Analysis of Methylated Patterns and Quality-Related Genes in Tobacco (Nicotiana tabacum) Cultivars

Methylation-sensitive amplified polymorphism was used in this study to investigate epigenetic information of four tobacco cultivars: Yunyan 85, NC89, K326, and Yunyan 87. The DNA fragments with methylated information were cloned by reamplified PCR and sequenced. The results of Blast alignments showed that the genes with methylation information included chitinase, nitrate reductase, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase, ribulose carboxylase, and promoter sequences. Homologous comparison in three cloned gene sequences (nitrate reductase, ornithine decarboxylase, and ribulose decarboxylase) indicated that geographic factors had significant influence on the whole genome methylation. Introns also contained different information in different tobacco cultivars. These findings suggest that synthetic mechanisms for tobacco aromatic components could be affected by different environmental factors leading to variation of noncoding regions in the genome, which finally results in different fragrance and taste in different tobacco cultivars.     

B型烟粉虱在6个烟草品种上的取食行为

【背景】不同烟草品种会影响B型烟粉虱的取食选择性,但具体的机制尚不明确。刺吸电波图（EPG）技术是研究刺吸式昆虫对寄主植物取食选择性的重要手段,通过烟粉虱取食产生的电信号,可以分析其对不同烟草品种的取食差异。【方法】应用EPG技术对B型烟粉虱雌成虫在6个烟草品种（翠碧1号、K326、闽烟7号、云烟85、云烟87、红花大金元）上的取食行为进行比较研究。【结果】6个烟草品种上有效记录的雌成虫数均为18头,在翠碧1号、K326、闽烟7号、云烟87、云烟85和红花大金元叶片上能够到达韧皮部的个体数分别为15、15、12、13、15和15头,能够持续吸食的个体数分别为13、14、8、12、14和15头。烟粉虱雌成虫在6个烟草品种上的非刺探（np）波所占的比例差异显著,在红花大金元上所占的比例最小,显著低于其他5个品种。雌成虫在红花大金元和K326上E1波的总持续时间显著短于闽烟7号和云烟87。雌成虫在红花大金元上E2波的总持续时间显著长于其他5个品种,而在闽烟7号、云烟87和云烟85上显著短于其他3个品种。【结论与意义】在6个烟草品种中,B型烟粉虱雌成虫最喜食红花大金元,最不喜食闽烟7号和云烟87。因此,在福建省B型烟粉虱的发生区,选择烟草品种时可根据实际情况减少红花大金元的种植,增多闽烟7号和云烟87等抗B型烟粉虱品种的种植。     

烟草内生细菌种群动态研究

经过对 7个田间种植的烟草品种不同栽培时期、不同组织内生细菌种群动态研究表明 ,不同品种内生细菌种群有一定程度差异。同一品种中有的内生细菌为常住菌群 ,有的为暂居菌群 ,带菌量根中最高 ,茎次之 ,叶中最低。在整个生育期中 ,7个品种内生细菌数量表现出从种子到出苗期大幅增加 ,从出苗期到十字期又大幅度下降 ,随后从缓苗期到伸根期再一次急剧增加并维持在一个较高水平。通过对烟草这一重要经济作物内生细菌种群动态变化研究 ,可为烟草病虫害生物防治和促生增产研究提供理论基础。     

Musa methylated DNA sequences associated with tolerance toMycosphaerella fijiensis toxins

DNA methylation is an epigenetic phenomenon associated with gene silencing in transgenic plants, retrotransposons and virus infection. Expression analysis of specific genes in Arabidopsis methylation mutants showed an association between DNA methylation and gene expression. To determine whether DNA methylation is associated with resistance to black Sigatoka (BS) andMycosphaerella fijiensis (MF), we used anin vitro assay of mesophyll cell suspensions of reference cultivars with known resistance to BS. Methylation of CC^mGG sequences was evaluated by methylation-sensitive amplification polymorphism (MSAP) markers of reference cultivars and somaclonal variants to identify molecular markers associated with resistance to MF toxins and BS. Four MSAP markers were associated with resistance (MAR) to MF toxins. The MSAP markers show a high degree of sequence similarity with resistance gene analog and with retrotransposon sequences. The MSAP markers are useful as molecular indicators of tolerance to MF toxins and resistance to BS.     

烟草种子内生细菌群落结构与多样性

【目的】为了解烟草种子内生细菌的群落结构和多样性。【方法】分别对3个品种(K326、云烟85、云烟87)烟草种子内生细菌的16S rRNA基因V3–V4区进行扩增,采用Illumina MiSeq测序技术对扩增片段进行高通量测序,并对3个品种烟草种子内生细菌群落结构和多样性进行分析。【结果】3个品种种子共获得的V3–V4区高质量序列片段128558条,Shannon指数计算为2.03–3.73,K326和云烟85内生菌多样性指数高于云烟87。3品种烟草种子内生细菌的优势门均为变形菌门Proteobacteria、放线菌门Actinobacteria、厚壁菌门Firmicute和拟杆菌门Bacteroidete。3个品种烟草种子内生细菌共有菌属共有27个,K326和云烟85的最优势菌属为假单胞菌属(Pseudomonas),云烟87的最优势菌属为大肠杆菌志贺菌属(Escherichia-shigella)。16S功能预测显示各种子中产生了丰度较高的蛋白质、核苷酸、糖类、辅酶及代谢产物合成的有益功能信息。【结论】烟草种子内生细菌多样性丰富,不同品种种子细菌群落组成基本相似,其丰度存在一定差异性。种子中存在的潜在有益细菌包括假单胞菌、类芽孢杆菌、根瘤菌、马赛菌、藤黄单胞菌、萨勒河菌、Lelliottia菌等,具有大量代谢相关的有益功能。研究结果为今后烟草种子内生菌的功能研究和利用以及种子病害生物防控提供参考信息。     

Surveying CpG methylation at 5′-CCGG in the genomes of rice cultivars

We investigated the CpG methylation status of the sequence CCGG in the rice genome by using methylation-sensitive AFLP and subsequent Southern analyses with the isolated AFLP fragments as probes. CpGs located in single- or low-copy-sequence regions could be grouped into two classes on the basis of their methylation status: methylation status at the class 1 CpG sites was conserved among genetically diverse rice cultivars, whereas cultivar-specific differential methylation was frequently detected among the cultivars at the class 2 CpG sites. The frequency of occurrence of methylation polymorphism between a pair of cultivars was not related to the genetic distance between the two. Through mapping, five class 2 CpG sites were localized on different chromosomes and were not clustered together in the genome. Segregation analysis of the cultivar-specific methylations with their target sites indicated that the differential methylation was stably inherited in a Mendelian fashion over 6 generations, although alterations in the methylation status at the class 2 CpG sites were observed with a low frequency.     

Newly developed MSAP analysis reveals the different polymorphism patterns in transgenic tobacco plants with the dsRNA MET1 gene

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we isolated NtMET1 from Nicotiana tabacum cv. Havana (SR1) and obtain transgenic plants that reduced MET1 expression level with the double-strand RNA (dsRNA) MET1 gene. Transgenic tobacco plants showed dwarf and abnormal flower development when compared with the wild type. Using methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in transformed plants and the wild type were compared. MseI/HpaII selection primers showed an interesting polymorphism, and 153 DNA bands of interest were detected. Among these, 30 selective fragments were sequenced and analyzed with a BLAST search by successful MSAP modifications. The homology search showed that the transposons and tandem repeated sequences were related to the phenotypes. These results suggested that the decreased degree of methylation by dsRNA strategy caused abnormal growth and development in N. tabacum.     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.     

干旱胁迫对烟草两品种幼苗生理生化特性及NtDEGP5基因表达的影响

以烟草Nicotianatabacum抗旱品种NC82和干旱敏感品种云烟87幼苗为材料,利用PEG-6000对幼苗进行干旱胁迫处理,研究干旱胁迫对烟草不同品种Fv/Fm、SOD活性、POD活性、MDA含量等生理生化指标及NtDEGP5基因表达的影响,为进一步开展NtDEGP5基因的功能研究提供依据。结果表明,干旱胁迫对烟苗的Fv/Fm、SOD活性、POD活性、MDA含量有影响,随着干旱的持续,Fv/Fm呈逐渐下降趋势,POD、SOD活性和MDA含量呈先升高后降低的趋势;不同品种对干旱胁迫的响应有差异,在干旱胁迫0～9 h,云烟87幼苗的Fv/Fm降幅大于NC82,POD、SOD活性、MDA含量的增幅小于NC82,胁迫12h后与对照差异不显著。干旱胁迫下不同品种、不同器官间的Nt DEGP5基因表达有差异,云烟87幼苗根、茎、叶中的表达量均较低,NC82幼苗根中的表达量较低,但干旱胁迫9～12 h后叶和茎的表达量较高。NC82幼苗叶片NtDEGP5基因表达量与Fv/Fm、POD活性、MDA含量呈显著正相关。Fv/Fm、SOD活性、POD活性、MDA含量可作为烟草苗期抗旱性评价指标,NtD...     

MSAP-based analysis of DNA methylation diversity in tobacco exposed to different environments and at different development phases

The DNA methylation phenomenon, which plays an important role in the regulation of gene expression, exists in most eukaryotes. In this study, we employed methylation-sensitive amplification polymorphism (MSAP) technology to investigate the extent and patterns of methylation in tobacco planted in five different areas of Dali County, Yunnan Province. All of the samples were collected during the vegetative and reproductive growth phases. The data obtained from the MSAP analysis showed that full-methylation and non-methylation modifications were predominant and that the hemi-methylation modification ratio was markedly lower. The PCA and UPGMA analysis results indicated that the samples collected from different areas during the vegetative and reproductive growth phases exhibit certain differences: development had a more significant impact on the methylation diversity than environmental factors, although the samples collected at the same growth stages showed that the geographical environment may also affect the methylation status. The sequencing and BLASTn analyses indicated that most of the bands that showed significant polymorphisms are highly homologous to some methylation-sensitive-related sequences discovered in tobacco or other plant species and that many of these sequences are closely related to growth regulation or environmental factors.     

Comparative Analysis of DNA Methylation Polymorphism in Drought Sensitive (HPKC2) and Tolerant (HPK4) Genotypes of Horse Gram (Macrotyloma uniflorum)

DNA methylation is known as an epigenetic modification that affects gene expression in plants. Variation in CpG methylation behavior was studied in two natural horse gram (Macrotyloma uniflorum [Lam.] Verdc.) genotypes, HPKC2 (drought-sensitive) and HPK4 (drought-tolerant). The methylation pattern in both genotypes was studied through methylation-sensitive amplified polymorphism. The results revealed that methylation was higher in HPKC2 (10.1%) than in HPK4 (8.6%). Sequencing demonstrated sequence homology with the DRE binding factor (cbf1), the POZ/BTB protein, and the Ty1-copia retrotransposon among some of the polymorphic fragments showing alteration in methylation behavior. Differences in DNA methylation patterns could explain the differential drought tolerance and the epigenetic signature of these two horse gram genotypes.     

基因与环境互作对云南保山烤烟主要潜香型物质的影响

为研究云南保山烤烟不同品种潜香型物质在不同环境条件下的稳定性问题,将云南保山3个烤烟常栽品种(K326、Y87、Y99)种植在3个海拔的2种土壤上,应用AMMI模型对烤烟主要潜香型物质进行基因型(G)、环境(E)和基因型与环境(G×E)互作分析.结果表明： 基因型、环境及其互作对烤烟叶黄素含量、β-胡萝卜素含量、绿原酸含量的影响均达到显著水平;芸香苷含量主要受基因型影响.烤烟主要潜香型物质含量及其稳定性受G×E互作影响显著,叶黄素含量、β-胡萝卜素含量、绿原酸含量随海拔的增加而显著增加,其稳定性也不同程度得到加强;K326适种在中低海拔区,Y87、Y99适种在中高海拔区.
     

Inheritance and Variation of Cytosine Methylation in Three Populus Allotriploid Populations with Different Heterozygosity

DNA methylation is an epigenetic mechanism with the potential to regulate gene expression and affect plant phenotypes. Both hybridization and genome doubling may affect the DNA methylation status of newly formed allopolyploid plants. Previous studies demonstrated that changes in cytosine methylation levels and patterns were different among individual hybrid plant, therefore, studies investigating the characteristics of variation in cytosine methylation status must be conducted at the population level to avoid sampling error. In the present study, an F1 hybrid diploid population and three allotriploid populations with different heterozygosity [originating from first-division restitution (FDR), second-division restitution (SDR), and post-meiotic restitution (PMR) 2n eggs of the same female parent] were used to investigate cytosine methylation inheritance and variation relative to their common parents using methylation-sensitive amplification polymorphism (MSAP). The variation in cytosine methylation in individuals in each population exhibited substantial differences, confirming the necessity of population epigenetics. The total methylation levels of the diploid population were significantly higher than in the parents, but those of the three allotriploid populations were significantly lower than in the parents, indicating that both hybridization and polyploidization contributed to cytosine methylation variation. The vast majority of methylated status could be inherited from the parents, and the average percentages of non-additive variation were 6.29, 3.27, 5.49 and 5.07% in the diploid, FDR, SDR and PMR progeny populations, respectively. This study lays a foundation for further research on population epigenetics in allopolyploids.     

Characterization and sequence of a novel nitrate reductase from barley

Summary Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.Scientific Paper No. 9101-14. College of Agriculture and Home Economics Research Center, Washington State University, Research Project Nos. 0233 and 0745     

Physiological dissection revealed that both uptake and assimilation are the major components regulating different growth responses of two tobacco cultivars to nitrogen nutrition

• K326 and HD represent major tobacco cultivars in China, which required large N fertiliser input but at different application rates. To understand primary components affecting tobacco N use physiology, we adopted these two varieties as valuable genetic material to assess their growth response to N nutrition.
• We established a hydroponic culture system to grow plants supplied with different N regimes. Plant biomass, N, ammonium, nitrate, arginine, GS and NR activity, N transfer and use efficiency as well as root uptake were examined.
• Our data revealed the preference of K326 and HD to utilise nitrate or ammonium nitrate but not ammonium alone, with 2 mm N supply probably sufficient and economical to achieve good biomass production at the vegetative stage. Moreover, both varieties were very sensitive to ammonium, perhaps due to lack of or abnormal signalling related to nitrate and/or arginine rather than impairment of N acquisition and initial assimilation; this was supported by measurements of the plant content of N, ammonium and activities of GS and NR. Notably, short‐term ¹⁵N root influx studies identified differential uptake kinetics of K326 and HD, with distinct affinities and transport rates for ammonium and nitrate.
• The data suggest that the growth adaptation of K326 or HD to higher or lower N may be ascribed to different competences for effective N uptake/translocation and assimilation. Thus, our work provides valuable information to prompt deeper investigation of the molecular basis controlling plant N use efficiency.

     

Effect of cryopreservation on apple genetic resources at morphological, chromosomal, and molecular levels.

Shoot tips of three apple genotypes, namely, Malus pumila cv. M26, Gala, and Hokkaido No. 9, were successfully cryopreserved using a modified encapsulation-dehydration method. As a result, in addition to a high survival rate and regeneration rate, the capacity of shoots regenerated from cryopreserved samples to root was enhanced. Eight M26 single-bud sibling lines were used to assess genetic stability. Although cytological examination revealed a ploidy difference in the noncryopreserved control, the ploidy constitution remained relatively stable during the period of cryopreservation. Amplified fragment length polymorphism (AFLP) assay was performed to detect DNA-level variation. No change in DNA fragment pattern and number was observed between the control and the cryopreserved samples. In addition, methylation-sensitive amplified polymorphism (MSAP) assay was carried out to investigate the DNA methylation status during the period of cryopreservation. It was found that cryopreservation induced a decrease in DNA methylation level.     

Association of the NAD(P)H-bispecific nitrate reductase structural gene with the Nar7 locus in barley.

The NADH-specific and NAD(P)H-bispecific nitrate reductase genes from barley have been cloned and sequenced. To determine if the Nar7 locus encodes the NAD(P)H-bispecific nitrate reductase structural gene, a cross was made between a wild-type cultivar, Morex (Nar7 Nar7), and Az70 (nar7w nar7w), a mutant from the cultivar Steptoe that is deficient in NAD(P)H-bispecific nitrate reductase activity. A probe specific to the NAD(P)H-bispecific nitrate reductase structural gene detected restriction fragment length polymorphism between the parents. This probe was used to classify selected F2 progeny for restriction fragment length genotype. All the NAD(P)H nitrate reductase deficient F2 progeny (24/101) possessed the Az70 restriction fragment genotype. The absence of recombination between the NAD(P)H-bispecific nitrate reductase deficient genotype and the NAD(P)H-bispecific nitrate reductase restriction fragment length genotype indicates that the two traits are closely associated in inheritance and that Nar7 is probably the NAD(P)H-bispecific nitrate reductase structural gene.     

The negative influence of <Emphasis Type="Italic">N</Emphasis>-mediated TMV resistance on yield in tobacco: linkage drag versus pleiotropy

Resistance to tobacco mosaic virus (TMV) is controlled by the single dominant gene N in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) by interspecific hybridization and backcrossing, but has historically been associated with reduced yields and/or quality in flue-cured tobacco breeding materials. Past researchers have suggested the role of pleiotropy and/or linkage drag effects in this unfavorable relationship. Introduction of the cloned N gene into a TMV-susceptible tobacco genotype (cultivar ‘K326’) via plant transformation permitted investigation of the relative importance of these possibilities. On average, yield and cash return ($ ha⁻¹) of 14 transgenic NN lines of K326 were significantly higher relative to an isoline of K326 carrying N introduced via interspecific hybridization and backcrossing. The negative effects of tissue culture-induced genetic variation confounded comparisons with the TMV-susceptible cultivar, K326, however. Backcrossing the original transgenic lines to non-tissue cultured K326 removed many of these unfavorable effects, and significantly improved their performance for yield and cash return. Comparisons of the 14 corresponding transgenic NN backcross-derived lines with K326 indicated that linkage drag is the main factor contributing to reduced yields in TMV-resistant flue-cured tobacco germplasm. On average, these transgenic lines outyielded the conventionally-developed TMV-resistant K326 isoline by 427 kg ha⁻¹ (P < 0.05) and generated $1,365 ha⁻¹ more (P < 0.05). Although transgenic tobacco cultivars are currently not commercially acceptable, breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N may increase the likelihood of developing high-yielding TMV-resistant flue-cured tobacco cultivars.