Genome-wide identification of the Pyrus R2R3-MYB gene family and PhMYB62 regulation analysis in Pyrus hopeiensis flowers at low temperature.

The R2R3-MYB gene family play an important role in plant growth, development and stress responses. In this study, a total of 122 PcoR2R3-MYB genes were identified and grouped into 26 clades in pear. And these PcoMYBs were unevenly distributed among 17 chromosomes. The sequence characteristics, conversed motifs, exon/intron structures, classification, duplication events and cis-acting elements were also investigated. The gene duplication events showed that segmental duplication may play key roles in expansion of the PcoMYB gene family. Pyrus hopeiensis, which is a valuable wild resource, has strong cold resistance. An integrative analyses of miRNA and mRNA showed that PhMYB62 was involved in regulating low-temperature stress in P. hopeiensis flower organs. Subcellular localization analysis showed that PhMYB62 protein was specifically localized to the nucleus. The result of DAP-seq showed that PhMYB62 responded to low-temperature stress in P. hopeiensis by regulating TFs, which were associated with plant stress resistance, and POD, GAUT12, AUX28 and CHS genes. Subsequently, yeast one-hybrid verified that PhMYB62 could bind and activate the promoter of POD gene. The current study would provide a comprehensive information for further functional research on the stress-responsive R2R3-MYB gene candidates in pear, and may help to identify the genes associated with cold resistance for the cultivation of cold-resistant pear varieties.

Complete chloroplast genomes and comparative analysis of Ligustrum species.

In this study, we assembled and annotated the chloroplast (cp) genomes of four Ligustrum species, L. sinense, L. obtusifolium, L. vicaryi, and L. ovalifolium 'Aureum'. Including six other published Ligustrum species, we compared various characteristics such as gene structure, sequence alignment, codon preference, and nucleic acid diversity, and performed positive-selection genes screening and phylogenetic analysis. The results showed that the cp genome of Ligustrum was 162,185-166,800 bp in length, with a circular tetrad structure, including a large single-copy region (86,885-90,106 bp), a small single-copy region (11,446-11,499 bp), and a pair of IRa and IRb sequences with the same coding but in opposite directions (31,608-32,624 bp). This structure is similar to the cp genomes of most angiosperms. We found 132-137 genes in the cp genome of Ligustrum, including 89-90 protein-coding genes, 35-39 tRNAs, and 8 rRNAs. The GC content was 37.93-38.06% and varied among regions, with the IR region having the highest content. The single-nucleotide (A/T)n was dominant in simple-sequence repeats of the Ligustrum cp genome, with an obvious A/T preference. Six hotspot regions were identified from multiple sequence alignment of Ligustrum; the ycf1 gene region and the clpP1 exon region can be used as potential DNA barcodes for the identification and phylogeny of the genus Ligustrum. Branch-site model and Bayes empirical Bayes (BEB) analysis showed that four protein-coding genes (accD, clpP, ycf1, and ycf2) were positively selected, and BEB analysis showed that accD and rpl20 had positively selected sites. A phylogenetic tree of Oleaceae species was constructed based on the whole cp genomes, and the results were consistent with the traditional taxonomic results. The phylogenetic results showed that genus Ligustrum is most closely related to genus Syringa. Our study provides important genetic information to support further investigations of the phylogenetic development and adaptive evolution of Ligustrum species.

Cold Resistance of Euonymus japonicus Beihaidao Leaves and Its Chloroplast Genome Structure and Comparison with Celastraceae Species.

Euonymus japonicus Beihaidao is one of the most economically important ornamental species of the Euonymus genus. There are approximately 97 genera and 1194 species of plants worldwide in this family (Celastraceae). Using E. japonicus Beihaidao, we conducted a preliminary study of the cold resistance of this species, evaluated its performance during winter, assembled and annotated its chloroplast genome, and performed a series of analyses to investigate its gene structure GC content, sequence alignment, and nucleic acid diversity. Our objectives were to understand the evolutionary relationships of the genus and to identify positive selection genes that may be related to adaptations to environmental change. The results indicated that E. japonicus Beihaidao leaves have certain cold resistance and can maintain their viability during wintering. Moreover, the chloroplast genome of E. japonicus Beihaidao is a typical double-linked ring tetrad structure, which is similar to that of the other four Euonymus species, E. hamiltonianus, E. phellomanus, E. schensianus, and E. szechuanensis, in terms of gene structure, gene species, gene number, and GC content. Compared to other Celastraceae species, the variation in the chloroplast genome sequence was lower, and the gene structure was more stable. The phylogenetic relationships of 37 species inferred that members of the Euonymus genus do not form a clade and that E. japonicus Beihaidao is closely related to E. japonicus and E. fortunei. A total of 11 functional positive selected genes were identified, which may have played an important role in the process of Celastraceae species adapting to environmental changes. Our study provides important genetic information to support further investigations into the phylogenetic development and adaptive evolution of Celastraceae species.

Complete chloroplast genome structure of four Ulmus species and Hemiptelea davidii and comparative analysis within Ulmaceae species.

In this study, the chloroplast (cp) genomes of Hemiptelea davidii, Ulmus parvifolia, Ulmus lamellosa, Ulmus castaneifolia, and Ulmus pumila 'zhonghuajinye' were spliced, assembled and annotated using the Illumina HiSeq PE150 sequencing platform, and then compared to the cp genomes of other Ulmus and Ulmaceae species. The results indicated that the cp genomes of the five sequenced species showed a typical tetrad structure with full lengths ranging from 159,113 to 160,388 bp. The large single copy (LSC), inverted repeat (IR), and small single copy (SSC) lengths were in the range of 87,736-88,466 bp, 26,317-26,622 bp and 18,485-19,024 bp, respectively. A total of 130-131 genes were annotated, including 85-86 protein-coding genes, 37 tRNA genes and eight rRNA genes. The GC contents of the five species were similar, ranging from 35.30 to 35.62%. Besides, the GC content was different in different region and the GC content in IR region was the highest. A total of 64-133 single sequence repeat (SSR) loci were identified among all 21 Ulmaceae species. The (A)n and (T)n types of mononucleotide were highest in number, and the lengths were primarily distributed in 10-12 bp, with a clear AT preference. A branch-site model and a Bayes Empirical Bayes analysis indicated that the rps15 and rbcL had the positive selection sites. Besides, the analysis of mVISTA and sliding windows got a lot of hotspots such as trnH/psbA, rps16/trnQ, trnS/trnG, trnG/trnR and rpl32/trnL, which could be utilized as potential markers for the species identification and phylogeny reconstruction within Ulmus in the further studies. Moreover, the evolutionary tree of Ulmaceae species based on common protein genes, whole cp genome sequences and common genes in IR region of the 23 Ulmaceae species were constructed using the ML method. The results showed that these Ulmaceae species were divided into two branches, one that included Ulmus, Zelkova and Hemiptelea, among which Hemiptelea was the first to differentiate and one that included Celtis, Trema, Pteroceltis, Gironniera and Aphananthe. Besides, these variations found in this study could be used for the classification, identification and phylogenetic study of Ulmus species. Our study provided important genetic information to support further investigations into the phylogenetic development and adaptive evolution of Ulmus and Ulmaceae species.

Effects of three regeneration methods on the growth and bacterial community diversity of Populus × euramericana.

To study the effects of different regeneration methods on the growth and bacterial community diversity of Populus × euramericana cv. '74/76' (poplar 107), we investigated the growth of poplar 107 trees under three regeneration methods in 2017 and 2020, and sequenced the 16S rDNA V5-V7 regions in stem endophytic, root endophytic, and rhizosphere soil bacteria present in samples from the three regeneration methods using the Illumina high-throughput sequencing platform. The growth analysis showed that stump grafting regeneration (ST) and stump sprouting regeneration (SP) presented similar tree height and diameter at breast height (DBH), which were significantly lower by planted seedling regeneration (CK). The high-throughput sequencing results showed that the rhizosphere soil bacteria appeared to be significantly more diverse and rich than the root and stem endophytic bacteria. Cluster analysis showed that the similarity of bacterial community structure among the rhizosphere soil, root, and stem was small. Thus, the three sample types showed significant differences in bacteria. While comparing the two years, 2020 was significantly more diverse and rich than 2017. With the increase in stand age, the abundance of Proteobacteria increased and the abundance of Acidobacteria decreased. Among the three regeneration methods, ST significantly increased the diversity of stem endophytic bacteria. Chthoniobacter was enriched in SP, which promoted the decomposition of organic matter, and more plant growth promoting rhizobacteria (PGPR) were accumulated in the rhizosphere of SP and ST. The composition of the bacterial community was similar in the three regeneration methods, but the community composition was different. Regeneration and transformation of poplar plantations can be better carried out by stump grafting and stump sprouting.

Comparative analysis of the dust retention capacity and leaf microstructure of 11 Sophora japonica clones.

We used fresh leaves of Sophora japonica L. variety 'Qingyun 1' (A0) and 10 superior clones of the same species (A1-A10) to explore leaf morphological characteristics and total particle retention per unit leaf area under natural and artificial simulated dust deposition treatments. Our objectives were to explore the relationship between the two methods and to assess particle size distribution, X-ray fluorescence (XRF) heavy metal content, and scanning electron and atomic force microscopy (SEM and AFM) characteristics of leaf surface microstructure. Using the membership function method, we evaluated the dust retention capacity of each clone based on the mean degree of membership of its dust retention index. Using correlation analysis, we selected leaf morphological and SEM and AFM indices related significantly to dust retention capacity. Sophora japonica showed excellent overall dust retention capacity, although this capacity differed among clones. A5 had the strongest overall retention capacity, A2 had the strongest retention capacity for PM2.5, A9 had the strongest retention capacity for PM2.5-10, A0 had the strongest retention capacity for PM>10, and A2 had the strongest specific surface area (SSA) and heavy metal adsorption capacity. Overall, A1 had the strongest comprehensive dust retention ability, A5 was intermediate, and A7 had the weakest capacity. Certain leaf morphological and SEM and AFM characteristic indices correlated significantly with the dust retention capacity.

The Distribution and Origins of Pyrus hopeiensis-"Wild Plant With Tiny Population" Using Whole Genome Resequencing.

Pyrus hopeiensis is a valuable but endangered wild resource in the genus Pyrus. It has been listed as one of the 120 wild species with tiny population in China. The specie has been little studied. A preliminary study of propagation modes in P. hopeiensis was performed through seed propagation, hybridization, self-crossing trials, bud grafting, branch grafting, and investigations of natural growth. The results showed that the population size of P. hopeiensis was very small, the distribution range was limited, and the habitat was extremely degraded. In the wild population, natural hybridization and root tiller production were the major modes of propagation. Whole genome re-sequencing of the 23 wild and cultivated accessions from Pyrus species collected was performed using an Illumina HiSeq sequencing platform. The sequencing depth range was 26.56x-44.85x and the average sequencing depth was 32x. Phylogenetic tree and principal component analyses (PCA) based on SNPs showed that the wild Pyrus species, such as PWH06, PWH07, PWH09, PWH10, PWH13, and PWH17, were closely related to both P. hopeiensis HB-1 and P. hopeiensis HB-2. Using these results in combination with morphological characteristics, it speculated that P. hopeiensis populations may form a natural hybrid group with frequent gene exchanges between and within groups. A selective elimination analysis on the P. hopeiensis population were performed using Fst and π radio and a total of 381 overlapping genes including SAUR72, IAA20, HSFA2, and RKP genes were obtained. These genes were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) function enrichment. And four KEGG pathways, including lysine degradation, sphingolipid metabolism, other glycan degradation, and betaine biosynthesis were significantly enriched in the P. hopeiensis population. Our study provided information on genetic variation, evolutionary relationships, and gene enrichment in P. hopeiensis population. These data will help reveal the evolutionary history and origin of P. hopeiensis and provide guidelines for subsequent research on the locations of functional genes.

Niches and Seasonal Changes, Rather Than Transgenic Events, Affect the Microbial Community of Populus × euramericana 'Neva'.

Exploring the complex spatiotemporal changes and colonization mechanism of microbial communities will enable microbial communities to be better used to serve agricultural and ecological operations. In addition, evaluating the impact of transgenic plants on endogenous microbial communities is necessary for their commercial application. In this study, microbial communities of Populus × euramericana 'Neva' carrying Cry1Ac-Cry3A-BADH genes (ECAA1 line), Populus × euramericana 'Neva' carrying Cry1Ac-Cry3A-NTHK1 genes (ECAB1 line), and non-transgenic Populus × euramericana 'Neva' from rhizosphere soil, roots, and phloem collected in different seasons were compared and analyzed. Our analyses indicate that the richness and diversity of bacterial communities were higher in the three Populus × euramericana 'Neva' habitats than in those of fungi. Bacterial and fungal genetic-distance-clustering results were similar; rhizosphere soil clustered in one category, with roots and phloem in another. The diversity and evenness values of the microbial community were: rhizosphere soil > phloem > root system. The bacterial communities in the three habitats were dominated by the Proteobacteria, and fungal communities were dominated by the Ascomycota. The community composition and abundance of each part were quite different; those of Populus × euramericana 'Neva' were similar among seasons, but community abundance fluctuated. Seasonal fluctuation in the bacterial community was greatest in rhizosphere soil, while that of the fungal community was greatest in phloem. The transgenic lines ECAA1 and ECAB1 had a bacterial and fungal community composition similar to that of the control samples, with no significant differences in community structure or diversity among the lines. The abundances of operational taxonomic units (OTUs) were low, and differed significantly among the lines. These differences did not affect the functioning of the whole specific community. Sampling time and location were the main driving factors of changes in the Populus × euramericana 'Neva' microbial community. Transgenic events did not affect the Populus × euramericana 'Neva' rhizosphere or endophytic microbial communities. This study provides a reference for the safety evaluation of transgenic plants and the internal colonization mechanism of microorganisms in plants.

Comparative Analyses of Euonymus Chloroplast Genomes: Genetic Structure, Screening for Loci With Suitable Polymorphism, Positive Selection Genes, and Phylogenetic Relationships Within Celastrineae.

In this study, we assembled and annotated the chloroplast (cp) genome of the Euonymus species Euonymus fortunei, Euonymus phellomanus, and Euonymus maackii, and performed a series of analyses to investigate gene structure, GC content, sequence alignment, and nucleic acid diversity, with the objectives of identifying positive selection genes and understanding evolutionary relationships. The results indicated that the Euonymus cp genome was 156,860-157,611bp in length and exhibited a typical circular tetrad structure. Similar to the majority of angiosperm chloroplast genomes, the results yielded a large single-copy region (LSC) (85,826-86,299bp) and a small single-copy region (SSC) (18,319-18,536bp), separated by a pair of sequences (IRA and IRB; 26,341-26,700bp) with the same encoding but in opposite directions. The chloroplast genome was annotated to 130-131 genes, including 85-86 protein coding genes, 37 tRNA genes, and eight rRNA genes, with GC contents of 37.26-37.31%. The GC content was variable among regions and was highest in the inverted repeat (IR) region. The IR boundary of Euonymus happened expanding resulting that the rps19 entered into IR region and doubled completely. Such fluctuations at the border positions might be helpful in determining evolutionary relationships among Euonymus. The simple-sequence repeats (SSRs) of Euonymus species were composed primarily of single nucleotides (A)n and (T)n, and were mostly 10-12bp in length, with an obvious A/T bias. We identified several loci with suitable polymorphism with the potential use as molecular markers for inferring the phylogeny within the genus Euonymus. Signatures of positive selection were seen in rpoB protein encoding genes. Based on data from the whole chloroplast genome, common single copy genes, and the LSC, SSC, and IR regions, we constructed an evolutionary tree of Euonymus and related species, the results of which were consistent with traditional taxonomic classifications. It showed that E. fortunei sister to the Euonymus japonicus, whereby E. maackii appeared as sister to Euonymus hamiltonianus. Our study provides important genetic information to support further investigations into the phylogenetic development and adaptive evolution of Euonymus species.

Structural and Comparative Analysis of the Complete Chloroplast Genome of Pyrus hopeiensis-"Wild Plants with a Tiny Population"-and Three Other Pyrus Species.

Pyrus hopeiensis is a valuable wild resource of Pyrus in the Rosaceae. Due to its limited distribution and population decline, it has been listed as one of the "wild plants with a tiny population" in China. To date, few studies have been conducted on P. hopeiensis. This paper offers a systematic review of P. hopeiensis, providing a basis for the conservation and restoration of P. hopeiensis resources. In this study, the chloroplast genomes of two different genotypes of P. hopeiensis, P. ussuriensis Maxin. cv. Jingbaili, P. communis L. cv. Early Red Comice, and P. betulifolia were sequenced, compared and analyzed. The two P. hopeiensis genotypes showed a typical tetrad chloroplast genome, including a pair of inverted repeats encoding the same but opposite direction sequences, a large single copy (LSC) region, and a small single copy (SSC) region. The length of the chloroplast genome of P. hopeiensis HB-1 was 159,935 bp, 46 bp longer than that of the chloroplast genome of P. hopeiensis HB-2. The lengths of the SSC and IR regions of the two Pyrus genotypes were identical, with the only difference present in the LSC region. The GC content was only 0.02% higher in P. hopeiensis HB-1. The structure and size of the chloroplast genome, the gene species, gene number, and GC content of P. hopeiensis were similar to those of the other three Pyrus species. The IR boundary of the two genotypes of P. hopeiensis showed a similar degree of expansion. To determine the evolutionary history of P. hopeiensis within the genus Pyrus and the Rosaceae, 57 common protein-coding genes from 36 Rosaceae species were analyzed. The phylogenetic tree showed a close relationship between the genera Pyrus and Malus, and the relationship between P. hopeiensis HB-1 and P. hopeiensis HB-2 was the closest.