Haoru Tang

Somatic embryogenesis and organogenesis from immature embryo cotyledons of three sour cherry cultivars (Prunus cerasus L.)

Immature cotyledons of open-pollinated fruits from three sour cherry cultivars (Prunus cerasus L.) were excised and cultured on Murashige and Skoog medium supplemented with various combinations of auxin and cytokinin to induce somatic embryogenesis. Somatic embryogenesis occurred principally when using the combinations of 2,4-dichlorophenoxyacetic acid plus kinetin. Using a-naphthaleneacetic acid or 6-benzylaminopurine reduced the incidence of somatic embryogenesis. Conversely, formation of cotyledon-like structures, leaves, shoots and roots was enhanced. The addition of 0.1 mg l ˇ1 3-indolebutyric acid to the inductive medium was beneficial to the induction of somatic embryogenesis. In a few cases, secondary somatic embryos formed and well-developed somatic embryos germinated. Of the three cultivars tested, ‘Scharo ¨’ was less responsive than ‘Gerema’ and ‘Schattenmorelle’ when cultured under equivalent conditions. After trisectioning the cotyledons of cultivar ‘Gerema’, morphogenic gradients were apparent in shoot and leaf formation but not in root and somatic embryo formation. The embryonic axes attached to the cotyledons of cultivar ‘Schattenmorelle’ had an inhibitory effect on morphogenesis. # 2000 Elsevier Science B.V. All rights reserved.

Genetic Diversity and Population Structure Patterns in Chinese Cherry (Prunus pseudocerasus Lindl) Landraces

Chinese cherry (Prunus pseudocerasus Lindl.) is an ancient fruit crop with highly economic and ornamental values. It originated in China and the cultivation history can be traced back to 3,000 - 4,000 years ago. Over such a long-term domestication process, a large number of genetic variations have been accumulated in different landraces. However, their utilization for cultivar improvement is limited by the scarcity of information involving genetic diversity and population structure. Here, 17 populations comprised of 140 individuals were collected from four geographic areas: Sichuan Basin (SC), Qinglin Mountain (QL), Yungui Plateau (YG) and North of China (NC), and analyzed using a set of 20 microsatellite markers. In total, 126 polymorphic loci were generated, with 6.3 loci per primer. The global expected heterozygosity (He = 0.63) and Shannon information index (I = 1.23) implied a moderately high level of genetic variation. Two major clusters (cluster 1 and cluster 2) were demonstrated based on population structure analysis, which implied the presence of two potential domestication sites of Chinese cherry landraces. Individuals from SC were assigned to cluster 1 and those from QL, YG and NC were grouped into cluster 2. Samples from QL region contained the most plentiful admixture genetic components, implied the possibility of being one transition region of genetic variation. Moreover, botanical characteristics, such as long lifespan, inbreeding preference as well as vegetative propagation, might lead to a relatively low level but significant genetic divergence among populations. Finally, conservation strategies were proposed to protect these valuable natural germplasm based on these results.

Phylogenetic insight into subgenera Idaeobatus and Malachobatus (Rubus, Rosaceae) inferring from ISH analysis

BackgroundRubus is a large and taxonomically complex genus exhibiting agamospermy, polyploidy and frequent hybridization. The objective of this work was to elucidate rDNA disrtibution pattern and investigate genomic composition of polyploids in 16 Rubus taxa (2n = 2x, 3x, 4x, 8x) of two subgenera Idaeobatus and Malachobatus by ISH method.ResultsThe basic Rubus genome had one 45S rDNA locus, and all the polyploids (except R. setchuenensis) had the expected multiples of this number. Diploid and tetraploid Rubus taxa carried two 5S rDNA, whereas the triploid and octoploid species only had three. The duplicated 45S rDNA sites tended to be conserved, whereas those of 5S rDNA tended to be eliminated after polyploidization. The accession R03-20 was an autotriploid R. parvifolius, while R03-27 and R03-57 were naturally-occurred triploid hybrids between R. parvifolius and R. coreanus. GISH results suggested that R. parvifolius had close relationship with polyploids from Malachobatus.ConclusionsThe polyploids from Malachobatus were probable allopolyploid. In addition, Rubus parvifolius might be involved in hybridization, polyploidization and speciation of some Idaeobatus and Malachobatus species.

Phylogeography of Chinese cherry (Prunus pseudocerasus Lindl.) inferred from chloroplast and nuclear DNA: insights into evolutionary patterns and demographic history.

Chinese cherry (Prunus pseudocerasus Lindl.) is a commercially valuable fruit crop in China. In order to obtain new insights into its evolutionary history and provide valuable recommendations for resource conservation, phylogeographic patterns of 26 natural populations (305 total individuals) from six geographic regions were analyzed using chloroplast and nuclear DNA fragments. Low levels of haplotype and nucleotide diversity were found in these populations, especially in landrace populations. It is likely that a combined effect of botanical characteristics impact the effective population size, such as inbreeding mating system, long life span, as well as vegetative reproduction. In addition, strong bottleneck effect caused by domestication, together with founder effect after dispersal and subsequent demographic expansion, might also accelerate the reduction of the genetic variation in landrace populations. Interestingly, populations from Longmen Mountain (LMM) and Daliangshan Mountain (DLSM) exhibited relatively higher levels of genetic diversity, inferring the two historical genetic diversity centers of the species. Moreover, moderate population subdivision was also detected by both chloroplast DNA (GST = 0.215; NST = 0.256) and nuclear DNA (GST = 0.146; NST = 0.342), respectively. We inferred that the episodes of efficient gene flow through seed dispersal, together with features of long generation cycle and inbreeding mating system, were likely the main contributors causing the observed phylogeographic patterns. Finally, factors that led to the present demographic patterns of populations from these regions and taxonomic varieties were also discussed.

Meiotic configuration and rDNA distribution patterns in Six Rubus taxa

More than 200 Rubus species described so far have been included in subgenera Idaeobatus and Malachobatus. Tremendous amount of genetic diversity exists in Rubus due to inter- or intra-species hybridization, polyploidization, or agamospermy make it a typical taxonomically challenging but genetically intriguing genus among land plants. In present study, meiotic pairing behavior and the distribution patterns of 45S and 5S ribosomal DNA (rDNA) arrays were assessed by fluorescence in situ hybridization (FISH) in six Rubus taxa with ploidy ranging from diploid (2n=14) to octoploid (2n=56). At all ploidy levels, bivalent pairing is the most predominant form in meiotic configuration. Very few multivalents were observed in tetraploid or octoploid samples. FISH analysis revealed two 45S rDNA sites in each of the two diploid taxa. Each of the polyploids (2n=28) studied had the expected multiples of this number except the tetraploid R. setchuenensis (with only three sites). On the contrary, both diploid and tetraploid taxa had two 5S rDNA loci, while the octoploid R. buergeri carried only three sites. Both meiotic behavior observations and FISH results support that Rubus polyploids might be of allopolyploid in origin.

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.

Genomic in situ hybridization analysis between Rubus coreanus and its relatives in Rubus (Sect. Idaeobatus)

Abstract To further investigate the phylogenetic relationship between Rubus coreanus and its relatives in the section Idaeobatus, we used genomic in situ hybridization (GISH) to ascertain the degree of their genomic homology. Genomic DNA from R. parvifolius and R. inopertus hybridized throughout the centromeric and sub-terminal regions on 14 and 12 chromosomes of R. coreanus, respectively. The probes from R. niveus and R. ellipticus var. obcordatus gave robust signals at the same region of eight chromosomes. R. ellipticus and R. pinfaensis generated strong signals at the centromeric and sub-terminal parts of six chromosomes. The hybridization signals from the R. tsangii and R. corchorifolius probes existed only at the telomeric parts of four chromosomes. The two signals at the sub-terminal region on chromosome 6 of R. coreanus might be 45S rDNA repeats. These results indicated that R. coreanus and R. parvifolius shared many repeat sequences. It could be deduced that the genome of R. parvifolius was most closely related to that of R. coreanus among the species tested, R. inopertus came next, while R. tsangii and R. corchorifolius showed the farthest relationship. The phylogenetic relationships between R. parvifolius and R. coreanus, as well as among the five subsections were mainly discussed.

Phylogenetic Insights into Chinese Rubus (Rosaceae) from Multiple Chloroplast and Nuclear DNAs.

Rubus L. is a large and taxonomically complex genus, species of which exhibit apomixis, polyploidy, and frequent hybridization. Most of Chinese Rubus are assigned in two major sections, Idaeobatus and Malachobatus. To explore the phylogenetic relationships within Chinese Rubus, inferences upon three chloroplast DNA (rbcL, rpl20-rps12, and trnG-trnS), nuclear ribosomal ITS, and two low-copy nuclear markers (GBSSI-2 and PEPC) were deduced in 142 Rubus taxa from 17 subsections in 6 sections. nrITS and GBSSI-2 were the most informative among the six DNA regions assessed. Phylogenetic relationships within Rubus were well-resolved by combined nuclear datasets rather than chloroplast markers. The phylogenetic inferences strongly supported that section Idaeobatus was a polyphyletic group with four distant clades. All samples of sect. Malachobatus formed a monophyletic clade, in which R. tsangorum and R. amphidasys of sect. Dalibardastrum, and R. peltatus from subsection Peltati of sect. Idaeobatus were always nested. Rubus pentagonus (2n = 2x = 14) from subsect. Alpestres of sect. Idaeobatus was a sister group to the polyploid sect. Malachobatus, as well as the polytomy of three sect. Cyalctis members. This suggests that some polyploids of Malachobatus might originate from common ancestors, via polyploidization of hybrids between R. pentagonus and sect. Cylactis species. They had experienced species explosion in a short time. Section Dalibardastrum species have potential parental lineages from subsects. Moluccani and Stipulosi of sect. Malachobatus. Based on molecular phylogenies, we also provided recommendations for the taxonomic treatments of four taxa. In addition, our results showed certain incongruence between chloroplast and nuclear markers, which might be due to hybridization and introgression.

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.

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.