Phylogenomics and integrative taxonomy reveal two new species of Amana (Liliaceae).

Until now the genus Amana (Liliaceae), known as 'East Asian tulips', has contained just seven species. In this study, a phylogenomic and integrative taxonomic approach was used to reveal two new species, Amana nanyueensis from Central China and A. tianmuensis from East China. A. nanyueensis resembles Amana edulis in possessing a densely villous-woolly bulb tunic and two opposite bracts, but differs in its leaves and anthers. Amana tianmuensis resembles Amana erythronioides in possessing three verticillate bracts and yellow anthers, but differs in aspects of its leaves and bulbs. These four species are clearly separated from each other in principal components analysis based on morphology. Phylogenomic analyses based on plastid CDS further support the species delimitation of A. nanyueensis and A. tianmuensis and suggests they are closely related to A. edulis. Cytological analysis shows that A. nanyueensis and A. tianmuensis are both diploid (2n = 2x = 24), different from A. edulis, which is either diploid (northern populations) or tetraploid (southern populations, 2n = 4x = 48). The pollen morphology of A. nanyueensis is similar to other Amana species (single-groove germination aperture), but A. tianmuensis is quite different because of the presence of a sulcus membrane, which creates the illusion of double grooves. Ecological niche modelling also revealed a niche differentiation between A. edulis, A. nanyueensis and A. tianmuensis.

Tuliposides H-J and Bioactive Components from the Bulb of Amana edulis.

Three new tuliposides H-J (1-3) and 11 known compounds were obtained from the methanolic extracts of the bulbs of Amana edulis for the first time. Their structures were elucidated by NMR, MS, and IR spectroscopic data, optical rotation, and Mosher's method. The melanogenesis properties of all the isolates were evaluated in B16 melanoma cells. Consequently, tributyl citrate (9) had anti-melanogenesis activity but was cytotoxic toward B16. (+)-Pyroglutamic acid (4), (+)-butyl 5-oxopyrrolidine-2-carboxylate (6), (-)-3-hydroxy-2-methylbutyrolactone (10), and 5-(hydroxymethyl)furfural (12) had increased melanin productions and tyrosinase activities. Those active components could be further studied as the candidates against melanoma and vitiligo for skin diseases or whitening/hypopigmentation for hair.

[Cloning and expression of AeMYB4 gene related to stolon development of Amana edulis].

Stolon is an important organ for reproduction and regeneration of Amana edulis. Previous analysis of transcriptome showed that MYB was one of the most active transcription factor families during the development of A. edulis stolon. In order to study the possible role of MYB transcription factors in stolon development, the authors screened out an up-regulated MYB gene named AeMYB4 was by analyzing the expression profile of MYB transcription factors. In the present study, sequence analysis demonstrated that AeMYB4 contained an open reading frame of 756 bp encoding 251 amino acids, and domain analysis revealed that the predicted amino acids sequence contained two highly conserved SANT domains and binding sites for cold stress factor CBF. By multiple sequence alignment and phylogenetic analysis, it is indicated that AeMYB4 clustered with AtMYB15 from Arabidopsis thaliana, belonging to subgroup S2 of R2 R3-MYB. And most of the transcription factors in this subfamily are related to low temperature stress. The GFP-AeMYB4 fusion protein expression vector for subcellular localization was constructed and transferred into Agrobacterium tumefaciens to infect the leaves of Nicotiana benthamiana, and the results showed the protein was located in the nucleus. To investigate the transcriptional activation, the constructed pGBKT7-AeMYB4 fusion expression vector was transferred into Y2 H Gold yeast cells, which proved that AeMYB4 was a transcription activator with strong transcriptional activity. Real-time quantitative PCR was used to detect the expression of AeMYB4 gene in three different development stages of stolon and in leaves, flowers, and bulbs of A. edulis, which indicated that AeMYB4 transcription factor was tissue-specific in expression, mainly in the stolon development stage, and that the expression was the most active in the middle stage of stolon development, suggesting that AeMYB4 gene may play an important role in stolon development. This study contributes to the further research on the function of AeMYB4 transcription factor in stolon development of A. edulis.

[Selection and validation of reference genes for quantitative Real-time PCR analysis in Amana edulis].

Amana edulis is a traditional Chinese medicinal plant with low propagation coefficient. In recent years, the increasing demands of A. edulis lead to a shortage of its wild resources. In order to analyze the expression of related functional genes in A. edulis, the selection of suitable internal reference genes is crucial to improve the accuracy of experimental results. Eight genes(ACT, TUA, CYP, GAPDH, UBQ, UBI, EF1a, UBC)were chosen as candidate reference genes based on the RNA-Seq. Real-time fluorescence quantitative technique was used to detect the expression level of candidate internal reference genes in different organs(bulb, leaf, flo-wer) and stolons at different development stages of A. edulis. Then GeNorm, NormFinder, BestKeeper softwares and RefFinder website were used for a comprehensive analysis of the expression stability of the candidate genes.The results showed that among the 8 candidate reference genes, the variation range of Ct value of UBC was the smallest, and the expression level was stable, which was suitable for an reference gene. GeNorm and NormFinder software analysis showed that UBC and UBI were the optimal reference genes. BestKeeper analysis showed that CYP and UBC expression were relatively stable. Comprehensive evaluation of RefFinder website showed that UBC and UBI were the most stable genes, and ACT displayed the lowest stability in all software evaluation, indicating UBC and UBI were suitable for reference genes. Additionally, the most stable UBC, UBI and the most unstable ACT were used as internal reference genes to detect the expression of GBSS gene in A. edulis, and expression pattern of GBSS gene was the same under the calibration of UBC and UBI. The expression data of GBSS gene confirmed that UBC and UBI genes were reliable for A. edulis qRT-PCR as internal reference genes. The results would benefit future studies on related gene expression of A. edulis.

Cloning and expression of AeMYB4 gene related to stolon development of Amana edulis / 中国中药杂志

Stolon is an important organ for reproduction and regeneration of Amana edulis. Previous analysis of transcriptome showed that MYB was one of the most active transcription factor families during the development of A. edulis stolon. In order to study the possible role of MYB transcription factors in stolon development, the authors screened out an up-regulated MYB gene named AeMYB4 was by analyzing the expression profile of MYB transcription factors. In the present study, sequence analysis demonstrated that AeMYB4 contained an open reading frame of 756 bp encoding 251 amino acids, and domain analysis revealed that the predicted amino acids sequence contained two highly conserved SANT domains and binding sites for cold stress factor CBF. By multiple sequence alignment and phylogenetic analysis, it is indicated that AeMYB4 clustered with AtMYB15 from Arabidopsis thaliana, belonging to subgroup S2 of R2 R3-MYB. And most of the transcription factors in this subfamily are related to low temperature stress. The GFP-AeMYB4 fusion protein expression vector for subcellular localization was constructed and transferred into Agrobacterium tumefaciens to infect the leaves of Nicotiana benthamiana, and the results showed the protein was located in the nucleus. To investigate the transcriptional activation, the constructed pGBKT7-AeMYB4 fusion expression vector was transferred into Y2 H Gold yeast cells, which proved that AeMYB4 was a transcription activator with strong transcriptional activity. Real-time quantitative PCR was used to detect the expression of AeMYB4 gene in three different development stages of stolon and in leaves, flowers, and bulbs of A. edulis, which indicated that AeMYB4 transcription factor was tissue-specific in expression, mainly in the stolon development stage, and that the expression was the most active in the middle stage of stolon development, suggesting that AeMYB4 gene may play an important role in stolon development. This study contributes to the further research on the function of AeMYB4 transcription factor in stolon development of A. edulis.

Selection and validation of reference genes for quantitative Real-time PCR analysis in Amana edulis / 中国中药杂志

Amana edulis is a traditional Chinese medicinal plant with low propagation coefficient. In recent years, the increasing demands of A. edulis lead to a shortage of its wild resources. In order to analyze the expression of related functional genes in A. edulis, the selection of suitable internal reference genes is crucial to improve the accuracy of experimental results. Eight genes(ACT, TUA, CYP, GAPDH, UBQ, UBI, EF1a, UBC)were chosen as candidate reference genes based on the RNA-Seq. Real-time fluorescence quantitative technique was used to detect the expression level of candidate internal reference genes in different organs(bulb, leaf, flo-wer) and stolons at different development stages of A. edulis. Then GeNorm, NormFinder, BestKeeper softwares and RefFinder website were used for a comprehensive analysis of the expression stability of the candidate genes.The results showed that among the 8 candidate reference genes, the variation range of Ct value of UBC was the smallest, and the expression level was stable, which was suitable for an reference gene. GeNorm and NormFinder software analysis showed that UBC and UBI were the optimal reference genes. BestKeeper analysis showed that CYP and UBC expression were relatively stable. Comprehensive evaluation of RefFinder website showed that UBC and UBI were the most stable genes, and ACT displayed the lowest stability in all software evaluation, indicating UBC and UBI were suitable for reference genes. Additionally, the most stable UBC, UBI and the most unstable ACT were used as internal reference genes to detect the expression of GBSS gene in A. edulis, and expression pattern of GBSS gene was the same under the calibration of UBC and UBI. The expression data of GBSS gene confirmed that UBC and UBI genes were reliable for A. edulis qRT-PCR as internal reference genes. The results would benefit future studies on related gene expression of A. edulis.

The complete chloroplast genome of Amana baohuaensis (Liliaceae).

Amana baohuaensis is a new species that was just named in 2019. Here, we obtained the complete chloroplast (cp) genome of A. baohuaensis using the Illumina paired-end sequencing technology. The cp genome has a typical quadripartite structure with 150,757 bp in length, containing a large single-copy (LSC) region of 81,757 bp, a small single-copy (SSC) region of 16,962 bp, and two inverted repeat (IR) regions of 26,019 bp. The total GC content is 36.73%, of which, the GC content of LSC, SSC and IR regions are 34.63%, 30.11% and 42.20%, respectively. The cp genome of A. baohuaensis contains 111 unique genes, including 78 protein-coding genes, 29 tRNA genes, and four rRNA genes. The Maximum Parsimony (MP) phylogenetic analysis suggested that A. baohuaensis had the closest relationship with A. wanzhensis, and all Amana species grouped together with high bootstrap support.

Effects of sulfated, phosphorylated and carboxymethylated modifications on the antioxidant activities in-vitro of polysaccharides sequentially extracted from Amana edulis.

In this study, four sequentially extracted polysaccharides (AEPs) from Amana edulis were modified by sulphation, phosphorylation, and carboxylation modifications (S-AEPs, P-AEPs, C-AEPs), and compared for their anti-oxidant activities. After modification, sugar and protein contents were decreased and uronic acid content was increased in comparison to native AEPs. UV absorption showed similar maximum absorption peaks of modified derivatives which indicated their homogeneous nature. FTIR spectra confirmed the conversion of hydroxyl groups to OS, COO, and POH bonds, respectively. The phosphorylated derivatives (P-AEPs) displayed the highest DPPH, hydroxyl radical, and ferrous ions radical scavenging abilities. Sulfated polysaccharides (S-AEPs) were observed with high reducing ability. The C-AEPs maintained the stable antioxidant properties after carboxylation modification. Our results indicated that the chemical modification of different polysaccharide components has significantly affected their antioxidant potential for their use in food industry and human health.

The rheological properties and emulsifying behavior of polysaccharides sequentially extracted from Amana edulis.

The rheological properties and emulsifying behavior of four polysaccharides (HBSS, CHSS, DASS, and CASS) sequentially extracted from Amana edulis (AEPs) were investigated under various concentrations, temperatures, pH levels, and ionic strengths. The apparent viscosity of the four AEPs solutions at 1% (w/w) concentration were found to be CHSS > DASS > HBSS > CASS. When the AEPs were heated to 100 οC, they showed lower colloidal viscosity, whereas after refrigeration and chilling, higher apparent viscosities were observed. The apparent viscosity of four AEPs at pH 10 or pH 4 was lower than that at pH 7. The apparent viscosity increased at a lower sodium ion concentration and then declined with an increase in ion concentration. The storage modulus (G') and loss modulus (G″) increased with an increase in oscillation frequency. The emulsifying activity and stability were enhanced as the concentration of the four AEPs increased. The emulsifying activity and stability of the AEPs were steady within the pH range of 2-10 and NaCl concentration range of 0-0.4 mol/L. Our results implied that these polysaccharides can be utilized as a novel hydrocolloid source for natural thickeners in the food industry.

Physicochemical and antioxidant potential of polysaccharides sequentially extracted from Amana edulis.

Amana edulis polysaccharides (AEPs) specifically HBSS, CHSS, DASS, and CASS were sequentially extracted with four different solvents. The present study characterized the AEPs with particular focus on their physicochemical and anti-oxidant based functional properties. Initially, monosaccharide analysis revealed arabinose (31.7%, 32.5%, 36.5%) as the main sugar in HBSS, CHSS, and DASS whereas, galactose (31.4%) in CASS besides their respective molecular weights of 6.29 × 102, 1.5 × 102, 8.1 × 102, and 2.6 × 103 kD. HBSS showed the maximum solubility, while, CASS was observed for higher foam capacity and foam stability. Among all the fractions, DASS was observed with higher thermal stability. HBSS showed the highest ABTS+ scavenging activity. HBSS and CASS had higher DPPH and OH- scavenging activities. DASS depicted the highest chelation and reducing ability. To summarize, these polysaccharides fractions may be further utilized for their enormous prospective in functional foods preparation.

Comparative Genomics and Phylogenomics of East Asian Tulips (Amana, Liliaceae).

The genus Amana Honda (Liliaceae), when it is treated as separate from Tulipa, comprises six perennial herbaceous species that are restricted to China, Japan and the Korean Peninsula. Although all six Amana species have important medicinal and horticultural uses, studies focused on species identification and molecular phylogenetics are few. Here we report the nucleotide sequences of six complete Amana chloroplast (cp) genomes. The cp genomes of Amana range from 150,613 bp to 151,136 bp in length, all including a pair of inverted repeats (25,629-25,859 bp) separated by the large single-copy (81,482-82,218 bp) and small single-copy (17,366-17,465 bp) regions. Each cp genome equivalently contains 112 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 78 protein coding genes. Gene content, gene order, AT content, and IR/SC boundary structure are nearly identical among all Amana cp genomes. However, the relative contraction and expansion of the IR/SC borders among the six Amana cp genomes results in length variation among them. Simple sequence repeat (SSR) analyses of these Amana cp genomes indicate that the richest SSRs are A/T mononucleotides. The number of repeats among the six Amana species varies from 54 (A. anhuiensis) to 69 (Amana kuocangshanica) with palindromic (28-35) and forward repeats (23-30) as the most common types. Phylogenomic analyses based on these complete cp genomes and 74 common protein-coding genes strongly support the monophyly of the genus, and a sister relationship between Amana and Erythronium, rather than a shared common ancestor with Tulipa. Nine DNA markers (rps15-ycf1, accD-psaI, petA-psbJ, rpl32-trnL, atpH-atpI, petD-rpoA, trnS-trnG, psbM-trnD, and ycf4-cemA) with number of variable sites greater than 0.9% were identified, and these may be useful for future population genetic and phylogeographic studies of Amana species.

New data on the ecology and geographic distribution of Saguinus inustus Schwarz, 1951 (Primates, Callitrichidae) / Novos dados sobre a ecologia e distribuição geográfica de Saguinus inustus Schwarz, 1951 (Primates, Callitrichidae)

Saguinus inustus (Schwarz, 1951) is one of the neotropical primates least studied. The distribution of the species ranges from the north of the Solimões River, between the Negro and Japurá Rivers in Brazil, and Guayabero-Guaviare Rivers in Colombia. Nevertheless, due to the low number of specimens collected from the lower Japurá and lower Negro Rivers areas, the geographic distribution is so far poorly delineated. In this study, field data was composed of sightings and the collection of specimens during a survey of mammal diversity in the Amana Sustainable Development Reserve (ASDR). For this survey, two 40-day expeditions were carried out in 2004. The first one occurred during the flooded season in June and July, and the second was during the peak of the dry season in October. Direct sightings were made through hiking along transects, navigation along water channels with a 30-hp speedboat, and gliding along flooded trails in the forest. New records of S. inustus were made in 11 different localities in ASDR. The study has confirmed the presence of the species in the Amanã area, carrying out the first records of the species in flooded forest habitats.

Saguinus inustus (Schwarz, 1951) é um dos primatas neotropicais menos estudados. No Brasil, a espécie ocorre ao norte do Rio Amazonas entre os Rios Negro e Japurá (Caquetá), e Guayabero-Guaviare na Colômbia. No entanto, devido ao pequeno número de espécimes coletados entre o baixo Japurá e o baixo Negro a distribuição geográfica é mal delineada. No presente estudo, os dados de campo são compostos por observações e coletas realizadas durante o levantamento da diversidade de mamíferos da Reserva de Desenvolvimento Sustentável Amanã (RDSA). Para este levantamento, duas expedições de 40 dias foram realizadas em 2004. A primeira ocorreu durante a estação da cheia em junho e julho, e a segunda durante o pico da estação seca em outubro. Observações diretas foram feitas através de deslocamentos a pé em transecções, de lancha 30 hp ao longo de cursos d'água, e de canoas durante a cheia na área de várzea. Novos registros de S. inustus foram realizados em 11 diferentes localidades na RDSA. Este estudo confirmou a presença da espécie em Amanã, realizando os primeiros registros da espécie em florestas alagadas.