genes participate in wood production [21,22]and the control of ?owering time [23–26],while only a few have been associated with the regulation of meristem activity and identity.Due to the lack of genomic and transcriptome data,information about transcriptome reprogramming during tree aging is still limited.Thus the availability of transcriptome data for some trees,of which the genome has not been sequenced,would enable global analysis of transcriptome reprogramming during https://www.wendangku.net/doc/8c5312929.html,rix kaempferi is a forest tree of important ecological and economic value widely-grown in the northern hemi-sphere,and its somatic embryogenesis has been used to study the regulatory mechanisms of plant development [27–35].With the advantage of next-generation sequen-cing technologies,deep sequencing of the transcriptome has become rapid and economical,making it easier to develop molecular markers at the genome scale.Recently,transcriptome sequencing of L.kaempferi embryogenic

Received April 10,2014;accepted May 5,2014Correspondence:shougong.zhang@https://www.wendangku.net/doc/8c5312929.html,

Front.Agr.Sci.Eng.2014,1(1):77–84DOI:10.15302/J-FASE-2014010

?The Author(s)2014.This article is published with open access at https://www.wendangku.net/doc/8c5312929.html,

cell cultures and L.gmelinii needles using the Roche454 and Illumina Solexa sequencing platforms has been reported[36,37],greatly adding to availability of tran-scriptome information for Larix.However,the Larix transcriptome is still one of the least explored among conifers[38]and remains insuf?cient for tree-aging studies.Here,we sequenced the transcriptome of the stems of1-,2-,5-,10-,25-and50-year-old L.kaempferi, and assembled it de novo with the transcriptome of the embryogenic cell cultures for global analysis of transcrip-tome resources and the development of genome-wide markers.

2Materials and methods

2.1Plant material

The materials were sampled in July2011at Dagujia seed orchard(42°22′N,124°51′E),Liaoning Province,in North-east China.The uppermost main stems produced in the current year from three trees were harvested from2-, 5-,10-,25-and50-year-old L.kaempferi trees and ten stems from1-year-old trees,which had been grown from seed.After removing the needles,the stems from trees of the same age were pooled,frozen in liquid nitrogen,and stored at–80°C for RNA extraction.We selected these ages because they constitute an entire rotation period from establishment to harvest,and include the vegetative and reproductive phases of L.kaempferi.

2.2CDNA library preparation and transcriptome sequencing

CDNA(cDNA)library preparation and transcriptome sequencing were performed according to the manufac-turer’s instructions(Illumina,San Diego,CA).Brie?y, total RNA was extracted from each sample using plant RNA puri?cation reagent according to the manufacturer’s instructions(Invitrogen,San Diego,CA).RNA was quanti?ed on an ND-1000Spectrophotometer(Thermo Fisher Scienti?c,Inc.,Wilmington,DE).One microgram of total RNA from each sample was pooled and5μg of the mixed total RNA was used to generate an RNA library for Illumina paired-end sequencing.After isolation from5μg of the mixed total RNA,the mRNA was broken into short https://www.wendangku.net/doc/8c5312929.html,ing these short fragments as templates,a random hexamer primer was used to synthesize?rst-strand cDNA.Then second-strand cDNA was synthesized.The double-stranded cDNA was cleaned up and used for end repair and addition of poly A.Thereafter,these strands were connected to sequencing adapters and the desired cDNAwas isolated through a2%agarose gel.To enrich the desired cDNA,PCR ampli?cation was performed.PCR products were isolated on2%agarose gel and puri?ed using a PCR column puri?cation kit.Finally,the library was sequenced using the Illumina HiSeq TM2000platform.

2.3Transcriptome assembly

The raw Illumina RNA sequencing reads were?rst preprocessed by discarding reads with adaptors,with unknown nucleotides>5%,of low-quality(quality score<20),or<20bp.Then,de novo assembly was performed using Trinity software[39]with default parameters.The Roche454raw reads(accession number: SRX110249)[36]were?rst preprocessed by trimming adaptors and Poly A,and then the preprocessed sequences were assembled using Newbler 2.6(Roche;cDNA assembly mode)with default parameters.Another3,874 expressed sequence tags(ESTs)[40,41]were used to increase the transcriptome coverage.Any vector contam-ination of the ESTs was removed using the seqclean program(https://www.wendangku.net/doc/8c5312929.html,/tgi/software/). The assembled Illumina transcripts,Roche454sequencing transcripts and ESTs were further assembled using CAP3 software[42]with default parameters.

2.4Transcriptome annotation

To identify the L.kaempferi protein-coding genes,blastx (the basic local alignment search tool,search protein databases using a translated nucleotide query)[43]was used to search our assembled sequences against four sets of protein sequences from Arabidopsis thaliana(http://plants. https://www.wendangku.net/doc/8c5312929.html,/Arabidopsis_thaliana/Info/Index),Picea abies(https://www.wendangku.net/doc/8c5312929.html,/),Populus trichocarpa(http:// https://www.wendangku.net/doc/8c5312929.html,/poplar.php)and Vitis vinifera(http:// https://www.wendangku.net/doc/8c5312929.html,/Vitis_vinifera/Info/Index/)with an e-value of1e-5.The sequences with no blastx hits were then searched against the NR database(NCBI non-redundant protein database‘nr’)with an e-value of1e-5.To identify the biologic processes enriched in the L.kaempferi transcriptome,we assigned the Gene Ontology(GO) terms associated with the top hits in the four protein databases to the annotated transcripts.

2.5Evolutionary analysis

To do evolutionary analysis,we extracted likely coding regions from the L.kaempferi annotated transcripts using TransDecoder,included with the Trinity software.First, protein sequences longer than30amino-acids from A. thaliana,L.kaempferi,P.abies,P.trichocarpa and V. vinifera were collected.Second,blastp(the basic local alignment search tool,simply compares a protein query to a protein database)[43]was used against a database containing a protein data set of the?ve species with the e-value of1e-7.Then,the single-copy orthologous genes among the?ve species were used to construct a

78Front.Agr.Sci.Eng.2014,1(1):77–84

phylogenetic tree using PhyML[44]with default para-

meters.

2.6Molecular marker development

To aid Larix breeding and genetic diversity studies,we

identi?ed single nucleotide polymorphism(SNP),simple

sequence repeat(SSR),and insertion and deletion(InDel)

markers using the transcriptome data obtained in this work.

To identify SNPs,the sequencing reads were aligned with

the representative transcripts in the CLC genomic work-

bench(https://www.wendangku.net/doc/8c5312929.html,/products/clc-genomics-

workbench/).The general alignment parameters were set

to defaults except that non-speci?c matches were ignored

to minimize read-alignment ambiguities.To capture

reliable SNPs,we adjusted the minimum read coverage

to5.

SSRs were identi?ed using Msat?nder(http://www.

https://www.wendangku.net/doc/8c5312929.html,/msat?nder/).The repeat thresholds for

mononucleotide,dinucleotide,trinucleotide,tetranucleo-

tide,pentanucleotide,and hexanucleotide motifs were set

at12,8,5,5,5,and5,respectively.Only SSRs with ?anking sequences longer than50bp on both sides were collected.

BWA and SAMtools software[45,46]were used to align

reads to transcriptome reference and call InDels.The ?ltering threshold was set as follows:read depth no less than10and quality score no less than20.The default parameter was used for quality control of?anking sequences in the SAMtools mpileup step.

3Results

3.1Transcriptome assembly

From the stem transcriptome sequencing of1-,2-,5-,10-, 25-and50-year-old L.kaempferi,a total of26074916 reads were generated and have been deposited in the NCBI SRA database(accession number:SRR1107838).Another 591759Roche454sequencing reads and3874ESTs were also included in the L.kaempferi transcriptome sequence assembly.Assembly of26670549reads generated146786 transcripts,ranging from200to16701bp,with a mean length of849bp and an N50length of1538bp(Table1).3.2Transcriptome annotation

Transcripts were?rst annotated by blastx of four protein

databases from A.thaliana,P.abies,P.trichocarpa,and V.

vinifera,and then the NR database(e-value<1e-5).

Altogether,79182(53.9%)of the146786transcripts had

signi?cant matches,at least one hit in these databases.

Among the79182annotated transcripts,74744(94.4%)

were matched to P.abies,56573(71.4%)to A.thaliana,

55807(70.5%)to V.vinifera,54687(69.1%)to P. trichocarpa,and3022(3.8%)to the NR database.

GO annotation was performed for the79182annotated

transcripts in terms of‘biologic process’,‘molecular

function’and‘cellular component’.In total,55349

(69.9%)transcripts were assigned to a total of2024491

GO terms:53241transcripts were assigned to the biologic

process category,53015to molecular function,and51800

to cellular component.In the biologic process category,‘response to stress’was the most abundant GO term (21280,40.0%),followed by‘biosynthetic process’

(20855,39.2%),and‘development of an anatomical

structure’(17558,33.0%)(Fig.1).

3.3Evolutionary analysis

Seventy-two single-copy gene families across A.thaliana, L.kaempferi,P.abies,P.trichocarpa and V.vinifera were obtained.The gene families were concatenated to?ve super-peptides(100084peptide sites)to construct a phylogenetic tree.The phylogenetic tree analysis showed that L.kaempferi was closely related to P.abies(Fig.2), consistent with their taxonomic classi?cation and evolu-tionary relationship.

3.4Identi?cation of gene-associated markers

In total,463482high-quality SNPs from48578transcripts were identi?ed,including272417transitions and191065 transversions(Table2).The minor allele frequencies of SNPs were estimated from the sequence data(Fig.3a).The distribution of SNPs per transcript was estimated(Fig.3b) and the overall frequency of all types of SNPs in the transcriptome was one per108bp.Among these SNPs, 364227(78.6%)were identi?ed from transcripts with annotation information,and they were distributed in32453 known genes(Table2).

The4756SSRs detected in4438transcripts(Table2) included2165(45.5%)mononucleotide,569(12.0%) dinucleotide,1941(40.8%)trinucleotide,57(1.2%) tetranucleotide,17(0.4%)pentanucleotide and7(0.1%) hexanucleotide motifs.Apart from the mononucleotide motifs,the most abundant was ACG\CGT(9.7%), followed by AT\AT(8.5%),and AAG\CTT(8.5%)(Fig. 3c).Suitable PCR primers were designed for3595SSRs using primer3[47].

Table1Summary of transcriptome assembly

Types of data Total sequences Total bases/bp Mean length/bp

Illumina260749162607491600100

Roche454591759211291630357

Sanger38742956198763

Total26670549

Assembled transcripts146786124640235849

Wanfeng LI et https://www.wendangku.net/doc/8c5312929.html,rix transcriptome and molecular markers79

A total of 12434InDels were identi ?ed in 10357transcripts (Table 2),including 4080deletions and 8354insertions.Only 1640(15.8%)of the sequences had more than one InDel.Among the 4080deletions,>68%were mononucleotide deletions,followed by dinucleotide (16.7%),trinucleotide (11.0%),tetranucleotide (3.1%),and pentanucleotide (1.0%)deletions.Among the 8354

insertions,mononucleotide insertions were also prevalent (59.8%),while insertions of dinucleotide (19.7%),trinu-cleotide (13.6%),tetranucleotide (4.3%),and pentanucleo-tide (2.6%)accounted for small proportions.

4

Discussion

Environmental signals control tree growth and develop-ment.Notably,age affects their responses to these environmental signals [3,48].Studying the underlying mechanisms is important not only for understanding the adaptation of trees in the context of global climate change,but also for forest breeding and management.For example,in one rotation period of Larix,the rate of increase in height peaks at the age of about 15[49];so determining the optimal time to harvest forest trees is important.Here,large-scale identi ?cation of the transcripts associated with ‘response to stress ’(21280),‘response to salt stress ’(8016),‘response to cadmium ion ’(6166),‘response to cold ’(5313),‘response to water deprivation ’(4999),‘response to heat ’(2933),‘response to oxidative stress ’(2566),and ‘response to osmotic stress ’(2544)will facilitate studies of the environmental regulation of tree growth and development and the mechanisms of age control of the responses to these environmental signals.As a tree ages,?oral meristems develop.Based on the functional annotation by GO,4851transcripts

were

Fig.1Representation of Gene Ontology (GO)classi ?cation in terms of ‘biologic processes

’

Fig.2Phylogenetic tree analysis from the orthologous data set across A.thaliana ,L.kaempferi ,P .abies ,P .trichocarpa and V .vinifera

Table 2Molecular markers identi ?ed from L.kaempferi

Marker information

SNP SSR

InDel

Total number of identi ?ed markers

463482475612434Number of markers within annotated transcripts 3642273520

8723

Number of transcripts with markers

48578443810357Number of annotated transcripts with markers

32453

3264

7035

80Front.Agr.Sci.Eng.2014,1(1):77–84

assigned to the GO term ‘vegetative to reproductive phase transition of meristem ’,suggesting that the transcriptome in meristem is reprogrammed during aging.The transcripts assigned to ‘regulation of transcription,DNA-dependent ’(6391),‘positive regulation of transcription,DNA-depen-dent ’(4275),and ‘chromosome organization ’(3350)might participate in these transcriptome reprogramming processes.These results provide important information for studies of the phase-transition of meristem from vegetative to reproductive during tree aging.

When we collected the samples for sequencing,the L.kaempferi trees were actively growing.Active cambium cell division and wood formation were re ?ected by the assignation of 8439transcripts to ‘cell differentiation ’,6901to ‘cell wall organization or biogenesis ’,4112to ‘cell cycle ’,3390to ‘cell division ’,3213to ‘cytokinesis by cell plate formation ’,2997to ‘cell wall organization ’and 2568to ‘plant-type cell wall organization ’.Hormones control cambium activity and wood formation [50,51],and this was supported by the assignation of 4541transcripts to ‘jasmonic acid mediated signaling pathway ’,3855to ‘response to auxin stimulus ’,3386to ‘response to jasmonic acid stimulus ’,and 3006to ‘response to ethylene stimulus ’.These results will help to understand the molecular basis of wood formation and the regulation of cambium activity by hormones.

Somatic embryogenesis is not only a valuable technique in clonal propagation,but also provides a useful experi-mental system to study the regulatory mechanisms of plant development [52–54].Hormonal signals also play regula-tory roles in somatic embryogenesis.For example,abscisic acid promotes the maturation of the somatic embryo,regulates the synchronization of its development,main-tains its dormant state,and suppresses its germination [29,31,35,55,56].Based on the functional annotation by GO,17558transcripts were assigned to the GO term ‘anatomical structure development ’,6878to ‘cell mor-phogenesis ’,6604to ‘response to abscisic acid stimulus ’,6403to ‘embryo development ending in seed dormancy ’,5668to ‘embryo development ’,and 3455to ‘abscisic acid mediated signaling pathway ’.Consistent with the reorga-nization of meristem during somatic embryogenesis,4179transcripts were assigned to ‘regulation of meristem growth ’and 2932to ‘meristem initiation ’.These data depict the features of somatic embryogenesis and give prominence to the regulation of somatic embryo develop-ment by abscisic acid.

Molecular markers are important for the assessment of genetic diversity [57–61],pedigree and mating system analyses [62],hybrid identi ?cation [63],the development of genetic maps,and marker-assisted breeding.In Larix ,EST-SSR markers have been developed [64,65]and two highly-informative SSRs have also been identi ?ed recently [66],but other types of markers,such as SNPs and InDels,have not been well developed.Here,using Larix transcriptome sequences,we identi ?ed 463482SNPs,4756SSRs,and 12434InDels,which will have consider-able utility for the assessment of genetic diversity and marker-assisted breeding in Larix .

5Conclusions

Here,we present an analysis of Larix transcriptomes.All of these data enrich the transcriptome resources of Larix ,are publicly available online,and will serve as useful

tools

Fig.3Summary analysis of single-nucleotide polymorphisms (SNPs)and single-nucleotide polymorphisms (SSRs).(a)Dis-tribution of minor allele frequencies of SNPs identi ?ed in L.kaempferi .The x -axis represents the SNP sequence-derived minor allele frequency,while the y -axis represents the number of SNPs with a given minor allele frequency;(b)distribution of SNPs per transcript.The x -axis represents the number of SNPs per transcript,while the y -axis represents the number of transcripts with a given number of SNPs;(c)frequency of SSRs.The x -axis represents the SSR types and the y -axis represents the percentage of a given SSR type.

Wanfeng LI et https://www.wendangku.net/doc/8c5312929.html,rix transcriptome and molecular markers 81

for understanding the molecular basis of tree growth and development and for breeding and genetic diversity studies of Larix.

Acknowledgements This work was supported by the National Natural Science Foundation of China(31200464and31330017),and the National High Technology Research and Development Program of China (2011AA100203and2013AA102704).The authors thank Dr.IC Bruce (Zhejiang University)and Dr.Yong Guo(Institute of Crop Science,Chinese Academy of Agricultural Sciences)for critical reading of the manuscript,and Dr.Tao Wu for sample collection.

Compliance with ethics guidelines Wanfeng Li,Suying Han,Liwang Qi and Shougong Zhang declare that they have no con?ict of interest or?nancial con?icts to disclose.

This article does not contain any studies with human or animal subjects performed by the any of the authors.

1.Rossi S,Deslauriers A,Anfodillo T,Carrer M.Age-dependent

xylogenesis in timberline conifers.New Phytologist,2008,177(1): 199–208

2.Rossi S,Deslauriers A,Gri?ar J,Seo J W,Rathgeber C B K,

Anfodillo T,Morin H,Levanic T,Oven P,Jalkanen R.Critical temperatures for xylogenesis in conifers of cold climates.Global Ecology and Biogeography,2008,17(6):696–707

3.Begum S,Nakaba S,Oribe Y,Kubo T,Funada R.Cambial

sensitivity to rising temperatures by natural condition and arti?cial heating from late winter to early spring in the evergreen conifer Cryptomeria japonica.Trees-Structure and Function,2010,24(1): 43–52

4.Li X,Liang E,Gri?ar J,Prislan P,Rossi S,?ufar K.Age

dependence of xylogenesis and its climatic sensitivity in Smith?r on the south-eastern Tibetan Plateau.Tree Physiology,2013,33(1):48–56

5.Li W F,Ding Q,Chen J J,Cui K M,He X Q.Induction of PtoCDKB

and PtoCYCB transcription by temperature during cambium reactivation in Populus tomentosa Carr.Journal of Experimental Botany,2009,60(9):2621–2630

6.Little C H A,Bonga J M.Rest in cambium of Abies balsamea.

Canadian Journal of Botany,1974,52(7):1723–1730

7.Mwange K N,Wang X W,Cui K M.Mechanism of dormancy in the

buds and cambium of Eucommia ulmoides.Acta Botanica Sinica, 2003,45(6):698–704

8.Mwange K N,Hou H W,Wang Y Q,He X Q,Cui K M.Opposite

patterns in the annual distribution and time-course of endogenous abscisic acid and indole-3-acetic acid in relation to the periodicity of cambial activity in Eucommia ulmoides Oliv.Journal of Experi-mental Botany,2005,56(413):1017–1028

9.Sundberg B,Little C H A.Tracheid production in response to

changes in the internal level of indole-3-acetic Acid in1-year-old shoots of scots pine.Plant Physiology,1990,94(4):1721–1727 10.Baba K,Karlberg A,Schmidt J,Schrader J,Hvidsten T R,Bako L,

Bhalerao R P.Activity-dormancy transition in the cambial meristem involves stage-speci?c modulation of auxin response in hybrid aspen.Proceedings of the National Academy of Sciences of the

United States of America,2011,108(8):3418–3423

11.Nilsson J,Karlberg A,Antti H,Lopez-Vernaza M,Mellerowicz E,

Perrot-Rechenmann C,Sandberg G,Bhalerao R P.Dissecting the molecular basis of the regulation of wood formation by auxin in hybrid aspen.Plant Cell,2008,20(4):843–855

12.Savidge R A,Wareing P F.A tracheid-differentiation factor from

pine needles.Planta,1981,153(5):395–404

13.Little C H A,Sundberg B.Tracheid production in response to

indole-3-acetic-acid varies with internode age in Pinus sylvestris stems.Trees-Structure and Function,1991,5(2):101–106

14.Savidge R A.The role of plant hormones in higher plant cellular

differentiation.II.Experiments with the vascular cambium,and sclereid and tracheid differentiation in the pine,Pinus contorta.

Histochemical Journal,1983,15(5):447–466

15.Alvarez C,Valledor L.R.H,Sanchez-Olate M,Ríos D.Variation in

gene expression pro?le with aging of Pinus radiata D.Don.BMC Proceedings,2011,5(Suppl7):62

16.Busov V B,Johannes E,Whetten R W,Sederoff R R,Spiker S L,

Lanz-Garcia C,Goldfarb B.An auxin-inducible gene from loblolly pine(Pinus taeda L.)is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein.Planta,2004,218(6):916–927

17.Carlsbecker A,Tandre K,Johanson U,Englund M,Engstr?m P.The

MADS-box gene DAL1is a potential mediator of the juvenile-to-adult transition in Norway spruce(Picea abies).Plant Journal, 2004,40(4):546–557

18.Diego L B,Berdasco M,Fraga M F,Ca?al M J,Rodríguez R,

Castresana C.A Pinus radiata AAA-ATPase,the expression of which increases with tree ageing.Journal of Experimental Botany, 2004,55(402):1597–1599

19.Fernández-Oca?a A,Carmen García-López M,Jiménez-Ruiz J,

Saniger L,Macías D,Navarro F,Oya R,Belaj A,de la Rosa R, Corpas F J,Bautista Barroso J,Luque F.Identi?cation of a gene involved in the juvenile-to-adult transition(JAT)in cultivated olive trees.Tree Genetics&Genomes,2010,6(6):891–903

20.Hutchison K W,Sherman C D,Weber J,Smith S S,Singer P B,

Greenwood M S.Maturation in larch:II.effects of age on photosynthesis and gene expression in developing foliage.Plant Physiology,1990,94(3):1308–1315

21.Li X,Wu H X,Southerton S G.Seasonal reorganization of the

xylem transcriptome at different tree ages reveals novel insights into wood formation in Pinus radiata.New Phytologist,2010,187(3): 764–776

22.Li X,Wu H X,Southerton S G.Transcriptome pro?ling of wood

maturation in Pinus radiata identi?es differentially expressed genes with implications in juvenile and mature wood variation.Gene, 2011,487(1):62–71

23.Wang J W,Park M Y,Wang L J,Koo Y,Chen X Y,Weigel D,

Poethig R S.miRNA control of vegetative phase change in trees.

PLOS Genetics,2011,7(2):e1002012

24.Hsu C Y,Liu Y,Luthe D S,Yuceer C.Poplar FT2shortens the

juvenile phase and promotes seasonal?owering.Plant Cell,2006, 18(8):1846–1861

25.Hsu C Y,Adams J P,Kim H,No K,Ma C,Strauss S H,Drnevich J,

Vandervelde L,Ellis J D,Rice B M,Wickett N,Gunter L E,Tuskan

G A,Brunner A M,Page G P,Barakat A,Carlson J E,DePamphilis

82Front.Agr.Sci.Eng.2014,1(1):77–84

C W,Luthe

D S,Yuceer C.FLOWERING LOCUS T duplication

coordinates reproductive and vegetative growth in perennial poplar.

Proceedings of the National Academy of Sciences of the United States of America,2011,108(26):10756–10761

26.B?hlenius H,Huang T,Charbonnel-Campaa L,Brunner A M,

Jansson S,Strauss S H,Nilsson O.CO/FT regulatory module controls timing of?owering and seasonal growth cessation in trees.

Science,2006,312(5776):1040–1043

27.Li S G,Li W F,Han S Y,Yang W H,Qi L W.Stage-speci?c

regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos.Gene,2013, 522(2):177–183

28.Li W F,Zhang S G,Han S Y,Wu T,Zhang J H,Qi L W.Regulation

of LaMYB33by miR159during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.)Carr.Plant Cell,Tissue and Organ Culture,2013,113(1): 131–136

29.Zhang J,Zhang S,Han S,Li X,Tong Z,Qi L.Deciphering small

noncoding RNAs during the transition from dormant embryo to germinated embryo in Larches(Larix leptolepis).PLoS ONE,2013, 8(12):e81452

30.Zhang J,Zhang S,Han S,Wu T,Li X,Li W,Qi L.Genome-wide

identi?cation of microRNAs in larch and stage-speci?c modulation of11conserved microRNAs and their targets during somatic embryogenesis.Planta,2012,236(2):647–657

31.Zhang L F,Li W F,Han S Y,Yang W H,Qi L W.cDNA cloning,

genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP)gene from Japanese larch(Larix leptolepis).Gene,2013, 529(1):150–158

32.Zhang S,Zhou J,Han S,Yang W,Li W,Wei H,Li X,Qi L.Four

abiotic stress-induced miRNA families differentially regulated in the embryogenic and non-embryogenic callus tissues of Larix leptole-pis.Biochemical and Biophysical Research Communications,2010, 398(3):355–360

33.Zhang S G,Han S Y,Yang W H,Wei H L,Zhang M,Qi L W.

Changes in H2O2content and antioxidant enzyme gene expression during the somatic embryogenesis of Larix leptolepis.Plant Cell, Tissue and Organ Culture,2010,100(1):21–29

34.Li W F,Zhang S G,Han S Y,Wu T,Zhang J H,Qi L W.The post-

transcriptional regulation of LaSCL6by miR171during main-tenance of embryogenic potential in Larix kaempferi(Lamb.).Carr.

Tree Genetics&Genomes,2014,10(1):223–229

35.Zhang J H,Zhang S G,Li S G,Han S Y,Li W F,Li X M,Qi L W.

Regulation of synchronism by abscisic-acid-responsive small noncoding RNAs during somatic embryogenesis in larch(Larix leptolepis).Plant Cell,Tissue and Organ Culture,2014,116(3): 361–370

36.Zhang Y,Zhang S,Han S,Li X,Qi L.Transcriptome pro?ling and

in silico analysis of somatic embryos in Japanese larch(Larix leptolepis).Plant Cell Reports,2012,31(9):1637–1657

37.Men L,Yan S,Liu G.De novo characterization of Larix gmelinii

(Rupr.)Rupr.transcriptome and analysis of its gene expression induced by jasmonates.BMC Genomics,2013,14(1):548

38.Mackay J,Dean J F,Plomion C,Peterson D G,Cánovas F M,Pavy

N,Ingvarsson P K,Savolainen O,Guevara Má,Fluch S,Vinceti B,

Abarca D,Díaz-Sala C,Cervera M T.Towards decoding the conifer giga-genome.Plant Molecular Biology,2012,80(6):555–569 39.Grabherr M G,Haas B J,Yassour M,Levin J Z,Thompson D A,

Amit I,Adiconis X,Fan L,Raychowdhury R,Zeng Q,Chen Z, Mauceli E,Hacohen N,Gnirke A,Rhind N,di Palma F,Birren B W, Nusbaum C,Lindblad-Toh K,Friedman N,Regev A.Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nature Biotechnology,2011,29(7):644–652

40.Zhang L,Qi L W,Han S Y.[Differentially expressed genes during

Larix somatic embryomaturation and the expression pro?le of partial genes.Hereditas,2009,31(5):540–545(in Chinese)

41.Zhang L,Qi L,Han S.Construction and analysis of differentially

expressed cDNA library of larch somatic embryo at the stage of proembryogenic mass.Molecular Plant Breeding,2008,6(4):675–682(in Chinese)

42.Huang X,Madan A.CAP3:A DNA sequence assembly program.

Genome Research,1999,9(9):868–877

43.Altschul S F,Gish W,Miller W,Myers E W,Lipman D J.Basic

local alignment search tool.Journal of Molecular Biology,1990, 215(3):403–410

44.Guindon S,Dufayard J F,Lefort V,Anisimova M,Hordijk W,

Gascuel O.New algorithms and methods to estimate maximum-likelihood phylogenies:assessing the performance of PhyML3.0.

Systematic Biology,2010,59(3):307–321

45.Li H,Durbin R.Fast and accurate short read alignment with

Burrows-Wheeler transform.Bioinformatics,2009,25(14):1754–1760

46.Li H,Handsaker B,Wysoker A,Fennell T,Ruan J,Homer N,Marth

G,Abecasis G,Durbin R.The Sequence Alignment/Map format and SAMtools.Bioinformatics,2009,25(16):2078–2079

47.Untergasser A,Cutcutache I,Koressaar T,Ye J,Faircloth B C,

Remm M,Rozen S G.Primer3–new capabilities and interfaces.

Nucleic Acids Research,2012,40(15):e115

48.Carrer M,Urbinati C.Age-dependent tree-ring growth responses to

climate in Larix decidua and Pinus cembra.Ecology,2004,85(3): 730–740

49.Tian Z H,Dong J,Wang X W,Huang G R.Silviculture of Larix

kaempferi.1995,Beijing:Beijing Agriculture University Press94–105(in Chinese)

50.Sorce C,Giovannelli A,Sebastiani L,Anfodillo T.Hormonal

signals involved in the regulation of cambial activity,xylogenesis and vessel patterning in trees.Plant Cell Reports,2013,32(6):885–898

51.Sehr E M,Agusti J,Lehner R,Farmer E E,Schwarz M,Greb T.

Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation.Plant Journal,2010,63(5):811–822

52.Cairney J,Pullman G S.The cellular and molecular biology of

conifer embryogenesis.New Phytologist,2007,176(3):511–536 53.Quiroz-Figueroa F R,Rojas-Herrera R,Galaz-Avalos R M,Loyola-

Vargas V M.Embryo production through somatic embryogenesis can be used to study cell differentiation in plants.Plant Cell,Tissue and Organ Culture,2006,86(3):285–301

54.Zimmerman J L.Somatic embryogenesis:a model for early

development in higher plants.Plant Cell,1993,5(10): 1411–1423

Wanfeng LI et https://www.wendangku.net/doc/8c5312929.html,rix transcriptome and molecular markers83

55.Gutmann M,vonAderkas P,Label P,Lelu M A.Effects of abscisic

acid on somatic embryo maturation of hybrid larch.Journal of Experimental Botany,1996,47(12):1905–1917

56.Rai M K,Shekhawat N S,Harish,Gupta A K,Phulwaria M,Ram K,

Jaiswal U.Harish,Gupta A K,Phulwaria M,Ram K,Jaiswal U.The role of abscisic acid in plant tissue culture:a review of recent progress.Plant Cell,Tissue and Organ Culture,2011,106(2):179–190

57.Khasa D P,Jaramillo-Correa J P,Jaquish B,Bousquet J.Contrasting

microsatellite variation between subalpine and western larch,two closely related species with different distribution patterns.Mole-cular Ecology,2006,15(13):3907–3918

58.Pluess A R.Pursuing glacier retreat:genetic structure of a rapidly

expanding Larix decidua population.Molecular Ecology,2011,20

(3):473–485

59.Oreshkova N V,Belokon M M,Jamiyansuren S.Genetic diversity,

population structure,and differentiation of Siberian larch,Gmelin larch and Cajander larch on SSR-markers data.Russian Journal of Genetics,2013,49(2):178–186

60.Kozyrenko M M,Artyukova E V,Reunova G D,Levina E A,

Zhuravlev Y N.Genetic diversity and relationships among Siberian and Far Eastern larches inferred from RAPD analysis.Russian Journal of Genetics,2004,40(4):401–40961.Yu X M,Zhou Q,Qian Z Q,Li S,Zhao G F.Analysis of genetic

diversity and population differentiation of Larix potaninii var.

chinensis using microsatellite DNA.Biochemical Genetics,2006, 44(11–12):483–493

62.Funda T,Chen C,Liewlaksaneeyanawin C,Kenawy A M A,El-

Kassaby Y A.C..Liewlaksaneeyanawin C,Kenawy A M A,El-Kassaby Y A..Pedigree and mating system analyses in a western larch(Larix occidentalis Nutt.)experimental population.Annals of Forest Science,2008,65(7):705

63.AcheréV,Faivre Rampant P,Paques L E,Prat D.Chloroplast and

mitochondrial molecular tests identify European?Japanese larch hybrids.Theoretical and Applied Genetics,2004,108(8):1643–1649

64.Yang X,Sun X,Zhang S,Xie Y,Han H.Development of EST-SSR

markers and genetic diversity analysis of the second cycle elite population in Larix kaempferi.Scientia Silvae Sinicae,2011,47(11): 52–58(in Chinese)

65.Liu C,Zhang L P,Wang C G,Song W Q,Chen C B.Development

and Characterization of EST-SSR Molecular Markers in Larix kaempferi.Forest reseach,2013,26(S1):60–68(in Chinese)

66.Wagner S,Gerber S,Petit R J.Two highly informative dinucleotide

SSR multiplexes for the conifer Larix decidua(European larch).

Molecular ecology resources,2012,12(4):717–25

84Front.Agr.Sci.Eng.2014,1(1):77–84