The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers     

Accelerated Evolution of Cytochrome b in Simian Primates: Adaptive Evolution in Concert with Other Mitochondrial Proteins?

We have sequenced the cytochrome b gene of Horsfield's tarsier, Tarsius bancanus, to complete a data set of sequences for this gene from representatives of each primate infraorder. These primate cytochrome b sequences were combined with those from representatives of three other mammalian orders (cat, whale, and rat) in an analysis of relative evolutionary rates. The nonsynonymous nucleotide substitution rate of the cytochrome b gene has increased approximately twofold along lineages leading to simian primates compared to that of the tarsier and other primate and nonprimate mammalian species. However, the rate of transversional substitutions at fourfold degenerate sites has remained uniform among all lineages. This increase in the evolutionary rate of cytochrome b is similar in character and magnitude to that described previously for the cytochrome c oxidase subunit II gene. We propose that the evolutionary rate increase observed for cytochrome b and cytochrome c oxidase subunit II may underlie an episode of coadaptive evolution of these two proteins in the mitochondria of simian primates. Received: 15 December 1997 / Accepted: 24 February 1998     

PCR-SSP技术对广东汉族人HLA-DR基因分型

探索具有高分辨率、高特异性和简捷快速的方法对ＨＬＡ－ＤＲ基因分型,为临床器官移植配型和疾病相关性分析提供实用的方法和基础资料．利用ＤＲ１～ＤＲｗ１８序列特异性的１９组引物及１对内参照引物进行ＰＣＲ扩增即ＰＣＲ－ＳＳＰ对ＨＬＡ－ＤＲ进行基因分型,扩增产物经琼脂糖凝胶电泳,溴乙锭染色,在紫外光下观察分型结果．每个被检个体的ＤＲ型别可由特异引物扩增出现的电泳谱带直接判断．双盲检测２２例的结果１００％正确．在１０２例中国广东地区汉族人中,ＤＲ９和ＤＲ２的基因频率最高,分别为０．２２０５和０．１９１２,ＤＲ１０为最低（０．００９８）．与用ＰＣＲ－ＳＳＯ方法分型获得的结果比较,基因型别分布基本一致,但一些等位基因的频率有差异,表明ＨＬＡ－ＤＲ基因频率的分布在不同地区、不同种族的人群间存在着差异．ＰＣＲ－ＳＳＰ法分辨率和特异性虽不及ＰＣＲ－ＳＳＯ法但比血清学方法精细,分型的全过程只需２～４ｈ能满足临床器官移植配型的要求．基因频率调查结果为器官移植配型和疾病相关性分析提供了基础资料．     

用万能引物PCR法从YAC中分离制备荧光原位杂交探针的研究

报道了一种从微量且未知碱基顺序的YAC DNA中制备FISH探针的新方法——万能引物PCR(UP PCR),即把复性后的UP-Linker连接于PFGE分离后经Alu Ⅰ酶切的YAC DNA上,再用UP对其进行PCR扩增和标记。由于Alu Ⅰ的广谱性和UP与Linker长度不同等,使该法能根据PCR效率调节扩增产物的广泛性向特异性的转变,且产物能覆盖YAC嵌入DNA的全长。此法制备的人21号染色体FISH探针,特异性强,杂交信号明亮,21号染色体的检出率高。该法稳定简便。     

Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences

Honeybee workers express a pronounced age‐dependent polyethism switching from various indoor duties to foraging outside the hive. This transition is accompanied by tremendous changes in the sensory environment that sensory systems and higher brain centers have to cope with. Foraging and age have earlier been shown to be associated with volume changes in the mushroom bodies (MBs). Using age‐ and task‐controlled bees this study provides a detailed framework of neuronal maturation processes in the MB calyx during the course of natural behavioral maturation. We show that the MB calyx volume already increases during the first week of adult life. This process is mainly driven by broadening of the Kenyon cell dendritic branching pattern and then followed by pruning of projection neuron axonal boutons during the actual transition from indoor to outdoor duties. To further investigate the flexible regulation of division of labor and its neuronal correlates in a honeybee colony, we studied the modulation of the nurse‐forager transition via a chemical communication system, the primer pheromone ethyl oleate (EO). EO is found at high concentrations on foragers in contrast to nurse bees and was shown to delay the onset of foraging. In this study, EO effects on colony behavior were not as robust as expected, and we found no direct correlation between EO treatment and synaptic maturation in the MB calyx. In general, we assume that the primer pheromone EO rather acts in concert with other factors influencing the onset of foraging with its effect being highly adaptive. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1368–1384, 2015     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.     

Assessing the effects of primer specificity on eliminating numt coamplification in DNA barcoding: a case study from Orthoptera (Arthropoda: Insecta)

DNA barcoding is a diagnostic method of species identification based on sequencing a short mitochondrial DNA fragment of cytochrome oxidase I (COI), but its ability to correctly diagnose species is limited by the presence of nuclear mitochondrial pseudogenes (numts). Numts can be coamplified with the mitochondrial orthologue when using universal primers, which can lead to incorrect species identification and an overestimation of the number of species. Some researchers have proposed that using more specific primers may help eliminate numt coamplification, but the efficacy of this method has not been thoroughly tested. In this study, we investigate the taxonomic distribution of numts in 11 lineages within the insect order Orthoptera, by analysing cloned COI sequences and further test the effects of primer specificity on eliminating numt coamplification in four lineages. We find that numts are coamplified in all 11 taxa using universal (barcoding) primers, which suggests that numts may be widespread in other taxonomic groups as well. Increased primer specificity is only effective at reducing numt coamplification in some species tested, and only eliminates it in one species tested. Furthermore, we find that a number of numts do not have stop codons or indels, making it difficult to distinguish them from mitochondrial orthologues, thus putting the efficacy of barcoding quality control measures under question. Our findings suggest that numt coamplification is a serious problem for DNA barcoding and more quality control measures should be implemented to identify and eliminate numts prior to using mitochondrial barcodes for species diagnoses.