Gap-free genome assembly and comparative analysis reveal the evolution and anthocyanin accumulation mechanism of Rhodomyrtus tomentosa

This is a correction to:Fangping Li,Shiqiang Xu,Zitong Xiao,Jingming Wang,Yu Mei,Haifei Hu,Jingyu Li,Jiey-ing Liu,Zhuangwei Hou,Junliang Zhao,Shaohai Yang,Jihua Wang,Gap-free genome assembly and comparative analysis reveal the evolution and anthocyanin accumu-lation mechanism of Rhodomyrtus tomentosa,Horticul-ture Research,Volume 10,Issue 3,March 2023,uhad005,https://doi.org/10.1093/hr/uhad005.

Selection of Media for Hardwood Cuttings Container Seedling-raising of Triploid Clones of Populus tomentosa

[Objective] The experiment was aimed to select effective and economical media for container seedling of triploid clones of Populus tomentosa that was carried out. [Method] The sandy loam, peat, perlite, vermiculite, riversand, sludge were taken as media of hardwood cutting and survival rate, seedling height were taken as indexes to select media for container seedling of triploid clones of Populus tomentosa. [Result] Different mixedmedia had great influence on survival rates of container seedlings. Taking peat and vermiculite with the proportion of 5∶2 (M10) or peat ,vermiculite with the proportion of 7∶2 (M11) or sandy loam (M1) as media would generate higher cutting survival rate that was higher than 90.0%. There were significant differece in height increments of container seedlings. Taking sandy loam, peat and vermiculite with the proportion of 6∶2∶2(M5)or sandy loam (M1), seedling height of 60-days the seedling was over 37.0 cm. [Conclusion] According to cost analysis of nursery medium, the optimum medium for hardwood cuttings container seedling-raising of triploid clones of Populus tomentosa was sandy loam.

Cloning of cDNA Encoding CCoAOMT from Populus tomentosa and Down-regulation of Lignin Content in Transgenic Plant Expressing Antisense Gene

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula x P. alba mediated by Agrobacterium tumefaciens ( Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.

Establishment of High Frequency Regeneration System of Populus tomentosa

The establishment of high frequency regeneration system is a foundation for Agrobacterium mediated genetic transformation. In this work, several important factors influencing the efficiency of regeneration of plants, such as concentration of plant growth regulators, leaf explant orientation, leaf growth sequence and leaf segment, were studied. The results indicated that the differentiation rate of adventitious shoots was 90% on basal MS medium only supplemented with 1 5?mg·L -1 BA (6 benzyladenine) and reached the highest level(95%) when 1 0?mg·L -1 BA and 0 3?mg·L -1 NAA (naphthaleneacetic acid) were added to MS medium. 90% of differentiation rate of adventitious roots were obtained when 0 3?mg·L -1 NAA was only added to MS medium. It was also found that more adventitious shoots were regenerated from the lower segment of leaf (with petiole) than the other segments, the number of adventitious shoots decreased from top to base of leaf growth sequence and the percentage of adventitious shoot induction with adaxial side downward was higher than that with adaxial side upward.

Role of CaCl_(2) in Cold Acclimation-Induced Freezing Resistance of Populus tomentosa Cuttings

Populus tomentosa cuttings were treated with 1mmol·L -1 , 5mmol·L -1 , 10mmol·L -1 or 15mmol·L -1 of CaCl 2 for 1\|7 d, respectively, for studying the effects of different concentrations of CaCl 2 on freezing resistance. Results indicated that 10?mmol·L -1 of CaCl 2 has greater effect than other concentrations on the enhancement of freezing resistance, and the optimum time of pretreatment was 5?d. In addition, cuttings used for cold acclimation at -3℃ were pretreated with or without 10?mmol·L -1 of CaCl 2, 3?mmol·L -1 of Ca 2+ chelator EGTA, 0 05?mmol·L -1 of CaM antagonist CPZ or 0 1?mmol·L -1 of Ca 2+ channel inhibitor LaCl 3 The changes in CaM and freezing resistance of all cuttings were investigated. The results showed that cold acclimation at -3℃ increased CaM content and decreased the minimum temperature for 100% survival. The CaCl 2 pretreatment enhanced the effect of cold acclimation and obviously increased CaM content and decreased the minimum temperature for 100% survival, but this effect was strongly inhibited by the EGTA, CPZ or LaCl 3 It is concluded that the effect of CaCl 2 on freezing resistance is associated with its concentration and time of pretreatment, Ca 2+ CaM may be involved in the induction of freezing resistance of the cuttings.

Effects of Cold Acclimation and CaCl_2 on Total Soluble Protein, CaM and Freezing Resistance of Populus tomentosa Seedlings

Populus tomentosa seedlings used for cold acclimation at -3℃ were pretreated with or without 10?mmol·L -1 CaCl 2, 3?mmol·L -1 of Ca 2+ chelator EGTA, 0 1?mmol·L -1 of Ca 2+ channel inhibitor LaCl 3,and 0 05?mmol·L -1 of CaM antagonist CPZ. The changes in the contents of total soluble protein and CaM, and freezing resistance in all pretreated seedlings in various periods ( viz: following cold acclimation, chilling stress and recovery) were investigated. Results showed that cold acclimation increased the contents of total soluble protein and CaM, and freezing resistance of seedlings, which could be strongly reduced by the pretreatments of EGTA CPZ and LaCl 3 Cold acclimation combined with CaCl 2 pretreatment enhanced the effect of cold acclimation on freezing resistance, and obviously increased the contents of total soluble protein and CaM, reduced the declining degree of the contents of total soluble protein and CaM caused by chilling stress as compared with cold acclimation, augmented the increase in the level of total soluble protein and CaM during the recovery periods. Further analysis found that an increase in total soluble protein content during cold acclimation with or without CaCl 2 pretreatment mainly resulted from the increase in content of heat stable protein in total soluble protein. It is suggested that Ca 2+ calmodulin may be involved in the synthesis of total soluble protein, and the induction of freezing resistance of seedlings.

Cold Acclimation-Induced Changes in Total Soluble Protein, RNA, DNA, RNase and Freezing Resistance in Populus tomentosa Cuttings

The changes in the contents of total soluble protein and RNA, the activity of RNase in leaves and branches of Populus tomentosa cuttings at various periods (viz: cold acclimation, deacclimation, chilling stress and the recovery after chilling stress), and the survival rate and the freezing resistance of cuttings during cold acclimation at -3℃ were investigated. Results showed that cold acclimation not only increased the contents of total soluble protein and RNA, the survival rates and the freezing resistance of cuttings, decreased the activity of RNase, but also reduced the declining degree of total soluble protein and RNA contents, and the increasing level of RNase caused by chilling stress as compared with the controls. In addition, cold acclimation augmented the increase in the level of total soluble protein and RNA, and facilitated the decrease of RNase during the recovery periods. Further analysis found that the DNA content of all treatments kept relative stability at various periods. The changes in total soluble protein, RNA and RNase were closely related to the freezing resistance of cuttings. It appears that the increase of RNA content caused by cold acclimation induced decrease of RNase activity may be involved in the accumulation of total soluble protein and the induction of freezing resistance of cuttings.

Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr

Acyl-ACP thioesterases （FATs） terminates the fatty acid synthesis and allow the transport of fatty acids out of the plastids, which are the important determinants of cellular metabolism. FATB is a member of FAT enzymes that has been described previously in most of the plants. In silico cloning is a new method that utilizes the bioinformatics on the complete genome and available EST database. In this study, a full-length cDNA clone of PtFATB gene was isolated from Populus tomentosa using this approach. It is 1,450 bp in length and the open reading frame encodes a peptide of 421 amino acids. The predicted amino acid sequence shows significant homology with those from other plant species, which contain typical domains owned by FATB proteins. The transcripts of PtFATB were abundant in leaves, and less in roots detected by using semiquantitative RT-PCR. When the shoots were subjected to the stress treatments （cold, dry, NaC1） and ABA （Abscisic acid）, the expression of PtFATB was only slightly reduced under the treatment of low temperature. This suggests that the expression of PtFATB is in a constitutive fashion. This study provides the basis not only for the identification and characterization of this gene but also for the improvement of cold tolerance by controlling the expression of the PtFATB gene in trees in near future.

Seasonal Changes in the Ultrastructure of the Vascular Cambium in Shoots of Populus tomentosa

Wood is the product of cambial activity in trees, and the seasonal activity style of cambium directly influences wood biomass production, structures and properties. The seasonal changes in the ultrastructure of the vascular cambium activity of Populus tonientosa Carr. planted in Beijing area were examined in shoot tissues collected during 15 months by means of transmission electron microscopy. Before xylem mother cells reactivated completely, the dividing fusiform cells in cambium and new phloem cells had appeared at the same time. The initiation of cambial activity may be related to the bud sprouting and the young leaf growth in shoots. More details about the ultrastructural changes of cambial cells at the onset of cambial activity have been gained. When the large vacuole in active cambial cells divided into smaller ones during the dormant phase, proteinaceous material that disappeared in active cambial cells refilled many of these small vactioles. In addition, lipid droplets and starch granules had the same cycles as proteinaceous material. The plasmalemma invaginations of fusiform cells were observed not only in active phase but also in dormancy. The endomembrane system consisting of nuclear membrane, endoplasmic reticulum (ER), dictyosomes and their secretory vesicles, changed in form and distribution at different phases during a cycle and performed important roles at the onset of active cambium and during the wall formation process of secondary xylem cells. The tangential walls remained relatively thin throughout the year but the radial walls thickened markedly when the cambium was dormant. During the transition from dormancy to activity, a partial autolysis occurred in the radial walls of the cambial cells, especially at the cell wall junctions. A notable feature of the cells at the onset of cambial activity was the thinning of the radial walls.

Effect of Sugar on the Process of Cold-Acclimation-Induced Freezing Tolerance of Populus tomentosa

Populus tomentosa seedlings for cold-acclimating were pretreated wit h or without 20% saccharose. Changes in the concentrations of total soluble sugar , the survival rates, and freezing tolerance of seedlings during cold acclimatio n were investigated. The results showed that cold acclimation increased the conc entrations of total soluble sugar, the survival rates and freezing tolerance. Co ld acclimation, combined with the saccharose-pretreatment, enhanced the above- ment ioned effect of cold acclimation, and obviously increased the concentrations of total soluble sugar, the survival rates and freezing tolerance of seedlings. Fur ther analysis found that the concentrations of total soluble sugar in branches i ncreased greater than that in leaves during both cold acclimation with or withou t the pretreatment of saccharose. Moreover, an increase of the concentrations of total soluble sugar in branches and leaves was closely related to the freezing tolerance of seedlings. The results indicate the accumulation of soluble sugar i n seedlings induced by cold acclimation may be involved in the induction of free zing tolerance .

Segregation of AFLP Markers in A ( Populus tomentosa× P. bolleana) ×P. tomentosa Carr. BC_1 Family

To investigate the levels of polymorphisms and Mendelian segregation ratio in clone “TB01” (P. tomentosa×P. bolleana) ×clone “LM50” (P. tomentosa) BC 1 population at the entire genome level, amplified fragment length polymorphisms (AFLPs) analysis was conducted for both parents and 120 progenies. Forty one pairs of selective primers were used to detect 2?707 bands, of which 712 (26.4%) were polymorphic. Chi\|square tests were performed to examine if the observed genotypic frequencies of AFLP loci deviated from expected 1∶1 Mendelian segregation ratio ( P <0.01) in BC 1 population. Among the 712 loci 571 (80.2%) fit to Mendelian 1∶1 segregation ratio, corresponding to DNA polymorphisms heterozygous in one parent and a null in the other. The result shows that the AFLP markers are very suitable for fingerprinting and genetic mapping in the Chinese white poplar (Populus tomentosa Carr.).

Genetic Control of Air-Dried Wood Density, Mechanical Properties and Its Implication for Veneer Timber Breeding of New Triploid Clones in Populus tomentosa Carr

The wood samples of 9 triploid clones of Populus tomentosa Carr. taken from a 9 year old clonal test site were analyzed in order to investigate the genetic variation of wood properties, including air dried wood density and some mechanical properties. The results showed that significant or extremely significant difference in air dried wood density and the mechanical properties existed among the clones, this means these wood properties were under moderate or strong genetic controls and could be improved by genetic manipulations. The radial and vertical variation patterns of air dried wood density were also studied and the results were found to coordinate with other previous research results. The vertical variation patterns of most mechanical properties within the individual tree also conformed to the general wood theories except the modulus of elasticity and cross section hardness. Among the mechanical properties, modulus of elasticity (MOE) and tangent section hardness were under strong genetic control, with the clonal repeatabilities being 0 90 and 0 80, respectively. However, the clonal repeatabilities of other mechanical properties under study were a little lower than above two indexes. Genetic correlation analysis indicated that super clonal selection and breeding for veneer timber could be realized through indirect selection of wood density and form indexes.

Selection of female parents with high fertility and high combining abilities for cross-breeding Populus tomentosa

To select maternal parents with high fertility and high combining ability during cross breeding of Populus tomentosa Carr., the ploidy level of 75 female clones was determined using flow cytometry. In addition, genetic variations and phenotypic correlations of seed traits and early growth traits, as well as the general combining ability(GCA) for seedling height(SH) and basal diameter(BD) of all diploid half-sib families were examined. A total of 26 natural triploid clones were identified. For all diploid families, family effects were significant for inflorescence length(IL), thousand kernel weight(TKW), seedling emergence rate(SER), and for SH and BD. There was a positive correlation between TKW and other traits,excluding IL. The seed-bearing coefficient(NS 9 SER)ranged from zero to 32.4%. Clones 3-10-2, 2-8, 3119,3206, and 3-10-1 had the best performance of the GCA for SH and BD. Based on the fertility and GCA for SH and BD, clone 3-10-2 and clone 3119 could be used as female parents for controlled cross-breeding of P. tomentosa.

In vitro regeneration of Populus tomentosa from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ＇fasta-3＇. A high frequency of callus induction （〉97 %） was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on 1/2MS basal medium supplemented with 0.25 mg/L BA, 0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and 0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/ L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.

Improvement in salt tolerance of Populus tomentosa from transformation by a mtl-D gene

Transgenic lines were achieved by transforming the E. coli 1-phosphate mannitol dehydrogenase gene （mtl-D） into the Populus tomentosa Cart. genome. An Agrobacterium tumefaciens strain （AGL1）, constructed by cloning mtl-D into the disarmed plasmid pBin438, was used to infect leaves of the clone YW2. The infected leaf discs were cultured on a medium containing 30 mg.L 1 kanamycin and 500 mg.L 1 cefotaxime. Transgenic plantlets regenerated from the infected leaves, rooted on the medium con- taining 30 mg.L 1 kanamycin. PCR and a Southern blotting test verified that the exogenous mtl-D gene had integrated into the trans- formation plants of the P. tomentosa genome. The mannitol content in control plant was 69 gg.gl FW, and the mannitol contents of the transgenic lines T1 to T5 ranged between 103.7 and 289.5 μg·g^-1 FW. Of the shoots of the control plants 20% survived; on the medium containing 0.6% NaCl, 60% and 70% of two transgenic shoots survived on a medium containing 0.8% NaCI.

Physiological, histopathological and cellular immune effects of Pergularia tomentosa extract on Locusta migratoria nymphs

The migratory locust Locusta migratoria(Orthoptera,Acrididae)is one of the most important pests due to its extensive and severe damage to crops in large parts of Africa and Asia.Biodegradable and ecologically natural products such as bota nical insecticides are emergi ng can didates for replaceme nt of usually applied chemical pesticides.The crude methanolic extract of Pergularia tomentosa(PME)was investigated for their toxicity and physiological aspects on L.migratoria nymphs.Results showed that treatment of newly emerged fourth and fifth instar nymphs resulted in significant mortality and significant repellent activity with an LC50 value of 0.18 and 0.38%,respectively,after seven days of treatment.The PME toxicity was also dem on strated by histopathological cha nges in the alime ntary canal resulting in con siderable disorganization and severe damage of the caeca and proventriculus structure.The extract induced cellular immune reactions which manifested by a significant decrease in the number of the differential haemocyte counts(prohemocytes and plasmatocytes)and important cell lysis.Data of biochemical analyses showed that the PME reduced the activity of acetylcholinesterase and induced the glutathione S-transferases.The neurotoxic effect was confirmed by the histological alterations in the brain structure,particularly in the neurosecretory cells showing typical signs of cell necrosis.

Genome-wide search for segregation distortion loci associated with the expression of complex traits in Populus tomentosa

Segregation distortion of molecular markers has been reported in a broad range of organisms. It has been detected in an interspecific BC1 Populus pedigree established by controlled crossing between clone ＂LM50＂ （Populus tomentosa） and its hybrid clone ＂TB01＂ （P. tomentosa × p. bolleana）. The study with a total of 150 AFLP markers （approximately 18.9% of the total loci） exhibited significant deviation from the Mendelian ratio （1：1） （p〈0.01）. Twenty-five percent of the markers were mapped on the parental specific genetic linkage maps of clones ＂LM50＂ and ＂TB01＂ with a pseudo-test-cross mapping strategy. Twelve linkage groups had markers with skewed segregation ratios, but the major regions were on linkage groups TLG2, TLG4 and TLG6 in the linkage map of clone ＂LM50＂. We also analyzed the association between distorted loci and expression of complex traits with Mapmaker/QTL software. A total of 16 putative QTLs affecting 12 traits were identified in the distorted regions on seven linkage groups. Therefore we could detect the distribution of skewed loci along the entire genome and identify the association between quantitative traits and segregation loci via genetic mapping in an interspecific BC1 P. tomentosa family. Furthermore, the genetic nature and possible causes of these segregation distortions for differentiation between female and male parents were also discussed.

大火草(Anemone tomentosa)形态解剖的研究

为进一步保护和利用野生花卉,丰富大火草(Anemone tomentosa)的基础资料,试验对引种栽培的大火草进行了形态学方面的研究。在光学显微镜和扫描电镜下对其叶片解剖结构和种子形态进行了观察。结果表明,大火草的叶片为典型的异面叶,由表皮、叶肉和叶脉三部分组成。上表皮细胞为不规则型,垂周壁成浅波浪状;下表皮细胞不规则型,垂周壁浅波浪状,平周壁平滑。上下表皮均被毛。气孔器在下表皮有分布,为不规则型;保卫细胞肾形,不具副卫细胞。其果实为聚合瘦果,球形;种子小,褐色椭圆形,有细柄,密被棉毛。大火草种子的千粒重为0.3163 g。

Isolation, Crystal Structure, and Anti-inflammatory Activity of Sakuranetin from Populus tomentosa

The title compound of sakuranetin, a main active flavanone component, was first isolated from Populus tomentosa Carr. and characterized by X-ray single-crystal diffraction analysis It crystallizes in the monoclinic lattice, space group P21/c with a = 12.8531（12）, b = 5.7141（3）, c = 18.0355（12） A, β = 97.333（8）°, V = 1313.77（16） A^3, Z= 4, Mr = 286.27, C16H14O5,μ（MoKa） = 0.108 mm^-1, Dc = 1.447 g/cm^3, F（000） = 600, the final R = 0.0350 and wR = 0.0859 for 2571 independent reflections （Rint = 0.0246） which were used in all calculations. The molecular crystal structure of sakuranetin shows relative stereochemistry of 5,4′-dihydroxy-7-methoxyflavanone. Intermolecular hydrogen bonds together with the continuous π-π interactions construct the three-dimensional architecture of the title compound. The preliminary bioassay reveals that the title compound exhibits moderate anti-inflammatory activity in vitro against the nitric oxide release.

Cloning and RNAi Construction of a LEAFY Homologous Gene from Populus tomentosa and Preliminary Study in Tobacco

PtLFY, a LEAFY （LFY） gene, was cloned from Populus tomentosa （LM50） by PCR. Sequencing analysis indicated that PtLFY was 2629 bp long, composed of three exons and two introns and encoded 378 amino acids. The splice donor sites and the splice acceptor sites were in identical positions to the LFY and its homologues. The amino acid sequence inferred was 68%-99% homologous to those of LFY and its homologues by blast analysis in GenBank. The Southern blot analysis indicated that there was a single copy of the PtLFY gene in genomic DNA of male and female P. tomentosa （LM50 and 5082）. The pBI121-Ptalfy （reverse）-intron-Ptlfy-GUS-nos was constructed using RNA interference （RNAi） technique and verified by PCR and digestion identification and transformed into tobacco. Some transgenic tobacco plants were obtained by PCR and PCR-Southern identification. The growth was generally repressed in transgenic tobacco plants compared with wild-type ones and some phenotypic differences were observed.