Molecular sexing of birds: A comparative review of polymerase chain reaction (PCR)-based methods

Accurate identification of sex in birds is important for the management and conservation of avian wildlife in several ways, namely in the development of population, behavioral and ecological studies, as well as in the improvement of ex situ captive breeding programs. In general, nestlings, juveniles and adult birds of a wide number of sexually monomorphic species cannot be sexed based on phenotypic traits. The development of molecular methodologies for avian sexing overcame these difficulties, allowing a reliable gender differentiation for these species. The polymerase chain reaction (PCR)-based methods have been widely applied in molecular sexing of birds, using a large diversity of sex-linked markers. During the last 15 yrs, there was a continuous improvement in the PCR-based protocols for bird sexing, increasing the accuracy, speed and high-throughput applicability of these techniques. The recent advances in real-time PCR platforms and whole genome analysis methods provided new resources for the detection and analysis of novel specific markers and protocols. This review presents a comparative guide of classical and recent advances in PCR-based methods for avian molecular sexing, highlighting its strengths and limitations. Future research opportunities in this field are also addressed.     

Co-localisation of host plant resistance QTLs affecting the performance and feeding behaviour of the aphid Myzus persicae in the peach tree

The architecture and action of quantitative trait loci (QTL) contributing to plant resistance mechanisms against aphids, the largest group of phloem-feeding insects, are not well understood. Comparative mapping of several components of resistance to the green peach aphid (Myzus persicae) was undertaken in Prunus davidiana, a wild species related to peach. An interspecific F(1) population of Prunus persica var. Summergrand × P. davidiana clone P1908 was scored for resistance (aphid colony development and foliar damage) and 17 aphid feeding behaviour traits monitored by means of the electrical penetration graph technique. Seven resistance QTLs were detected, individually explaining 6.1-43.1% of the phenotypic variation. Consistency was shown over several trials. Nine QTLs affecting aphid feeding behaviour were identified. All resistance QTLs except one co-located with QTLs underlying aphid feeding behaviour. A P. davidiana resistance allele at the major QTL was associated with drastic reductions in phloem sap ingestion by aphids, suggesting a phloem-based resistance mechanism. Resistance was also positively correlated with aphid salivation into sieve elements, suggesting an insect response to restore the appropriate conditions for ingestion after phloem occlusion. No significant QTL was found for traits characterising aphid mouthpart activity in plant tissues other than phloem vessels. Two QTLs with effects on aphid feeding behaviour but without effect on resistance were identified. SSR markers linked to the main QTLs involved in resistance are of potential use in marker-assisted selection for aphid resistance. Linking our results with the recent sequencing of the peach genome may help clarify the physiological resistance mechanisms.     

What makes transposable elements move in the Drosophila genome?

Transposable elements (TEs), by their capacity of moving and inducing mutations in the genome, are considered important drivers of species evolution. The successful invasions of TEs in genomes, despite their mutational properties, are an apparent paradox. TEs' transposition is usually strongly regulated to low value, but in some cases these elements can also show high transposition rates, which has been associated sometimes to changes in environmental conditions. It is evident that factors susceptible to induce transpositions in natural populations contribute to TE perpetuation. Different factors were proposed as causative agents of TE mobilization in a wide range of organisms: biotic and abiotic stresses, inter- and intraspecific crosses and populational factors. However, there is no clear evidence of the factors capable of inducing TE mobilization in Drosophila, and data on laboratory stocks show contradictory results. The aim of this review is to have an update critical revision about mechanisms promoting transposition of TEs in Drosophila, and to provide to the readers a global vision of the dynamics of these genomic elements in the Drosophila genome.     

Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes

? Premise of the study: The development of three low-copy nuclear markers for low taxonomic level phylogenies in Asteraceae with emphasis on the subtribe Hieraciinae is reported. ? Methods and Results: Marker candidates were selected by comparing a Lactuca complementary DNA (cDNA) library with public DNA sequence databases. Interspecific variation and phylogenetic signal of the selected genes were investigated for diploid taxa from the subtribe Hieraciinae and compared to a reference phylogeny. Their ability to cross-amplify was assessed for other Asteraceae tribes. All three markers had higher variation (2.1-4.5 times) than the internal transcribed spacer (ITS) in Hieraciinae. Cross-amplification was successful in at least seven other tribes of the Asteraceae. Only three cases indicating the presence of paralogs or pseudogenes were detected. ? Conclusions: The results demonstrate the potential of these markers for phylogeny reconstruction in the Hieraciinae as well as in other Asteraceae tribes, especially for very closely related species.     

Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.     

The proteasome is responsible for caspase-3-like activity during xylem development

Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven β subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin β-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.     

The sunflower (Helianthus annuus L.) genome reflects a recent history of biased accumulation of transposable elements

Aside from polyploidy, transposable elements are the major drivers of genome size increases in plants. Thus, understanding the diversity and evolutionary dynamics of transposable elements in sunflower (Helianthus annuus L.), especially given its large genome size (～3.5 Gb) and the well‐documented cases of amplification of certain transposons within the genus, is of considerable importance for understanding the evolutionary history of this emerging model species. By analyzing approximately 25% of the sunflower genome from random sequence reads and assembled bacterial artificial chromosome (BAC) clones, we show that it is composed of over 81% transposable elements, 77% of which are long terminal repeat (LTR) retrotransposons. Moreover, the LTR retrotransposon fraction in BAC clones harboring genes is disproportionately composed of chromodomain‐containing Gypsy LTR retrotransposons (‘chromoviruses’), and the majority of the intact chromoviruses contain tandem chromodomain duplications. We show that there is a bias in the efficacy of homologous recombination in removing LTR retrotransposon DNA, thereby providing insight into the mechanisms associated with transposable element (TE) composition in the sunflower genome. We also show that the vast majority of observed LTR retrotransposon insertions have likely occurred since the origin of this species, providing further evidence that biased LTR retrotransposon activity has played a major role in shaping the chromatin and DNA landscape of the sunflower genome. Although our findings on LTR retrotransposon age and structure could be influenced by the selection of the BAC clones analyzed, a global analysis of random sequence reads indicates that the evolutionary patterns described herein apply to the sunflower genome as a whole.     

Diversification of lindsaeoid ferns and phylogenetic uncertainty of early polypod relationships

We analysed one nuclear gene (18S) and seven plastid markers [five protein coding (atpA, atpB, rbcL, rpoC1, rps4) and two non‐coding (trnH‐psbA, trnL‐trnF] for 31 members of Polypodiales and four outgroup taxa. We focused our sampling on the lindsaeoids and associated ferns in order to obtain a better understanding of the diversification of the early polypods. However, the exact phylogenetic position of Saccoloma and Cystodium remained uncertain. Based on relaxed molecular clock analyses, it appears that the crown group lindsaeoids diversified in the Caenozoic, more or less simultaneously with the main radiation of other Polypodiales, even though the original divergence between the lindsaeoid and non‐lindsaeoid polypods occurred before the end of the Jurassic. The current pantropical distribution of lindsaeoids can be explained by either long‐distance dispersal across the oceans or vicariance caused by the retreat of previously widely distributed tropical forests from higher to lower latitudes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 489–503.     

Heterozygosity-fitness correlations in zebra finches: microsatellite markers can be better than their reputation

Numerous studies have reported associations between heterozygosity in microsatellite markers and fitness-related traits (heterozygosity-fitness correlations, HFCs). However, it has often been questioned whether HFCs reflect general inbreeding depression, because a small panel of microsatellite markers does not reflect very well an individual's inbreeding coefficient (F) as calculated from a pedigree. Here, we challenge this prevailing view. Because of chance events during Mendelian segregation, an individual's realized proportion of the genome that is identical by descent (IBD) may substantially deviate from the pedigree-based expectation (i.e. F). This Mendelian noise may result in a weak correlation between F and multi-locus heterozygosity, but this does not imply that multi-locus heterozygosity is a bad estimator of realized IBD. We examined correlations between 11 fitness-related traits measured in up to 1192 captive zebra finches and three measures of inbreeding: (i) heterozygosity across 11 microsatellite markers, (ii) heterozygosity across 1359 single-nucleotide polymorphism (SNP) markers and (iii) F, based on a 5th-generation pedigree. All 11 phenotypic traits showed positive relationships with measures of heterozygosity, especially traits that are most closely related to fitness. Remarkably, the small panel of microsatellite markers produced equally strong HFCs as the large panel of SNP markers. Both marker-based approaches produced stronger correlations with phenotypes than the pedigree-based F, and this did not seem to result from the shortness of our pedigree. We argue that a small panel of microsatellites with high allelic richness may better reflect an individual's realized IBD than previously appreciated, especially in species like the zebra finch, where much of the genome is inherited in large blocks that rarely experience cross-over during meiosis.