Characteristics of Amorphophallus konjac as indicated by its genome.

Amorphophallus konjac, belonging to the genus Amorphophallus of the Araceae family, is an economically important crop widely used in health products and biomaterials. In the present work, we performed the whole-genome assembly of A. konjac based on the NovaSeq platform sequence data. The final genome assembly was 4.58 Gb with a scaffold N50 of 3212 bp. The genome includes 39,421 protein-coding genes, and 71.75% of the assemblies were repetitive sequences. Comparative genomic analysis showed 1647 gene families have expanded and 2685 contracted in the A. konjac genome. Likewise, genome evolution analysis indicated that A. konjac underwent whole-genome duplication, possibly contributing to the expansion of certain gene families. Furthermore, we identified many candidate genes involved in the tuber formation and development, cellulose and lignification synthesis. The genome of A. konjac obtained in this work provides a valuable resource for the further study of the genetics, genomics, and breeding of this economically important crop, as well as for evolutionary studies of Araceae family.

Comparative analysis of buds transcriptome and identification of two florigen gene AkFTs in Amorphophallus konjac.

Leaves and flowers of Amorphophallus konjac do not develop simultaneously thus unique features can be elucidated through study of flowering transformation in A. konjac. In this study, transcriptome libraries of A. konjac leaf buds (LB) and flower buds (FB) were constructed followed by high-throughput sequencing. A total of 68,906 unigenes with an average length of 920 bp were obtained after library assembly. Out of these genes, 24,622 unigenes had annotation information. A total of 6859 differentially expressed genes (DEGs) were identified through differential expression analysis using LB as control. Notably, 2415 DEGs were upregulated whereas 4444 DEGs were downregulated in the two transcriptomes. Go and KEGG analysis showed that the DEGs belonged to 44 functional categories and were implicated in 98 metabolic pathways and 38 DEGs involved in plant hormone signal transduction. Several genes were mined that may be involved in A. konjac flower bud differentiation and flower organ development. Eight DEGs were selected for verification of RNA-seq results using qRT-PCR analysis. Two FLOWERING LOCUS T (FT) genes named AkFT1 and AkFT2 were identified though homologous analysis may be the florigen gene implicated in modulation of A. konjac flowering. These genes were significantly upregulated in flower buds compared with the expression levels on leaf buds. Overexpression of AkFT genes though heterologous expression in Arabidopsis showed that the transgenics flowered at a very early stage relative to wild type plants. These findings indicate that AkFT1 and AkFT2 function as regulation genes in A. konjac flowering development and the two genes may present similar functions during flowering transition.

A novel neutral thermophilic ß-mannanase from Malbranchea cinnamomea for controllable production of partially hydrolyzed konjac powder.

Partially hydrolyzed konjac powder (PHKP) can be used to increase the daily intake of dietary fibers of consumers. To produce PHKP by enzymatic hydrolysis, a novel ß-mannanase gene (McMan5B) from Malbranchea cinnamomea was expressed in Pichia pastoris. It showed a low identity of less than 52% with other GH family 5 ß-mannanases. Through high cell density fermentation, the highest ß-mannanase activity of 42200 U mL-1 was obtained. McMan5B showed the maximal activity at pH 7.5 and 75 °C, respectively. It exhibited excellent pH stability and thermostability. Due to the different residues (Phe214, Pro253, and His328) in catalytic groove and the change of ß2-α2 loop, McMan5B showed unique hydrolysis property as compared to other ß-mannanases. The enzyme was employed to hydrolyze konjac powder for controllable production of PHKP with a weight-average molecular weight of 22000 Da (average degree of polymerization 136). Furthermore, the influence of PHKP (1.0%-4.0%) on the qualities of steamed bread was evaluated. The steamed bread adding 3.0% PHKP had the maximum specific volume and the minimum hardness, which showed 11.0% increment and 25.4% decrement as compared to the control, respectively. Thus, a suitable ß-mannanase for PHKP controllable production and a fiber supplement for steamed bread preparation were provided in this study. KEY POINTS: • A novel ß-mannanase gene (McMan5B) was cloned from Malbranchea cinnamomea and expressed in Pichia pastoris at high level. • McMan5B hydrolyzed konjac powder to yield partially hydrolyzed konjac powder (PHKP) instead of manno-oligosaccharides. • PHKP showed more positive effect on the quality of steamed bread than many other dietary fibers including konjac powder.

Odor polymorphism in deceptive Amorphophallus species - a review.

Some plant lineages, such as Araceae and Orchidaceae, have independently evolved deceptive flowers. These exploit the insect's perception and deceive the insects into believing to have located a suitable opportunity for reproduction. The scent compounds emitted by the flowers are the key signals that dupe the insects, guiding them to the right spots that in turn ensure flower pollination. Most species of the genus Amorphophallus of the Araceae emit scent compounds that are characteristic of a deceit, suggesting a specific plant pollinator interaction and according odors. However, only a few clear evolutionary trends in regard to inflorescence odors in Amorphophallus could be traced in previous studies - an intriguing result, considered the multitude of characteristic scent compounds expressed in Amorphophallus as well as the key function of scent compounds in deceptive floral systems in general. At least two factors could account for this result. (1) The deceptive pollinator-attraction floral system, including the emitted scent compounds, is less specific than assumed. (2) An evolutionary trend cannot be discerned if the intraspecific scent variation (odor polymorphism) exceeds the interspecific odor variation. Therefore, we discuss the potential deceptive function of the emitted scent compounds, in particular those that are related to cadaveric decomposition. Moreover, we review the data about emitted scent compounds in Amorphophallus with a focus on putative odor polymorphism. Upon examination, it appears that the emitted scent compounds in Amorphophallus are highly mimetic of decomposing organic materials. We show that several species display odor polymorphism, which in turn might constitute an obstacle in the analysis of evolutionary trends. An important odor polymorphism is also indicated by subjective odor perceptions. Odor polymorphism may serve several purposes: it might represent an adaptation to local pollinators or it might assumingly prevent insects from learning to distinguish between a real decomposing substrate and an oviposition-site mimic.

A mixed ploidy natural population of Amorphophallus muelleri provides an opportunity to trace the evolution of Amorphophallus karyotype.

Amorphophallus, a perennial herb belongs to the family Araceae, and is widely distributed in Asia and Africa. As an agricultural crop, it has been cultivated and consumed for ~2000 years in China. Previous studies have found that there are chromosome number and ploidy changes in this genus, but there are a few reports on the evolution of different karyotypes. For this study, we collected 37 samples of a wild population of Amorphophallus muelleri from Myanmar and analysed their karyotypes. The karyotype analysis showed that it is a population with mixed chromosome numbers and ploidy, with four karyotypes of 2n = 24, 26, 28 and 39. Combining the results of this study with previous literature, we speculate that karyotypes with 2n = 26 may be the common ancestor, and further the other three karyotypes were evolved from this by various ways. As far as we know, this is the first attempt to put forward the hypothesis of the evolution of those four karyotypes together. On the other hand, by using inter-simple sequence repeat marker-based unweighted pair group method with arithmetic mean cluster analysis, we found that these individuals of four karyotypes can be divided into four corresponding categories, indicating that they have been differentiated at the genome, providing a theoretical basis for future use of these wild germplasm resources.

Genetic diversity and population structure of Amorphophallus albus, a plant species with extremely small populations (PSESP) endemic to dry-hot valley of Jinsha River.

BACKGROUND: Amorphophallus albus P. Y. Liu & J. F. Chen (Araceae) is a plant species with extremely small populations (PSESP) and an important economic crop endemic to dry-hot valleys along the Jinsha River. In order to gain information for sustaining the development and conservation of A. albus, we studied the genetic diversity and population structure of this species using microsatellite markers (SSR). In this study, we analysed 364 individuals belonging to 24 populations, including four wild populations and three ex-situ cultivated populations, collected in the provinces Yunnan, Sichuan and Hubei. RESULTS: The population genetic analyses indicated that A. albus possesses moderate genetic diversity with the percentage of polymorphic loci (PPL) from 69.23 to 100%, an expected heterozygosity (He) of 0.504 and an average Shannon's Information Index (I) 0.912. Analysis of molecular variance (AMOVA) indicated that most of the variance (71%) resided within populations and the estimated gene flow (Nm) was 0.61. The results of UPGMA cluster tree, STRUCTURE analyses together with the Mantel test (R2 = 0.352, P < 0.01) indicated that geographically closely located populations are clustered together with some exceptions. CONCLUSIONS: Our results showed that A. albus still possesses moderate genetic variation in most of the studied populations, and for now, most cultivated populations were naturally distributed but still some reintroduction exists. For sustaining the present genetic variation, some protections measures are necessary for the wild populations and also for the cultivated ones with high genetic diversity.

First report on DNA content of three species of Amorphophallus.

The Amorphophallus genus is a perennial herb which belongs to the family Araceae. There are more than 170 species in this genus, which is widely distributed in tropical and subtropical areas. As a kind of food and medicine Amorphophallus has been used for more than 2000 years in China. Because of the high content of konjac glucomannan (KGM) and dietary fiber, it has attracted more attention worldwide. In this article, the DNA contents of A. konjac, A. albus and A. bulbifer in China, A. albus, A. paeoniifolius and A. muelleri in Indonesia were estimated by using flow cytometry. In the samples of China, the DNA contents were 12.95 ± 0.73 pg/2C in A. konjac, 10.51 ± 0.05 pg/2C in A. albus and 17.61 pg/2C in A. bulbifer, and for Indonesia, 14.16 ± 0.48 pg/2C in A. albus (flowering), 8.49 ± 0.2 pg/2C in A. paeoniifolius and 17.84 ± 1.46 pg/2C in A. muelleri were used. Interspecific variation was found significantly (P<0.01), suggesting that DNA content might be a parameter that can be used to differentiate the species. Intraspecific variation has also been found significantly (P<0.01), whether in the same region or between two regions. As far as we know, this is the first report ongenome size estimation of the A. konjac, A. albus and A. muelleri using flow cytometry. Understanding the genome size of Amorphophallus species will help to sequence the genome and analyse the genetic diversity, evolutionary relationship and geographical variation pattern of Amorphophallus species.

Comparative analysis of complete chloroplast genome sequences of four major Amorphophallus species.

Amorphophallus (Araceae) contains more than 170 species that are mainly distributed in Asia and Africa. Because the bulbs of Amorphophallus are rich in glucomannan, they have been widely used in food, medicine, the chemical industry and so on. To better understand the evolutionary relationships and mutation patterns in the chloroplast genome of Amorphophallus, the complete chloroplast genomes of four species were sequenced. The chloroplast genome sequences of A. albus, A. bulbifer, A. konjac and A. muelleri ranged from 162,853 bp to 167,424 bp. The A. albus chloroplast (cp) genome contains 113 genes, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The A. bulbifer cp genome contains 111 genes, including 78 protein-coding genes, 29 tRNA genes and 4 rRNA genes. A. muelleri contains 111 and 113 genes, comprising 78 and 80 protein-coding genes, respectively, 29 tRNA genes and 4 rRNA genes. The IR (inverted repeat) region/LSC (long single copy) region and IR/SSC (short single copy) region borders of the four Amorphophallus cp genomes were compared. In addition to some genes being deleted, variations in the copy numbers and intron numbers existed in some genes in the four cp genomes. One hundred thirty-four to 164 SSRs (simple sequence repeats) were detected in the four cp genomes. In addition, the highest mononucleotide SSRs were composed of A and T repeat units, and the majority of dinucleotides were composed of AT and TA. SNPs (single nucleotide polymorphisms) and indels (insertion-deletions) were calculated from coding genes and noncoding genes, respectively. These divergences comprising SSRs, SNPs and indel markers will be useful in testing the maternal inheritance of the chloroplast genome, identifying species differentiation and even in breeding programs. Furthermore, the regression of ndhK was detected from four Amorphophallus cp genomes in our study. Complete cp genome sequences of four Amorphophallus species and other plants were used to perform phylogenetic analyses. The results showed that Amorphophallus was clustered in Araceae, and Amorphophallus was divided into two clades; A. albus and A. konjac were clustered in one clade, and A. bulbifer and A. muelleri were clustered in another clade. Phylogenetic analysis among the Amorphophallus genus was conducted based on matK and rbcL. The phylogenetic trees showed that the relationships among the Amorphophallus species were consistent with their geographical locations. The complete chloroplast genome sequence information for the four Amorphophallus species will be helpful for elucidating Amorphophallus phylogenetic relationships.

Members of the DUF231 Family are O-Acetyltransferases Catalyzing 2-O- and 3-O-Acetylation of Mannan.

Mannans are hemicellulosic polysaccharides commonly found in the primary and secondary cell walls of land plants, and their mannosyl residues are often acetylated at O-2 and O-3. Currently, little is known about the genes responsible for the acetylation of mannans. In this report, we investigated the roles of a subgroup of DUF231 proteins including 11 from Arabidopsis thaliana and one from Amorphophallus konjac in mannan acetylation. Acetyltransferase activity assays of their recombinant proteins revealed that four Arabidopsis DUF231 proteins possessed an enzymatic activity capable of transferring acetyl groups from acetyl-CoA onto the mannohexaose acceptor, and thus were named mannan O-acetyltransferases (MOAT1, MOAT2, MOAT3 and MOAT4). Their close homolog from A. konjac (named AkMOAT1) also exhibited mannan O-acetyltransferase activity. Structural analysis of the MOAT-catalyzed reaction products demonstrated that these MOATs catalyzed 2-O- and 3-O-monoacetylation of mannosyl residues, an acetyl substitution pattern similar to that of Arabidopsis glucomannan. Site-directed mutagenesis showed that mutations of the conserved residues in the GDS and DXXH motifs of MOAT3 abolished its acetyltransferase activity, indicating the essential roles of these motifs in its activity. In addition, simultaneous RNA interference (RNAi) inhibition of the expression of the four Arabidopsis MOAT genes led to a drastic reduction in the degree of acetyl substitutions in glucomannan, further corroborating their role in glucomannan acetylation. Together, these results present the first lines of biochemical and genetic evidence demonstrating that these four Arabidopsis DUF231 members and their close A. konjac homolog are mannan O-acetyltransferases.

Gene cloning of a neutral ceramidase from the sphingolipid metabolic pathway based on transcriptome analysis of Amorphophallus muelleri.

Amorphophallus is a perennial herbaceous plant species mainly distributed in the tropics or subtropics of Asia and Africa. It has been used as a traditional medicine for a long time and now is utilized for the pharmaceutical, chemical and agriculture industries as a valued economic crop. Recently, Amorphophallus has attracted tremendous interest because of its high ceramide content. However, the breeding and genome studies are severely limited by the arduous whole genome sequencing of Amorphophallus. In this study, the transcriptome data of A. muelleri was obtained by utilizing the high-throughput Illumina sequencing platform. Based on this information, the majority of the significant genes involved in the proposed sphingolipid metabolic pathway were identified. Then, the full-length neutral ceramidase cDNA was obtained with the help of its candidate transcripts, which were acquired from the transcriptome data. Furthermore, we demonstrated that this neutral ceramidase was a real ceramidase by eukaryotic expression in the yeast double knockout mutant Δypc1 Δydc1, which lacks the ceramidases-dihydroCDase (YDC1p), phytoCDase (YPC1p). In addition, the biochemical characterization of purified A. muelleri ceramidase (AmCDase) exhibited classical Michaelis-Menten kinetics with an optimal activity ranging from pH 6.5 to 8.0. Based on our knowledge, this study is the first to report the related information of the neutral ceramidase in Amorphophallus. All datasets can provide significant information for related studies, such as gene expression, genetic improvement and application on breeding in Amorphophallus.

Genetic diversity and phylogenetic relationships of seven Amorphophallus species in southwestern China revealed by chloroplast DNA sequences.

Plants species in the genus Amorphophallus are of great economic importance, as they are the only plants known to produce glucomannan. Although southwestern China has been recognized as one of the origin centres of Amorphophallus, only a few studies assessing its genetic diversity have been reported. To aid in the utilization and conservation of Amorphophallus species, we evaluated the genetic diversity and phylogenetic relationships among seven edible Amorphophallus species using three chloroplast DNA regions (rbcL, trnL and trnK-matK). The results showed that the genetic diversity at the population level was relatively low, with over half of the populations harbouring only one haplotype. The widely scattered species, A. konjac, had the largest genetic diversity, while the narrow endemic species, A. yuloensis, possessed only one haplotype. Phylogeny analysis identified three well-supported major lineages. Our study suggested that habitat fragmentation might be a driver of the genetic variation patterns within and between populations of Amorphophallus. A conservation strategy consisting of in situ conservation and germplasm collection is recommended.

Genetic diversity and structure of wild and cultivated Amorphophallus paeoniifolius populations in southwestern China as revealed by RAD-seq.

Amorphophallus paeoniifolius, is a commercially important vegetable crop because of its high production potential. In this study, we generated a total of 166 Gb of genomic data from 16 wild and 20 cultivated A. paeoniifolius individuals in southwestern China using restriction site associated DNA sequencing (RAD-seq). We compared the genome-wide variations between the wild and cultivated populations. Wild populations exhibited higher genetic diversity than did cultivated populations based on private allele number, expected heterozygosity, observed heterozygosity and nucleotide diversity. STRUCTURE analysis, principal component analysis (PCA) and a maximum likelihood (ML) tree indicated that A. paeoniifolius populations could be divided into three groups (a cultivated group and two wild groups) with significant genetic differentiation. The low genetic diversity and shallow genetic differentiation found within cultivated populations are likely caused by continuous selection and the clonal propagation methods used during domestication. The significant differentiation between the wild populations may suggest strong genetic drift due to small populations and human disturbance. The genome-wide single nucleotide polymorphisms (SNPs) identified in our study will provide a valuable resource for further breeding improvement and effective use of the germplasm.

Phylogenetic trends in the evolution of inflorescence odours in Amorphophallus.

The chemical composition of inflorescence odours of 80 species of Amorphophallus (Araceae) were determined by headspace-thermal desorption GC-MS. When compared to published molecular phylogenies of the genus, the data reveal evidence both of phylogenetic constraint and plasticity of odours. Dimethyl oligosulphides were found as common constituents of Amorphophallus odours and were the most abundant components in almost half of the species studied. Odours composed mainly of dimethyl oligosulphides, and perceived as being 'gaseous', were only found among Asian species, and some of these species clustered in certain clades in molecular phylogenies; e.g. in two clades in Amorphophallus subgenus Metandrium. However, some species with gaseous odours were found to be closely related to species producing odours more reminiscent of rotting meat in which various minor components accompany the dominant dimethyl oligosulphides. These two broad types of odours have co-evolved with other inflorescence characteristics such as colour, with species with rotting meat odours having darker inflorescences. Species producing pleasant odours characterised by benzenoid compounds constitute two broad groups that are not related in published phylogenies. Species having fruity odours containing 1-phenylethanol derivatives mainly occur in a clade in subgenus Metandrium while those with anise odours composed almost solely of the 2-phenylethanol derivative 4-methoxyphenethyl alcohol are restricted to a clade in subgenus Scutandrium. Phylogenetic mapping of odours also indicates that the evolution of some odour types is likely to have been influenced by ecological factors. For example, species producing fishy odours dominated by trimethylamine and occurring in N and NE Borneo are not all closely related. Conversely, two sister species, A. mossambicensis and A. abyssinicus, which are morphologically very similar and have overlapping geographical distribution, produce odours which are very different chemically. The pressure of pollinator resource has therefore been a factor influencing the evolution of odours in Amorphophallus, driving both the divergence of odour types in some taxa and the convergence of odour types in others.

Population structure of elephant foot yams (Amorphophallus paeoniifolius (Dennst.) Nicolson) in Asia.

The corms and leaves of elephant foot yams (Amorphophallus paeoniifolius (Dennst.) Nicolson) are important foods in the local diet in many Asian regions. The crop has high productivity and wide agroecological adaptation and exhibits suitability for the agroforestry system. Although the plant is assumed to reproduce via panmixia, a comprehensive study on the genetic background across regions to enhance wider consumer palatability is still lacking. Here, ten informative microsatellites were analyzed in 29 populations across regions in India, Indonesia and Thailand to understand the genetic diversity, population structure and distribution to improve breeding and conservation programs. The genetic diversity was high among and within regions. Some populations exhibited excess heterozygosity and bottlenecking. Pairwise FST indicated very high genetic differentiation across regions (FST = 0.274), and the Asian population was unlikely to be panmictic. Phylogenetic tree construction grouped the populations according to country of origin with the exception of the Medan population from Indonesia. The current gene flow was apparent within the regions but was restricted among the regions. The present study revealed that Indonesia and Thailand populations could be alternative centers of the gene pool, together with India. Consequently, regional action should be incorporated in genetic conservation and breeding efforts to develop new varieties with global acceptance.

Genetic variation in wild populations of the tuber crop Amorphophallus konjac (Araceae) in central China as revealed by AFLP markers.

Amorphophallus konjac is an economically important crop. In order to provide baseline information for sustainable development and conservation of the wild plant resources of A. konjac, we studied the genetic diversity and population structure of this species using amplified fragment length polymorphism (AFLP) molecular markers. We sampled 139 individuals from 10 wild populations of A. konjac in central China. Using five AFLP primer combinations, we scored a total of 270 DNA fragments, most of which were polymorphic (98.2%). Percentage of polymorphic loci, Nei's genetic diversity index, and Shannon's information index showed high levels of genetic variation within A. konjac populations. Analysis of molecular variance indicated that most of the variance (68%) resided within populations. The coefficient of genetic differentiation between populations was 0.348 and the estimated gene flow was 0.469, indicating that there was limited gene flow among the populations. Unweighted pair group method with arithmetic mean cluster analysis and principal coordinates analysis indicated that geographically close populations were more likely to cluster together. The Mantel test revealed a significant correlation between geographic and genetic distances (R2 = 0.2521, P < 0.05). The special insect-pollination system of A. konjac and the complex geography of central China are likely to have contributed to the current pattern of genetic variation of this species. In the present study, we provide several suggestions on the future protection of the wild plant genetic resources of A. konjac.

De novo transcriptome and small RNA analyses of two amorphophallus species.

Konjac is one of the most important glucomannan crops worldwide. The breeding and genomic researches are largely limited by the genetic basis of Amorphophallus. In this study, the transcriptomes of A. konjac and A. bulbifer were constructed using a high-throughput Illumina sequencing platform. All 108,651 unigenes with average lengths of 430 nt in A. konjac and 119,678 unigenes with average lengths of 439 nt were generated from 54,986,020 reads and 52,334,098 reads after filtering and assembly, respectively. A total of 54,453 transcripts in A. konjac and 55,525 in A. bulbifier were annotated by comparison with Nr, Swiss-Prot, KEGG, and COG databases after removing exogenous contaminated sequences. A total of 80,332 transcripts differentially expressed between A. konjac and A. bulbifer. The majority of the genes that are associated with konjac glucomannan biosynthetic pathway were identified. Besides, the small RNAs in A. konjac leaves were also obtained by deep sequencing technology. All of 5,499,903 sequences of small RNAs were obtained with the length range between 18 and 30 nt. The potential targets for the miRNAs were also predicted according to the konjac transcripts. Our study provides a systematic overview of the konjac glucomannan biosynthesis genes that are involved in konjac leaves and should facilitate further understanding of the crucial roles of carbohydrate synthesis and other important metabolism pathways in Amorphophallus.

Development of microsatellite markers by transcriptome sequencing in two species of Amorphophallus (Araceae).

BACKGROUND: Amorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of West Africa and South Asia. Its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. Genetic studies of Amorphophallus have been hindered by a lack of genetic markers. A large number of molecular markers are required for genetic diversity study and improving disease resistance in Amorphophallus. Here, we report large scale of transcriptome sequencing of two species: Amorphophallus konjac and Amorphophallus bulbifer using deep sequencing technology, and microsatellite (SSR) markers were identified based on these transcriptome sequences. RESULTS: cDNAs of A. konjac and A. bulbifer were sequenced using Illumina HiSeq™ 2000 sequencing technology. A total of 135,822 non-redundant unigenes were assembled from about 9.66 gigabases, and 19,596 SSRs were identified in 16,027 non-redundant unigenes. Di-nucleotide SSRs were the most abundant motif (61.6%), followed by tri- (30.3%), tetra- (5.6%), penta- (1.5%), and hexa-nucleotides (1%) repeats. The top di- and tri-nucleotide repeat motifs included AG/CT (45.2%) and AGG/CCT (7.1%), respectively. A total of 10,754 primer pairs were designed for marker development. Of these, 320 primers were synthesized and used for validation of amplification and assessment of polymorphisms in 25 individual plants. The total of 275 primer pairs yielded PCR amplification products, of which 205 were polymorphic. The number of alleles ranged from 2 to 14 and the polymorphism information content valued ranged from 0.10 to 0.90. Genetic diversity analysis was done using 177 highly polymorphic SSR markers. A phenogram based on Jaccard's similarity coefficients was constructed, which showed a distinct cluster of 25 Amorphophallus individuals. CONCLUSION: A total of 10,754 SSR markers have been identified in Amorphophallus using transcriptome sequencing. One hundred and seventy-seven polymorphic markers were successfully validated in 25 individuals. The large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in Amorphophallus.

Isolation and characterization of microsatellite loci for the herbaceous tuber crop, Amorphophallus konjac (Araceae).

Amorphophallus konjac is an herbaceous tuber crop with tremendous potential for commercial development. We report the development of microsatellite primers for this important crop species. Thirteen polymorphic microsatellite markers were developed and tested in two populations of A. konjac from the Wuling Mountain Region (WL population) and the Yunnan Province (YN population) in China. The number of alleles per locus ranged from 1 to 7; the observed and expected heterozygosities ranged from 0 to 1 and from 0 to 0.844, respectively, in the two populations. These microsatellite markers will facilitate further studies in population genetics and utilization of A. konjac.

Deep sequencing of voodoo lily (Amorphophallus konjac): an approach to identify relevant genes involved in the synthesis of the hemicellulose glucomannan.

A Roche 454 cDNA deep sequencing experiment was performed on a developing corm of Amorphophallus konjac--also known as voodoo lily. The dominant storage polymer in the corm of this plant is the polysaccharide glucomannan, a hemicellulose known to exist in the cell walls of higher plants and a major component of plant biomass derived from softwoods. A total of 246 mega base pairs of sequence data was obtained from which 4,513 distinct contigs were assembled. Within this voodoo lily expressed sequence tag collection genes representing the carbohydrate related pathway of glucomannan biosynthesis were identified, including sucrose metabolism, nucleotide sugar conversion pathways for the formation of activated precursors as well as a putative glucomannan synthase. In vivo expression of the putative glucomannan synthase and subsequent in vitro activity assays unambiguously demonstrate that the enzyme has indeed glucomannan mannosyl- and glucosyl transferase activities. Based on the expressed sequence tag analysis hitherto unknown pathways for the synthesis of GDP-glucose, a necessary precursor for glucomannan biosynthesis, could be proposed. Moreover, the results highlight transcriptional bottlenecks for the synthesis of this hemicellulose.

Transgenic Amorphophallus konjac expressing synthesized acyl-homoserine lactonase (aiiA) gene exhibit enhanced resistance to soft rot disease.

Amorphophallus konjac is an important economic crop widely used in health products and biomaterials. However, this monocotyledonous plant's production is seriously restricted by soft rot disease. Some Bacillus thuringiensis strains generate an endocellular acyl homoserine lactonase (AiiA), which has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain transgenic A. konjac expressing AiiA protein and exhibiting resistance to soft rot. But till now, there is not any report about exogenous gene transformation in A. konjac. In this research, an Agrobacterium-mediated genetic transformation system was constructed. An aiiA gene was synthesized according to the codon usage in A. konjac. Embryogenic callus was infected with the A. tumefaciens strain EHA105 harboring the plant transformation plasmid pU1301 plus synthesized aiiA gene. After antibiotics screening, 34 plants were obtained. PCR analysis showed that positive amplified fragments were present in 21 out of these 34 lines. Southern blot analysis indicated that aiiA gene had integrated into the genome of A. konjac. Western blotting demonstrated that the target protein of interest was reactive with the antibody against AiiA. Further disease resistance detection revealed that all of the tested transgenic A. konjac lines exhibited high resistance to soft rot bacteria Erwinia carotovora subsp. Carotovora (Ecc) SCG1. The protocol is useful for the quality improvement of A. konjac through genetic transformation.