Identification of Flowering Regulatory Genes in Allopolyploid Brassica juncea
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摘要
Brassica juncea is an allopolyploid originating from the interspecific hybridization between Brassica rapa and Brassica nigra, which is of multiple usage as a vegetable, oilseed and condiment worldwide. Both vernalization and non-vernalization under long-day photoperiod can promote floral transition in B. juncea suggesting merged flowering pathways of its ancestors and better environmental adaptability. We identified genomewide flowering regulatory genes in B. juncea, which include 84 and 79 genes from A and B sub-genomes, respectively. Ka/Ks analysis revealed a purification effect on both photoperiod and vernalization flowering regulation pathways during evolution. Expression profile of those genes during long-day and vernalization treatments suggested Bju ACO4, Bju AFT1, Bju BFT4, Bju ASOC1 and Bju ASOC4 may be the major functional copies of B. juncea flowering regulation. Further functional studies about Bju COs showed three days delayed flowering time in Bju ACO4 or Bju BCO3 silenced plants. Increased transcription of all BjuFLCs in Bju ACO4 or Bju BCO3 silenced plants suggested interactions between photoperiod and vernalization pathways governing flowering time. Our findings provided flowering regulating networks in allopolyploid B. juncea.
Brassica juncea is an allopolyploid originating from the interspecific hybridization between Brassica rapa and Brassica nigra, which is of multiple usage as a vegetable, oilseed and condiment worldwide. Both vernalization and non-vernalization under long-day photoperiod can promote floral transition in B. juncea suggesting merged flowering pathways of its ancestors and better environmental adaptability. We identified genomewide flowering regulatory genes in B. juncea, which include 84 and 79 genes from A and B sub-genomes, respectively. Ka/Ks analysis revealed a purification effect on both photoperiod and vernalization flowering regulation pathways during evolution. Expression profile of those genes during long-day and vernalization treatments suggested Bju ACO4, Bju AFT1, Bju BFT4, Bju ASOC1 and Bju ASOC4 may be the major functional copies of B. juncea flowering regulation. Further functional studies about Bju COs showed three days delayed flowering time in Bju ACO4 or Bju BCO3 silenced plants. Increased transcription of all BjuFLCs in Bju ACO4 or Bju BCO3 silenced plants suggested interactions between photoperiod and vernalization pathways governing flowering time. Our findings provided flowering regulating networks in allopolyploid B. juncea.
Brassica juncea is an allopolyploid originating from the interspecific hybridization between Brassica rapa and Brassica nigra, which is of multiple usage as a vegetable, oilseed and condiment worldwide. Both vernalization and non-vernalization under long-day photoperiod can promote floral transition in B. juncea suggesting merged flowering pathways of its ancestors and better environmental adaptability. We identified genomewide flowering regulatory genes in B. juncea, which include 84 and 79 genes from A and B sub-genomes, respectively. Ka/Ks analysis revealed a purification effect on both photoperiod and vernalization flowering regulation pathways during evolution. Expression profile of those genes during long-day and vernalization treatments suggested Bju ACO4, Bju AFT1, Bju BFT4, Bju ASOC1 and Bju ASOC4 may be the major functional copies of B. juncea flowering regulation. Further functional studies about Bju COs showed three days delayed flowering time in Bju ACO4 or Bju BCO3 silenced plants. Increased transcription of all BjuFLCs in Bju ACO4 or Bju BCO3 silenced plants suggested interactions between photoperiod and vernalization pathways governing flowering time. Our findings provided flowering regulating networks in allopolyploid B. juncea.
引文
Bouché,F.,Woods,D.P.,Amasino,R.M.,Wisconsin,F.B.,2017.Winter memory throughout the plant kingdom:different paths to flowering.Plant Physiol,173:27-35.
Bowers,J.E.,Chapman,B.A.,Rong,J.,Paterson,A.H.,2003.Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events.Nature,422:433-438.
Cai,X.,Cui,Y.,Zhang,L.,Wu,J.,Liang,J.,Cheng,L.,Wang,X.,Cheng,F.,2018.Hotspots of independent and multiple rounds of LTR-retrotransposon burst in Brassica species.Horticultural Plant J,4:165-174.
Cho,L.,Yoon,J.,An,G.,2017.The control of flowering time by environmental factors.Plant J,172:708-719.
Csorba,T.,Questa,J.I.,Sun,Q.,Dean,C.,2014.Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization.Proc Natl Acad Sci,111:16160-16165.
Duan,W.,Song,X.,Liu,T.,Huang,Z.,Ren,J.,Hou,X.,Du,J.,Li,Y.,2014.Patterns of evolutionary conservation of ascorbic acid-related genes following whole-genome triplication in Brassica rapa.Genome Biol Evol,7:299-313.
Duan,W.,Zhang,H.,Zhang,B.,Wu,X.,Shao,S.,Li,Y.,Hou,X.,Liu,T.,2017.Role of vernalization-mediated demethylation in the floral transition of Brassica rapa.Planta,245:227-233.
Fornara,F.,Montaigu,A.,Coupland,G.,2010.SnapShot:control of flowering in Arabidopsis.Cell,141:3-5.
Gaeta,R.T.,Pires,J.C.,Iniguez-Luy,F.,Leon,E.,Osborn,T.C.,2007.Genomic changes in resynthesized Brassica napus and their effect on gene expression and phenotype.Plant Cell,19:3403-3417.
Green,R.M.,2002.Circadian rhythms confer a higher level of fitness to Arabidopsis plants.Plant Physiol,129:576-584.
Garner,W.W.,Allard,H.A.,1930.Effect of the relative length of day and night and other factors of the environment on growth and reproduction in Plants.Jap Comp Educ Soc,15:7-8.
Jian,H.,Zhang,A.,Ma,J.,Wang,T.,Yang,B.,Shuang,L.S.,Liu,M.,Li,J.,Xu,X.,Paterson,A.H.,Liu,L.,2019.Joint QTL mapping and transcriptome sequencing analysis reveal candidate flowering time genes in Brassica napus L.BMC Genomics,1:20-21.
Johansson,M.,Staiger,D.,2015.Time to flower:interplay between photoperiod and the circadian clock.J Exp Bot,66:719-730.
Kim,D.H.,Sung,S.,2014.Genetic and epigenetic mechanisms underlying vernalization.Arabidopsis Book,12:e0171.
Laughton,A.M.,O’Connor,C.O.,Knell,R.J.,2017.Responses to a warming world:integrating life history,immune investment,and pathogen resistance in a model insect species.Ecol Evol,7:9699-9710.
Lee,J.,Oh,M.,Park,H.,Lee,I.,2008.SOC1 translocated to the nucleus by interaction with AGL24 directly regulates LEAFY.Plant J,55:832-843.
Lee,S.,Kim,J.,Han,J.J.,Han,M.J.,An,G.,2004.Functional analyses of the flowering time gene OsMADS50,the putative suppressor of overexpression of SOC1/Agamous-Like 20(SOC1/AGL20)ortholog in rice.Plant J,38:754-764.
Livak,K.J.,Schmittgen,T.D.,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-CTmethod.Methods,25:402-408.
Levy,A.A.,2002.The impact of polyploidy on grass genome evolution.Plant Physiol,130:1587-1593.
Liu,C.,Zhou,J.,Bracha-Drori,K.,Yalovsky,S.,Ito,T.,Yu,H.,2007.Specification of Arabidopsis floral meristem identity by repression of flowering time genes.Development,134:1901-1910.
Liu,L.J.,Zhang,Y.C.,Li,Q.H.,Sang,Y.,Mao,J.,Lian,H.L.,Wang,L.,Yang,H.Q.,2008.COP1-mediated ubiquitination of CONSTANS is implicated in cryptochrome regulation of flowering in Arabidopsis.Plant Cell,20:292-306.
Lv,X.,Lan,S.,Guy,K.M.,Yang,J.,Zhang,M.,Hu,Z.,2016.Global expressions landscape of NAC transcription factor family and their responses to abiotic stresses in Citrullus lanatus.Sci Rep,6:1-14.
Mayee,P.,Singh,A.,2016.Natural genetic variation in Brassica homologs of FLOWERING LOCUS T and characterization of its expression domains.J Plant Biochem Biot,25:270-277.
Melzer,S.,Lens,F.,Gennen,J.,Vanneste,S.,Rohde,A.,Beeckman,T.,2008.Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana.Nat Genet,40:1489-1492.
Michaels,S.D.,2005.Integration of flowering signals in winter-annual Arabidopsis.Plant Physiol,137:149-156.
Nakamura,T.,Song,I.J.,Fukuda,T.,Yokoyama,J.,Maki,M.,Ochiai,T.,Kameya,T.,Kanno,A.,2005.Characterization of TrcMADS1 gene of Trillium camtschatcense(Trilliaceae)reveals functional evolution of the SOC1/TM3-like gene family.J Plant Res,118:229-234.
Pflieger,S.,Blanchet,S.,Camborde,L.,Drugeon,G.,Rousseau,A.,Noizet,M.,Planchais,S.,Jupin,I.,2008.Efficient virus-induced gene silencing in Arabidopsis using a“one-step”TYMV-derived vector.Plant J,56:678-690.
Qüesta,J.I.,Song,J.,Geraldo,N.,An,H.,2016.The sequence specific transcriptional repressor VAL1 triggers Polycomb silencing at FLC.Science,1:1-5.
Ryan,P.T.,Maoiléidigh,D.S.ó.,Drost,H.,Kwa,K.,Gabel,A.,Grosse,I.,Graciet,E.,Quint,M.,Wellmer,F.,2015.Patterns of gene expression during Arabidopsis flower development from the time of initiation to maturation.BMC Biol,16:488.
Samach,A.,Onouchi,H.,Gold,S.E.,Ditta,G.S.,Schwarz-Sommer,Z.,Yanofsky,M.F.,Coupland,G.,2000.Distinct roles of constans target genes in reproductive development of Arabidopsis.Science,288:1613-1616.
Schiessl,S.,Huettel,B.,Kuehn,D.,Reinhardt,R.,Snowdon,R.J.,2017.Targeted deep sequencing of flowering regulators in Brassica napus reveals extensive copy number variation.Sci Data,4:1-10.
Searle,I.,He,Y.,Turck,F.,Vincent,C.,Fornara,F.,Kr?ber,S.,Amasino,R.A.,Coupland,G.,2006.The transcription factor FLCconfers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis.Genes Dev,20:898-912.
Sheldon,C.C.,Rouse,D.T.,Finnegan,E.J.,Peacock,W.J.,Dennis,E.S.,2000.The molecular basis of vernalization:the central role of FLOWERING LOCUS C(FLC).Proc Natl Acad Sci,97:3753-3758.
Shim,J.S.,Imaizumi,T.,2015.Circadian clock and photoperiodic response in Arabidopsis:from seasonal flowering to redox homeostasis.Biochemistry,54:157-170.
Shindo,C.,Aranzana,M.J.,Lister,C.,Baxter,C.,Nicholls,C.,Nordborg,M.,Dean,C.,2005.Role of FRIGIDA and FLOWERING LOCUSC in determining variation in flowering time.Plant Physiol,138:1163-1173.
Swiezewski,S.,Liu,F.,Magusin,A.,Dean,C.,2009.Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target.Nature,462:799-802.
Tyagi,S.,Mazumdar,P.A.,Mayee,P.,Shivaraj,S.M.,Anand,S.,Singh,A.,Madhurantakam,C.,Sharma,P.,Das,S.,Kumar,A.,Singh,A.,2018.Natural variation in Brassica FT homeologs influences multiple agronomic traits including flowering time,silique shape,oil profile,stomatal morphology and plant height in B.juncea.Plant Sci,277:251-266.
Thompson,J.D.,Gibson,T.,Higgins,D.G.,2002.Multiple sequence alignment using ClustalW and ClustalX.Curr Protoc Bioinformatics,1:2-3.
Valverde,F.,2011.CONSTANS and the evolutionary origin of photoperiodic timing of flowering.J Exp Bot,62:2453-2463.
Wagner,D.,2016.Making flowers at the right time.De Cell,37:208-210.
Wu,F.,Sedivy,E.J.,Price,W.B.,Haider,W.,Hanzawa,Y.,2017.Evolutionary trajectories of duplicated FT homologues and their roles in soybean domestication.Plant J,90:941-953.
Xi,X.,Wei,K.,Gao,B.,Liu,J.,Liang,J.,Cheng,F.,Wang,X.,Wu,J.,2018.BrFLC5:a weak regulator of flowering time in Brassica rapa.Theor Appl Genet,131:2107-2116.
Xu,F.,Rong,X.,Huang,X.,Cheng,S.,2012.Recent advances of Flowering Locus T gene in higher plants.Int J Mol Sci,13:3773-3781.
Yang,J.,Liu,D.,Wang,X.,Ji,C.,Cheng,F.,Liu,B.,Hu,Z.,Chen,S.,Pental,D.,Ju,Y.,Yao,P.,Li,X.,Xie,K.,Zhang,J.,Wang,J.,Liu,F.,Ma,W.,Shopan,J.,Zheng,H.,Mackenzie,S.A.,Zhang,M.,2016.The genome sequence of allopolyploid Brassica juncea and analysis of differential homeolog gene expression influencing selection.Nat Genet,48:1225-1232.
Yang,J.,Song,N.,Zhao,X.,Qi,X.,Hu,Z.,Zhang,M.,2014.Genome survey sequencing provides clues into glucosinolate biosynthesis and flowering pathway evolution in allotetrapolyploid Brassica juncea.BMC Genomics,15:107.
Yang,J.,Zhang,C.,Zhao,N,Hu,Z.,Chen,S,Zhang,M.,2018.Chinese root-type mustard provides phylogenomic insights into the evolution of the multi-use diversified allopolyploid Brassica juncea.Mol Plant,11:512-514.
Zhang,Z.,Li,J.,Zhao,X.Q.,Wang,J.,Wong,G.K.S.,Yu,J.,2006.KaKs calculator:calculating Ka and Ks through model selection and model averaging.GPB,4:259-263.
Bowers,J.E.,Chapman,B.A.,Rong,J.,Paterson,A.H.,2003.Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events.Nature,422:433-438.
Cai,X.,Cui,Y.,Zhang,L.,Wu,J.,Liang,J.,Cheng,L.,Wang,X.,Cheng,F.,2018.Hotspots of independent and multiple rounds of LTR-retrotransposon burst in Brassica species.Horticultural Plant J,4:165-174.
Cho,L.,Yoon,J.,An,G.,2017.The control of flowering time by environmental factors.Plant J,172:708-719.
Csorba,T.,Questa,J.I.,Sun,Q.,Dean,C.,2014.Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization.Proc Natl Acad Sci,111:16160-16165.
Duan,W.,Song,X.,Liu,T.,Huang,Z.,Ren,J.,Hou,X.,Du,J.,Li,Y.,2014.Patterns of evolutionary conservation of ascorbic acid-related genes following whole-genome triplication in Brassica rapa.Genome Biol Evol,7:299-313.
Duan,W.,Zhang,H.,Zhang,B.,Wu,X.,Shao,S.,Li,Y.,Hou,X.,Liu,T.,2017.Role of vernalization-mediated demethylation in the floral transition of Brassica rapa.Planta,245:227-233.
Fornara,F.,Montaigu,A.,Coupland,G.,2010.SnapShot:control of flowering in Arabidopsis.Cell,141:3-5.
Gaeta,R.T.,Pires,J.C.,Iniguez-Luy,F.,Leon,E.,Osborn,T.C.,2007.Genomic changes in resynthesized Brassica napus and their effect on gene expression and phenotype.Plant Cell,19:3403-3417.
Green,R.M.,2002.Circadian rhythms confer a higher level of fitness to Arabidopsis plants.Plant Physiol,129:576-584.
Garner,W.W.,Allard,H.A.,1930.Effect of the relative length of day and night and other factors of the environment on growth and reproduction in Plants.Jap Comp Educ Soc,15:7-8.
Jian,H.,Zhang,A.,Ma,J.,Wang,T.,Yang,B.,Shuang,L.S.,Liu,M.,Li,J.,Xu,X.,Paterson,A.H.,Liu,L.,2019.Joint QTL mapping and transcriptome sequencing analysis reveal candidate flowering time genes in Brassica napus L.BMC Genomics,1:20-21.
Johansson,M.,Staiger,D.,2015.Time to flower:interplay between photoperiod and the circadian clock.J Exp Bot,66:719-730.
Kim,D.H.,Sung,S.,2014.Genetic and epigenetic mechanisms underlying vernalization.Arabidopsis Book,12:e0171.
Laughton,A.M.,O’Connor,C.O.,Knell,R.J.,2017.Responses to a warming world:integrating life history,immune investment,and pathogen resistance in a model insect species.Ecol Evol,7:9699-9710.
Lee,J.,Oh,M.,Park,H.,Lee,I.,2008.SOC1 translocated to the nucleus by interaction with AGL24 directly regulates LEAFY.Plant J,55:832-843.
Lee,S.,Kim,J.,Han,J.J.,Han,M.J.,An,G.,2004.Functional analyses of the flowering time gene OsMADS50,the putative suppressor of overexpression of SOC1/Agamous-Like 20(SOC1/AGL20)ortholog in rice.Plant J,38:754-764.
Livak,K.J.,Schmittgen,T.D.,2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-CTmethod.Methods,25:402-408.
Levy,A.A.,2002.The impact of polyploidy on grass genome evolution.Plant Physiol,130:1587-1593.
Liu,C.,Zhou,J.,Bracha-Drori,K.,Yalovsky,S.,Ito,T.,Yu,H.,2007.Specification of Arabidopsis floral meristem identity by repression of flowering time genes.Development,134:1901-1910.
Liu,L.J.,Zhang,Y.C.,Li,Q.H.,Sang,Y.,Mao,J.,Lian,H.L.,Wang,L.,Yang,H.Q.,2008.COP1-mediated ubiquitination of CONSTANS is implicated in cryptochrome regulation of flowering in Arabidopsis.Plant Cell,20:292-306.
Lv,X.,Lan,S.,Guy,K.M.,Yang,J.,Zhang,M.,Hu,Z.,2016.Global expressions landscape of NAC transcription factor family and their responses to abiotic stresses in Citrullus lanatus.Sci Rep,6:1-14.
Mayee,P.,Singh,A.,2016.Natural genetic variation in Brassica homologs of FLOWERING LOCUS T and characterization of its expression domains.J Plant Biochem Biot,25:270-277.
Melzer,S.,Lens,F.,Gennen,J.,Vanneste,S.,Rohde,A.,Beeckman,T.,2008.Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana.Nat Genet,40:1489-1492.
Michaels,S.D.,2005.Integration of flowering signals in winter-annual Arabidopsis.Plant Physiol,137:149-156.
Nakamura,T.,Song,I.J.,Fukuda,T.,Yokoyama,J.,Maki,M.,Ochiai,T.,Kameya,T.,Kanno,A.,2005.Characterization of TrcMADS1 gene of Trillium camtschatcense(Trilliaceae)reveals functional evolution of the SOC1/TM3-like gene family.J Plant Res,118:229-234.
Pflieger,S.,Blanchet,S.,Camborde,L.,Drugeon,G.,Rousseau,A.,Noizet,M.,Planchais,S.,Jupin,I.,2008.Efficient virus-induced gene silencing in Arabidopsis using a“one-step”TYMV-derived vector.Plant J,56:678-690.
Qüesta,J.I.,Song,J.,Geraldo,N.,An,H.,2016.The sequence specific transcriptional repressor VAL1 triggers Polycomb silencing at FLC.Science,1:1-5.
Ryan,P.T.,Maoiléidigh,D.S.ó.,Drost,H.,Kwa,K.,Gabel,A.,Grosse,I.,Graciet,E.,Quint,M.,Wellmer,F.,2015.Patterns of gene expression during Arabidopsis flower development from the time of initiation to maturation.BMC Biol,16:488.
Samach,A.,Onouchi,H.,Gold,S.E.,Ditta,G.S.,Schwarz-Sommer,Z.,Yanofsky,M.F.,Coupland,G.,2000.Distinct roles of constans target genes in reproductive development of Arabidopsis.Science,288:1613-1616.
Schiessl,S.,Huettel,B.,Kuehn,D.,Reinhardt,R.,Snowdon,R.J.,2017.Targeted deep sequencing of flowering regulators in Brassica napus reveals extensive copy number variation.Sci Data,4:1-10.
Searle,I.,He,Y.,Turck,F.,Vincent,C.,Fornara,F.,Kr?ber,S.,Amasino,R.A.,Coupland,G.,2006.The transcription factor FLCconfers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis.Genes Dev,20:898-912.
Sheldon,C.C.,Rouse,D.T.,Finnegan,E.J.,Peacock,W.J.,Dennis,E.S.,2000.The molecular basis of vernalization:the central role of FLOWERING LOCUS C(FLC).Proc Natl Acad Sci,97:3753-3758.
Shim,J.S.,Imaizumi,T.,2015.Circadian clock and photoperiodic response in Arabidopsis:from seasonal flowering to redox homeostasis.Biochemistry,54:157-170.
Shindo,C.,Aranzana,M.J.,Lister,C.,Baxter,C.,Nicholls,C.,Nordborg,M.,Dean,C.,2005.Role of FRIGIDA and FLOWERING LOCUSC in determining variation in flowering time.Plant Physiol,138:1163-1173.
Swiezewski,S.,Liu,F.,Magusin,A.,Dean,C.,2009.Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target.Nature,462:799-802.
Tyagi,S.,Mazumdar,P.A.,Mayee,P.,Shivaraj,S.M.,Anand,S.,Singh,A.,Madhurantakam,C.,Sharma,P.,Das,S.,Kumar,A.,Singh,A.,2018.Natural variation in Brassica FT homeologs influences multiple agronomic traits including flowering time,silique shape,oil profile,stomatal morphology and plant height in B.juncea.Plant Sci,277:251-266.
Thompson,J.D.,Gibson,T.,Higgins,D.G.,2002.Multiple sequence alignment using ClustalW and ClustalX.Curr Protoc Bioinformatics,1:2-3.
Valverde,F.,2011.CONSTANS and the evolutionary origin of photoperiodic timing of flowering.J Exp Bot,62:2453-2463.
Wagner,D.,2016.Making flowers at the right time.De Cell,37:208-210.
Wu,F.,Sedivy,E.J.,Price,W.B.,Haider,W.,Hanzawa,Y.,2017.Evolutionary trajectories of duplicated FT homologues and their roles in soybean domestication.Plant J,90:941-953.
Xi,X.,Wei,K.,Gao,B.,Liu,J.,Liang,J.,Cheng,F.,Wang,X.,Wu,J.,2018.BrFLC5:a weak regulator of flowering time in Brassica rapa.Theor Appl Genet,131:2107-2116.
Xu,F.,Rong,X.,Huang,X.,Cheng,S.,2012.Recent advances of Flowering Locus T gene in higher plants.Int J Mol Sci,13:3773-3781.
Yang,J.,Liu,D.,Wang,X.,Ji,C.,Cheng,F.,Liu,B.,Hu,Z.,Chen,S.,Pental,D.,Ju,Y.,Yao,P.,Li,X.,Xie,K.,Zhang,J.,Wang,J.,Liu,F.,Ma,W.,Shopan,J.,Zheng,H.,Mackenzie,S.A.,Zhang,M.,2016.The genome sequence of allopolyploid Brassica juncea and analysis of differential homeolog gene expression influencing selection.Nat Genet,48:1225-1232.
Yang,J.,Song,N.,Zhao,X.,Qi,X.,Hu,Z.,Zhang,M.,2014.Genome survey sequencing provides clues into glucosinolate biosynthesis and flowering pathway evolution in allotetrapolyploid Brassica juncea.BMC Genomics,15:107.
Yang,J.,Zhang,C.,Zhao,N,Hu,Z.,Chen,S,Zhang,M.,2018.Chinese root-type mustard provides phylogenomic insights into the evolution of the multi-use diversified allopolyploid Brassica juncea.Mol Plant,11:512-514.
Zhang,Z.,Li,J.,Zhao,X.Q.,Wang,J.,Wong,G.K.S.,Yu,J.,2006.KaKs calculator:calculating Ka and Ks through model selection and model averaging.GPB,4:259-263.