Cryopreservation of shoot tips,evaluations of vegetative growth,and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’ (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis ‘Jinmi’, respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.     

贵阳地区蝉花微生态系统中细菌种群结构与功能

【目的】探索自然生态下蝉花内菌核、菌膜和环境细菌群落结构、功能及其相互关系。【方法】对细菌16S rRNA扩增片段进行高通量测序,分析贵阳市大将山和贵阳森林公园的蝉花内菌核、菌膜及其生境土壤的细菌群落组成、多样性及潜在功能。【结果】蝉花内菌核样本共检测到细菌562个属,菌膜样本521个属,菌际土样本578个属。两地的各组样本细菌群落结构相似,内菌核样本中假单胞菌属Pseudomonas、沙雷氏菌属Serratia占优势地位;菌膜样本中假单胞菌属和氨基杆菌属Aminobacter占优势;土壤样本以未分类的酸杆菌纲Acidobacteria和黄杆菌科Xanthobacteraceae为优势属。Venn图分析显示,菌际土样本包括了菌膜样本的大多数属,内菌核样本拥有较多的特有属,如沃尔巴克氏体属Wolbachia和立克次氏体Rickettsia等。PICRUSt功能预测结果显示,共计24个基因功能家族,主要与物质能量的代谢运输、细胞的行为发生及调控等功能相关。【结论】蝉花及其微生境中细菌具有丰富多样性,它们的潜在功能可能与营养物质的新陈代谢有关,对蝉花的个体生长发育有重要作用。研究结果对蝉花虫草的生态信息数据补充和仿野生栽培具有参考价值。     

CRISPR-Cas9技术在细菌耐药性防控研究进展

近年来,多种新型耐药基因的出现和全球性流行,严重威胁了全球公众健康。CRISPR-Cas9系统(clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 system)是细菌的一种适应性免疫系统,可切割耐药基因、抵御外来核酸入侵,现已作为一种新型基因编辑工具应用于防控细菌耐药性研究。本团队已建立了一种单质粒介导靶向mcr-1基因的CRISPR-Cas9系统,能有效并特异性消除黏菌素耐药大肠杆菌中的mcr-1,恢复其对黏菌素的敏感性。同时也发现在临床中应用还需要优化其递送方式。本文对近几年该技术在细菌耐药性防控方面的研究进展进行了综述,包括CRISPR-Cas9系统的发现过程、作用机制、递送方式、在体外检测实验结果的进展以及当前存在的问题等方面,以期为防控细菌耐药性提供新思路。     

Rapid screenhouse assessment of bacterial blight in cassava genotypes in Nigeria

Bacterial blight disease caused by Xanthomonas axonopodis pv. manihotis (Berthet-Bondar) Dye was assessed in 11 artificially inoculated cassava genotypes in a screenhouse. Disease progress was estimated at intervals of 3 days by measuring the length of necrotic lesions on stems and leaves, as well as estimating the average disease score and area under disease progress curve (AUDPC). Based on the average disease scores, cassava genotypes 30572, TME 1, TME 7 and TME 9 were classified as resistant to bacterial blight, genotypes 4(2)1425, TME 2, TME 4 and TME 12 were tolerant while cassava genotypes 30001, TME 3, and TME 28 were susceptible. Direct correlations, statistically significant at p < 0.05, were obtained between stem necrosis, leaf necrosis, average disease scores and AUDPC in the 11 cassava genotypes. Screenhouse experiments afford rapid assessment of resistance status of cassava genotypes to bacterial blight in Nigeria.     

Races of Xanthomonas citri subsp. malvacearum,the causal organism of bacterial blight of cotton in northern Nigeria

Xanthomonas citri subsp. malvacearum (Xcm) causes severe qualitative and quantitative losses to farmers in cotton-growing areas of the world. Isolates of Xcm were extracted from cotton seeds obtained from five ginneries located in Funtua, Malumfashi, Gusau and Zaria and standardised to 10^?5?cfu/ml. One isolate per location was used to inoculate three sets of 10 cotton differential lines known to differentiate races of Xcm through possession of B-genes for resistance to bacterial blight. Each cotton differential line was inoculated with the isolates at the six-leaf stage and SDW was inoculated as control. One hundred and sixty pots used were arranged in completely randomised design on the screen house bench. Four different pathogenic races were identified namely race 1, race 12, race 13 and race 16. This confirms the existence of an evolution of the species across northern Nigeria.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

Phenotype and Micro-array characterization of duplication 11q22.1-q25 and review of the literature

Partial duplication of 11q is related to several malformations like growth retardation, intellectual disability, hypoplasia of corpus callosum, short nose, palate defects, cardiac, urinary tract abnormalities and neural tube defects. We have studied the clinical and molecular characteristics of a patient with severe intellectual disabilities, dysmorphic features, congenital inguinal hernia and congenital cerebral malformation which is referred to as cytogenetic exploration. We have used FISH and array CGH analysis for a better understanding of the double chromosomic aberration involving a 7p microdeletion along with a partial duplication of 11q due to adjacent segregation of a paternal reciprocal translocation t(7;11)(p22;q21) revealed after banding analysis. The patient's karyotype formula was: 46,XY,der(7)t(7;11)(p22;q21)pat. FISH study confirmed these rearrangement and array CGH technique showed precisely the loss of at least 140 Kb on chromosome7p22.3pter and 33.4 Mb on chromosome11q22.1q25. Dysmorphic features, severe intellectual disability and brain malformations could result from the 11q22.1q25 trisomy. Our study provides an additional case for better understanding and delineating the partial duplication 11q.     

Prenatal diagnosis and molecular cytogenetic characterization of a de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14)

We present prenatal diagnosis of de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14) and molecular cytogenetic characterization of the deletion using uncultured amniocytes. We review the phenotypic abnormalities of previously reported patients with similar proximal interstitial 4p deletions, and we discuss the functions of the genes of RBPJ, CCKAR, STIM2, PCDH7 and ARAP2 that are deleted within this region.     

Physical size,condition, and demography of chamois (Rupicapra rupicapra L.) in the Avoca River region,Canterbury, New Zealand

Abstract

The physical and demographic characteristics of chamois in the Avoca region are evaluated from 306 animals shot and autopsied between 1975 and 1978. These data are compared with published and unpublished information for chamois populations in Westland and Canterbury. Avoca chamois were large-framed, but weighed less than Westland chamois. The weight difference suggests better habitat condition and food resources for Westland animals, but the large skeletal size of Avoca chamois is unexplained.

High rates of juvenile mortality were caused by acute bacterial-pneumonia infections (Pasteurella). These deaths and other losses by natural causes were offset by the good breeding success of adult females so that stable population numbers were maintained.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.