Selection and Evaluation of Reference Genes for RT-qPCR Analysis in Amorphophallus Konjac Based on Transcriptome Data.

Combined with the Konjac transcriptome database of our laboratory and internal reference genes commonly used in plants, the eight candidate internal reference genes were screened and detected. They are the 25S ribosomal RNA gene (25S rRNA), 18S ribosomal RNA gene (18S rRNA), actin gene (ACT), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), ubiquitin gene (UBQ), ß-tubulin gene (ß-TUB), eukaryotic elongation factor 1-αgene(eEF-1α), and eukaryotic translation initiation factor 4α-1 gene (eIF-4α). The results of GeNorm, Normfinder, and BestKeeper were analyzed comprehensively. The data showed that the expression levels of 25S rRNA, 18S rRNA, and ACT at the reproductive periods, eEF-1α and eIF-4α at the nutritional periods, and eEF-1α, UBQ, and ACT at different leaf developmental periods were stable. These identified and stable internal reference genes will provide the basis for the subsequent molecular biology-related studies of Konjac.

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.

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.

Mimicking Livor Mortis: a Well-Known but Unsubstantiated Color Profile in Sapromyiophily.

By emitting strong scents resembling rotting organic materials suitable for oviposition and/or foraging of flies, sapromyiophilous flowers mimic the substrates that attract flies as pollinators. It has been suggested that the wide range of volatile organic compounds emitted by this deceptive pollination system reflects the trophic preferences of flies to different types of substrate, including herbivore and carnivore feces, carrion, and fruiting bodies of fungi. Previous studies suggest that floral scents play a particularly important role in sapromyiophily. However, few studies on the relative importance of floral color or synergy between visual and olfactory cues in sapromyiophily have been substantiated. In this study, we analyzed fetid floral odor, floral pigment composition, and reflectance of an Amorphophallus konjac C. Koch inflorescence, and we conducted bioassays with different visual and/or olfactory cues to explore an unsubstantiated color profile in sapromyiophily: mimicking livor mortis. Our analysis showed A. konjac can emit oligosulphide-dominated volatile blends similar to those emitted by carrion. Necrophagous flies cannot discriminate between the color of an inflorescence, livor mortis, and floral pigments. We concluded that mimicking livor mortis may represent a common tactic of pollinator attraction in "carrion flower" systems within angiosperms.