The large single-copy(LSC) region functions as a highly effective and efficient molecular marker for accurate authentication of medicinal Dendrobium species

Having great medicinal values,Dendrobium species of"Fengdou"(DSFs)are a taxonomically complex group in Dendrobium genus including many closely related and recently diverged species.Traditionally used DNA markers have been proved to be insufficient in authenticating many species of this group.Here,we investigated 101 complete plastomes from 23 DSFs,comprising 72 newly sequenced and 29 documented,which all exhibited well-conserved genomic organization and gene order.Plastomewide comparison showed the co-occurrence of single nucleotide polymorphisms(SNPs)and insertions/deletions(indels),which can be explained by both the repeat-associated and indel-associated mutation hypotheses.Moreover,guanine-cytosine(GC)content was found to be negatively correlated with the three divergence variables(SNPs,indels and repeats),indicating that GC content may reflect the level of the local sequence divergence.Our species authentication analyses revealed that the relaxed filtering strategies of sequence alignment had no negative impact on species identification.By assessing the maximum likelihood(ML)trees inferred from different datasets,we found that the complete plastome and large single-copy(LSC)datasets both successfully identified all 23 DSFs with the maximum bootstrap values.However,owing to the high efficiency of LSC in species identification,we recommend using LSC for accurate authentication of DSFs.

Isolation, Mapping, DNA Sequence and RFLPs Studies of Random Single-Copy DNA Segments on Human X Chromosome

Using the total human/mouse DNA as the probe, screening has been carried out three times with in situ plaque hybridization to obtain the single-copy DNA sequence from the human X chromosome genomic library. The effective rate of screening is 1. 45%. DNAs from clones containing single-copy inserts have been analyzed by a panel of hybrid cells with or without human X chromosome. Three segments, designated by DXFD52,73,75, are mapped to the X chromosome. DXFD52 has been precisely localized on Xq12-q13 with in situ chromosomal hybridization. DXFD52 has been partially sequenced. The results indicate that DXFD52 is a new isolated single-copy segment on the X chromosome. Great progress in the RFLPs study with DXFD52 has been achieved in the population of Chongqing, Sichuan Province. The results show that the DXFD52 can be used to detect the RFLP with Hind Ⅲ, Bgl Ⅱ, and Hinf Ⅰ. DXFD52 will be a potential "landmark" for the construction of the complete linkage map of human genome and the analysis of genomic

Regular Production of Transgenic Wheat Mediated by Agrobacterium tumefaciens

Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0 - 1.5mm in size, C58cl of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production, shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptll ELISA (nptII enzyme assay), PCR, Southern blot and leaf bleach, 29 positive plants were identified from two genotypes of Bobwhite and Yangmai 10, with an average transformation efficiency of 0.82%. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.

Systematic arrangement within the family Clitocybaceae (Tricholomatineae, Agaricales): phylogenetic and phylogenomic evidence, morphological data and muscarine-producing innovation

The Clitocybaceae is a recently established family.Currently,the infrafamilial divisions and relationships within the family are vague due to limited sampling and genes employed for phylogenetic analysis.Some mushrooms of the family contain the neurotoxic muscarine,which has caused many severe and even deadly poisonings worldwide.However,the taxonomic distribution and evolution of the toxin within the family is largely unknown.In this study,phylogenetic analyses based on nucleotide sequences of ITS and of six molecular loci(ITS,LSU,TEF1,RPB1,RPB2 and ATP6),plus a phylogenomic analysis based on 485 single-copy orthologous genes,were performed to reconstruct the framework of Clitocybaceae.BEAST analysis was used to estimate the divergence times within the family.Additionally,biochemical analysis for muscarine was conducted of 32 representative species.Based on these analyses,an updated classification of Clitocybaceae into six genera(Clitocybe,Collybia,Dendrocollybia,Lepista,Pseudolyophyllum,and Singerocybe)is proposed.The genus Collybia is emended to accommodate four subgenera(Collybia,Crassicybe,Leucocalocybe,and Macrosporocybe).Seventeen new Chinese species and 15 new combinations are proposed.Keys to the genera of Clitocybaceae and the subgenera of Collybia,as well as to the known species of Clitocybe and Collybia subgen.Collybia in China,are presented.In addition,muscarine was detected in 18 species,and these muscarine-containing species formed a major monophyletic clade within Collybia subgen.Collybia.Finally,our phylogenetic,phylogenomic,chemotaxonomic and molecular dating results indicate that the Clitocybaceae is a natural group estimated to have arisen some 60 million years ago,and in this family,muscarine has evolved only once circa 20 million years ago without later losses.

Phylogenomic conflict analyses in the apple genus Malus s.l.reveal widespread hybridization and allopolyploidy driving diversification,with insights into the complex biogeographic history in the Northern Hemisphere

Phylogenomic evidence from an increasing number of studies has demonstrated that different data sets and analytical approaches often reconstruct strongly supported but conflicting relationships.In this study,785 single-copy nuclear genes and 75 complete plastomes were used to infer the phylogenetic relationships and estimate the historical biogeography of the apple genus Malus sensu lato,an economically important lineage disjunctly distributed in the Northern Hemisphere and involved in known and suspected hybridization and allopolyploidy events.The nuclear phylogeny recovered the monophyly of Malus s.l.(including Docynia);however,the genus was supported to be biphyletic in the plastid phylogeny.An ancient chloroplast capture event in the Eocene in western North America best explains the cytonuclear discordance.Our conflict analysis demonstrated that ILS,hybridization,and allopolyploidy could explain the widespread nuclear gene tree discordance.One deep hybridization event(Malus doumeri)and one recent event(Malus coronaria)were detected in Malus s.l.Furthermore,our historical biogeographic analysis integrating living and fossil data supported a widespread East Asianwestern North American origin of Malus s.l.in the Eocene,followed by several extinction and dispersal events in the Northern Hemisphere.We also propose a general workflow for assessing phylogenomic discordance and biogeographic analysis using deep genome skimming data sets.

Testing the systematic status of Homalictus and Rostrohalictus with weakened cross-vein groups within Halictini(Hymenoptera:Halictidae)using low-coverage whole-genome sequencing

The halictid genus Lasioglossum,as one of the most species-rich bee groups with persistently contentious subgeneric boundaries,is one of the most challenging bee groups from a systematic standpoint.An enduring question is the relationship of La-sioglossum and Homalictus,whether all halictine bees with weakened distal wing ve-nation comprise one or multiple genera.Here,we analyzed the phylogenetic relation-ships among the subgroups within Lasioglossum s.l.based on thousands of single-copy orthologs and ultraconserved elements,which were extracted from 23 newly sequenced low-coverage whole genomes alongside a published genome(22 ingroups plus 2 out-groups).Both marker sets provided consistent results across maximum likelihood and coalescent-based species tree approaches.The phylogenetic and topology test results show that the Lasioglossum and Hemihalictus series are reciprocally monophyletic and Homa-lictus and Rostrohalictus are valid subgenera of Lasioglossum.Consequently,we lower Homalictus to subgenus status within Lasioglossum again,and we also raise Rostrohalic-tus to subgenus status from its prior synonymy with subgenus Hemihalictus.Lasioglossum przewalskyi is also transferred to the subgenus Hemihalictus.Ultimately,we redefine La-sioglossum to include all halictine bees with weakened distal wing venation.