BACKGROUND
sucrose transporters  are important for both phloem loading in source tissue and sucrose uptake into some sink cells  <cit> .
suts are considered to be the control points for sucrose storage in plant because they can carry sucrose across cell membranes and play an important role in loading sucrose into phloem systems in a series of steps  <cit> .
plant suts belong to the glycoside-pentoside-hexuronide  cation symporter family  that is part of the major facilitator super-family   <cit> .
transporters in the gph family have the primary characteristics of mfs proteins that contain  <dig> transmembrane spanning domains with n- and c-termini in the cytoplasm  <cit> .
since the first plant sucrose transporter sosut <dig> was functionally identified using an elegant yeast complementation strategy from spinach   <cit> , suts have been demonstrated to effect on the multiple aspects of plant development such as biomass partitioning, pollen germination, restraining plant growth, fruit size reduction and ethylene biosynthesis .
comprehensive understanding of the molecular structure and evolution of a gene family in plant species is the first step towards understanding their physiological roles and metabolic mechanism involved in different growth phases.
recent studies have revealed that sut was a small gene family that consisted of at least four sut genes in the most plant species.
the sut family members have been identified from a number of plant species such as arabidopsis  <cit> , rice   <cit> , wheat   <cit> , populus  <cit> , sorghum   <cit>  and pineapple   as well.
phylogenetic analysis of sut family suggested that the plant sut could be divided into five subgroups including two monocot specific subgroups, one dicot specific and two monocot-dicot subgroups.
sut family members in many plant species are divergent in function and deferentially expressed in different tissues types or at different plant developmental stages.
for instance, in rice, ossut <dig> expression has been detected in germinated seeds, leaf blades, leaf sheaths and panicles ; the expressions of both ossut <dig> and ossut <dig> are dramatically lower in embryos than those of ossut <dig>  ossut <dig>  and ossut <dig>  <cit> ; ossut <dig> has been detected in most tissues such as roots, leaves, and panicles  <cit> , and could play a role in sucrose loading into the sheath phloem of the upper leaves during the post-heading period for sucrose transport to developing grains  <cit> .
in arabidopsis  <cit> , atsuc <dig> has been detected in phloem and companion cells in source leaves  <cit>  and functions in loading sucrose into the phloem sieve elements , correspond with the result identified by tissue-specific complementation of different promoters  <cit>  and by 14c labeling studies  <cit> ; atsuc <dig> and atsuc <dig> are expressed in minor veins of source leaves of mature plants .
sugarcane  is one of the world’s most produced crops , and contributes to about 80 % of the world sugar and about 40 % of ethanol production worldwide.
modern sugarcane cultivar has one of the most complex genome among all the crops; by being both aneupoid and autopolypoid with an extreme ploidy level that can range from octoploidy  to dodecaploidy .
approximately 80 % of cultivars’ chromosomes are derived from s. officinarum and 10–20 % is derived from s.spontaneum with the remained from interspecific recombination .
sugarcane is not only an economically important crop species, but also serves as an important model crop for studying sucrose transporters because its remarkable ability to accumulate vast amounts of sucrose in its stems that can reach close to 700 mm or in excess of 50 % of the dry weight    <cit> .
but to date, limited works in characterizing these sut genes have been reported, except for sut <dig> .
in an earlier study based on sugarcane expressed sequence tag  database survey, sut <dig> was revealed to be more abundant in the mature internodes than the other tissues  <cit> .
this result was confirmed by a study for hawaiian sugarcane cultivar, in which the sut <dig> transcript levels increased during maturation and sucrose storage, whereas, sut <dig> expression was observed to be not affected by sugarcane yellow leaf virus  infection in sugarcane  <cit> .
biochemical analysis of sugarcane sut <dig> suggested that sut <dig> was highly selective for sucrose, but had a relatively low affinity for sucrose, inhibited by sucralose and played key role in sucrose loading from the vascular tissues into the storage sites in parenchyma cells of sugarcane stems  <cit> .
besides sut <dig>  the gene family of suts is not understood in sugarcane due to the formidable challenge caused by its high degree of polyploidy and heterozygosity genome.
in this study, to gain comprehensive understandings of the molecular and evolutionary characterization as well as the possible functions of sut family in sugarcane, based on combination of comparative genomics strategies and high genome coverage of bacterial artificial chromosomes  libraries resources, we identified and characterized sut gene families in saccharum species and investigated their transcriptional expression patterns.
the analysis in this study mainly focused on:  identifying the gene members and allele haplotypes of the sut gene family in sugarcane;  analyzing evolutionary relationship, exon/intron organization of the sut gene family; and  characterizing the expression patterns of the sut gene family in three progenitor saccharum species.
methods
plant materials
three varieties of saccharum species were used in the study: la-purple , molokai <dig>  and ses <dig>   <cit> .
plants were grown in plastic pots under greenhouse conditions and standard growing practices.
tissue samples were obtained from 10-month old plants  for leaf roll, leaf, top immature internode , premature internode  and mature internode .
the internodes were numbered from top to bottom according to the method of moore  <cit> .
stem and leaf tissues from seedlings of the three species were collected at 35 days after planting.
for drought stress treatment, the 35 day-old seedlings were treated with peg <dig>  for 48 h, and stem and leaf tissues were collected.
the tissues were immediately frozen using liquid nitrogen and stored at −80 °c prior to rna isolation.
bac libraries
the haploid of s. spontaneum ses <dig>  ap85- <dig> , was used to construct the bac library.
nuclei were isolated from the young leaf tissues of ap85- <dig> following the method described by ming et al.
<cit> .
the high molecular weight dna embedded in agarose was partially digested using hindiii.
fractions at approximately 100 kb were recovered and cloned into psmart bac vector .
<dig>  clones from ap85- <dig> bac libraries were picked and stored in  <dig> 384-well plates with freezing medium.
bac clones were grided onto performa ii nylon filters  using q-pix  <dig> .
database search for the sut gene family and phylogenetic analyses
the sequence data used in this study were collected using the keyword “sucrose transporter” and a query search in the genbank using the known sut gene sequences from sorghum  <cit> , rice  <cit>  and arabidopsis  <cit> .
matches achieved similarity scores of  <dig>  and probability scores > <dig>  and e-value <10− <dig> were collected.
the amino acid sequences of sucrose transporter gene family members in  <dig> monocotyledons  and  <dig> dicotyledons  identified by searching public databases available at various resources.
the phylogenetic trees were constructed with the mega <dig> .
<dig> program with clustalw alignment using default parameter.
identification and sequencing of sut families from bac library
bac library screening was carried out as described by yu et al.
<cit> .
the bac clones representing different haplotypes were selected.
the insert size of bac clones was estimated by comparing with standard size markers using chef gel electrophoresis.
the bac dnas were isolated using phasepreptm tmbac dna kit  and the sequencing libraries were prepared individually with unique barcode for each clone.
the sequencing libraries were then pooled and sequenced with 150 bp, pair-end reads on illumina hiseq <dig> at center for genomics and biotechnology in fujian agriculture and forestry university.
the raw reads were then assembled using spades genome assember v.  <dig> .
<dig> .
genomic sequence annotation and functional prediction
the genomic sequences of sut genes were annotated by dna subway , and the corresponding cds sequences were translated into protein by the expasy-translate tool .
the exon-intron structures were graphed using online tool gsds .
the putative conserved domains of sucrose transporter protein were detected by using blastp  and interpro .
the protein transmembrane helices domain was predicted using tmpred .
the isoelectric point and relative molecular mass of the protein were predicted using expasy .
analysis of sucrose transporter gene co-expression profiling
the sorghum gene models were used as reference to align the saccharum rna-seq database by using novoalign  with default parameter.
the normalization and statistical evaluation of differential gene expression has been performed using edge-r with a p-value cut-off of  <dig>  and using the benjamini-hochberg  <cit>  method for multiple testing corrections.
the raw data was normalized according to the default procedure of the differential expression analysis package used.
the dispersion was estimated using the pooled setting.
the expression values were log-transformed, and cluster analyses were performed using a software cluster with euclidean distances and the hierarchical cluster method of “complete linkage clustering”.
the clustering tree was constructed and viewed in java treeview.
experimental validation of expression levels of sut gene by qrt-pcr
the expression levels of six sut genes in three tissues  of three saccharum species were validated by qrt-pcr.
gene-specific primer pairs were designed by using integrated dna technologies  .
after treated with dnase i , two microgram of rna was used in reverse transcription with the superscript vilo cdna synthesis kit  according to the manufacturer’s guidelines.
the real-time qpcr was performed by using multicolor real-time pcr detection system  with conditions for all reactions were 95 °c for 30s, 40 cycles of 95 °c for 5 s, followed by 60 °c for 30s, and 95 °c for 10s.
melting curve analysis were performed to confirm the pcr specificity.
the glyceraldehyde-3-phosphate dehydrogenase gene  and eukaryotic elongation factor 1a  were selected as internal standard for normalization  <cit> , and three replicates were completed for each sample.
the relative expression level for each sut gene in different tissues of three saccharum species were calculated by using the 2-ΔΔct method.
the correlation coefficient was calculated between the transcript accumulation levels obtained by rnaseq and qrt-pcr using excel.
RESULTS
identification of six sut genes in sugarcane
<dig> positive bac clones from ap85- <dig> were identified using  <dig> probes  designed from six well-annotated suts genomic regions in sorghum bicolor .
to determine haplotypes, the pcr fragments of the six suts were cloned by using these probe primers and sequenced, which confirmed  <dig> of these  <dig> bac clones contains different paralogous and homologous haplotypes.
the six s.spontaneum suts were referred to sssut1-sssut <dig> according to sequence similarity with sorghum suts  <cit> .
in the  <dig> sut sequences, both sssut <dig> and sssut <dig>  both sssut <dig> and sssut <dig> and remaining two suts have  <dig>   <dig> and  <dig> allelic haplotypes, respectively.
the allelic haplotypes of each suts were indicated additional“-h1” to “-h4” to the gene name end.
using the gene model sequences of the annotated sut genes as queries, both the in-house est and genbank database were extensively searched.
the results showed that all the sssuts had the corresponding ests in the genbank database except sssut <dig> .table  <dig> information of the putative sut genes in sorghum
sbsut1
sbsut2
sbsut3
sbsut4
sbsut5
sbsut6
the six sssuts containing complete orfs  with the predicted molecular weights ranged from  <dig>  to  <dig>  kda in sugarcane .
comparing with the sut family from sorghum, sssut <dig> showed a lower molecular weight, and the remaining gene pairs between these two species were consistent.
sssut <dig> and sssut <dig> shared a higher similarity of protein sequences  in contrast to the other  <dig> pairwise sequences between the remaining four genes in the sut families  .
the analyses of the deducted protein sequences of the sut genes in sugarcane revealed that all these gene families had highly conserved mfs domains and contained  <dig> membrane-spanning helices .
a conserved histidine residue was presented in the first loop domain corresponding to his- <dig>  <cit>  and amino acids which corresponded to the g-x-x-x-d/e-r/k-x-g--r/k-r/k motif reside in the second and eighth loop domains  <cit> .
additionally, sssut <dig> contained a lxxll motif in the n-terminal domain, indicating that it might be targeted to the tonoplast  <cit> .table  <dig> comparison of the characterization of the suts between sugarcane and sorghum
sbsut1
sssut1
sbsut2
sssut2
sbsut3
sssut3
sbsut4
sssut4
sbsut5
sssut5
sbsut6
sssut6
sssut1
sssut2
sssut3
sssut4
sssut5
sssut6
sssut2
sssut3
sssut4
sssut5
sssut6
allelic haplotype analysis of sssuts
genomic sequence comparisons within the allelic haplotypes from the four suts revealed that these allelic haplotypes shared very high similarity above 99 %.
slight variations were observed within the allelic haplotypes of sssuts.
the deducted protein sequences were compared within the four sssuts.
the results showed that, for the protein sequence of allelic haplotypes, both sssut <dig> and sssut <dig> shared identities of  <dig>  %, while, sssut <dig> and sssut <dig> had  <dig>  and  <dig>  % sequence similarity, respectively.
the specific amino acids variations were discovered through the alignment of the allelic haplotypes from each suts.
<dig>   <dig>   <dig> and  <dig> amino acids within sssut <dig>  sssut <dig>  sssut <dig>  sssut <dig> respectively were observed to be vary among the allelic haplotypes .
in addition, in sssut <dig>  compared with h <dig> and h <dig>  h <dig> and h <dig> had shorter peptides with  <dig> amino acids deletion caused by the shift of exon structure .
furthermore, in sssut <dig>   <dig> of the variant amino acids were located at the sixth loop domain  and transmembrane helixes , indicating that potential functional variation existed among these allelic haplotypes .table  <dig> the variation of deducted amino acid sequences among allelic haplotypes within the four sssut
sssut1
sssut2
sssut3
sssut4
sssut5
sssut6
fig.
<dig> alignment of the amino acid sequences of sssut haplotypes.
amino acid sequences of haplotypes were aligned using the dnaman program.
similarity in amino acids across all the sequences is indicated by stars.
the difference between haplotypes was shown in a red box
in general, gene structures of the sssuts allelic haplotypes presented high conservation for exon/intron numbers and exon sizes, while, for intron size, in sssut <dig>  sssut3-h3’s first intron was larger than other allelic haplotypes; and in sssut <dig>  both sssut6-h <dig> and sssut6-h <dig> contained a smaller second exon than sssut6-h <dig> and sssut6-h <dig> .fig.
<dig> comparison of the allelic gene structures of sssuts.
boxes represent exons, triangles represent transposons
nonsynonymous to synonymous substitution ratio  was analyzed to investigate evolutionary function constraint in s.spontaneum.
to identify the evolutionary forces acting on four sut genes having alleles  , the ka/ks was calculated.
within the coding regions, the ka/ks ratio was much less than  <dig>  indicating that purifying selection was the dominant force driving the evolution of sssut genes.fig.
<dig> the ka/ks of sssut haplotypes and sbsut-sssut.
the lower value of ka/ks was indicated by stars
comparative analysis of gene structure between sssut and other plant sut
the gene structures of sssut family has a great variation with exon numbers ranging from five to fourteen, and their introns were aligned accordance with the gt-ag rule for splicing sites.
sssut <dig>  sssut <dig> and sssut <dig> had larger first introns than the other sut genes.
both sssut <dig> and sssut <dig> only have five exons that were lesser than the other sssuts .
in addition, comparative analysis of sssut families suggested that, the fifth exon in sssut <dig> were presumed to split into 3– <dig> exons, and the first, second, and fifth exons in sssut <dig> were presumed to evolve into 2– <dig> exons .
sssut <dig> and sssut <dig> shared highest similarity of exon/intron pattern in spite of the great size variation between the second introns, which correlated to their amino acids similarities .fig.
<dig> comparison of the gene structure of the six members of the sssut gene family.
three monocotyledons  and dicotyledons  sut gene family are also shown for comparison.
boxes represent exons, triangles represent transposons
to investigate the evolutionary mechanisms underlying the genesis of gene families, we performed comparative analyses of the sssut structures with the sut families from sorghum, rice, arabidopsis , maize, grape  and potato  .
the suts from these species could be divided into five groups sut <dig> to sut <dig>  which were consistent with previous studies  <cit> .
the results showed that suts in the same group had the similar gene structures.
in the dicot specific group sut <dig>  most of the genes had two to four exons, in which the first exons were large and the second ones were small.
in this group, the only exception was stsut <dig>  which had a smaller first exon, due to the possible exon splitting comparing with the other dicot species.
in group sut <dig> that was closely related to group sut <dig>  all the genes had first two large exons that were likely originated from sut <dig> first exon splitting based on sequence comparison.
it was interesting that in sut <dig> group, the dicot genes and the monocot genes had siminar number of exons,  <dig> and  <dig>  respectively, and the sequences of the fifth and sixth exons in dicot were presumed to originated from the monocot fifth exon splitting.
furthermore, based on the genomic and amino acids sequence comparisons among the  <dig> sequences from the seven plant species in group sut <dig> and sut <dig>  the common ancestral gene of monocots and dicots were suggested to have two exons and the exon members have differentiated in a later period of evolution caused by exon splits and partial exon fusions.
the genes in group containing sut <dig> had similar exon number of  <dig> or  <dig> for both dicot and monocot species.
gene size expansion due to intron size stretching in dicot plant grape and potato were observed in this group.
in the sut <dig> group, besides zmsut <dig> with an additional small intron caused by the first exon splitting, the remaining sut <dig> group genes harbor large first introns, which included wheat tasut1d  <cit>  and tomato lesut <dig>  <cit> .
in contrast to sut <dig> group, the sut <dig> group had varied number of exons ranging from  <dig> to 14; among them, sssut <dig>  sbsut <dig> and zmsut <dig> had six exons, the other genes in the subfamilies contained 10– <dig> exons; both of ossuts in this group had more exons number than their orthologous genes.
in monocot specific group sut <dig>  similar to the high identities shown by the alignment of amino acid sequence of sssut <dig> and sssut <dig>  high conservation of exon/intron structures were observed from the schematic representation; sssut <dig> had larger second exon than the other genes; similar to sut <dig> group, ossut had more exon number than their orthologous genes.
scrutiny of the exon/intron structure of the  <dig> genes in branch with sut3/sut2/sut <dig> revealed that the exons could be corresponding to six exons, in which the first and the last two exons were observed to be fused/spited.
these results suggested that the genes in this branch might originate from common ancestral gene containing six exons for both monocots and dicots.
phylogenetic analysis of sssut and other plant sut homologs
to comprehensively analyze the evolutionary relationships of sut families between s. spontaneum and other plant species, we aligned  <dig> plant amino acid sequences from  <dig> dicots and  <dig> monocots including s.spontaneum, using clustalx to construct an unrooted tree with neighbor-joining method .
same as the distribution above, the suts were phylogenetically distributed into five groups, sut <dig>  sut <dig>  sut <dig>  sut <dig> and sut <dig>  respectively.
among these five groups, sut <dig> genes are only found in dicotyledonous, in contract, genes in sut <dig> and sut <dig> groups are only from monocotyledonous.
whereas, the remaining two groups, sut <dig> and sut <dig>  are found in both dicot and monocot, and could be well classified into two distinct subclades.
these results strongly suggested that plant suts were diverged from a recent evolutionary event after the common ancestor of dicots and monocots.
in addition, the sut families could be divided into two branches in the phylogenetic tree, with sut <dig> and sut <dig> groups in one branch and the other three groups in another branch, indicting two ancestral genes were the origins of suts in both dicot and monocot.fig.
<dig> phylogenetic analysis of sssut and other plant sut homologs.
unrooted phylogenetic tree of plant sut proteins constructed using the neighbour-joining method with mega  <dig> .
<dig> program.
zmsut: zea mays, sbsut: sorghum bicolor, bdsut: brachypodium distachyon, ossut: oryza sativa, atsut: arabidopsis thaliana, cssut: citrus sinensis, gmsut: glycine max, stsut: solanum tuberosum,vvsut: vitis vinifera, sisut: setaria italica, sssut:saccharum spontaneum
in dicot specific sut <dig> group, the paralogous genes from each of the dicot species were observed to be closely related, indicting recent gene duplications after the divergence of dicotyledons .
in addition, both sut gene number and sequences had great variations among the dicot plant species, suggesting rapid evolutionary dynamics exist in the dicotyledonous sut families .
in sut <dig> group, all dicots and monocots had only one gene member, which the phylogenetic distribution were generally consistent with the plant species taxonomy .
in sut <dig>  as description above, the genes from dicot and monocot species could be further classified into two subclades, respectively.
in this group, dicot plants have d 1- <dig> gene members, while, all the monocot plants only hadone gene member, which suggestted that dicot plants sut <dig> group were undergoing expansion.
in the monocot specific sut <dig>  the examined monocot species consisted of two paralogous genes from two separated clades, suggesting the gene duplication event occurred before the divergence of these dicot plants.
in contract to sut <dig> group, in sut <dig> group, recent gene duplication events were observed in the andropogoneae plants  and bambusa oldhamiias shown by the closest phylogenetic distribution of genes within these plant species.fig.
<dig> the distribution of sut family member in monocotyledon  and dicotyledon
in the sssut family, sssut <dig> was the solo member in a branch, but sssut <dig>  sssut <dig>  sssut <dig>  sssut <dig> and sssut <dig> were clustered together and shared a more recent common ancestral gene.
thus, sssut <dig> was suggested to be the oldest gene, while, both of sssut <dig> and sssut <dig> should be younger than the splitting of trib, andropogoneae dumort and zea mays.
in addition, sssut <dig> and sssut <dig> were observed to be undergoing rapid evolution as shown by lower amino acids sequences similarity than other orthologous genes between the sorghum and saccharum .
gene expression of suts among three saccharum species
to investigate the possible physiological functions for suts, we performed comparative transcriptome profiling among three saccharum species at different developmental stages of seedling and five different tissues from the mature leaf  and stalks  by using rna-seq method.
the rna-seq results were verified by qrt-pcr in three tissues  from each of the three saccharum speices .
there is a significant positive relationship  between the reads per kilobases per million reads  based on rna-seq and the relative expression level based on qrt-pcr .fig.
<dig> the sut gene family expression based on rpkm in different tissues of different stage in three saccharum species.
the expression level of  <dig> sut genes in seeding stage and mature stage of nature condition.
a correlation coefficient between rnaseq  and qrt-pcr  of six sut genes.
b, and in seeding stage of drought stress condition .
in, internode; lr, leaf roll.
internnodes  <dig>   <dig>   <dig>  internodes  <dig>   <dig>   <dig> and internodes  <dig>   <dig>   <dig> were from saccharum officinarum , saccharum robustum  and saccharum spontaneum , respectively
in sut families, the transcription of sut <dig> was undetectable in all the examined tissues from sacchaurm plants, which was consistent with that of sut <dig> in sorghum  <cit> .
the other gene expression levels had significant variations with a clear trend of transcript levels from higher to lower sut <dig>  sut <dig>  sut <dig>  sut <dig> and sut <dig>  of them, sut <dig> and sut <dig> had predominant expression levels among the gene families, indicating that the two genes were the fundamental members in sut families.
sut <dig> transcripts were abundant in both source and sink organs, and were the most expressed gene in all tissues except for the mature leaf, indicating sut <dig> was the key member.
the gene displayed a higher expression level in sink tissues  than the source tissue  in mature plant.
however, it was the opposite in seedling plants, suggesting the gene was more important in phloem loading for seedlings than the mature plant.
comparing with both s. sponteanum and s. robustum, sut <dig> displayed a lower expression level in the high sucrose s. officinarum , in all examined tissues.
in addition, under drought stress, sut <dig> was observed to be down regulated in s. robustum.
sut <dig> was expressed in all the organs examined for the three saccharum species.
in the seedling plants, similar expressions were observed in both stem and leaves among these saccharum species.
in the mature plants, sut <dig> displayed a lower expression level in the source tissue of leave and leafroll for two lower sucrose accumulating saccahurm species, s. sponteneum and s. robustum, while, high sugar s. officinarum had a more uniform sut <dig> expression level in all of the examined tissues .
under drought stress, twofold lower levels of expressions were observed in the three saccharum species than their control .
sut <dig> had higher expression level in the seedling than the mature plants , suggesting it might contribute more for sucrose loading at the early age of plant development; whereas, sut <dig> displayed similar expression level in the examined tissues from both seedling and mature plants.
obviously, sut <dig> was not correlated to the sucrose content differential among the saccharum species.
under drought stress, in contrast to sut <dig> and sut <dig>  sut <dig> was up regulated in the leaf of three saccharum species.
sut <dig> displayed dramatically higher expression level in sink tissues  than in source  in the seeding plant; in contract, in the mature plants, the expression level were lower in leave than the other tissues.
similar to sut <dig>  sut <dig> showed up-regulation under water stress.
sut <dig> was undetectable in the seeding plants and had similar expression pattern as sut <dig> in the mature plants that gene expression exhibited higher in leave than the other tissues.
discussions
genomic study for the gene families is the first step toward the gene functional study.
however, the identifications of gene family in sugarcane is still a formidable challenge caused by its complex genomes.
recently, the whole genome sequencing of sorghum and other relative species of saccharum provides the references for comparative genomics to identify the gene families in saccharum species.
in previous studies, based on comparative genomics, the gene families of phosphoenol pyruvate carboxylase gene  <cit> , sucrose synthase  <cit> , sucrose phosphate synthase  <cit> , atp-dependent phosphofructokinase  <cit>  were identified by using the est database, dna fragment, and cdna cloning.
however, none of these studies has investigated on the genomics of whole gene families because of the lack of genomic sequences for sugarcane.
our study through comparative genomics and bacs sequencing is the first report for the structure of a gene family and their gene allelic haplotypes in saccharum.
evolutionary conservation and divergence of sssut
plant suts had been well documented in previous studies for gene phylogenetic analyses two classifications  <cit> .
one classification was to divide plant suts into three types, type i, type ii and type iii, with the reference of arabidopsis  <cit> , in which, type i and type ii suts were localized to the plasma membrane, while type iii suts were associated with vacuolar membrane  <cit> .
another classification was to group the suts into five groups sut <dig>  sut <dig>  sut <dig>  sut <dig> and sut <dig>  <cit> .
comparing these two classifications, sut <dig> was included in type i, sut <dig> was in type ii, and sut <dig>  sut <dig> and sut <dig> were in type iii.
the former classification is likely associated with dicot plants studies, while, the latter is used for both dicot and monocot plants, especially for gene evolution studies.
in this study, we used large amount of plant suts for phylogenetic analysis, and the results confirmed the later classification, by revealing the existence of one dicot specific and two monocots specific groups and the independence evolution process in plant sut families .
furthermore, the comparative analysis of these plant suts showed that genes in sut <dig> group were more conservative than the genes in sut <dig> group, and further provided the direct evidence of the recent duplication event occurred after monocot/dicot divergence.
the evolution history of sssuts, which was sorted by age in duplicated descending order, sssut <dig>  sssut <dig>  sssut2/sssut <dig>  and sssut5/sssut <dig>
the comparative analyses could be used to predict the number of sut gene family members in saccharum.
without the whole genome sequences for saccharum, it could be debatable to conclude that we have discovered all suts in sacchaurm, in spite of our high coverage bacs libraries.
in this study, both phylogenetic analysis and sequences comparison revealed that saccharum spontaneum and sorghum bicolor were both composed of six suts members and each gene pairs were orthologous between the two species .
beside sut <dig> group, which was dicot specific, monocot plants have the same gene numbers in sut <dig>  sut <dig> and sut <dig> groups, the remaining group sut <dig> had the same gene number in andropogoneae plants, which were found to be the result of a recent duplication .
therefore, it is less likely that any sut gene duplication events had occurred after the diverging of sorghum and saccharum, knowing no additional gene in this study.
hence, we concluded that the six sssuts comprise the sut family in the saccarhum spontanum genome.
further experiments such as southern blot could be used to verify the conclusion.
the exon–intron structure differences were demonstrated to be accomplished by three main types of mechanisms, exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion  <cit> .
in this study, comparative analyses of gene structures for sssut made it possible to evaluate the sut gene structure evolution in plant .
all suts genes, including sssuts, had a great variation of exon numbers, ranging from  <dig> to  <dig>  comparing with the gene structure variation, the protein sequences were more conserved among the paralogous genes in sacchaurm, as shown by all sut members containing  <dig> membrane-spanning helices and similar protein sizes.
a common feature of plant suts was that the  <dig> membrane-spanning helices were distributed roughly uniformly in the deducted peptides of each gene in conserved position.
thus, the gene structure evolution after these plant divergences did not cause significant coding region variations.
therefore, the sut structures variations were mainly evolved from intron gain/lost and insertion/deletion but not from exonization/pseudoexonization.
sorghum is one of the closest relative diploid genera of sacchaurm.
comparative analysis of the orthologous between sssuts and sbsuts made it possible to investigate the specific evolutionary events after the polyploidzation of saccharum.
in sssuts, sssut <dig> harbored a much larger second intron than its closest paralogous sssut <dig> and the other orthologs .
similarly, sssut <dig> contained a larger first intron than its orthologous gene sbsut <dig>  in addition, sssut <dig> were observed to have a putative te  insertion in the last intron .
our results suggested that the sssut families were undergoing gene extension following polyploidization in s.spontaneum.
the estimated monoploid genome size of s. spontaneum  and s. officinarum  were both larger than the monoploid genome size of sorghum at 760 mb  <cit> , supporting the conclusion that the genome of saccharum expanded in general after polyploidization.
our results demonstrated the first case that expansion in intron regions of a gene families contribute to genome expansion in sacchaurm.
in this study, allelic haplotype sequences for sssut <dig>   <dig>   <dig> and  <dig> were comparatively analyzed for gene structures and ka/ks.
relative conservative gene structures were observed among allelic haplotypes within each of the four sssuts, whereas, sssut3-h <dig> contained a larger first intron than the other three haplotypes, both sssut6-h <dig> and sssut6-h <dig> had larger second exons .
in addition, the ka/ks ratios, which are all under  <dig> , revealed that all the suts allelic haplotypes were under strong purifying selection .
multiple alleles in polypoidy are considered to be functional redundant at the time of origin ; the conservation and constraint purification within the allelic haplotypes of sssuts were likely due to the key function of sut in saccharum.
it would be worthy to note that the transcription of sssut <dig> was undetectable in the examined tissues while its haplotypes were under constrain selection, indicating that sssut <dig> might have a necessary function for sacchaurm.
in paleopolyploids , and recent allopolyploid species, such as wheat  <cit>  and tragopogon  <cit> , eliminations and pseudogenizations of key functional genes after polyploidzation have been well documented.
the allelic gene variations supposed to the key topics for studying the genome dosage of sacchaurm species and further investigation for them would provide the foundation to understand the molecular basis of sugarcane genetics.
gene expression and functions of suts in saccharum
examination of sut gene expressions in saccharum species in source and sink tissues of seedling and mature plants proves an insightful indication regarding the roles of gene functions.
previous studies of gene expression were mostly done on saccharum hybrids with combined genetic background of s.officinarum and s.sponteneum.
to simplify genetic backgrounds, in this study, three saccharum species, high-sucrose s.offcinarum, low-sucrose s.robustum  and stress-tolerant s.spontaneum were used for studying the suts expression profiles.
sut <dig> was the only gene in sut family, which had been well documented in saccharum hybrid  <cit> .
in these previous studies, shsut <dig> had been shown to have high expression level in premature stem tissue, and decreased expression level in mature internodes  <cit> .
shsut <dig> was demonstrated to be highly selective for sucrose, inhibited by sucralose and had the function in loading sucrose from the vascular tissue into the stem parenchyma cells  <cit> .
consistent with previous study  <cit> , our results suggested that the expression of sut <dig> was higher in mature stems than the mature leave from all three saccharum species , and thus supported the previous conclusions.
moreover, saccharum sut1s displayed much higher expression level in the seedling leave tissues than in both of the seedling stems and all the mature tissues, suggesting sut <dig> may play an enhanced role in directing sucrose in source tissue before sucrose accumulation in sacchaurm species.
similar to the expression of sbsut <dig> in sorghum  <cit> , the sut family expression analysis revealed that sut <dig> was the most expressed among suts in saccharum species.
nevertheless, the only direct evidence for the sut <dig> function was from the saccharum close related maize sut <dig> mutant, which exhibited a phenotype of shorter stature and carbohydrate accumulation in their source leaves  <cit> .
sut <dig> in saccharum may have similar function as zmsut <dig> since the two orthologs shared high sequence similarity.
comparative analyses of sut expression among the three saccharum species revealed that sut <dig> had lower expression level in all tissue types from high sugar species  than the lower sugar species , which was consistent to sorghum that sut <dig> had lower gene expression in high sucrose rio.
than in the grain type genotype rtx <dig>  <cit> .
similar to sut <dig>  besides the leaf tissues, sut <dig> was more abundant in s.spontaneum and s.robustum than in s.officinarum .
these results supported the notion that sinks demand in the mature plant might be stronger than in the lower sucrose content plants of andropogoneae tribe.
we compared the expression level of suts of all saccharum species in both mature and seedling plant.
in mature plants, sut <dig> had higher expression level in sink than source tissues; in contrast, sut <dig> had a lower expression level in sink than source tissues.
sut <dig> showed a higher expression level in seedling than the mature plants.
sut <dig> and sut <dig> accounted for above 70 % of transcripts in this gene family .
these results indicated that sucrose transport were active before the sucrose accumulation and both sut <dig> and sut <dig> were involved in the plant development in saccharum.fig.
<dig> total accumulative expression level of the suts in tissues and in stage of three saccharum species.
in, internode; lr, leaf roll.
internnodes  <dig>   <dig>   <dig>  internodes  <dig>   <dig>   <dig> and internodes  <dig>   <dig>   <dig> were from saccharum officinarum la-purple, saccharum robustum molokai <dig> and saccharum spontaneum ses <dig> respectively
in the saccharum species, sut <dig> showed similar expression level in both seedling and mature stage tissues.
these results were different from its close orthologs including ossut <dig>  <cit> , ptasut <dig>  <cit> , and sbsut <dig>  <cit> .
thus, sut <dig> might contribute to the characteristics of sucrose accumulation in saccharum species.
the orthologous of sut <dig> phylogenetic group was proved to be localized in the tonoplast .
in model plant oryza sativa  <cit>  and arabidopsis  <cit> , sut <dig> played a role for transporting sucrose from mesophyll vacuoles to their cytoplasm.
this information may not be sufficient for discovering the gene function of sut <dig>  but is an indication for further functional study of sut <dig> in sugarcane.
both sut <dig> and sut <dig> in sut <dig> group  were revealed to be recent duplication and through rapid evolution accompanied by the multiple amino acid differences in their allelic haplotypes , suggesting that these two genes may have similar gene expression profiles.
gene expression analysis showed that these two genes have much lower gene expression level among the suts families and had similar gene expression profiles in the mature tissues , supporting that the two genes were derived from a recent duplication.
nevertheless, in the seedling plants, sut <dig> was absent while sut <dig> had higher expression in stems than leaves , hence, sut <dig> may contribute to phloem loading before the sucrose accumulation in saccharum.
in sorghum, great variation of gene expression level for sbsut <dig> and sbsut <dig> were observed among the tissues from of vegetative stages and anthesis  <cit> .
of them, sbsut <dig> showed higher expression in spikelet tissue and inflorescence, and thus was suggested to play a role for inflorescence development; similarly, sut <dig> in saccharum species was more abundant in leaves than the other tissues  <cit> .
based on above genomic analysis, sut <dig> and sut <dig> have gone through rapid evolution after the split of sorghum and saccharum, suggesting that these two genes have functional divergence between these two species.
these two gene expression profiles in saccharum and sorghum were different from their closest orthologous ossut <dig>  which exhibited broad expression level across source and sink tissues as well as in filling rice grains  <cit> .
phylogenetic and comparative analysis revealed that there was a single sut in group sut <dig> from rice.
these differences can be explained by the single gene ossut <dig> in rice response for function of two duplication genes in the andropogoneae tribe.
soluble sugar such as sucrose usually increases in plant under drought stress.
to identify which sut gene responsible to drought stress, we examined the suts expression level under drought stress in the seedling plant leaves of three saccharum species.
under drought stress, in the four detectable suts in the seeding, sut <dig> and sut <dig> were down regulated, in contrast, sut <dig> and sut <dig> were up regulated, indicating that the sut <dig> and sut <dig> are important in response to drought stress and may involved in transporting sugar into cell for osmotic adjustment.
sut families in saccharum presented a great gene expression diversity in response to drought stress.
sut <dig> was the predominant expression member in the suts families in saccharum, therefore, the up-regulation of sut <dig> expression resulted in the higher total suts transcript level.
a possible explanation for this phenomenon could be that the source tissue reduced the sucrose product level under drought stress thus down regulated the sut expression level.
an expression profile for saccharum plants under water stress with different time points could be further used for verifying this notion.
similar experiment was performed for the five sut members in rice, which revealed that ossut <dig> was only member display up-regulated during drought and salinity treatments  <cit> .
therefore, it is most likely that plant sut gene members possess diversity pattern in response to stress tolerance.
CONCLUSIONS
in this study, we presented the first report of a gene family consists of six suts in saccharum.
we provided the comprehensive evaluation of the evolutionary genesis, gene allelic haplotypes, phylogenetic relationships, gene structure, and gene expression pattern for the sut gene family in saccharum species.
our results revealed that sssut <dig> and sssut <dig> were recent duplication genes companied by rapid evolution, while, sssut <dig> and sssut <dig> were the ancient members in the families.
gene size extensions caused by sequence insertions in introns were observed in the sut families.
despite the high polyploidy level, the examined suts exhibited conserved gene structures and amino acid sequences among the allelic haplotypes.
both sut <dig> and sut <dig> had predominant expression in saccharum sut families.
sut <dig>  which displayed lower expression in the high sucrose species s.officinarum, might be involved in saccharide unloading in the sink tissue of mature plant, phloem loading in early developmental stage of saccharum.
sut <dig> likely contributed to both phloem loading and sink development.
sut <dig> was more important at early developmental stage than in mature plants.
both sut <dig> and sut <dig> had lower expression level than other gene members, and had higher expression in source leaves than in other tissues, thus, supposed to play roles in phloem loading.
in the seedling plant leaves with drought stress treatment, four genes sut <dig>  sut <dig>  sut <dig> and sut <dig> were detectable, among which, sut <dig> and sut <dig> were down regulated, while, sut <dig> and sut <dig> were up regulated.
to further reveal these genes’ roles under stress, experiments such as, characterizing the spatio-temporal expression dissection, enzyme activity assay, and gene editing technology like crispr-cas <dig> system, would be necessary.
the results offered useful foundation and framework for future research for understanding the physiological roles for each sut gene and molecular mechanisms of sucrose metabolism in sugarcane.
availability of supporting data
the  <dig> sequences of sssuts were deposited into genbank .
phylogenetic data  have been deposited to treebase and are accessible via the url: http://purl.org/phylo/treebase/phylows/study/tb2:s <dig>
additional files
additional file 1:
the probe primers for sut bac hybridization in
s. spontaneum
.
additional file 2:
blast results for
sssuts
est in ncbi database.
additional file 3:
alignment of the amino acid sequences of
sssut
haplotypes.
additional file 4:
the qrt-pcr primers for
suts
in this study.
additional file 5:
qrt-pcr verification of sut gene expressions in partial issues of three saccharum species.
abbreviations
bacsbacterial artificial chromosomes
eef-1aeukaryotic elongation factor 1a
estexpressed sequence tag
gapdhglyceraldehyde-3-phosphate dehydrogenase gene
rpkmreads per kilobasesper million reads
sutssucrose transporters
competing interests
the authors declare that they have no competing interests.
authors’ contributions
qz and jz conceived the study and designed the experiments.
qz,wh, fz, lw, qy, rm and jz carried out the experiments and analyzed the data.
qz and jz wrote the manuscript.
all authors read and approved the final paper.

 sucrose transporters are important for both phloem loading in source tissue and sucrose uptake into some sink cells. suts are considered to be the control points for sucrose storage in plant because they can carry sucrose across cell membranes and play an important role in loading sucrose into phloem systems in a series of steps. plant suts belong to the glycoside-pentoside-hexuronide cation symporter family that is part of the major facilitator super-family. transporters in the gph family have the primary characteristics of mfs proteins that contain transmembrane spanning domains with n- and c-termini in the cytoplasm. since the first plant sucrose transporter sosut was functionally identified using an elegant yeast complementation strategy from spinach, suts have been demonstrated to effect on the multiple aspects of plant development such as biomass partitioning, pollen germination, restraining plant growth, fruit size reduction and ethylene biosynthesis. comprehensive understanding of the molecular structure and evolution of a gene family in plant species is the first step towards understanding their physiological roles and metabolic mechanism involved in different growth phases. recent studies have revealed that sut was a small gene family that consisted of at least four sut genes in the most plant species. the sut family members have been identified from a number of plant species such as arabidopsis, rice, wheat, populus, sorghum and pineapple as well. phylogenetic analysis of sut family suggested that the plant sut could be divided into five subgroups including two monocot specific subgroups, one dicot specific and two monocot-dicot subgroups. sut family members in many plant species are divergent in function and deferentially expressed in different tissues types or at different plant developmental stages. for instance, in rice, ossut expression has been detected in germinated seeds, leaf blades, leaf sheaths and panicles; the expressions of both ossut and ossut are dramatically lower in embryos than those of ossut ossut and ossut; ossut has been detected in most tissues such as roots, leaves, and panicles, and could play a role in sucrose loading into the sheath phloem of the upper leaves during the post-heading period for sucrose transport to developing grains. in arabidopsis, atsuc has been detected in phloem and companion cells in source leaves and functions in loading sucrose into the phloem sieve elements, correspond with the result identified by tissue-specific complementation of different promoters and by 14c labeling studies; atsuc and atsuc are expressed in minor veins of source leaves of mature plants. sugarcane is one of the world’s most produced crops, and contributes to about 80 % of the world sugar and about 40 % of ethanol production worldwide. modern sugarcane cultivar has one of the most complex genome among all the crops; by being both aneupoid and autopolypoid with an extreme ploidy level that can range from octoploidy to dodecaploidy. approximately 80 % of cultivars’ chromosomes are derived from s. officinarum and 10–20 % is derived from s.spontaneum with the remained from interspecific recombination. sugarcane is not only an economically important crop species, but also serves as an important model crop for studying sucrose transporters because its remarkable ability to accumulate vast amounts of sucrose in its stems that can reach close to 700 mm or in excess of 50 % of the dry weight. but to date, limited works in characterizing these sut genes have been reported, except for sut. in an earlier study based on sugarcane expressed sequence tag database survey, sut was revealed to be more abundant in the mature internodes than the other tissues. this result was confirmed by a study for hawaiian sugarcane cultivar, in which the sut transcript levels increased during maturation and sucrose storage, whereas, sut expression was observed to be not affected by sugarcane yellow leaf virus infection in sugarcane. biochemical analysis of sugarcane sut suggested that sut was highly selective for sucrose, but had a relatively low affinity for sucrose, inhibited by sucralose and played key role in sucrose loading from the vascular tissues into the storage sites in parenchyma cells of sugarcane stems. besides sut the gene family of suts is not understood in sugarcane due to the formidable challenge caused by its high degree of polyploidy and heterozygosity genome. in this study, to gain comprehensive understandings of the molecular and evolutionary characterization as well as the possible functions of sut family in sugarcane, based on combination of comparative genomics strategies and high genome coverage of bacterial artificial chromosomes libraries resources, we identified and characterized sut gene families in saccharum species and investigated their transcriptional expression patterns. the analysis in this study mainly focused on: identifying the gene members and allele haplotypes of the sut gene family in sugarcane; analyzing evolutionary relationship, exon/intron organization of the sut gene family; and characterizing the expression patterns of the sut gene family in three progenitor saccharum species. methods plant materials three varieties of saccharum species were used in the study: la-purple, molokai and ses. plants were grown in plastic pots under greenhouse conditions and standard growing practices. tissue samples were obtained from 10-month old plants for leaf roll, leaf, top immature internode, premature internode and mature internode. the internodes were numbered from top to bottom according to the method of moore. stem and leaf tissues from seedlings of the three species were collected at 35 days after planting. for drought stress treatment, the 35 day-old seedlings were treated with peg for 48 h, and stem and leaf tissues were collected. the tissues were immediately frozen using liquid nitrogen and stored at −80 °c prior to rna isolation. bac libraries the haploid of s. spontaneum ses ap85-, was used to construct the bac library. nuclei were isolated from the young leaf tissues of ap85- following the method described by ming et al.. the high molecular weight dna embedded in agarose was partially digested using hindiii. fractions at approximately 100 kb were recovered and cloned into psmart bac vector. clones from ap85- bac libraries were picked and stored in 384-well plates with freezing medium. bac clones were grided onto performa ii nylon filters using q-pix. database search for the sut gene family and phylogenetic analyses the sequence data used in this study were collected using the keyword “sucrose transporter” and a query search in the genbank using the known sut gene sequences from sorghum, rice and arabidopsis. matches achieved similarity scores of and probability scores > and e-value <10− were collected. the amino acid sequences of sucrose transporter gene family members in monocotyledons and dicotyledons identified by searching public databases available at various resources. the phylogenetic trees were constructed with the mega. program with clustalw alignment using default parameter. identification and sequencing of sut families from bac library bac library screening was carried out as described by yu et al.. the bac clones representing different haplotypes were selected. the insert size of bac clones was estimated by comparing with standard size markers using chef gel electrophoresis. the bac dnas were isolated using phasepreptm tmbac dna kit and the sequencing libraries were prepared individually with unique barcode for each clone. the sequencing libraries were then pooled and sequenced with 150 bp, pair-end reads on illumina hiseq at center for genomics and biotechnology in fujian agriculture and forestry university. the raw reads were then assembled using spades genome assember v... genomic sequence annotation and functional prediction the genomic sequences of sut genes were annotated by dna subway, and the corresponding cds sequences were translated into protein by the expasy-translate tool. the exon-intron structures were graphed using online tool gsds. the putative conserved domains of sucrose transporter protein were detected by using blastp and interpro. the protein transmembrane helices domain was predicted using tmpred. the isoelectric point and relative molecular mass of the protein were predicted using expasy. analysis of sucrose transporter gene co-expression profiling the sorghum gene models were used as reference to align the saccharum rna-seq database by using novoalign with default parameter. the normalization and statistical evaluation of differential gene expression has been performed using edge-r with a p-value cut-off of and using the benjamini-hochberg method for multiple testing corrections. the raw data was normalized according to the default procedure of the differential expression analysis package used. the dispersion was estimated using the pooled setting. the expression values were log-transformed, and cluster analyses were performed using a software cluster with euclidean distances and the hierarchical cluster method of “complete linkage clustering”. the clustering tree was constructed and viewed in java treeview. experimental validation of expression levels of sut gene by qrt-pcr the expression levels of six sut genes in three tissues of three saccharum species were validated by qrt-pcr. gene-specific primer pairs were designed by using integrated dna technologies. after treated with dnase i, two microgram of rna was used in reverse transcription with the superscript vilo cdna synthesis kit according to the manufacturer’s guidelines. the real-time qpcr was performed by using multicolor real-time pcr detection system with conditions for all reactions were 95 °c for 30s, 40 cycles of 95 °c for 5 s, followed by 60 °c for 30s, and 95 °c for 10s. melting curve analysis were performed to confirm the pcr specificity. the glyceraldehyde-3-phosphate dehydrogenase gene and eukaryotic elongation factor 1a were selected as internal standard for normalization, and three replicates were completed for each sample. the relative expression level for each sut gene in different tissues of three saccharum species were calculated by using the 2-ΔΔct method. the correlation coefficient was calculated between the transcript accumulation levels obtained by rnaseq and qrt-pcr using excel.  identification of six sut genes in sugarcane positive bac clones from ap85- were identified using probes designed from six well-annotated suts genomic regions in sorghum bicolor. to determine haplotypes, the pcr fragments of the six suts were cloned by using these probe primers and sequenced, which confirmed of these bac clones contains different paralogous and homologous haplotypes. the six s.spontaneum suts were referred to sssut1-sssut according to sequence similarity with sorghum suts. in the sut sequences, both sssut and sssut both sssut and sssut and remaining two suts have and allelic haplotypes, respectively. the allelic haplotypes of each suts were indicated additional“-h1” to “-h4” to the gene name end. using the gene model sequences of the annotated sut genes as queries, both the in-house est and genbank database were extensively searched. the results showed that all the sssuts had the corresponding ests in the genbank database except sssut.table information of the putative sut genes in sorghum sbsut1 sbsut2 sbsut3 sbsut4 sbsut5 sbsut6 the six sssuts containing complete orfs with the predicted molecular weights ranged from to  kda in sugarcane. comparing with the sut family from sorghum, sssut showed a lower molecular weight, and the remaining gene pairs between these two species were consistent. sssut and sssut shared a higher similarity of protein sequences in contrast to the other pairwise sequences between the remaining four genes in the sut families. the analyses of the deducted protein sequences of the sut genes in sugarcane revealed that all these gene families had highly conserved mfs domains and contained membrane-spanning helices. a conserved histidine residue was presented in the first loop domain corresponding to his- and amino acids which corresponded to the g-x-x-x-d/e-r/k-x-g--r/k-r/k motif reside in the second and eighth loop domains. additionally, sssut contained a lxxll motif in the n-terminal domain, indicating that it might be targeted to the tonoplast.table comparison of the characterization of the suts between sugarcane and sorghum sbsut1 sssut1 sbsut2 sssut2 sbsut3 sssut3 sbsut4 sssut4 sbsut5 sssut5 sbsut6 sssut6 sssut1 sssut2 sssut3 sssut4 sssut5 sssut6 sssut2 sssut3 sssut4 sssut5 sssut6 allelic haplotype analysis of sssuts genomic sequence comparisons within the allelic haplotypes from the four suts revealed that these allelic haplotypes shared very high similarity above 99 %. slight variations were observed within the allelic haplotypes of sssuts. the deducted protein sequences were compared within the four sssuts. the results showed that, for the protein sequence of allelic haplotypes, both sssut and sssut shared identities of  %, while, sssut and sssut had and  % sequence similarity, respectively. the specific amino acids variations were discovered through the alignment of the allelic haplotypes from each suts. and amino acids within sssut sssut sssut sssut respectively were observed to be vary among the allelic haplotypes. in addition, in sssut compared with h and h h and h had shorter peptides with amino acids deletion caused by the shift of exon structure. furthermore, in sssut of the variant amino acids were located at the sixth loop domain and transmembrane helixes, indicating that potential functional variation existed among these allelic haplotypes.table the variation of deducted amino acid sequences among allelic haplotypes within the four sssut sssut1 sssut2 sssut3 sssut4 sssut5 sssut6 fig. alignment of the amino acid sequences of sssut haplotypes. amino acid sequences of haplotypes were aligned using the dnaman program. similarity in amino acids across all the sequences is indicated by stars. the difference between haplotypes was shown in a red box in general, gene structures of the sssuts allelic haplotypes presented high conservation for exon/intron numbers and exon sizes, while, for intron size, in sssut sssut3-h3’s first intron was larger than other allelic haplotypes; and in sssut both sssut6-h and sssut6-h contained a smaller second exon than sssut6-h and sssut6-h.fig. comparison of the allelic gene structures of sssuts. boxes represent exons, triangles represent transposons nonsynonymous to synonymous substitution ratio was analyzed to investigate evolutionary function constraint in s.spontaneum. to identify the evolutionary forces acting on four sut genes having alleles, the ka/ks was calculated. within the coding regions, the ka/ks ratio was much less than indicating that purifying selection was the dominant force driving the evolution of sssut genes.fig. the ka/ks of sssut haplotypes and sbsut-sssut. the lower value of ka/ks was indicated by stars comparative analysis of gene structure between sssut and other plant sut the gene structures of sssut family has a great variation with exon numbers ranging from five to fourteen, and their introns were aligned accordance with the gt-ag rule for splicing sites. sssut sssut and sssut had larger first introns than the other sut genes. both sssut and sssut only have five exons that were lesser than the other sssuts. in addition, comparative analysis of sssut families suggested that, the fifth exon in sssut were presumed to split into 3– exons, and the first, second, and fifth exons in sssut were presumed to evolve into 2– exons. sssut and sssut shared highest similarity of exon/intron pattern in spite of the great size variation between the second introns, which correlated to their amino acids similarities.fig. comparison of the gene structure of the six members of the sssut gene family. three monocotyledons and dicotyledons sut gene family are also shown for comparison. boxes represent exons, triangles represent transposons to investigate the evolutionary mechanisms underlying the genesis of gene families, we performed comparative analyses of the sssut structures with the sut families from sorghum, rice, arabidopsis, maize, grape and potato. the suts from these species could be divided into five groups sut to sut which were consistent with previous studies. the results showed that suts in the same group had the similar gene structures. in the dicot specific group sut most of the genes had two to four exons, in which the first exons were large and the second ones were small. in this group, the only exception was stsut which had a smaller first exon, due to the possible exon splitting comparing with the other dicot species. in group sut that was closely related to group sut all the genes had first two large exons that were likely originated from sut first exon splitting based on sequence comparison. it was interesting that in sut group, the dicot genes and the monocot genes had siminar number of exons, and respectively, and the sequences of the fifth and sixth exons in dicot were presumed to originated from the monocot fifth exon splitting. furthermore, based on the genomic and amino acids sequence comparisons among the sequences from the seven plant species in group sut and sut the common ancestral gene of monocots and dicots were suggested to have two exons and the exon members have differentiated in a later period of evolution caused by exon splits and partial exon fusions. the genes in group containing sut had similar exon number of or for both dicot and monocot species. gene size expansion due to intron size stretching in dicot plant grape and potato were observed in this group. in the sut group, besides zmsut with an additional small intron caused by the first exon splitting, the remaining sut group genes harbor large first introns, which included wheat tasut1d and tomato lesut. in contrast to sut group, the sut group had varied number of exons ranging from to 14; among them, sssut sbsut and zmsut had six exons, the other genes in the subfamilies contained 10– exons; both of ossuts in this group had more exons number than their orthologous genes. in monocot specific group sut similar to the high identities shown by the alignment of amino acid sequence of sssut and sssut high conservation of exon/intron structures were observed from the schematic representation; sssut had larger second exon than the other genes; similar to sut group, ossut had more exon number than their orthologous genes. scrutiny of the exon/intron structure of the genes in branch with sut3/sut2/sut revealed that the exons could be corresponding to six exons, in which the first and the last two exons were observed to be fused/spited. these results suggested that the genes in this branch might originate from common ancestral gene containing six exons for both monocots and dicots. phylogenetic analysis of sssut and other plant sut homologs to comprehensively analyze the evolutionary relationships of sut families between s. spontaneum and other plant species, we aligned plant amino acid sequences from dicots and monocots including s.spontaneum, using clustalx to construct an unrooted tree with neighbor-joining method. same as the distribution above, the suts were phylogenetically distributed into five groups, sut sut sut sut and sut respectively. among these five groups, sut genes are only found in dicotyledonous, in contract, genes in sut and sut groups are only from monocotyledonous. whereas, the remaining two groups, sut and sut are found in both dicot and monocot, and could be well classified into two distinct subclades. these results strongly suggested that plant suts were diverged from a recent evolutionary event after the common ancestor of dicots and monocots. in addition, the sut families could be divided into two branches in the phylogenetic tree, with sut and sut groups in one branch and the other three groups in another branch, indicting two ancestral genes were the origins of suts in both dicot and monocot.fig. phylogenetic analysis of sssut and other plant sut homologs. unrooted phylogenetic tree of plant sut proteins constructed using the neighbour-joining method with mega. program. zmsut: zea mays, sbsut: sorghum bicolor, bdsut: brachypodium distachyon, ossut: oryza sativa, atsut: arabidopsis thaliana, cssut: citrus sinensis, gmsut: glycine max, stsut: solanum tuberosum,vvsut: vitis vinifera, sisut: setaria italica, sssut:saccharum spontaneum in dicot specific sut group, the paralogous genes from each of the dicot species were observed to be closely related, indicting recent gene duplications after the divergence of dicotyledons. in addition, both sut gene number and sequences had great variations among the dicot plant species, suggesting rapid evolutionary dynamics exist in the dicotyledonous sut families. in sut group, all dicots and monocots had only one gene member, which the phylogenetic distribution were generally consistent with the plant species taxonomy. in sut as description above, the genes from dicot and monocot species could be further classified into two subclades, respectively. in this group, dicot plants have d 1- gene members, while, all the monocot plants only hadone gene member, which suggestted that dicot plants sut group were undergoing expansion. in the monocot specific sut the examined monocot species consisted of two paralogous genes from two separated clades, suggesting the gene duplication event occurred before the divergence of these dicot plants. in contract to sut group, in sut group, recent gene duplication events were observed in the andropogoneae plants and bambusa oldhamiias shown by the closest phylogenetic distribution of genes within these plant species.fig. the distribution of sut family member in monocotyledon and dicotyledon in the sssut family, sssut was the solo member in a branch, but sssut sssut sssut sssut and sssut were clustered together and shared a more recent common ancestral gene. thus, sssut was suggested to be the oldest gene, while, both of sssut and sssut should be younger than the splitting of trib, andropogoneae dumort and zea mays. in addition, sssut and sssut were observed to be undergoing rapid evolution as shown by lower amino acids sequences similarity than other orthologous genes between the sorghum and saccharum. gene expression of suts among three saccharum species to investigate the possible physiological functions for suts, we performed comparative transcriptome profiling among three saccharum species at different developmental stages of seedling and five different tissues from the mature leaf and stalks by using rna-seq method. the rna-seq results were verified by qrt-pcr in three tissues from each of the three saccharum speices. there is a significant positive relationship between the reads per kilobases per million reads based on rna-seq and the relative expression level based on qrt-pcr.fig. the sut gene family expression based on rpkm in different tissues of different stage in three saccharum species. the expression level of sut genes in seeding stage and mature stage of nature condition. a correlation coefficient between rnaseq and qrt-pcr of six sut genes. b, and in seeding stage of drought stress condition. in, internode; lr, leaf roll. internnodes internodes and internodes were from saccharum officinarum, saccharum robustum and saccharum spontaneum, respectively in sut families, the transcription of sut was undetectable in all the examined tissues from sacchaurm plants, which was consistent with that of sut in sorghum. the other gene expression levels had significant variations with a clear trend of transcript levels from higher to lower sut sut sut sut and sut of them, sut and sut had predominant expression levels among the gene families, indicating that the two genes were the fundamental members in sut families. sut transcripts were abundant in both source and sink organs, and were the most expressed gene in all tissues except for the mature leaf, indicating sut was the key member. the gene displayed a higher expression level in sink tissues than the source tissue in mature plant. however, it was the opposite in seedling plants, suggesting the gene was more important in phloem loading for seedlings than the mature plant. comparing with both s. sponteanum and s. robustum, sut displayed a lower expression level in the high sucrose s. officinarum, in all examined tissues. in addition, under drought stress, sut was observed to be down regulated in s. robustum. sut was expressed in all the organs examined for the three saccharum species. in the seedling plants, similar expressions were observed in both stem and leaves among these saccharum species. in the mature plants, sut displayed a lower expression level in the source tissue of leave and leafroll for two lower sucrose accumulating saccahurm species, s. sponteneum and s. robustum, while, high sugar s. officinarum had a more uniform sut expression level in all of the examined tissues. under drought stress, twofold lower levels of expressions were observed in the three saccharum species than their control. sut had higher expression level in the seedling than the mature plants, suggesting it might contribute more for sucrose loading at the early age of plant development; whereas, sut displayed similar expression level in the examined tissues from both seedling and mature plants. obviously, sut was not correlated to the sucrose content differential among the saccharum species. under drought stress, in contrast to sut and sut sut was up regulated in the leaf of three saccharum species. sut displayed dramatically higher expression level in sink tissues than in source in the seeding plant; in contract, in the mature plants, the expression level were lower in leave than the other tissues. similar to sut sut showed up-regulation under water stress. sut was undetectable in the seeding plants and had similar expression pattern as sut in the mature plants that gene expression exhibited higher in leave than the other tissues. discussions genomic study for the gene families is the first step toward the gene functional study. however, the identifications of gene family in sugarcane is still a formidable challenge caused by its complex genomes. recently, the whole genome sequencing of sorghum and other relative species of saccharum provides the references for comparative genomics to identify the gene families in saccharum species. in previous studies, based on comparative genomics, the gene families of phosphoenol pyruvate carboxylase gene, sucrose synthase, sucrose phosphate synthase, atp-dependent phosphofructokinase were identified by using the est database, dna fragment, and cdna cloning. however, none of these studies has investigated on the genomics of whole gene families because of the lack of genomic sequences for sugarcane. our study through comparative genomics and bacs sequencing is the first report for the structure of a gene family and their gene allelic haplotypes in saccharum. evolutionary conservation and divergence of sssut plant suts had been well documented in previous studies for gene phylogenetic analyses two classifications. one classification was to divide plant suts into three types, type i, type ii and type iii, with the reference of arabidopsis, in which, type i and type ii suts were localized to the plasma membrane, while type iii suts were associated with vacuolar membrane. another classification was to group the suts into five groups sut sut sut sut and sut. comparing these two classifications, sut was included in type i, sut was in type ii, and sut sut and sut were in type iii. the former classification is likely associated with dicot plants studies, while, the latter is used for both dicot and monocot plants, especially for gene evolution studies. in this study, we used large amount of plant suts for phylogenetic analysis, and the results confirmed the later classification, by revealing the existence of one dicot specific and two monocots specific groups and the independence evolution process in plant sut families. furthermore, the comparative analysis of these plant suts showed that genes in sut group were more conservative than the genes in sut group, and further provided the direct evidence of the recent duplication event occurred after monocot/dicot divergence. the evolution history of sssuts, which was sorted by age in duplicated descending order, sssut sssut sssut2/sssut and sssut5/sssut the comparative analyses could be used to predict the number of sut gene family members in saccharum. without the whole genome sequences for saccharum, it could be debatable to conclude that we have discovered all suts in sacchaurm, in spite of our high coverage bacs libraries. in this study, both phylogenetic analysis and sequences comparison revealed that saccharum spontaneum and sorghum bicolor were both composed of six suts members and each gene pairs were orthologous between the two species. beside sut group, which was dicot specific, monocot plants have the same gene numbers in sut sut and sut groups, the remaining group sut had the same gene number in andropogoneae plants, which were found to be the result of a recent duplication. therefore, it is less likely that any sut gene duplication events had occurred after the diverging of sorghum and saccharum, knowing no additional gene in this study. hence, we concluded that the six sssuts comprise the sut family in the saccarhum spontanum genome. further experiments such as southern blot could be used to verify the conclusion. the exon–intron structure differences were demonstrated to be accomplished by three main types of mechanisms, exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion. in this study, comparative analyses of gene structures for sssut made it possible to evaluate the sut gene structure evolution in plant. all suts genes, including sssuts, had a great variation of exon numbers, ranging from to comparing with the gene structure variation, the protein sequences were more conserved among the paralogous genes in sacchaurm, as shown by all sut members containing membrane-spanning helices and similar protein sizes. a common feature of plant suts was that the membrane-spanning helices were distributed roughly uniformly in the deducted peptides of each gene in conserved position. thus, the gene structure evolution after these plant divergences did not cause significant coding region variations. therefore, the sut structures variations were mainly evolved from intron gain/lost and insertion/deletion but not from exonization/pseudoexonization. sorghum is one of the closest relative diploid genera of sacchaurm. comparative analysis of the orthologous between sssuts and sbsuts made it possible to investigate the specific evolutionary events after the polyploidzation of saccharum. in sssuts, sssut harbored a much larger second intron than its closest paralogous sssut and the other orthologs. similarly, sssut contained a larger first intron than its orthologous gene sbsut in addition, sssut were observed to have a putative te insertion in the last intron. our results suggested that the sssut families were undergoing gene extension following polyploidization in s.spontaneum. the estimated monoploid genome size of s. spontaneum and s. officinarum were both larger than the monoploid genome size of sorghum at 760 mb, supporting the conclusion that the genome of saccharum expanded in general after polyploidization. our results demonstrated the first case that expansion in intron regions of a gene families contribute to genome expansion in sacchaurm. in this study, allelic haplotype sequences for sssut and were comparatively analyzed for gene structures and ka/ks. relative conservative gene structures were observed among allelic haplotypes within each of the four sssuts, whereas, sssut3-h contained a larger first intron than the other three haplotypes, both sssut6-h and sssut6-h had larger second exons. in addition, the ka/ks ratios, which are all under, revealed that all the suts allelic haplotypes were under strong purifying selection. multiple alleles in polypoidy are considered to be functional redundant at the time of origin; the conservation and constraint purification within the allelic haplotypes of sssuts were likely due to the key function of sut in saccharum. it would be worthy to note that the transcription of sssut was undetectable in the examined tissues while its haplotypes were under constrain selection, indicating that sssut might have a necessary function for sacchaurm. in paleopolyploids, and recent allopolyploid species, such as wheat and tragopogon, eliminations and pseudogenizations of key functional genes after polyploidzation have been well documented. the allelic gene variations supposed to the key topics for studying the genome dosage of sacchaurm species and further investigation for them would provide the foundation to understand the molecular basis of sugarcane genetics. gene expression and functions of suts in saccharum examination of sut gene expressions in saccharum species in source and sink tissues of seedling and mature plants proves an insightful indication regarding the roles of gene functions. previous studies of gene expression were mostly done on saccharum hybrids with combined genetic background of s.officinarum and s.sponteneum. to simplify genetic backgrounds, in this study, three saccharum species, high-sucrose s.offcinarum, low-sucrose s.robustum and stress-tolerant s.spontaneum were used for studying the suts expression profiles. sut was the only gene in sut family, which had been well documented in saccharum hybrid. in these previous studies, shsut had been shown to have high expression level in premature stem tissue, and decreased expression level in mature internodes. shsut was demonstrated to be highly selective for sucrose, inhibited by sucralose and had the function in loading sucrose from the vascular tissue into the stem parenchyma cells. consistent with previous study, our results suggested that the expression of sut was higher in mature stems than the mature leave from all three saccharum species, and thus supported the previous conclusions. moreover, saccharum sut1s displayed much higher expression level in the seedling leave tissues than in both of the seedling stems and all the mature tissues, suggesting sut may play an enhanced role in directing sucrose in source tissue before sucrose accumulation in sacchaurm species. similar to the expression of sbsut in sorghum, the sut family expression analysis revealed that sut was the most expressed among suts in saccharum species. nevertheless, the only direct evidence for the sut function was from the saccharum close related maize sut mutant, which exhibited a phenotype of shorter stature and carbohydrate accumulation in their source leaves. sut in saccharum may have similar function as zmsut since the two orthologs shared high sequence similarity. comparative analyses of sut expression among the three saccharum species revealed that sut had lower expression level in all tissue types from high sugar species than the lower sugar species, which was consistent to sorghum that sut had lower gene expression in high sucrose rio. than in the grain type genotype rtx. similar to sut besides the leaf tissues, sut was more abundant in s.spontaneum and s.robustum than in s.officinarum. these results supported the notion that sinks demand in the mature plant might be stronger than in the lower sucrose content plants of andropogoneae tribe. we compared the expression level of suts of all saccharum species in both mature and seedling plant. in mature plants, sut had higher expression level in sink than source tissues; in contrast, sut had a lower expression level in sink than source tissues. sut showed a higher expression level in seedling than the mature plants. sut and sut accounted for above 70 % of transcripts in this gene family. these results indicated that sucrose transport were active before the sucrose accumulation and both sut and sut were involved in the plant development in saccharum.fig. total accumulative expression level of the suts in tissues and in stage of three saccharum species. in, internode; lr, leaf roll. internnodes internodes and internodes were from saccharum officinarum la-purple, saccharum robustum molokai and saccharum spontaneum ses respectively in the saccharum species, sut showed similar expression level in both seedling and mature stage tissues. these results were different from its close orthologs including ossut, ptasut, and sbsut. thus, sut might contribute to the characteristics of sucrose accumulation in saccharum species. the orthologous of sut phylogenetic group was proved to be localized in the tonoplast. in model plant oryza sativa and arabidopsis, sut played a role for transporting sucrose from mesophyll vacuoles to their cytoplasm. this information may not be sufficient for discovering the gene function of sut but is an indication for further functional study of sut in sugarcane. both sut and sut in sut group were revealed to be recent duplication and through rapid evolution accompanied by the multiple amino acid differences in their allelic haplotypes, suggesting that these two genes may have similar gene expression profiles. gene expression analysis showed that these two genes have much lower gene expression level among the suts families and had similar gene expression profiles in the mature tissues, supporting that the two genes were derived from a recent duplication. nevertheless, in the seedling plants, sut was absent while sut had higher expression in stems than leaves, hence, sut may contribute to phloem loading before the sucrose accumulation in saccharum. in sorghum, great variation of gene expression level for sbsut and sbsut were observed among the tissues from of vegetative stages and anthesis. of them, sbsut showed higher expression in spikelet tissue and inflorescence, and thus was suggested to play a role for inflorescence development; similarly, sut in saccharum species was more abundant in leaves than the other tissues. based on above genomic analysis, sut and sut have gone through rapid evolution after the split of sorghum and saccharum, suggesting that these two genes have functional divergence between these two species. these two gene expression profiles in saccharum and sorghum were different from their closest orthologous ossut which exhibited broad expression level across source and sink tissues as well as in filling rice grains. phylogenetic and comparative analysis revealed that there was a single sut in group sut from rice. these differences can be explained by the single gene ossut in rice response for function of two duplication genes in the andropogoneae tribe. soluble sugar such as sucrose usually increases in plant under drought stress. to identify which sut gene responsible to drought stress, we examined the suts expression level under drought stress in the seedling plant leaves of three saccharum species. under drought stress, in the four detectable suts in the seeding, sut and sut were down regulated, in contrast, sut and sut were up regulated, indicating that the sut and sut are important in response to drought stress and may involved in transporting sugar into cell for osmotic adjustment. sut families in saccharum presented a great gene expression diversity in response to drought stress. sut was the predominant expression member in the suts families in saccharum, therefore, the up-regulation of sut expression resulted in the higher total suts transcript level. a possible explanation for this phenomenon could be that the source tissue reduced the sucrose product level under drought stress thus down regulated the sut expression level. an expression profile for saccharum plants under water stress with different time points could be further used for verifying this notion. similar experiment was performed for the five sut members in rice, which revealed that ossut was only member display up-regulated during drought and salinity treatments. therefore, it is most likely that plant sut gene members possess diversity pattern in response to stress tolerance.  in this study, we presented the first report of a gene family consists of six suts in saccharum. we provided the comprehensive evaluation of the evolutionary genesis, gene allelic haplotypes, phylogenetic relationships, gene structure, and gene expression pattern for the sut gene family in saccharum species. our results revealed that sssut and sssut were recent duplication genes companied by rapid evolution, while, sssut and sssut were the ancient members in the families. gene size extensions caused by sequence insertions in introns were observed in the sut families. despite the high polyploidy level, the examined suts exhibited conserved gene structures and amino acid sequences among the allelic haplotypes. both sut and sut had predominant expression in saccharum sut families. sut which displayed lower expression in the high sucrose species s.officinarum, might be involved in saccharide unloading in the sink tissue of mature plant, phloem loading in early developmental stage of saccharum. sut likely contributed to both phloem loading and sink development. sut was more important at early developmental stage than in mature plants. both sut and sut had lower expression level than other gene members, and had higher expression in source leaves than in other tissues, thus, supposed to play roles in phloem loading. in the seedling plant leaves with drought stress treatment, four genes sut sut sut and sut were detectable, among which, sut and sut were down regulated, while, sut and sut were up regulated. to further reveal these genes’ roles under stress, experiments such as, characterizing the spatio-temporal expression dissection, enzyme activity assay, and gene editing technology like crispr-cas system, would be necessary. the results offered useful foundation and framework for future research for understanding the physiological roles for each sut gene and molecular mechanisms of sucrose metabolism in sugarcane. availability of supporting data the sequences of sssuts were deposited into genbank. phylogenetic data have been deposited to treebase and are accessible via the url: http://purl.org/phylo/treebase/phylows/study/tb2:s additional files additional file 1: the probe primers for sut bac hybridization in s. spontaneum. additional file 2: blast results for sssuts est in ncbi database. additional file 3: alignment of the amino acid sequences of sssut haplotypes. additional file 4: the qrt-pcr primers for suts in this study. additional file 5: qrt-pcr verification of sut gene expressions in partial issues of three saccharum species. abbreviations bacsbacterial artificial chromosomes eef-1aeukaryotic elongation factor 1a estexpressed sequence tag gapdhglyceraldehyde-3-phosphate dehydrogenase gene rpkmreads per kilobasesper million reads sutssucrose transporters competing interests the authors declare that they have no competing interests. authors’ contributions qz and jz conceived the study and designed the experiments. qz,wh, fz, lw, qy, rm and jz carried out the experiments and analyzed the data. qz and jz wrote the manuscript. all authors read and approved the final paper. 
sucrose transporters are critical for both phloem loading in source tissue and sucrose uptaking in sink tissue, and are considered to be the control points for regulating sucrose storage. gene expression analyses were performed for tissues from seedlings under drought stress and mature plants of three saccharum species. gene structure comparison demonstrated that the number and position of exons/introns in suts were highly conserved among the close orthologs; in contrast, there were variations among the paralogous suts in sacchuarm. though with the high polyploidy level, gene allelic haplotype comparative analysis showed that the examined four sssut members exhibited conservations of gene structures and amino acid sequences among the allelic haplotypes accompanied by variations of intron sizes. sut could be a low active member in the family because it is undetectable in our study, but it might not be a pseudogene because it harbored integrated gene structure. both of sut and sut had lower expression level than other gene member, and expressed stronger in source leaves and are likely to play roles in phloem loading. by using comparative genomics and bacterial artificial chromosomes, six sut genes were identified and characterized in s. spontaenum. sut had similar expression level in all of the examined tissues, but sut was undetectable. phylogenetic analyses revealed that the two pairs sssuts could be clustered together into two separate monocot specific sut groups, while sssut and sssut were separated into the other two groups, with members from both dicot and monocot species. saccharum speciessucrose transportergene familyallelic haplotypepolyploidyhttp://dx.doi.org/ /501100002855ministry of science and technology of the people's republic of china2013aa100604zhang jisen http://dx.doi.org/ /501100001809national natural science foundation of china 31201260zhang jisen fujian provincial department of educationja12082zhang jisen issue-copyright-statement© the author 2016 in this study, we presented the first comprehensive genomic study for a whole gene family, the sut family, in saccharum. out of the six members, sssuts, sssut and sssut were recent duplication genes accompanied by rapid evolution, while, sssut and sssut were the ancient members in the families. despite the high polypoidy genome, functional redundancy may not exist among the suts allelic haplotypes supported by the evidence of strong purifying selection of the gene allele. we speculated that there were six sut members in the s. spotaneum genome. however, due to sugarcane’s complicated genomes, it is still challenging to study the genetic basis of traits, such as sucrose accumulation. sut and sut were the main members for the sucrose transporter, while, these suts had sub-functional divergence in response to sucrose accumulation and plant development in saccharum. in the seeding plant leave under water stress, four genes sut sut sut and sut were detectable.
 sugarcane is an economically important crop contributing to about 80 % of the world sugar production. increasing efforts in molecular biological studies have been performed for improving the sugar yield and other relevant important agronomic traits. however, due to sugarcane’s complicated genomes, it is still challenging to study the genetic basis of traits, such as sucrose accumulation. sucrose transporters are critical for both phloem loading in source tissue and sucrose uptaking in sink tissue, and are considered to be the control points for regulating sucrose storage. however, no genomic study for sugarcane sucrose transporter families has been reported up to date.  by using comparative genomics and bacterial artificial chromosomes, six sut genes were identified and characterized in s. spontaenum. phylogenetic analyses revealed that the two pairs sssuts could be clustered together into two separate monocot specific sut groups, while sssut and sssut were separated into the other two groups, with members from both dicot and monocot species. gene structure comparison demonstrated that the number and position of exons/introns in suts were highly conserved among the close orthologs; in contrast, there were variations among the paralogous suts in sacchuarm. though with the high polyploidy level, gene allelic haplotype comparative analysis showed that the examined four sssut members exhibited conservations of gene structures and amino acid sequences among the allelic haplotypes accompanied by variations of intron sizes. gene expression analyses were performed for tissues from seedlings under drought stress and mature plants of three saccharum species. both sut and sut expressed abundantly at different conditions. sut had similar expression level in all of the examined tissues, but sut was undetectable. both of sut and sut had lower expression level than other gene member, and expressed stronger in source leaves and are likely to play roles in phloem loading. in the seeding plant leave under water stress, four genes sut sut sut and sut were detectable. in these detectable genes, sut and sut were down regulated, while, sut and sut were up regulated.  in this study, we presented the first comprehensive genomic study for a whole gene family, the sut family, in saccharum. we speculated that there were six sut members in the s. spotaneum genome. out of the six members, sssuts, sssut and sssut were recent duplication genes accompanied by rapid evolution, while, sssut and sssut were the ancient members in the families. despite the high polypoidy genome, functional redundancy may not exist among the suts allelic haplotypes supported by the evidence of strong purifying selection of the gene allele. sut could be a low active member in the family because it is undetectable in our study, but it might not be a pseudogene because it harbored integrated gene structure. sut and sut were the main members for the sucrose transporter, while, these suts had sub-functional divergence in response to sucrose accumulation and plant development in saccharum. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords saccharum speciessucrose transportergene familyallelic haplotypepolyploidyhttp://dx.doi.org/ /501100002855ministry of science and technology of the people's republic of china2013aa100604zhang jisen http://dx.doi.org/ /501100001809national natural science foundation of china 31201260zhang jisen fujian provincial department of educationja12082zhang jisen issue-copyright-statement© the author 2016 