Analysis of three types of triterpenoids in tetraploid white birches(Betula platyphylla Suk.) and selection of plus trees

Betulin, oleanolic acid, and betulinic acid are naturally occurring pentacyclic triterpenoids that have significant medicinal value. Considerable amounts of these triterpenoids are available in the outer bark of white birch. In this study, we used ultrasound-assisted extraction （UAE） to extract triterpenoids from birch bark rapidly and with high efficiency. Using high performance liquid chro- matography （HPLC）, three types of triterpenoids were separated and detected. We examined the differences among triterpenoids extracted from diploid versus tetra- ploid white birch. Then, we used factor analysis to screen out tetraploid white birches with comprehensively excel- lent performance. The results indicate that the optimum conditions for extraction include the use of ethanol as an extraction solvent, a solid-to-liquid ratio of 0.1 g/10 ml, ultrasonic power set at 100 W, a temperature of 60 ℃ and an extraction time of 15 min. A reversed-phase C18 col- umn （4.6 mm × 250 mm × 5 μm） with a column tem- perature of 30 ℃ and the mobile phase composed of A （acetonitrile） and B （0.1% aqueous phosphoric acid, v/v） at a flow rate of 0.5 ml/min were used, and the detection wavelength was 195 nm. No significant difference wasobserved between diploid and tetraploid white birch in terms of the content of three types of triterpenoids （at a confidence level of 0.05）. As triterpenoid content, height, and DBH （diameter at breast height） are strongly interre- lated, we used factor analysis to evaluate all individuals, and we screened out six plus trees with excellent com- prehensive characters.

Effect of MeJA and Light on the Accumulation of Betulin and Oleanolic Acid in the Saplings of White Birch (<i>Betula platyphylla</i>Suk.)

In this study, we investigated the effect of different types of light and MeJA treatment on the accumulation of betulin and oleanolic acid in various organs of white birch. Our results showed that betulin and oleanolic were accumulated mainly in the stalk skin. The content of both substances in the stalk skin was significantly affected by seasons with a peak accumulation in August. The content of oleanolic and betulin was significantly decreased in the stem skin treated with 4 types of light (red, yellow, blue and green) compared with the plant with normal illumination. In contrast, oleanolic acid in leaves was increased by 13.28 folds when the white birch was treated with green light. Betulin was increased by 1.959 folds in leaves of white birch treated with blue light. The highest content of betulin and oleanolic acid in various organs of birch with appropriate shading treatment (light transmittance: 50%) was increased by 45.09% and 30.50%, respectively, in comparison with those with non-shading treatment. Content of oleanolic acid and betulin can be significantly improved in various parts of birch after treatment with different concentration of MeJA. The study lays the foundation to metabolic regulation of oleanolic acid and betulin in birch.

Comparison, Selection and Progeny Test of Natural Populations of White Birch (Betula platyphylla Suk.) in Northern China

[Objectives]This study was conducted to rationally and effectively use natural genetic resources of white birch(Betula platyphylla Suk.),and improve the scientific and technological content of improved varieties.[Methods]With the progeny of the white birch populations naturally distributed in northern China as objects,based on genetic effect analysis and comprehensive evaluation,the genetic variation,heritability and genetic gain of the growth characters in the progeny forest were systematically analyzed,by which populations or individuals with good growth vigor and strong stress resistance and adaptability were finally selected.[Results]There were extremely significant differences in the growth characters between the natural populations of white birch in northern China.The variation was the smallest in tree height,followed by crown width,and largest in DBH,and the average coefficients of variation were 20.56%,32.25%and 33.00%,respectively.The differences in characters between progeny of the same population were also extremely significant.The growth characters of white birch were more controlled by genes,and the genetic gains were larger.The heritability of tree height,DBH and crown width was 0.971,0.816 and 0.576,respectively,and the genetic gains were 15.97%,21.54%and 13.87%,respectively.[Conclusions]With the growth characters as the main evaluation indexes,taking various influencing factors into account,the Shanxi[SX]population,Hebei[HB]population and Liaoning[LN]population were selected to be excellent populations,and LN305,SX516 and HB403 were excellent individuals,which are advised to be widely used in multi-generation genetic improvement breeding.

A study on photosynthetic characteristics of Betula platyphylla

Influences of temperature, humidity, and CO2 concentration on the photosynthesis and respiration of three-year-oldBetula platyphylla was investigated. Light compensation point, saturation point and CO2 compensation point were also determined. The results showed that the optimal temperature of photosynthesis and dark respiration was 24 °C and 30 °C, respectively, at ambient CO2. When relative humidity was 80%,Betula platyphylla could maintain strong photosynthesis. There was no significant correlation between respiration and relative humidity. The light compensation and saturation point was 25 μmol·m?2·s?1 and 1 375 μmol·m?2·s?1, respectively. The CO2 compensation point was 180 μL·L?1. The results showed thatBetula platyphylla still had potential to assimilate CO2 when CO2 concentration was above 2 400 μL·L?1.

干旱胁迫下白桦(Betula platyphylla)实生苗叶片的水分代谢与部分渗透调节物质的变化

在土壤干旱胁迫进程中 ,白桦实生苗叶片的自然含水量呈下降趋势 ;相对含水量总的变化趋势与自然含水量相同 ;束缚水含量及束缚水 /自由水比值显著升高 ,复水后 ,三者又重新恢复至正常水平。随着土壤水分逐渐减少 ,可溶性蛋白质的含量呈现减少趋势 ,复水后 ,可溶性蛋白质含量又可恢复至正常水平。白桦实生苗叶片可溶性糖含量增加 。

The effect of exogenous sugar solution and high concentration of CO_2 on the contents of sugar and protein of Betula platyphylla leaves

The content of total sugar, sucrose, fructose and protein in the leaves of3-yr.-old Betula platyphylla was measured after the treatment by three exogenous sugar solutions(sucrose, fructose, glucose) and three high concentrations of CO_2 (700, 1 400, 2 100 μL/L·L^(-1))for about a month in 1998. The results showed that spraying three exogenous sugar solutionsincreased markedly the content of sugar and protein of leaves under 700 μL·L^(-1) and 1 400μL·L^(-1) CO_2 The effect of spraying exogenous sucrose solution was the best among the threeexogenous sugars. The treatment of spraying exogenous sugar solution and 2 100 μL·L^(-1) CO_2constrained the accumulation of total sugar and protein of leaves. There was no difference inprotein content of leaves when spraying glucose and fructose solutions under 700 μL·L^(-1) and 1400 μL·L^(-1) CO_2. The treatment of 2 100 μL·L^(-1) CO_2 concentration significantly increasedthe contents of total sugar, sucrose, fructose, and protein of leaves compared with that of the 700μL·L^(-1) and 1 400 μL·L^(-1) CO_2 except the plants spraying fructose solution. There waspositive correlation between the content of sugar of leaves and CO_2 concentration when sprayingsame exogenous sugar solution.

Progeny test of tetraploid Betula platyphylla and preliminary selection of hybrid parents

Estimating genetic parameters of parental lines through progeny testing and choosing good hybrid parents are important for genetically improving seed orchard trees.In this study, 24 tetraploid progeny seedlings were used as experimental materials, which came from test cross design：six tetraploid Betula platyphylla lines（Q33, Q13, Q103,Q19, Q83 and Q14） as female parents and four individual B. platyphylla diploid lines（F3, F4, F9 and F11） as male parents were crossed. Variance analysis of height, diameter, height-to-diameter ratio, and internodal distance showed that the differences between hybrid combinations reached highly significant levels. Using multi-objective decisionmaking, we performed a comprehensive assessment of the various hybrid combinations. Using a selection rate of 20 % of the standard, five hybrids were selected;their genetic gains in average height, diameter, height-todiameter ratio, and internodal distance were 20.95, 6.07,13.07 and 8.96 %, respectively. We also analyzed the combining ability and genetic parameter effect values of parents and hybrid combinations. The combined analysis revealed that Q13, Q103, Q33 and Q83 were superior females; F3, F4, and F9 were superior males; and F3 × Q13, F4 × Q83 and F9 × Q33 were superior hybrid combinations. The heights and diameters of these progenies were 22.49 and 11.48 % greater than average,respectively.

Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence

Female inflorescence of Betula platyphylla was sampled at an interval of eachtwo days to analyze the background of gene expression in floral phase. On the basis of SMARTstrategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA wasfrom that of the others by RT-PCR which were subsequently used to construct a subtracted cDNAlibrary. The result of the ESTs (expression sequence tags) blastX showed that the genes in thesubtracted cDNA library could be mainly clustered into 5 groups related to metabolism,transportation and signal transduction, cell cycle, stress response, and regulation. Therelationship between gene expression and development was also discussed.

Identification of SSR loci in Betula luminifera using birch EST data

Expressed sequence tags（ESTs） are generated from single-pass sequencing of randomly picked cDNA clones and can be used for development of simple sequence repeat（SSR） markers or microsatellites.However,EST databases have been developed for only a small number of species.This paper provides a case study of the utility of freely available birch EST resources for the development of markers necessary for the genetic analysis of Betula luminifera.Based on birch EST data,primers for 80 EST-SSR candidate loci were developed and tested in birch.Of these,59 EST-SSR loci yielded single,stable and clear PCR products.We then tested the utility of those 59 markers in B.luminifera.The results showed 28（47.6%） yielded stable and clear PCR products for at least one B.luminifera genotype.In addition,this study describes a rapid and inexpensive alternative for the development of SSRs in species with scarce available sequence data.

A New Caffeoylquiniclactone, Neochlorgeniclatone from the Leaves of Betula platyphylla Suk.

A new caffeoylquiniclactone, named neochlorgeniclatone, was isolated from the leaves of Betula platyphylla Suk. The Structure was established by spectroscopic data.

A New Monoterpene Glucoside from the Leaves of Betula platyphylla Suk.

A new monoterpene glucoside. (2E,67.)-2.6-dimethyl-8-beta -D-glucosyloxy-2,6-octadienoic acid, was isolated from the leaves of Betula platyphylla Suk. The structure was established by spectroscopic data.

Plant hormone treatments to alleviate the effects of salt stress on germination of Betula platyphylla seeds

We analyzed the germination traits (germination rate, germination potential, and germination index) of seeds from 23 half-sib families of birch (Betula platyphylla). The germination rates of the 23 families ranged from 38.57 to 76.50%. Seeds from the eight families germinating at the highest rates were then used for germination experiments under salt stress. Germination rate, germination potential, and germination index of these eight half-sib families decreased with increasing salt concentrations. The effects of four different hormones (6-benzylaminopurine, 6BA;gibberellin, GA3;naphthalene acetic acid, NAA;and melatonin) at various concentrations on the germination of seeds from the eight half-sib birch families under salt stress were investigated. Treatments with 6BA and GA3 alleviated the effects of salt stress on seed germination, whereas treatments with NAA and melatonin aggravated the effects of salt stress. The most effective treatments for alleviating the effects of salt stress on birch seed germination were 10 mg/L 6BA and 300 mg/L GA3. This study provides practical information for screening birch families for salt tolerance at the seed germination stage, and for cultivation of birch in saline environments.

BpMADS12 gene role in lignin biosynthesis of Betula platyphylla Suk by transcriptome analysis

MADS-box transcription factors show highly diverse regulatory functions in a wide variety of organisms. In this study, we characterized a MADS-box gene （BpMADS12） from the white birch （Betula platyphylla Suk）. This gene is a member of the suppressor of overexpression of CO 1/tomato MADS 3 class of MADS-box genes. We generated lines overexpressing BpMADS12 and found that these had higher levels of lignin compared to that observed in nontransgenic lines. Transcriptome anal- ysis revealed numerous changes in gene expression patterns. In total, 8794 differentially expressed genes were identified, including 5006 upregulated unigenes and 3788 downregulated unigenes in BpMADS-overexpression lines. Differentially expressed genes involved in the pathways for lignin and brassinosteroid biosynthesis were significantly enriched and may have contributed to phenotypic changes. The results from a quantitative RT-PCR analysis were consistent those obtained with the transcriptome analysis.Our transcriptome analysis, in combination with measure- ment of lignin level, indicated that BpMADS12 promotes lignin synthesis through regulation of key enzymes in response to brassinosteroid signaling. These results suggest that this MADS-box protein is crucial to all subsequent structural events and provide a good foundation for studies aiming to elucidate the developmental mechanisms underlying formation of wood.

Hydrochemical Characteristics and Ion Transfer of Rainfall of Betula Platyphylla Secondary Forest in Xiaowutai Mountain

In order to study hydrochemical characteristics and ion transfer of annual rainfall of Betula platyphylla secondary forest,water samples of rainfall,throughfall,stemflow,litter infiltration water and slope runoff of Betula platyphylla secondary forest in the early growth stage,fastgrowing stage and growth decline stage in Shanjiankou watershed of Xiaowutai Mountain and nine indexes of pH,Ca^2+,Mg^2+,K+,Na+,Zn^2+,Mn^2+,Fe^2+ and Cu^2+ were determined.The results showed that:①The annual rainfall was weakly alkaline,and the ion concentration in different stages was obviously different.According to the ratio of the maximum concentration to the minimum concentration of the same ion,the rank was Mg^2+ > Mn^2+ >Na+ > K+ > Ca^2+ > Cu^2+ > Fe^2+ > Zn^2+.②Compared with the rainfall in the same period,in the early growth stage,Mg^2+,Mn^2+,Na+,K+,Ca^2+,Fe^2+ and Zn^2+ showed leaching loss in each distribution process.In the fastgrowing stage,Ca^2+,Mn^2+ and Fe^2+ showed leaching loss in each distribution process;Mg^2+ showed absorption,Na+ showed leaching loss in slope runoff and showed absorption in other process.K+ showed leaching loss in stemflow and showed absorption in other process.Zn^2+ showed absorption in stemflow and slope runoff and showed a little leaching loss in other process.In the late growth stage,Ca^2+,Mg^2+,Zn^2+,Mn^2+ and Fe^2+ showed leaching loss in each distribution process;Na+ and Cu^2+ showed absorption;K+ showed absorption in stemflow and showed leaching loss in other process.Most of the rainfall and metal ions in Betula platyphylla secondary forest were intercepted by litter layer.

Expression profiles of genes regulated by BplMYB46 in Betula platyphylla

The transcription factor BplMYB46 has been identified as a regulator of abiotic stress responses and promoter of secondary wall deposition in Betula platyphylla.To investigate the downstream targets of BplMYB46,the expression profiles of genes in stems from BplMYB46-overexpressing(OE)and BplMYB46-silencing(SE)plants were studied.In OE stems,952 genes were upregulated,and 1469 were downregulated in comparison to SE stems.In a KEGG pathway enrichment analysis of differentially expressed genes(DEGs),1387 differentially expressed genes were annotated for 117 metabolic pathways.DEGs were abundant for metabolic pathway,secondary metabolite biosynthesis,plant hormone signal transduction,phenylpropanoid and flavonoid biosynthesis.DEGs were implicated lignin or cellulose biosynthesis,cell wall modification,xylem development,disease resistance,stress responses,and anthocyanin biosynthesis.These results suggested that BplMYB46 regulates cell wall development and stress resistance by affecting the expression of these genes.Our study further elucidates the mechanism by which BplMYB46 mediates abiotic stress responses and secondary cell wall biosynthesis in birch.

Expression profiling of the BpIAA gene family and the determination of IAA levels in Betula platyphylla tetraploids

Betula platyphylla Sukaczev tetraploids have significantly larger leaf, fruit and stoma (gigantic phenotype) than diploids of the same species;however, the mechanism underlying this difference remains unclear. Tetraploid B. platyphylla transcriptome data have indicated that the expression of genes related to indole-3-acetic acid (IAA) biosynthesis and signal transduction was altered after genome duplication. IAA exerts pleiotropic effects on growth and development by inducing the expression of Aux/IAA. We identified 20 Aux/IAA genes (BpIAA1– BpIAA20) in B. platyphylla distributed across 10 chromosomes. Multiple alignment and motif analyses revealed that nine BpIAA proteins shared all four conserved domains. Phylogenetic analysis indicated that Aux/IAA families were divided into four subfamilies and that there were two pairs of BpIAA sister genes. The BpIAAs were differentially expressed in diploids and tetraploids. Moreover, the expression levels of the nine BpIAA genes were specifically up-regulated in tetraploids from June to September compared with May (except August 5th) in tetraploids, while they were down-regulated in diploids. IAA levels were more than twofold higher in tetraploids than diploids during the vegetative season. These results indicate that genome duplication of B. platyphylla caused the up-regulated of genes involved in IAA synthesis, and the increased concentration of IAA may induce the constitutive expression of 20 BpIAA genes. Therefore, the significant changes in the expression patterns of the BpIAAs contributed to the gigantic phenotype of tetraploids to some extent. Our research sheds light on the phenotypic variations observed in B. platyphylla tetraploids.

A phytosociological study on Betula Platyphylla forestsin Daxing'an Mountains of China

For the first time in this paper the forests of Betula platyphylla in Daxing’an Mountains are Subdivided by phytosociological methods. Three community types of B. platyphylla forest have been differentiated, e.g., Rhododendron dahuricum-Betula platyphylla Corylus, Corylus heterophylla-Betula platyphylla community and Arternisia stolonifera-Betula platyphylla community. The distributed elevation, stand height, differential species, major composition species of tree layer, shrub layer and herb layer for each community were detail

Growth Variation and Early Selection of Betula Platyphylla in Natural Stands

The stem analysis of Betula platyphylla in natural forest at the Maoershan Forest Experimental Station of Northeast Forestry Universitu. showed that the growth variation of the trees. including height and diameter breast height decreaseswith the increase of the age. There is the turning point for the growth variation of the tree from acute change to relative sta-bility when the trees are in eighteen years old. There are signiticant difttrences among the height. diameter and volumegrowth of the trees at that time. Theretbre. the optimal age for carly seleetion of this species in natural stands is eighteenyears old (18 a). Diameter is used as main index for eary seleclion and height growth as auxiliary one.

Analysis of EST-SSRs in silver birch(Betula pendula Roth.)

Simple sequence repeats（SSRs） defined as sequence repeat units between 1 and 6 bp occur abundantly in both coding and non-coding regions in eukaryotic genomes and these repeats can affect gene expression. In this study, ESTs（expressed sequence tags） of Betula pendula（silver birch） were analyzed for in silico mining of ESTSSRs, protein annotation, open reading frames（ORFs）,designing primers, and identifying codon repetitions. In B.pendula, the frequency of ESTs containing SSRs was 7.8 %with an average of 1SSR/4. 78 kb of EST sequences. A total of 188 SSRs was identified by using MISA software and dinucleotide SSR motifs（65.9 %） were found to be the most abundant type of repeat motif followed by tri-（27.1 %）,tetra-（4.8 %）, and penta-（2.2 %） motifs. Based on ORF analysis, 175 of 178 sequences were predicted as ORFs and the most frequent SSRs were detected in 50 UTR（58.43 %）,followed by in ORF（31.46 %） and in 30UTR（8.43 %）. 102 of 178 ESTs were annotated as ribosomal protein, transport protein, membrane protein, carrier protein, binding protein,and transferase protein. For a total of 102 SSRs（57.3 %）with significant matches, a set of 102 primers（100 %） with forward and reverse strands was designed by using Primer 3 software. Serine（Ser, 19.6 %） was predominant in putative encoded amino acids and most of amino acids showed nonpolar（35.3 %） nature. Our data provide resources for B.pendula and can be useful for in silico comparative analyses of Betulaceae species, including SSR mining.

Selection of elite lines of BpGH3.5-transgenic Betula platyphylla using growth adaptability analysis

White birch(Betula platyphylla)is precious material for pulpwood and widely distributed in 14 provinces of China.Previous study indicated that inhibited expression of a gene encoding an auxin amide synthase,BpGH3.5,in transgenic plants reduced the level of IAA–amino acid conjugation,resulting in more free IAA,thereby better growth of birch.Utilizing transgenic-B pGH3.5 lines to increase wood production in a wide range of environments is the goal for breeders.In three field trials here,we measured tree height,diameter at breast height,and volume of 16 BpGH3.5-transgenic 7-year-old white birch lines(including 12 antisense strand lines and 4 overexpression lines)and a wild-type white birch line from three sites that varied greatly in their environmental conditions.To select elite BpGH3.5-transgenic lines for each target environment,we used an additive main effects and multiplicative interaction model to analyze genotype by environment interaction,growth adaptability and stability.The selection criteria for elite transgenic lines were set as the average volume plus 0.75 times the standard deviation for the tested lines at each test site.Results showed that the effect of line and site for height was highly significant(P<0.01),and the effect of line×site was significant(P<0.05);selected as the elite lines were FG12,FG13 and FG27 at the Maoershan Experimental Forest Farm,FG13 and FG32 at the Shidaohe Forest Farm,and FG3 and FG31 at the Ecological Experiment Forest Farm.These seven high-yield,stable lines can now be tested in production trials or adjacent trial areas with similar environmental conditions,while the high-yield,unstable lines should be tested in production trials in areas deemed suitable for their growth.These results provide guidance on which released transgenic elite lines will grow best in a wide range of conditions.