The systematics of grasses has advanced through applications of plastome phylogenomics, although studies have been largely limited to subfamilies or other subgroups of Poaceae Here we present a plastome phylogenomic analysis of 250 complete plastomes (179 genera) sampled from 44 of the 52 tribes of Poaceae Plastome sequences were determined from high throughput sequencing libraries and the assemblies represent over 287 Mbases of sequence data Phylogenetic signal was characterized in 14 partitions, including (1) complete plastomes; (2) protein coding regions; (3) noncoding regions; and (4) three loci commonly used in single and multi-gene studies of grasses Each of the four main partitions was further refined, alternatively including or excluding positively selected codons and also the gaps introduced by the alignment All 76 protein coding plastome loci were found to be predominantly under purifying selection, but specific codons were found to be under positive selection in 65 loci The loci that have been widely used in multi-gene phylogenetic studies had among the highest proportions of positively selected codons, suggesting caution in the interpretation of these earlier results Plastome phylogenomic analyses confirmed the backbone topology for Poaceae with maximum bootstrap support (BP) Among the 14 analyses, 82 clades out of 309 resolved were maximally supported in all trees Analyses of newly sequenced plastomes were in agreement with current classifications Five of seven partitions in which alignment gaps were removed retrieved Panicoideae as sister to the remaining PACMAD subfamilies Alternative topologies were recovered in trees from partitions that included alignment gaps This suggests that ambiguities in aligning these uncertain regions might introduce a false signal Resolution of these and other critical branch points in the phylogeny of Poaceae will help to better understand the selective forces that drove the radiation of the BOP and PACMAD clades comprising more than 999% of grass diversity

A 250 plastome phylogeny of the grass family (Poaceae): topological support under different data partitions

Identification of effective DNA barcodes for Triticum plants through chloroplast genome-wide analysis

Environmental conditions in coastal salt marsh habitats have led to the development of specialist genetic adaptations We evaluated six DNA barcode loci of the 53 species of Poaceae and 15 species of Chenopodiaceae from China's coastal salt marsh area and inland area Our results indicate that the optimum DNA barcode was ITS for coastal salt-tolerant Poaceae and matK for the Chenopodiaceae Sampling strategies for ten common species of Poaceae and Chenopodiaceae were analyzed according to optimum barcode We found that by increasing the number of samples collected from the coastal salt marsh area on the basis of inland samples, the number of haplotypes of Arundinella hirta, Digitaria ciliaris, Eleusine indica, Imperata cylindrica, Setaria viridis, and Chenopodium glaucum increased, with a principal coordinate plot clearly showing increased distribution points The results of a Mann-Whitney test showed that for Digitaria ciliaris, Eleusine indica, Imperata cylindrica, and Setaria viridis, the distribution of intraspecific genetic distances was significantly different when samples from the coastal salt marsh area were included (P < 001) These results suggest that increasing the sample size in specialist habitats can improve measurements of intraspecific genetic diversity, and will have a positive effect on the application of the DNA barcodes in widely distributed species The results of random sampling showed that when sample size reached 11 for Chloris virgata, Chenopodium glaucum, and Dysphania ambrosioides, 13 for Setaria viridis, and 15 for Eleusine indica, Imperata cylindrica and Chenopodium album, average intraspecific distance tended to reach stability These results indicate that the sample size for DNA barcode of globally distributed species should be increased to 11-15

/pdf/evaluating-sampling-strategy-for-dna-barcoding-study-of-50yu7424ca.pdf

Evaluating sampling strategy for DNA barcoding study of coastal and inland halo-tolerant Poaceae and Chenopodiaceae: A case study for increased sample size.

Over 90% of global commercial trade occurs between seaports, which are initial points-of-entry for nonnative, potentially invasive propagules. As such, there is a need to develop means to both rapidly intercept and identify propagules as they arrive. Here, we focus on plant propagules that are assumed to be non-native, in seed form. Because standard morphological techniques alone are laborious and require taxonomic expertise, we sought to address if identification through barcoding of the plastid DNA (rbcL + matK genes) of plant seeds could improve current processes in the early detection and rapid response to prevent entry/establishment of nonnative plant species. This research conducted a preliminary foray to evaluate the utility of widely accepted plant plastid DNA barcodes to identify plant propagules (seeds, hereafter) collected from the air-intake grilles of refrigerated shipping containers of a single agricultural commodity arriving at the Port of Savannah, Georgia, USA. We ask four questions: (1) Can DNA barcoding be used to detect seeds collected from shipping containers at the port? (2) What is the genetic composition of propagules entering the port? (3) How do morphological identifications compare to those based on genetic analysis? (4) Are nonnative invasive plant species present on shipping containers entering the Port of Savannah? This research collected 11,044 seeds from 628 refrigerated shipping containers between 2015 and 2017. Seeds were then morphologically sorted into Seed Types. Barcoding of the matK and rbcL gene regions of the plastid genomes directly isolated from seeds resulted in poor amplification. This is likely due to a host of potential confounding factors. Therefore, we germinated seeds and utilized leaf-tissues for sequencing of these two gene regions. From BLASTn analyses, results returned top hits for a variety of species, with up to 22 possible nonnative plant species and one definite Federal Noxious Weed. This work investigates the interception application of DNA barcoding to improve agro- and bio-security issues posed by nonnative and invasive species. Though this study required the germination of the seeds to obtain leaf-tissue suitable for our DNA barcoding method, we effectively demonstrated seed viability. Our seed identification process was lengthy and understandably not feasible for real-time application. Therefore, we seek to improve our methods for future applications by testing other approaches that may better complement morphological identification. Next reasonable steps include improved extraction protocols, metabarcoding to generate DNA barcode sequences directly from groups of seeds harvested from shipping containers and implementing other next-generation sequencing techniques.

/pdf/preliminary-application-of-dna-barcoding-toward-the-y4iyhs1mm0.pdf

Preliminary application of DNA barcoding toward the detection of viable plant propagules at an initial, international point-of-entry in Georgia, USA

Grass pollen is one of the major causes of allergy. Aerobiological monitoring is a necessary element of the complex of anti-allergic measures, but the similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring. One of the solutions to this problem is the metabarcoding approach employing DNA barcodes for taxonomical identification of species in a mix by high-throughput sequencing of the pollen DNA. A diverse set of 14 grass species of different genera were selected to create a local reference database of nuclear ITS1, ITS2, 5′-ETS, and plastome trnL-F DNA barcodes. Sequences for the database were Sanger sequenced from live field and herbarium specimens and collected from GenBank. New Poaceae-specific primers for 5′-ETS were designed and tested to obtain a 5′-ETS region less than 600 bp long, suitable for high-throughput sequencing. The DNA extraction method for single-species pollen samples and mixes was optimized to increase the yield for amplification and sequencing of pollen DNA. Barcode sequences were analyzed and compared by the barcoding gap and intra- and interspecific distances. Their capability to correctly identify grass pollen was tested on artificial pollen mixes of various complexity. Metabarcoding analysis of the artificial pollen mixes showed that nuclear DNA barcodes ITS1, ITS2, and 5′-ETS proved to be more efficient than the plastome barcode in both amplification from pollen DNA and identification of grass species. Although the metabarcoding results were qualitatively congruent with the actual composition of the pollen mixes in most cases, the quantitative results based on read-counts did not match the actual ratio of pollen grains in the mixes.

/pdf/assessment-of-its1-its2-5-ets-and-trnl-f-dna-barcodes-for-2td190vj.pdf

Assessment of ITS1, ITS2, 5′-ETS, and trnL-F DNA Barcodes for Metabarcoding of Poaceae Pollen

Littledalea racemosa is an alpine perennial species in the grass (Poaceae) tribe Bromeae that is endemic to the Qinghai–Tibet Plateau and adjacent regions. In the present study, we sequenced the complete chloroplast (cp) genome of L. racemosa on the Illumina MiSeq platform. This represents the first complete cp sequence for the Bromeae. The total genome size was 135,330 bp in length with 38.5% GC content. It contains a pair of inverted repeat regions (21,429 bp) separated by a small single copy region (12,486 bp) and a large single copy region (79,986 bp). Within the newly sequenced cp genome, we annotated a total of 142 genes, which included 84 protein-coding genes, 38 tRNAs genes, and 8 rRNAs genes. The most of genes occur within the single copy regions. We used the cp genome of L. racemosa and 37 other chloroplast genomes to perform a phylogenetic analysis, which indicated that L. racemosa is closely related species in tribe Triticeae of subfamily Pooideae.

Characterization of the complete chloroplast genome sequence of Littledalea racemosa Keng (Poaceae: Bromeae)

Orinus is an alpine endemic genus of Poaceae. Because of the imperfect specimens, high level of intraspecific morphological variability, and homoplasies of morphological characters, it is relatively difficult to delimitate species of Orinus by using morphology alone. To this end, the DNA barcoding has shown great potential in identifying species. The present study is the first attempt to test the feasibility of four proposed DNA barcoding markers (matK, rbcL, trnH-psbA, and ITS) in identifying four currently revised species of Orinus from the Qinghai-Tibetan Plateau (QTP). Among all the single-barcode candidates, the differentiation power was the highest for the nuclear internal transcribed spacer (ITS), while the chloroplast barcodes matK (M), rbcL (R), and trnH-psbA (H) could not identify the species. Meanwhile, the differentiation efficiency of the nuclear ITS (I) was also higher than any two- or three-locus combination of chloroplast barcodes, or even a combination of ITS and any chloroplast barcode except H + I and R + I. All the combinations of chloroplast barcodes plus the nuclear ITS, H + I, and R + I differentiated the highest portion of species. The highest differentiation rate for the barcodes or barcode combinations examined here was 100% (H + I and R + I). In summary, this case study showed that the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions in differentiating Orinus species from the QTP. Moreover, combining the ITS region with chloroplast regions may improve the barcoding success rate.

/pdf/evaluation-of-candidate-barcoding-markers-in-orinus-poaceae-eiibcgxntq.pdf

Evaluation of candidate barcoding markers in Orinus (Poaceae).

Premise of the study: Transcriptomes were used to develop microsatellite markers for the plant genus Orinus (Poaceae), which comprises three species of grasses (O. thoroldii, O. kokonoricus, and O. intermedius) that are widely distributed in the Qinghai– Tibetan Plateau. Methods and Results: Primer pairs were developed for 16 high-quality simple sequence repeats (SSRs) using transcriptomes. SSRs were amplified in 248 individuals representing the three species of Orinus; the number of alleles per locus ranged from one to seven, with an average of 2.6. The expected and observed heterozygosity per locus varied from 0.00 to 0.83 and from 0.00 to 1.00, respectively, with respective mean values of 0.32 and 0.34. Conclusions: These newly developed SSR markers will be valuable for evaluating the population genetic structure of Orinus throughout its range.

https://bioone.org/journals/applications-in-plant-sciences/volume-5/issue-7/apps.1700029/Development-of-SSR-Markers-from-Transcriptomes-for-Orinus-Poaceae-an/10.3732/apps.1700029.pdf

Development of SSR markers from transcriptomes for Orinus (Poaceae), an endemic of the Qinghai–Tibetan Plateau

The utility model provides an appearance is scraped to leaf epidermis, it includes: operation panel and blade holder, the operation panel include the pillar and set up in the xarm at pillar top, be provided with the first runner that the control xarm reciprocated on the pillar, be provided with the blade fixed station that is used for fixed blade on the xarm and be used for controlling the second runner that the blade fixed station removed. The blade holder, its include with the blade correspond the blade retention platform that sets up and set up respectively in two fixed columns of blade retention platform both sides are equipped with the clamping piece and are used for control on the fixed column the fixed knob that the clamping piece reciprocated makes clamping piece and blade retention platform cooperation centre gripping blade. Scrape when scraping the blade, can also make a round trip to scrape to the blade surface utilizing this blade to scrape the appearance, make the vein by the scraping clear away more clean, the epidermis is more clean clean and tidy when under a microscope observes, the while more does benefit to tests the later stage and acquires data to analysis, the observation of picture.

Appearance is scraped to leaf epidermis

Biosystematic Relationships and Anatomical Characteristics of Leaf Epidermises of Three Sect' Plants in Elymus (Poaceae)

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