Ying Diao

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

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.

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 and characterisation of EST-SSR markers by transcriptome sequencing in taro ( Colocasia esculenta (L.) Schoot)

Taro (Colocasia esculenta) is an important crop with a long history of cultivation. In this study 5278 SSRs were identified in taro transcriptome data. A total of 2858 primer pairs were designed for marker development. 100 primers were randomly selected and synthesized. Among them, 72 primer pairs were successfully amplified and 62 were polymorphic in taro accessions. The number of alleles ranged from 2 to 14 for each different polymorphic locus and the polymorphism information content valued ranged from 0.01 to 0.82. The phylogenetic tree was also constructed to analyse the genetic diversity in 68 taro accessions. The large number of taro SSR markers developed in the present study will be useful in the researches of genetic diversity, germplasm characterization and molecular breeding etc.

Nuclear DNA content in Miscanthus sp. and the geographical variation pattern in Miscanthus lutarioriparius

The genome sizes of five Miscanthus species, including 79 accessions of M. lutarioriparius, 8 of M. floridulus, 6 of M. sacchariflorus, 7 of M. sinensis, and 4 of M. × giganteus were examined using flow cytometry. The overall average nuclear DNA content were 4.256 ± 0.6 pg/2C in M. lutarioriparius, 5.175 ± 0.3 pg/2C in M. floridulus, 3.956 ± 0.2 pg/2C in M. sacchariflorus, 5.272 ± 0.2 pg/2C in M. sinensis, and 6.932 ± 0.1 pg/2C in M. × giganteus. Interspecific variation was found at the diploid level, suggesting that DNA content might be a parameter that can be used to differentiate the species. Tetraploid populations were found in M. lutarioriparius, M. sacchariflorus, and M. sinensis, and their DNA content were 8.34 ± 1.2, 8.52, and 8.355 pg, respectively. The association between the DNA content of M. lutarioriparius, collected from representative ranges across the Yangtze River, and its geographic distribution was statistically analyzed. A consistent pattern of DNA content variation in 79 M. lutarioriparius accessions across its entire geographic range was found in this study. Along the Yangtze River, the DNA content of M. lutarioriparius tended to increase from the upstream to the downstream areas, and almost all tetraploids gathered in the upstream area extended to coastal regions.

Pedigree-based genome re-sequencing reveals genetic variation patterns of elite backbone varieties during modern rice improvement

Rice breeding has achieved great productivity improvements by semi-dwarf varieties and hybrid vigour. Due to poor understanding of genetic basis of elite backbone varieties, the continuous increasing in rice yield still faces great challenges. Here, 52 elite rice varieties from three historical representative pedigrees were re-sequenced with 10.1× depth on average, and ~6.5 million single nucleotide polymorphisms (SNPs) were obtained. We identified thousands of low-diversity genomic regions and 0-diversity genes during breeding. Using pedigree information, we also traced SNP transmission patterns and observed breeding signatures in pedigree. These regions included the larger number of key well-known functional genes. Besides, 35 regions spanning 0.16% of the rice gnome had been differentially selected between conventional and restorer pedigrees. These genes identified here will be useful to the further pedigree breeding. Our study provides insights into the genetic basis of backbone varieties and will have immediate implications for performing genome-wide breeding by design.

High embryogenic ability and regeneration from floral axis of Amorphophallus konjac (Araceae)

Abstract 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.

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.

Transcriptomics and proteomics reveal genetic and biological basis of superior biomass crop Miscanthus

Miscanthus is a rhizomatous C4 grass which is considered as potential high-yielding energy crop with the low-nutrient requirements, high water-use efficiency, and capability of C mitigation. To better understand the genetic basis, an integrative analysis of the transcriptome and proteome was performed to identify important genes and pathways involved in Miscanthus leaves. At the transcript level, 64,663 transcripts in M. lutarioriparius, 97,043 in M. sacchariflorus, 97,043 in M. sinensis, 67,323 in M. floridulus and 70,021 in M. × giganteus were detected by an RNA sequencing approach. At the protein level, 1964 peptide-represented proteins were identified and 1933 proteins differed by 1.5-fold or more in their relative abundance, as indicated by iTRAQ (isobaric tags for relative and absolute quantitation) analysis. Phylogenies were constructed from the nearly taxa of Miscanthus. A large number of genes closely related to biomass production were found. And SSR markers and their corresponding primers were derived from Miscanthus transcripts and 90% of them were successfully detected by PCR amplification among Miacanthus species. These similarities and variations on the transcriptional and proteomic level between Miscanthus species will serve as a resource for research in Miscanthus and other lignocellulose crops.

The cutting effect of male flower on the size of the fruit cob, the size of the fruit and seeds in Porang (Amorphophallus muelleri Blume)

The formation of fruits and seeds depends on pollination and fertilization. For the fruit itself, its formation and final size are closely correlated with the gibberellin content. It is known that pollen is one source of gibberellins. Porang is a member of the genus Amorphophallus, which has a compound fruit arranged on a cob and monoecious. Each fruit may contain a single seed or multiple seeds. The purpose of this study was to determine the effect of male flower removal on the size of the fruit cob, the number of fruit per ear, the seed number per ear, the fruit size and the proportion of fruit that contains single or multiple seeds. Removing male flowers is done by cutting the cluster of male flowers when the flower is very young and still wrapped with a sheath. The resulting data were analyzed using an unpaired t-test. Analysis of the results shows that the diameter and length of the cob of a cut-male flower are lower than the normal flower. The number of fruit or seed numbers per ear of the cut-male flower is lower than the normal flower. However, the length and diameter of the fruit of the cut-male flower is higher than the normal flower. The normal flower has higher numbers of individual fruit with single seeds than cut-male flowers, and cut-male flowers have higher incidences of fruit containing more than one seed (multiple seeds). The maximum seed number of individual fruit of normal flowers and cut-male flowers is 4 and 7 seeds respectively. In categories of fruit containing multiple seeds, the individual fruit containing 2 seeds has the highest proportion of cut-male flowers.

In vitro plant regeneration of lotus (Nelumbo nucifera)

Abstract In this study, an efficient and reproducible plant regeneration system for lotus (Nelumbo nucifera Gaertn.) was established using shoot apical meristems from the buds and one-week-old aseptically germinated embryos as explants. Multiple shoot clumps were induced on Murashige and Skoog (MS) basal medium supplemented with various combinations of N6-Benzylaminopurine (6-BA) and α-Naphthalene acetic acid (NAA). The maximum response was obtained with 2.22 μM 6-BA, and produced 21.33 shoots per explant after four weeks. After five subcultures, multiple shoot clumps were transferred to MS basal medium supplemented with various combinations of 3-Indolebutyric acid (IBA), NAA and sucrose for root induction. After four weeks, plantlets with well-developed roots were achieved on MS basal medium supplemented with 0.54 μM NAA and 30 g/L sucrose with 100% rooting rate. The successfully acclimated plantlets were transferred to pots with the addition of 2 g/L KMnO4 into the soils, and finally fertile plants with much bigger leaves were obtained in the greenhouse. The survival rate was 97.33%.