Whole genome duplication (WGD) or polyploidy event is a prominent process in Gramineae plants and has been significant in the evolution and separation history (Wendel, 2000). Interdisciplinary approaches combining phylogenetic and structural genomic data suggest that the Gramineae genes have undergo a WGD about 70 million years ago (Mya) before the divergence of the Gramineae (Paterson et al., 2004). In addition, it also found that the maize genome is the product of a genomic allotetraploid event approximately 12 Mya ago (Gaut and Doebley, 1997). Subsequently, the maize genome ‘diploidized’ by deleting most of the duplicated centromere regions and deleting or tolerating the degeneration of one number of most of its paired genes (Song and Messing, 2003; Brunner et al., 2005).

Starch is the main storage carbohydrate in plants and also by far the major carbon source in Gramineae seeds and is used as a primary store of energy for metabolism and biosynthesis. Starch is composed of glucose (Glc) polymer that occurs in two main forms: amylose and amylopectin. Both type starches are synthesized inside plastids in higher plants, and are achieved through the coordinated interactions of several of starch biosynthetic enzymes, including ADP glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE) (Ball and Morell, 2003). The duplicate sets of some genes which involved in the core pathway of starch biosynthesis were retained in rice, maize, and wheat following the ancient WGD event in Gramineae (Harn et al., 1998; Vrinten and Nakamura, 2000; Hirose and Terao, 2004; Dian et al., 2005).

The starch synthase (SS, EC 2.4.1.21) catalyzes the synthesis of the glucan polymers by transfering the glucosyl moiety from ADP-Glc to the nonreducing end of a preexisting α-1,4 linked glucan primer. Multiple isoforms of SSs are found in plants (Smith et al., 1997; Ball et al., 1998; Hirose and Terao, 2004). The granule-bound starch synthase family (GBSS) is responsible for amylose synthesis and is exclusively bound to the starch granule. The SSâ… -â…¢ isoforms are involved in amylopectin biosynthesis, while the SSâ…£ isoform in the control of granule numbers (Ball and Morell, 2003; Roldán et al., 2007).

SS isoforms have been identified in several Gramineae genomes through amino acid homology analysis. In rice (Oryza sativa L.), there are 10 SS isoforms separated into five types, including two GBSS genes (GBSSⅠ/WX and GBSSⅡ), one SSⅠ gene, three SSⅡ genes (SSⅡa, SSⅡb, and SSⅡc), two SSⅢ genes (SSⅢa and SSⅢb), and two SSⅣgenes (SSⅣa and SSⅣb) (Hirose and Terao, 2004). Compared to the rice genome through the current data, Maize (Zea mays L.) genome contains two SSⅡb and two SSⅢb genes, but only one SSⅣ gene (Yan et al., 2009). In wheat, seven SS genes, GBSSⅠ/WX, GBSSⅡ, SSⅠ, SSⅡa, SSⅡc, SSⅢa, and SSⅣb, had been cloned (Clark et al., 1991; Li et al., 1999a, 1999b, 2000; Leterrier et al., 2008; Yan et al., 2009).

In this study, we reported the cloning and characterization of the genes encoding GBSS, SSâ… , SSâ…¡, SSâ…¢ and SSâ…£ in sorghum, and the SSâ…¡b and SSâ…¢b genes in wheat. We found that the duplicator of SSâ…£ gene was lost in the sorghum, maize and wheat genomes. The expression patterns of the detected SS genes were analyzed and the evolution of the SS gene family in Gramineae was discussed.

1 Results and Discussion
1.1 Identification of the SS genes in sorghum and wheat
The previous studies reported the cloning of the genes encoding GBSSâ… , GBSSâ…¡ (Clark et al., 1991), SSâ…  (Li et al., 1999b), SSâ…¡a (Li et al., 999a), SSâ…¡c (Yan et al., 2009), SSâ…¢a (Li et al., 2000), SSâ…£ (Leterrier et al., 2008) in wheat, and GBSSâ… /WX (EF089858), SSâ…  (AF168786) in sorghum. In this study, we determined the complete coding domain sequences of the other six SS genes in sorghum and two in wheat. The designated gene names and GenBank accession numbers were SbGBSSâ…¡ (EF472254), SbSSâ…¡a (EU620718), SbSSâ…¡b (EU620719), SbSSâ…¡c (EU307275), SbSSâ…¢a (EU620720), SbSSâ…¢b (EU620721), TaSSâ…¡b (EU333947) and TaSSâ…¢b (EU333946). Only one SSâ…£ gene (XM_002440083) was detected in the public sorghum genomic sequences and EST database. No SSâ…£a gene was detected in wheat genomic sequences and EST database in NCBI and the raw Chinese spring genomic sequence reads using BLAST (http://www.cerealsdb.uk.net/ search_reads.htm). The domain orga- nization of SS proteins in Gramineae was listed in Table 1.

Table 1 Domain organization of the SS proteins in Gramineae

1.2 Phylogenetic relationships among the duplicated SS genes
In order to characterize the identified SS genes in sorghum and wheat, a phylogenetic tree was built with 58 SS amino acid sequences from some monocots and dicots (Figure 1, a bacterium glycogen synthase, EcGS, was as out group for the phylogenetic analysis). The phylogenetic tree indicated that the SS proteins are grouped into five clades of GBSS, SSâ… , SSâ…¡, SSâ…¢, and SSâ…£. In Gramineae, GBSS proteins were further divided into two subisoforms of GBSSâ…  and GBSSâ…¡, SSâ…¡ proteins into SSâ…¡a, SSâ…¡b and SSâ…¡c, SSâ…¢ proteins into SSâ…¢a and SSâ…¢b, SSâ…£ proteins into SSâ…£a and SSâ…£b, respectively (Figure 1). The present determined wheat SSâ…¡ and SSâ…¢ fall into the SSâ…¡b and SSâ…¢b subisoforms, respectively (Figure 1). The previous study found the WGD event occurred approximately 70 million years ago (Mya) prior to the divergence of the Gramineae (Paterson et al., 2004). Thus, it is likely that the duplicators of GBSS, SSâ…¡, and SSâ…¢b, but not SSâ…£, were retained in genomes of the observed Gramineae species.

Figure 1 Phylogenetic tree derived from the full amino acid sequences of starch synthesis proteins

1.3 Alignments of syntenic regions that contain the SSâ…£ gene among rice, maize, and sorghum genome
The two SSâ…£ genes in rice were located on chromosome 1 and chromosome 5, respectively. In order to determine whether the SSâ…£a gene is lost in sorghum and maize genomes, we compared the gene cluster on syntenic region containing the SSâ…£ genes among rice, sorghum, and maize, based on the rice-maize-sorghum synteny (http://www.gramene.org/).

The selected genomic fragment containing the SSâ…£a gene (GFa) was 544 kb on rice chromosome 1 (rC1) which contains 63 putative protein-coding genes (from Os01g0714900, a Ras-related gene, to Os01g0- 726100, an Ole e â…  family gene). The genomic fragment containing the SSâ…£b gene (GFb) was 296 Kb on rice chromosome 5 (rC5) which contains 34 putative protein-coding genes (from Os05g0531200, an Ole e I family gene, to Os05g0536900, a Ras- related gene) (http://rapdb.dna.affrc.go.jp/viewer/gbr-owse/build4/) (supplement and Figure 2). There are 16 paralogous between GFa and GFb. The OsSSâ…£a gene is located between a chlorophyll a-b binding protein gene and a serine acetyltransferase gene, while the OsSSâ…£b gene located between a serine acetyltrans- ferase gene and a NADH-ubiquinone oxidoreductase 20 kD subunit gene (supplement and Figure 2).

Figure 2 Comparison of the starch synthesis genes on the rice/ sorghum/maize synteny physical map

Based on the rice-sorghum synteny (http://www.gram-ene.org/), the syntenic region of rice GFa in sorghum is 436 Kb on sorghum chromosome 3 (sC3), while the syntenic region of rice GFb in sorghum is 248 Kb on sorghum chromosome 9 (sC9). In sorghum, the GFa contains 58 putative protein-coding genes (from Sb03g032810, a Ras-related gene, to Sb03g033370, an Ole e â…  family gene), while the GFb contains 36 putative protein-coding genes (from Sb09g026510, an Ole e I family gene, to Sb09g026820, a Ras-related gene) (http://rapdb.dna.affrc.go.jp/viewer/gbrowse/build4/) (supplement and Figure 2). GFa and GFb have 12 paralogous genes in sorghum. There are 44 orthologous genes in GFa, while 29 orthologous genes in GFb between in the rice genome and sorghum genome. Like the OsSSâ…£b in rice, the SbSSâ…£b gene was also located between a serine acetyltransferase 1 and a NADH-ubiquinone oxidoreductase 20 kDa subunit in sorghum. However, the SSâ…£a gene which located between the chlorophyll a-b binding protein gene and serine acetyltransferase gene in GFa in rice was lost in GFa in sorghum (supplement and Figure 2).

Based on the sorghum-maize synteny (http://www.gramene.org/), the syntenic region of sorghum GFa in maize is a 313 Kb on maize chromosome 3 (zC3), while the syntenic region of sorghum GFb in sorghum is a 64 Kb and 131 Kb on maize chromosome 6 (zC6) and chromosome 8 (zC8), respectively. In maize, the GFa contains 11 putative protein-coding genes (from GRMZM2G099166_T02, a DUF581 family protein, to GRMZM2G116282_T02, a RNA-binding protein), while the GFb on zC6 contains 6 putative protein- coding genes (from GRMZM2G033971_T01, a DUF212 family protein, to GRMZM2G043035_T02, an Ole e I family protein), and on zC8 contains 6 putative protein-coding genes (from GRMZM2G044- 866_T01, a DUF616 family protein, to GRMZM2G1- 21117_T02, a hypothetical protein) (http://rapdb.dna.affrc.go.jp/viewer/gbrowse/build4/) (supplement and Figure 2). There are 11 orthologous genes in GFa between in the rice and sorghum genome. There are 6 orthologous genes respectively in GFb on maize chromosome 6 and 8 between in the maize and sorghum genome. The ZmSSâ…£b was located between a hypothetical protein and a DUF212 family protein (supplement and Figure 2). Moreover, we can not found the SSâ…£a gene on chromosomr 3 regions in maize, and the chlorophyll a-b binding protein and the Serine acetyltransferase 3 also lost. These results strongly suggested that the SSâ…£a gene was lost in sorghum and maize genomes during their evolution.

1.4 Organ expression profile of the SS genes in sorghum and wheat
Expression divergence was often the first step in the functional divergence between duplicate genes, thus increasing the chance of retention of duplicated genes in a genome (Force et al., 1999). To define the function of the deteced SS genes, we investigated their expression in root, leaf and developing endosperm using RT-PCR in sorghum and wheat. The results indicated that the SbGBSSâ… , SbSSâ…¡a and SbSSâ…¢a genes were expressed mainly in endosperms, while the SbGBSSâ…¡, SbSSâ…¡b, and SbSSâ…¢b genes mainly in leaf in sorghum. The SbSSâ… , SbSSâ…¡c and SbSSâ…£ genes were constitutively expressed in sorghum. In addition, the SbGBSSâ…¡ and SbSSâ…¢b genes were also highly expressed in roots (Figure3A). TaSSâ…¡b and TaSSâ…¢b were mainly expressed in leaves, and moderately in roots and the early stage endosperms (Figure 3B).

Figure 3 The organ expression analysis of SS genes in sorghum and wheat

The mRNA for the TaSSâ…¡a was expressed in leaves and endosperms under the conditions used (Li et al., 1999a), but the SGP-B1 (TaSSâ…¡a) protein was not detected in leaf using monoclonal antibodies (Shimbata et al., 2005). This result is similar to the findings of OsSSâ…¡a in rice that the transcripts could be tested at a lower level in leaf, but its proteins could not be detected in leaf soluble or starch granule extracts using polyclonal antibodies (Jiang et al., 2004). The expression of TaSSâ…¡b and TaSSâ…¢b was mainly in leaves and roots, but lower in filling stage endosperms, suggests that TaSSâ…¡b and TaSSâ…¢b mainly function in leaves and roots, while TaSSâ…¡a and TaSSâ…¢a in endosperms as those in maize and rice (Jiang et al., 2004; Yan et al., 2009). These results indicated SS duplicators were also diverged in expression in wheat and sorghum as in rice and maize (Dian et al., 2003; Jiang et al., 2004; Yan et al., 2009). In addition, those SS duplication and expression divergence were similar to the starch legumes (mung bean and cowpea) genomes (Pan et al., 2009). In summary, the present study and previous reports indicate that duplicators of GBSSâ… , SSâ…¡, and SSâ…¢, but not SSâ…£, were remained in the genome and diverged in expression in all observed Gramineae species.

2 Materials and methods
2.1 Plant materials and growth conditions
The sorghum (Sorghum bicolor L.) and common wheat (Triticum aestivum L.) variety Shi4185 were planted in trail field in South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, and P. R. China. The seeds were harvested four times after flowering, while the roots were got from the fifth day after germinate (DAG) seeding and full expanded leaves were taken from seedling at one day after flowering. Samples were frozen under liquid nitrogen and stored at -72℃ until use. All samples were collected in a time period between 9:00 am to 10:00 am. 

2.2 cDNA cloning of GBSS, SSâ…¡, and SSâ…¢ genes in sorghum and wheat
Total RNA was isolated from sampled leaves with Plant RNAout kit (Tiandz Company, http://www.tian-dz.com). First-strand cDNA synthesis using M-MLV reverse transcriptase (Promega, http://www.promega.com) following manufacture's protocol. A specific fragment of sorghum and wheat GBSS, SSâ…¡, and SSâ…¢ genes were amplified with the primer pair (Table 2), designed based on the conserved regions of the corresponding genes from other higher plants. The purified fragments were cloned into a pMD18-T vector (TaKaRa, http://www.takara.com.cn) and confirmed by sequencing in the Invitrogen Company (http://www.invitrogen.com.cn). The 5'- and 3'-ends of sorghum GBSS, SSâ…¡, and SSâ…¢ genes were obtained with a 5' full RACE cDNA Amplification kit (Clontech) according to the user's instructions. Based on these sequences, 5’-end of the TaSSâ…¡b and TaSSâ…¢b cDNAs was determined by using the BD SMART™ RACE cDNA Amplification Kit (Invitrogen, Carlsbad, CA, USA). Putative transit peptide were identified using the ChloroP neural network analysis of the 100 amino acids at the N terminus of each sequence (http://www.cbs.dtu.dk/services/ChloroP).

Table 2 Primers used for sorghum in this study

2.3 Semi-quantitative RT-PCR analysis
Total RNA was extracted from roots, leaves and developing endosperms using the Plant RNAout kit. PCR amplifications were performed on the first cDNA strand using their specific primer sets (Table 2; Table 3). Primers (Table 2; Table 3) that amplify sorghum Actin (X79378) and wheat Actin (AY663392) were used as a control. PCR products were analyzed by 1% agarose gels, with ethidium bromide staining and take photo through Fluorescence Chemiluminescence & Visible Imaging System. Afterwards the purified fragments were cloned into the pMD18-T vector and the sequence were determined.

Table 3 Primers used for wheat in this study

2.4 Data analysis
Phylogenetic analysis was carried out using the conserved domains sequences of SS genes. Alignments of the SS sequences were aligned using the ClustalW program on EBI Web server. Phylogenetic trees were calculated using PhyML Online analysis (Guindon et al., 2005) (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=phyml) based on the JTT model with a constant rate of site variation (Guindon and Gascuel, 2003). Bootstrap values were calculated from 100 replicate analyses.

2.5 Gene loci comparison
To confirm their physical location, the gene loci on rice and sorghum were obtained by the alignment of the cDNA sequences from the NCBI database (http://www.ncbi.nlm.nih.gov/blast) to the corresponding chromosome-based pseudomolecules using PHYTO- ZOME blast (http://www.phytozome.net/search.php?show=blast). The location of the maize genes on the chromosome pseudomolecules was traced though the anchored corresponding BAC genome sequences or related markers (http://www.maizegdb.org). Finally, the loci of anchored rice, maize and sorghum starch synthesis genes were compared on the rice/maize/ sorghum synteny physical maps available at Gramene (http://www.gramene.org/).

Acknowledgements
This research was supported by funds received from the National Natural Science Foundation of China (No. 31070227) and the CAS/SAFEA International Partnership Program for Creative Research Teams.
 
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