BackgroundAmorphophallus 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.ResultscDNAs 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.ConclusionA 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.
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.
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.
Amorphophallus konjac (Araceae) a perennial herb, it has high medicinal and industrial value. In this study, a simple and efficient system for direct somatic embryogenesis and plantlet regeneration of Amorphophallus konjac was developed. The floral axis was used as the experimental material. The primary callus, developed from the floral axis grown on Murashige and Skoog (MS) medium supplemented with different hormone combination at different concentrations. The highest rate of embryogenic callus formation was observed on the MS medium containing 9.04 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 5.37 µM naphthalene acetic acid (NAA). The maximum induction rate was 79.8%, and the embryogenic calli were able to subculture on a medium containing similar hormone combination for over 1 year. The calli were also placed on different media for regeneration and it produced complete plants with shoots and root systems simultaneously. The highest differentiation rate of the embryogenic calli grown on differentiation medium supplemented with 8.88 µM 6-benzylaminopurine (6-BA) and 5.37 µM NAA was 95.6%. Flow cytometry analysis showed no ploidy variation in all the regenerate plantlets.
Twenty-three isolates of soft rot bacteria from konjac corms were examined for their diversity using 16S rDNAs and AFLP technology. Both methods clustered two groups, dependent on their biotype characterization of Pectobacterium carotovora subsp. carotovora (P.c.c) and Pectobacterium chrysanthemi (P.ch), respectively. Of all isolates, 17 (73.9%) belonged to P. ch, indicated as the main pathogenic bacteria of konjac producing areas in China. The genetic variation among isolates from the same biotype was also rich, not consistent with the distances of the geographic sources.
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