Application of Chloroplast Phylogenomics to Resolve Species Relationships Within the Plant Genus Amaranthus.

Amaranthus species are an emerging and promising nutritious traditional vegetable food source. Morphological plasticity and poorly resolved dendrograms have led to the need for well resolved species phylogenies. We hypothesized that whole chloroplast phylogenomics would result in more reliable differentiation between closely related amaranth species. The aims of the study were therefore: to construct a fully assembled, annotated chloroplast genome sequence of Amaranthus tricolor; to characterize Amaranthus accessions phylogenetically by comparing barcoding genes (matK, rbcL, ITS) with whole chloroplast sequencing; and to use whole chloroplast phylogenomics to resolve deeper phylogenetic relationships. We generated a complete A. tricolor chloroplast sequence of 150,027 bp. The three barcoding genes revealed poor inter- and intra-species resolution with low bootstrap support. Whole chloroplast phylogenomics of 59 Amaranthus accessions increased the number of parsimoniously informative sites from 92 to 481 compared to the barcoding genes, allowing improved separation of amaranth species. Our results support previous findings that two geographically independent domestication events of Amaranthus hybridus likely gave rise to several species within the Hybridus complex, namely Amaranthus dubius, Amaranthus quitensis, Amaranthus caudatus, Amaranthus cruentus and Amaranthus hypochondriacus. Poor resolution of species within the Hybridus complex supports the recent and ongoing domestication within the complex, and highlights the limitation of chloroplast data for resolving recent evolution. The weedy Amaranthus retroflexus and Amaranthus powellii was found to share a common ancestor with the Hybridus complex. Leafy amaranth, Amaranthus tricolor, Amaranthus blitum, Amaranthus viridis and Amaranthus graecizans formed a stable sister lineage to the aforementioned species across the phylogenetic trees. This study demonstrates the power of next-generation sequencing data and reference-based assemblies to resolve phylogenies, and also facilitated the identification of unknown Amaranthus accessions from a local genebank. The informative phylogeny of the Amaranthus genus will aid in selecting accessions for breeding advanced genotypes to satisfy global food demand.

Analysis of phylogenetic relationships and genome size evolution of the Amaranthus genus using GBS indicates the ancestors of an ancient crop.

The genus Amaranthus consists of 50-70 species and harbors several cultivated and weedy species of great economic importance. A small number of suitable traits, phenotypic plasticity, gene flow and hybridization made it difficult to establish the taxonomy and phylogeny of the whole genus despite various studies using molecular markers. We inferred the phylogeny of the Amaranthus genus using genotyping by sequencing (GBS) of 94 genebank accessions representing 35 Amaranthus species and measured their genome sizes. SNPs were called by de novo and reference-based methods, for which we used the distant sugarbeet Beta vulgaris and the closely related Amaranthus hypochondriacus as references. SNP counts and proportions of missing data differed between methods, but the resulting phylogenetic trees were highly similar. A distance-based neighbor joining tree of individual accessions and a species tree calculated with the multispecies coalescent supported a previous taxonomic classification into three subgenera although the subgenus A. Acnida consists of two highly differentiated clades. The analysis of the Hybridus complex within the A. Amaranthus subgenus revealed insights on the history of cultivated grain amaranths. The complex includes the three cultivated grain amaranths and their wild relatives and was well separated from other species in the subgenus. Wild and cultivated amaranth accessions did not differentiate according to the species assignment but clustered by their geographic origin from South and Central America. Different geographically separated populations of Amaranthus hybridus appear to be the common ancestors of the three cultivated grain species and A. quitensis might be additionally be involved in the evolution of South American grain amaranth (A. caudatus). We also measured genome sizes of the species and observed little variation with the exception of two lineages that showed evidence for a recent polyploidization. With the exception of two lineages, genome sizes are quite similar and indicate that polyploidization did not play a major role in the history of the genus.

Amaranthus species around Bangkok, Thailand and the release of allergenic proteins from their pollens.

BACKGROUND: Pollen of Amaranthus L., commonly known as careless weed or Phak-khom in Thai, has become one of the major causes of airway allergy in many countries including Thailand. Despite its recognized importance, there is no available information about which Amaranthus species are producing allergenic pollen more likely to affect Thai patients. Furthermore, only allergenic proteins released from pollen can cause allergy. OBJECTIVE: This study aimed to survey species of Amaranthus found in Bangkok and to investigate the impact of water on pollen damage and protein release from Amaranthus pollens. METHODS: Amaranthus inflorescences were sampled and identified using the identification key provided in "Flora of Thailand". Shed pollens were collected on day 1, 3 and 7 after shedding. Ten mg of pollens in distilled water including damaged pollens were counted under a light microscope. In addition, supernatant was analyzed for concentration of proteins released from pollens using Bradford's assay. Profiles of released proteins and IgE binding proteins were analyzed by SDS-PAGE and Western blot. RESULTS: Three species of Amaranthus-A. hybridus, A. spinosus, and A. viridis were identified. SDS-PAGE analysis revealed at least twelve protein bands with MW ranging from 10 to 80 kDa. Water caused more damage to pollens and higher amount of proteins were recovered from pollens 1 day after shedding than from 3- and 7-days old pollens. The results of Western blot showed IgE-bound proteins with MW ranging from 30 to 50 kDa. CONCLUSIONS: Water could damage pollens and time after shedding and significantly affected the amount of allergenic proteins released from pollen.

[Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus].

Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5'-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.

Chemical composition and ruminal nutrient degradability of fresh and ensiled amaranth forage.

BACKGROUND: Amaranth is a crop with potential as a source of forage for ruminants that has not been well characterized. A study was conducted to determine the impact of ensiling on the nutritional quality and ruminal degradability of forage from two amaranth cultivars adapted to North America (i.e. Plainsman and D136). In particular, quantification and some microscopic characterization of oxalate found in amaranth were performed as it is an antiquality compound of concern. RESULTS: There were limited interactions between cultivars and ensiling for most variables. Differences in chemical composition between amaranth cultivars were also limited. Ensiling reduced non-structural carbohydrate and true protein contents. The proportion of acid detergent protein was high in fresh and ensiled forages of both cultivars (average of 177 g kg(-1) crude protein). Total oxalate content averaged 30 and 25 g kg(-1) in fresh and ensiled forages respectively. Ensiling reduced soluble oxalate content. Crystals observed in amaranth were calcium oxalate druses found mostly in idioblast cells in leaf mesophyll and parenchyma of primary and secondary veins. In situ ruminal degradability data indicated that both fresh and ensiled amaranth are highly degradable in the rumen. CONCLUSION: This study confirms that amaranth is a suitable forage for ruminant animals. Its chemical composition is comparable, for most variables, to that of other commonly used forage species.

[Estimation of the genetic variability of amaranth collection (Amaranthus L.) with RAPD-analysis].

Genetic variability of amaranth collection was studied with RAPD-analysis. A high level of polymorphism of studied accessions was determined and amounted 85 % in mean. The unique bands characteristic only for the definite accessions, and 18 monomorphic loci proper for all amaranth accessions were detected with some primers. The genetic distances of Nei and Li were calculated. This index varied from 0,0009 to 0,0141. Cluster analysis was carried out. The amaranth accessions were classified into 2 clusters conformity with species belonging.

Germination and seed persistence of Amaranthus retroflexus and Amaranthus viridis: Two emerging weeds in Australian cotton and other summer crops.

Redroot pigweed (Amaranthus retroflexus L.) and slender amaranth (Amaranthus viridis L.) are becoming problematic weeds in summer crops, including cotton in Australia. A series of laboratory and field experiments were performed to examine the germination ecology, and seed persistence of two populations of A. retroflexus and A. viridis collected from the Goondiwindi and Gatton regions of Australia. Both populations of A. retroflexus and A. viridis behaved similarly to different environmental conditions. Initial dormancy was observed in fresh seeds of both species; however, germination reached maximum after an after-ripening period of two months at room temperature. Light was not a mandatory prerequisite for germination of both species as they could germinate under complete darkness. Although both species showed very low germination at the alternating day/night temperature of 15/5 C, these species germinated more than 40% between ranges of 25/15 C to 35/25 C. Maximum germination of A. retroflexus (93%) and A. viridis (86%) was observed at 35/25 C and 30/20, respectively. Germination of A. retroflexus and A. viridis was completely inhibited at osmotic potentials of -1.0 and -0.6 MPa, respectively. No germination was observed in both species at the sodium chloride concentration of 200 mM. A. retroflexus seedling emergence (87%) was maximum from the seeds buried at 1 cm while the maximum germination of A. viridis (72%) was observed at the soil surface. No seedling emergence was observed from a burial depth of 8 cm for both species. In both species, seed persistence increased with increasing burial depth. At 24 months after seed placement, seed depletion ranged from 75% (10 cm depth) to 94% (soil surface) for A. retroflexus, and ranged from 79% to 94% for A. viridis, respectively. Information gained from this study will contribute to an integrated control programs for A. retroflexus and A. viridis.

Amaranths.

Chance W. Riggins and Rita H. Mumm introduce the ancient amaranth genus, highlighting the ancient crop's controversial history and its contemporary use in improving food security.

Diversity in phenotypic and nutritional traits in vegetable amaranth (Amaranthus tricolor), a nutritionally underutilised crop.

BACKGROUND: Assessment of genetic diversity in a crop-breeding programme helps in the identification of diverse parental combinations to create segregating progenies with maximum genetic variability and facilitates introgression of desirable genes from diverse germplasm into the available genetic base. RESULTS: In the present study, 39 strains of vegetable amaranth (Amaranthus tricolor) were evaluated for eight morphological and seven quality traits for two test seasons to study the extent of genetic divergence among the strains. Multivariate analysis showed that the first four principal components contributed 67.55% of the variability. Cluster analysis grouped the strains into six clusters that displayed a wide range of diversity for most of the traits. CONCLUSION: Cluster analysis has proved to be an effective method in grouping strains that may facilitate effective management and utilisation in crop-breeding programmes. The diverse strains falling in different clusters were identified, which can be utilised in different hybridisation programmes to develop high-foliage-yielding varieties rich in nutritional components.

Species identification, phylogenetic analysis and detection of herbicide-resistant biotypes of Amaranthus based on ALS and ITS.

The taxonomically challenging genus Amaranthus (Family Amaranthaceae) includes important agricultural weed species that are being spread globally as grain contaminants. We hypothesized that the ALS gene will help resolve these taxonomic challenges and identify potentially harmful resistant biotypes. We obtained 153 samples representing 26 species from three Amaranthus subgenera and included in that incorporated ITS, ALS (domains C, A and D) and ALS (domains B and E) sequences. Subgen. Albersia was well supported, but subgen. Amaranthus and subgen. Acnida were not. Amaranthus tuberculatus, A. palmeri and A. spinosus all showed different genetic structuring. Unique SNPs in ALS offered reliable diagnostics for most of the sampled Amaranthus species. Resistant ALS alleles were detected in sixteen A. tuberculatus samples (55.2%), eight A. palmeri (27.6%) and one A. arenicola (100%). These involved Ala122Asn, Pro197Ser/Thr/Ile, Trp574Leu, and Ser653Thr/Asn/Lys substitutions, with Ala122Asn, Pro197Thr/Ile and Ser653Lys being reported in Amaranthus for the first time. Moreover, different resistant mutations were present in different A. tuberculatus populations. In conclusion, the ALS gene is important for species identification, investigating population genetic diversity and understanding resistant evolution within the genus Amaranthus.

Resistance to acetolactate synthase inhibitors is due to a W 574 to L amino acid substitution in the ALS gene of redroot pigweed and tall waterhemp.

Several Amaranthus spp. around the world have evolved resistance (and cross resistance) to various herbicide mechanisms of action. Populations of redroot pigweed (RRPW-R) and tall waterhemp (TW-R) in Mississippi, USA have been suspected to be resistant to one or more acetolactate synthase (ALS) inhibiting herbicides. Whole plant dose-response experiments with multiple ALS inhibitors, ALS enzyme assays with pyrithiobac, and molecular sequence analysis of ALS gene constructs were conducted to confirm and characterize the resistance profile and nature of the mechanism in the RRPW-R and TW-R populations. Two susceptible populations, RRPW-S and TW-S were included for comparison with RRPW-R and TW-R, correspondingly. The resistance index (R/S; the herbicide dose required to reduce plant growth by 50% of resistant population compared to the respective susceptible population) values of the RRPW-R population were 1476, 3500, and 900 for pyrithiobac, imazaquin, and trifloxysulfuron, respectively. The R/S values of the TW-R population for pyrithiobac, imazaquin, and trifloxysulfuron were 51, 950, and 2600, respectively. I50 values of RRPW-S and RRPW-R populations for pyrithiobac were 0.062 and 208.33 µM, indicating that the ALS enzyme of the RRPW-R population is 3360-fold more resistant to pyrithiobac than the RRPW-S population under our experimental conditions. The ALS enzyme of the TW-R population was 1214-fold resistant to pyrithiobac compared to the TW-S population, with the I50 values for pyrithiobac of ALS from TW-R and TW-S populations being 87.4 and 0.072 µM, correspondingly. Sequencing of the ALS gene identified a point mutation at position 574 of the ALS gene leading to substitution of tryptophan (W) residue with a leucine (L) residue in both RRPW-R and TW-R populations. Thus, the RRPW-R and TW-R populations are resistant to several ALS-inhibiting herbicides belonging to different chemical classes due to an altered target site, i.e., ALS. Resistance in Amaranthus spp. to commonly used ALS-inhibiting herbicides warrants an integrated weed management scheme incorporating chemical, mechanical, and cultural strategies by growers.

Evaluation of antioxidant activity of amaranth (Amaranthus cruentus) grain and by-products (flour, popping, cereal).

The objective of our study was evaluation antioxidant activity of Amaranthus cruentus grain and by-products (flour, cereals and popping). The evaluation was performed by FRAP, DPPH and ABTS methods. FRAP and ABTS assays gave comparable results, DPPH method gave lower values. Among by-products cereal had the highest activity as the least processed product. Additionally, antioxidant capacities of two cultivars of amaranth (varieties Aztek and Rawa) were compared and the influence of grain soaking on antioxidant properties was taken into account. It was found, that soaking decreased antioxidant activity of amaranth seed.

Germination Response of Four Alien Congeneric Amaranthus Species to Environmental Factors.

Seed germination is the key step for successful establishment, growth and further expansion of population especially for alien plants with annual life cycle. Traits like better adaptability and germination response were thought to be associated with plant invasion. However, there are not enough empirical studies correlating adaptation to environmental factors with germination response of alien invasive plants. In this study, we conducted congeneric comparisons of germination response to different environmental factors such as light, pH, NaCl, osmotic and soil burials among four alien amaranths that differ in invasiveness and have sympatric distribution in Jiangsu Province, China. The data were used to create three-parameter sigmoid and exponential decay models, which were fitted to cumulative germination and emergence curves. The results showed higher maximum Germination (Gmax), shorter time for 50% germination (G50) and the rapid slope (Grate) for Amaranthus blitum (low-invasive) and A. retroflexus (high-invasive) compare to intermediately invasive A. spinosus and A. viridis in all experimental regimes. It indicated that germination potential does not necessarily constitute a trait that can efficiently distinguish highly invasive and low invasive congeners in four Amaranthus species. However, it was showed that the germination performances of four amaranth species were more or less correlated with their worldwide distribution area. Therefore, the germination performance can be used as a reference indicator, but not an absolute trait for invasiveness. Our results also confirmed that superior germination performance in wide environmental conditions supplementing high seed productivity in highly invasive A. retroflexus might be one of the reasons for its prolific growth and wide distribution. These findings lay the foundation to develop more efficient weed management practice like deep burial of seeds by turning over soil and use of tillage agriculture to control these invasive weed species.

A quantitative assay for Amaranthus palmeri identification.

BACKGROUND: Amaranthus palmeri recently has been brought into the Midwestern USA as a contaminant in Conservation Reserve Program seeding mixes. Rapid species screening is required to mitigate the risk of continued species movement. RESULTS: Markers were developed for A. palmeri-specific nucleotide polymorphisms in the internal transcribed spacer of the ribosomal coding region. A quantitative polymerase chain reaction (qPCR) assay successfully identified A. palmeri from single-plant samples, simulated mixed-plant samples and seed mixtures. CONCLUSION: A qPCR assay for distinguishing A. palmeri from 12 other Amaranthus spp. was developed and validated. The assay can consistently detect a single A. palmeri seed when present in a pool of 100 total Amaranthus spp. seeds. © 2017 Society of Chemical Industry.

The Amaranth Genome: Genome, Transcriptome, and Physical Map Assembly.

Amaranth ( L.) is an emerging pseudocereal native to the New World that has garnered increased attention in recent years because of its nutritional quality, in particular its seed protein and more specifically its high levels of the essential amino acid lysine. It belongs to the Amaranthaceae family, is an ancient paleopolyploid that shows disomic inheritance (2 = 32), and has an estimated genome size of 466 Mb. Here we present a high-quality draft genome sequence of the grain amaranth. The genome assembly consisted of 377 Mb in 3518 scaffolds with an N of 371 kb. Repetitive element analysis predicted that 48% of the genome is comprised of repeat sequences, of which -like elements were the most commonly classified retrotransposon. A de novo transcriptome consisting of 66,370 contigs was assembled from eight different amaranth tissue and abiotic stress libraries. Annotation of the genome identified 23,059 protein-coding genes. Seven grain amaranths (, , and ) and their putative progenitor () were resequenced. A single nucleotide polymorphism (SNP) phylogeny supported the classification of as the progenitor species of the grain amaranths. Lastly, we generated a de novo physical map for using the BioNano Genomics' Genome Mapping platform. The physical map spanned 340 Mb and a hybrid assembly using the BioNano physical maps nearly doubled the N of the assembly to 697 kb. Moreover, we analyzed synteny between amaranth and sugar beet ( L.) and estimated, using analysis, the age of the most recent polyploidization event in amaranth.

A comparison of the bioactivity and phytochemical profile of three different cultivars of globe amaranth: red, white, and pink.

The phytochemical profiles and bioactivities of red, white and pink globe amaranth (Gomphrena haageana K., Gomphrena globosa var. albiflora and Gomphrena sp., respectively), much less studied than the purple species (G. globosa L.), were compared. The chemical characterization of the samples included the analysis of macronutrients and individual profiles of sugars, organic acids, fatty acids, tocopherols, and phenolic compounds. Their bioactivity was evaluated by determining the antioxidant and anti-inflammatory activities; the absence of cytotoxicity was also determined. Red and pink samples showed the highest sugar content. Otherwise, the white sample gave the highest level of organic acids, and together with the pink one showed the highest tocopherol and PUFA levels. Quercetin-3-O-rutinoside was the major flavonol in white and pink samples, whereas a tetrahydroxy-methylenedioxyflavone was the major compound in the red variety, which revealed a different phenolic profile. The pink globe amaranth hydromethanolic extract revealed the highest antioxidant activity, followed by those of red and white samples. The anti-inflammatory activity was more relevant in red and pink varieties. None of the samples presented toxicity in liver cells. Overall, these samples can be used in bioactive formulations against inflammatory processes and in free radical production.

[Direct identification of Celosia argentea from its confused species by FTIR].

OBJECTIVE: To directly and accurately identify Celosia argentea L. from its confused species. METHODS: Fourier Transform Infrared Spectrum. RESULT: Obvious characteristics for the identification in FTIR were revealed, which can be used to identify Celosia argentea L. and its confused species, such as Celosia cristata L., Amoranthus retroflexus. A. tricolor L. and A. patulus Bertol. CONCLUSION: Celosia argentea L. and its confused species were identified by FTIR directly, fastly and accurately.

Enhancement of Cd phytoextraction by two Amaranthus species with endophytic Rahnella sp. JN27.

Microbe-assisted phytoextraction shows a potential for the remediation of metal-contaminated soils. The aim of this study was to isolate, characterize, and evaluate the potential of endophytic bacteria in improving plant growth and metal uptake by Cd-hyperaccumulators-Amaranthus hypochondriacus and Amaranthus mangostanus. An endophytic bacterial strain JN27 isolated from roots of Zea mays displayed high tolerance and mobilization to Cd, and was identified as Rahnella sp. based on 16S rDNA sequencing. The strain also exhibited multiple plant growth beneficial features including the production of indole-3-acetic acid, siderophore, 1-aminocyclopropane-1-carboxylic acid deaminase and solubilization of insoluble phosphate. Subsequently, a pot trial was performed to elucidate the effects of inoculation with JN27 on plant growth and Cd uptake by A. hypochondriacus, A. mangostanus, Solanum nigrum and Z. mays grown in soils with different levels of Cd (25, 50, 100 mg Cd kg(-1)). The results revealed that inoculation with JN27 significantly increased the biomasses of all the tested plants and the Cd concentrations of all the tested plants except Z. mays in both above-ground and root tissues. Moreover, strain JN27 could successfully re-colonized in rhizosphere soils of all the tested plants and root interior of A. hypochondriacus and Z. mays. The present results indicated that the symbiont of A. hypochondriacus (or A. mangostanus) and strain JN27 can effectively improve the Cd uptake by plants and would be a new strategy in microbe-assisted phytoextraction for metal-contaminated soils.

Glyphosate-resistant and glyphosate-susceptible Palmer amaranth (Amaranthus palmeri S. Wats.): hyperspectral reflectance properties of plants and potential for classification.

BACKGROUND: Palmer amaranth (Amaranthus palmeri S. Wats.) is a troublesome agronomic weed in the southern United States, and several populations have evolved resistance to glyphosate. This paper reports on spectral signatures of glyphosate-resistant (GR) and glyphosate-sensitive (GS) plants, and explores the potential of using hyperspectral sensors to distinguish GR from GS plants. RESULTS: GS plants have higher light reflectance in the visible region and lower light reflectance in the infrared region of the spectrum compared with GR plants. The normalized reflectance spectrum of the GR and GS plants had best separability in the 400-500 nm, 650-690 nm, 730-740 nm and 800-900 nm spectral regions. Fourteen wavebands from within or near these four spectral regions provided a classification of unknown set of GR and GS plants, with a validation accuracy of 94% for greenhouse-grown plants and 96% for field-grown plants. CONCLUSIONS: GR and GS Palmer amaranth plants have unique hyperspectral reflectance properties, and there are four distinct regions of the spectrum that can separate the GR from GS plants. These results demonstrate that hyperspectral imaging has potential application to distinguish GR from GS Palmer amaranth plants (without a glyphosate treatment), with future implications for glyphosate resistance management. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Rutin and total quercetin content in amaranth (Amaranthus spp.).

The aim of the study was to confirm the presence of rutin, one of the most common quercetin glycosides, and other quercetin derivatives in plants of genus Amaranthus, to investigate the influence of the species and variety on rutin distribution in the plant and content changes during growing season. The rutin content was determined by micellar electrokinetic capillary chromatography in individual plant parts at the beginning of the growth, at the flowering stage and at the maturity stage of five Amaranthus species. The total quercetin content was determined by micellar electrokinetic capillary chromatography too. The rutin content in amaranth ranged from 0.08 (in seeds) to 24.5 g/kg dry matter (in leaves). Comparison of the determined total quercetin content and the calculated content of quercetin released from rutin did not prove important presence of quercetin or other quercetin derivatives than rutin. Only amaranth leaves sampled at the maturity stage probably contained quercetin or quercetin derivatives. Significant differences in the rutin content were established among species and as well varieties. Amaranthus hybrid and A. cruentus were the best sources of rutin.