Comparative Analysis of Transposable Elements and the Identification of Candidate Centromeric Elements in the Prunus Subgenus Cerasus and Its Relatives.

The subgenus Cerasus and its relatives include many crucial economic drupe fruits and ornamental plants. Repetitive elements make up a large part of complex genomes, and some of them play an important role in gene regulation that can affect phenotypic variation. However, the variation in their genomes remains poorly understood. This work conducted a comprehensive repetitive sequence identification across the draft genomes of eight taxa of the genus Prunus, including four of the Prunus subgenus Cerasus (Prunus pseudocerasus, P. avium, P. yedoensis and P. × yedoensis) as well as congeneric species (Prunus salicina, P. armeniaca, P. dulcis and P. persica). Annotation results showed high proportions of transposable elements in their genomes, ranging from 52.28% (P. armeniaca) to 61.86% (P. pseudocerasus). The most notable differences in the contents of long terminal repeat retrotransposons (LTR-RTs) and tandem repeats (TRs) were confirmed with de novo identification based on the structure of each genome, which significantly contributed to their genome size variation, especially in P. avium and P.salicina. Sequence comparisons showed many similar LTR-RTs closely related to their phylogenetic relationships, and a highly similar monomer unit of the TR sequence was conserved among species. Additionally, the predicted centromere-associated sequence was located in centromeric regions with FISH in the 12 taxa of Prunus. It presented significantly different signal intensities, even within the diverse interindividual phenotypes for Prunus tomentosa. This study provides insight into the LTR-RT and TR variation within Prunus and increases our knowledge about its role in genome evolution.

Evolution of Rosaceae Plastomes Highlights Unique Cerasus Diversification and Independent Origins of Fruiting Cherry.

Rosaceae comprises numerous types of economically important fruits, ornamentals, and timber. The lack of plastome characteristics has blocked our understanding of the evolution of plastome and plastid genes of Rosaceae crops. Using comparative genomics and phylogenomics, we analyzed 121 Rosaceae plastomes of 54 taxa from 13 genera, predominantly including Cerasus (true cherry) and its relatives. To our knowledge, we generated the first comprehensive map of genomic variation across Rosaceae plastomes. Contraction/expansion of inverted repeat regions and sequence losses of the two single-copy regions underlie large genomic variations in size among Rosaceae plastomes. Plastid protein-coding genes were characterized with a high proportion (over 50%) of synonymous variants and insertion-deletions with multiple triplets. Five photosynthesis-related genes were specially selected in perennial woody trees. Comparative genomic analyses implied divergent evolutionary patterns between pomaceous and drupaceous trees. Across all examined plastomes, unique and divergent evolution was detected in Cerasus plastomes. Phylogenomic analyses and molecular dating highlighted the relatively distant phylogenetic relationship between Cerasus and relatives (Microcerasus, Amygdalus, Prunus, and Armeniaca), which strongly supported treating the monophyletic true cherry group as a separate genus excluding dwarf cherry. High genetic differentiation and distinct phylogenetic relationships implied independent origins and domestication between fruiting cherries, particularly between Prunus pseudocerasus (Cerasus pseudocerasus) and P. avium (C. avium). Well-resolved maternal phylogeny suggested that cultivated P. pseudocerasus originated from Longmenshan Fault zone, the eastern edge of Himalaya-Hengduan Mountains, where it was subjected to frequent genomic introgression between its presumed wild ancestors and relatives.

The complete chloroplast genome sequence of a hybrid blackberry (Rubus spp.) cultivar.

Blackberry (Rubus spp.) is an important hybrid fruit crop popular in the US Pacific Northwest and the European region with complex origins. In this study, we report the complete chloroplast genome sequence of a hybrid blackberry cultivar 'Arapohol' using next-generation sequencing technology. The complete chloroplast genome size is 156,621 bp. The genome contains 134 genes, including 40 tRNA genes, 86 protein-coding genes, and 8 rRNA genes. Phylogenetic analysis based on 11 complete chloroplast genomes revealed that taxa is closely related to Rubus niveus. The complete chloroplast genome of this Rubus sp. provides valuable information for understanding the origination of this crop species.

Analysis of factors influencing the network teaching effect of college students in a medical school during the COVID-19 epidemic.

BACKGROUND: The purpose of this study is to understand the influencing factors of Chinese college students' satisfaction with online teaching and psychological pressure on learning during the novel coronavirus epidemic. METHODS: We assessed the effect of online teaching of 7084 medical students from wannan medical college in March 5 to April 2, 2020 using cluster sampling. The respondents were asked to complete a 7-item self-compiled online teaching satisfaction questionnaire. Chi-square test and multivariate logistic regression analysis are used. RESULTS: Sex is female (OR = 1.257, 95%CI: 1.132 ~ 1.396), grades are second and third grades (second grades: OR = 1.228, 95%CI: 1.080 ~ 1.397; third grades: OR = 1.197, 95%CI: 1.048 ~ 1.367), normal/unfamiliar learning platform operation (OR = 3.692, 95%CI: 3.321 ~ 4.103) were risk factors for satisfactory teaching effect. In addition, students whose school year system is four-year (OR = 0.870, 95%CI: 0.781 ~ 0.969) and grade 4 and above (OR = 0.594, 95%CI: 0.485 ~ 0.727) were more satisfied with the teaching effect of teachers. And, during the period of the COVID-19 epidemic, the risk factors for college students to have psychological stress were: female (OR = 1.258, 95%CI: 1.096 ~ 1.442), from rural areas (OR = 1.511, 95%CI: 1.312 ~ 1.740), and the academic year system is four-year system (OR = 1.191, 95%CI: 1.028 ~ 1.380), using mobile phones and other learning tools (OR = 1.388, 95%CI: 1.205 ~ 1.600), general/unfamiliar with learning platform operations (OR = 2.273), 95%CI: 1.888 ~ 2.735). While the protective factors for college students' psychological stress included: grade three and four and above (OR = 0.463, 95%CI: 0.387 ~ 0.554; OR = 0.232, 95%CI: 0.187 ~ 0.286), and they think that the teaching effect is satisfactory (OR = 0.314, 95%CI: 0.261 ~ 0.379). CONCLUSION: This survey shows that compared with male college students, female college students were more dissatisfied with the teaching effect of teachers and havd greater psychological pressure on learning. Psychological counseling should be strengthened for students in rural areas and those who were not familiar with the operating platform to relieve their psychological pressure on learning.

Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO.

The carotenoid isomerase gene (BoaCRTISO) of Chinese kale was targeted and edited using the CRISPR/Cas9 system in the present study. The results showed a high mutation rate (81.25%), and 13 crtiso mutants were obtained. Only two types of mutations, insertions and replacements, were found. Both the total and individual carotenoid and chlorophyll concentrations of the biallelic and homozygous mutants were reduced, and the total levels declined by 11.89-36.33%. The color of the biallelic and homozygous mutants changed from green to yellow, likely reflecting a reduction in the color-masking effect of chlorophyll on carotenoids. The expression levels of most carotenoid and chlorophyll biosynthesis-related genes, including CRTISO, were notably lower in the mutants than in the WT plants. In addition, the functional differences between members of this gene family were discussed. In summary, these findings indicate that CRISPR/Cas9 is a promising technique for the quality improvement of Chinese kale and other Brassica vegetables.

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts.

In this study, we report the isolation and purification of protoplasts from Chinese kale (Brassica oleracea var. alboglabra) hypocotyls, and their transient gene expression transformation and subcellular localization of BaMYB75 (Bol042409). The upshot is that the vintage protocol included 5-d hypocotyls that were enzymatically hydrolyzed for 8 h in enzyme solution (3.0% cellulase, 0.5% pectolase, and 0.5 M mannitol), and the protoplasts were purified by precipitation. The total yield of protoplasts was 8 × 105 protoplast g-1 fresh weight, and the protoplasts' viability was 90%. The maximum transformation efficiency obtained by using green fluorescent protein (GFP) as a detection gene was approximately 45% when the polyethylene glycol (PEG)4000 concentration was 40% and transformation time was 20 min. In addition, BaMYB75 was ultimately localized in the nucleus of Chinese kale hypocotyl protoplasts, verifying the validity and reliability of this transient transformation system. An effective and economical hypocotyl protoplast isolation, purification, and transformation system was established for Chinese kale in this study. This effectively avoided interference of chloroplast autofluorescence compared to using mesophyll cells, laying the foundation for future research in the molecular biology of Brassica vegetables.

Functional differences of BaPDS1 and BaPDS2 genes in Chinese kale.

This study presents a systematic analysis of the functional differences between two genes that encode phytoene desaturase (PDS) in Chinese kale. The promoter sequences of both BaPDS1 and BaPDS2 were amplified and cloned, and their lengths were 2005 bp and 2000 bp, respectively. The mining of cis-acting elements in the promoters showed that the two BaPDS genes are mainly associated with light and phytohormone responsiveness. Light quality, light intensity and plant hormone treatments were conducted in seedlings of Chinese kale, and the results indicated that the response of the two genes to different factors differed. Among them, BaPDSs collectively respond to the treatment with salicylic acid and abscisic acid. With regard to response differences, BaPDS1 is sensitive to red and blue light, blue light, and strong light, while BaPDS2 responds to blue light, weak light, darkness, gibberellin and methyl jasmonate. In addition, both BaPDS1 and BaPDS2 are likely targeted to the chloroplast. Furthermore, single and double mutants of BaPDSs were generated via CRISPR/Cas9 technology. Phenotypic analysis showed that the double mutant with edited PDS1 and PDS2 was a pure albino, while the single mutants with edited PDS1 or PDS2 were partly whitened. In summary, BaPDS1 and BaPDS2 genes played different and indispensable roles in Chinese kale, and their functions were partially complementary.

Allopolyploid origin in Rubus (Rosaceae) inferred from nuclear granule-bound starch synthase I (GBSSI) sequences.

BACKGROUND: Polyploidy and hybridization are ubiquitous in Rubus L., a large and taxonomically challenging genus. Chinese Rubus are mainly concentrated into two major sections, the diploid Idaeobatus and the polyploid Malachobatus. However, it remains unclear to be auto- or allo- polyploid origin of polyploids in Rubus. We investigated the homoeologs and the structure of the GBSSI-1 (granule-bound starch synthase I) gene in 140 Rubus individuals representing 102 taxa in 17 (out of the total 24) subsections of 7 (total of 12) sections at different ploidy levels. RESULTS: Based on the gene structure and sequence divergence, we defined three gene variants, GBSSI-1a, GBSSI-1b, and GBSSI-1c. When compared with GBSSI-1a, both GBSSI-1b and GBSSI-1c have a shorter fourth intron, and GBSSI-1c had an additional deletion in the fifth intron. For diploids, either GBSSI-1a or GBSSI-1b was detected in 56 taxa consisting of 82 individuals from sect. Idaeobatus, while both alleles existed in R. pentagonus and R. peltatus. Both homoeologs GBSSI-1a and GBSSI-1b were identified in 39 taxa (48 individuals) of Malachobatus polyploids. They were also observed in two sect. Dalibardastrum taxa, in one sect. Chamaebatus taxon, and in three taxa from sect. Cylactis. Interestingly, all three homoeologs were observed in the three tetraploid taxa. Phylogenetic trees and networks suggested two clades (I and II), corresponding to GBSSI-1a, and GBSSI-1b/1c sequences, respectively. GBSSI-1 homoeologs from the same polyploid individual were resolved in different well-supported clades, and some of these homoelogs were more closely related to homoelogs in other species than they were to each other. This implied that the homoeologs of these polyploids were donated by different ancestral taxa, indicating their allopolyploid origin. Two kinds of diploids hybridized to form most allotetraploid species. The early-divergent diploid species with GBSSI-1a or -1b emerged before polyploid formation in the evolutionary history of Rubus. CONCLUSION: This study provided new insights into allopolyploid origin and evolution from diploid to polyploid within the genus Rubus at the molecular phylogenetic level, consistent with the taxonomic treatment by Yü et al. and Lu.

Variations in the glucosinolates of the individual edible parts of three stem mustards (Brassica juncea).

The composition and content of glucosinolates were investigated in the edible parts (petioles, peel and flesh) of tuber mustard, bamboo shoots mustard and baby mustard by high-performance liquid chromatography to reveal the association between the different cooking methods and their glucosinolate profiles. Eight glucosinolates were identified from tuber mustard and baby mustard, including three aliphatic glucosinolates, four indole glucosinolates and one aromatic glucosinolate. Only six of the eight glucosinolates were detected in bamboo shoots mustard. The results show that the distribution and content of glucosinolates varied widely among the different tissues and species. The highest contents of glucosinolates in tuber mustard, bamboo shoots mustard and baby mustard were found in flesh, petioles and peel, respectively. The content of total glucosinolates ranged from 5.21 µmol g-1 dry weight in bamboo shoots mustard flesh to 25.64 µmol g-1 dry weight in baby mustard peel. Aliphatic glucosinolates were predominant in the three stem mustards, followed by indole and aromatic glucosinolates. Sinigrin was the predominant glucosinolate in the three stem mustards. Sinigrin content in tuber mustard was slightly higher than that in baby mustard and much higher than that in bamboo shoots mustard, suggesting that the pungent-tasting stem mustards contained more sinigrin. In addition, a principal components analysis showed that bamboo shoots mustard was distinguishable from the other two stem mustards. A variance analysis indicated that the glucosinolates were primarily influenced by a species × tissue interaction. The correlations among glucosinolates were also analysed.

Variations of Alkaloid Accumulation and Gene Transcription in Nicotiana tabacum.

To increase the understanding of alkaloid biosynthesis in Nicotiana tabacum during whole plant growth periods, variations of the contents of alkaloids and the transcription of key biosynthetic genes in fresh leaves were investigated in three varieties at five developmental stages. Six alkaloids were analyzed by gas chromatograph⁻mass spectrometry (GC⁻MS) and the most abundant alkaloid was observed during the upper leaves maturing stage in the varieties, among which the alkaloid content of K326 was the highest. Considering the genetic effect, variance analysis indicated that the developmental stage played a predominant role in alkaloid accumulation. Moreover, the levels of biosynthetic gene transcripts in the leaves at the vigorous growing stage might contribute to the contents of alkaloids in the leaves during the maturing stages. To further illuminate the metabolism of alkaloid biosynthesis, a correlation among alkaloids was also documented.

Metabolic profiles of Cuibi-1 and Zhongyan-100 flue-cured tobacco leaves in different growing regions by gas chromatography/mass spectrometry.

The metabolic profiles of tobacco leaves of two differential Chinese cultivars from different growing regions were analysed using gas chromatography-mass spectrometry (GC-MS). The results of principal component analysis, partial least-squares discriminant analysis and hierarchical cluster analysis showed significant differences in metabolome among three groups, identified 24 differential metabolites, and analysed the metabolic pathway in which the metabolites were involved. Among them, 13 metabolites were associated with geographical regions, including seven organic and fatty acids, four carbohydrates and two secondary metabolites. Four amino acids and two monosaccharides were associated with cultivars and the remaining five metabolites were associated with both. The relationships among the differential metabolites and the distinct characteristics of environment and cultivar were further discussed. In addition, correlation analysis indicated that most of the differential carbohydrates were negatively correlated with the differential amino acids and organic acids. Taken together, this study demonstrates the metabolite differences between two cultivars in different regions, and highlights the effect of environment and cultivar on tobacco leaf metabolism.

Dissection of the Mechanism for Compatible and Incompatible Graft Combinations of Citrus grandis (L.) Osbeck ('Hongmian Miyou').

'Hongmian miyou' (Citrus grandis L. Osbeck) is mutated from 'Guanxi miyou', with a different spongy layer coloration. Trifoliate orange (Poncirus trifoliata) is widely used as rootstocks in 'Guanxi miyou' grafting, whereas 'Hongmian miyou' is incompatible with available trifoliate orange rootstocks. To explore the reasons for the etiolation of leaves of 'Hongmian miyou'/trifoliate orange, anatomical differences among different graft unions, gene expression profiles, and auxin levels of scion were investigated in this study. A histological assay indicated that there was no significant difference in anatomical structure between the compatible and incompatible combinations. A total of 1950 significant differentially-expressed genes (DEGs) were identified and analyzed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that genes involved in carbohydrate metabolism, energy metabolism, amino acid metabolism, and plant hormone signal transduction were significantly enriched. Moreover, the expression of nine genes in the auxin pathway were upregulated and three were downregulated in compatible combinations compared with those in the incompatible group. Further experiments verified that indole-3-acetic acid (IAA) content increases in the compatible graft combination, which suggests that IAA might promote graft compatibility.

The complete chloroplast genome of Tomentosa cherry Prunus tomentosa (Prunoideae, Rosaceae).

Prunus tomentosa belongs to subgenus Lithocerasus in family Rosaceae. It is native to northern China and has become a staple back yard garden plant in Russia and much of Eastern Europe. In this study, de novo assembly with low coverage whole genome sequencing facilitated to generate the complete chloroplast (cp) genome of P. tomentosa. The genome size is 158,356 bp in length. It exhibited a typical quadripartite structure comprising a large single copy region (LSC, 86,630 bp), a small single copy region (SSC, 19,010 bp) and a pair of inverted repeat regions (IRs, 26,358 bp each). A total of 115 genes were predicted including 82 protein-coding genes, 29 tRNA genes, and four rRNA genes. Phylogenetic analysis indicated that P. tomentosa is most closely related to P. mume suggesting the phylogenetic relationship between P. tomentosa and subgenus prunpphora.

Characterization of complete chloroplast genome and phylogenetic analysis of sweet cherry Cerasus avium (L.) Moench (Prunoideae, Rosaceae).

Sweet cherry (Cerasus avium (L.) Moench) belonging to family Rosaceae, is an important economical fruit crop worldwide. In this study, the complete chloroplast (cp) genome of sweet cherry was generated by De novo assembly with low coverage whole-genome sequencing data. The genome size was 157,987 bp in length consisting of a typical quadripartite structure; a large single-copy region (LSC, 85,975 bp), a small single-copy region (SSC, 19,121 bp) and a pair of inverted repeat regions (IRs, 26,445 bp each). A total of 115 genes were predicted including 82 protein-coding genes, 29 tRNA genes and four rRNA genes. Phylogenetic analysis based on 12 reported complete chloroplast genome indicated the monophyly of the genus Creasus including newly sequenced C. avium, which is conform to the traditional classification.

Variation in the main health-promoting compounds and antioxidant activity of whole and individual edible parts of baby mustard (Brassica juncea var. gemmifera).

Despite being a brassicaceous vegetable that is widely consumed in winter and spring in Southwest China, there is lack of information available on baby mustard. The aim of this study was to determine the contents of soluble proteins, soluble sugars, chlorophylls, carotenoids, ascorbic acid, proanthocyanidins, flavonoids, total phenolics, and glucosinolates, as well as the antioxidant activity of the whole edible parts and seven individual edible parts (swollen stem: petioles, peel, flesh; lateral bud: leaves, petioles, peel, flesh) of baby mustard. The results showed that significant differences in health-promoting compounds and antioxidant activity existed between the different edible parts. The lateral bud of baby mustard possessed greater health-promoting compounds than the swollen stem. In particular, the lateral bud leaves possessed abundant antioxidant compounds and antioxidant activity, indicating that these should be conserved during harvesting due to their potential contribution to human health. Furthermore, aliphatic glucosinolates were predominant, and sinigrin was the most abundant glucosinolate in all the assessed parts of baby mustard, the content of which was 15.81 µmol g-1 dry weight, accounting for more than 87% of the total glucosinolate content. However, the sinigrin content in baby mustard is lower than tuber mustard, which explains the less pungent flavor of baby mustard and its better suitability as a fresh vegetable. In addition, antioxidant activity was highly correlated with total phenolics, whereas gluconasturtiin and soluble sugars were negatively correlated with the majority of antioxidants.

The complete chloroplast genome sequence of Maddenia hypoleuca koehne (Prunoideae, Rosaceae).

Maddenia hypoleuca Koehne belonging to family Rosaceae is a native species in China. The complete chloroplast (cp) genome was generated by de novo assembly using low coverage whole genome sequencing data and manual correction. The cp genome was 158 084 bp in length, with GC content of 36.63%. It exhibited a typical quadripartite structure: a pair of large inverted repeat regions (IRs, 26 246 bp each), a large single-copy region (LSC, 86 713 bp), and a small single-copy region (SSC, 18 879 bp). A total of 114 genes were predicted, which included 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analysis indicated that M. hypoleuca is most closely related to Prunus padus within the Prunoideae subfamily, which conforms to the traditional classification.

A Quantitative Real-Time PCR-Based Strategy for Molecular Evaluation of Nicotine Conversion in Burley Tobacco.

Nornicotine production in Nicotiana tabacum is undesirable because it is the precursor of the carcinogen N'-nitrosonornicotine. In some individual burley tobacco plants, a large proportion of the nicotine can be converted to nornicotine, and this process of nicotine conversion is mediated primarily by enzymatic N-demethylation of nicotine which is controlled mainly by CYP82E4. Here we report a novel strategy based on quantitative real-time polymerase chain reaction (qPCR) method, which analyzed the ratio of nicotine conversion through examining the transcript level of CYP82E4 in burley leaves and do not need ethylene induction before detected. The assay was linear in a range from 1 × 10¹ to 1 × 105 copies/mL of serially diluted standards, and also showed high specificity and reproducibility (93%-99%). To assess its applicability, 55 plants of burley cultivar Ky8959 at leaf maturing stage were analyzed, and the results were in accordance with those from gas chromatograph-mass spectrometry (GC-MS) method. Moreover, a linear correlation existed between conversion level and CYP82E4 transcript abundance. Taken together, the quantitative real-time PCR assay is standardized, rapid and reproducible for estimation of nicotine conversion level in vivo, which is expected to shed new light on monitoring of burley tobacco converter.