A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

Genetic imprints of grafting in wild iron walnut populations in southwestern China.

BACKGROUND: Anthropogenic activities are causing unprecedented loss of genetic diversity in many species. However, the effects on genetic diversity from large-scale grafting onto wild plants of crop species are largely undetermined. Iron walnut (Juglans sigillata Dode) is a deciduous nut tree crop endemic to southwestern China with a long history of cultivation. Due to the rapid expansion of the walnut industry, many natural populations are now being replaced by cultivars grafted onto wild rootstocks. However, little is known about the potential genetic consequences of such action on natural populations. RESULTS: We sampled the scion and the rootstock from each of 149 grafted individuals within nine wild populations of J. sigillata from Yunnan Province which is the center of walnut diversity and cultivation in China, and examined their genetic diversity and population structure using 31 microsatellite loci. Scions had lower genetic diversity than rootstocks, and this pattern was repeated in seven of the nine examined populations. Among those seven populations, AMOVA and clustering analyses showed a clear genetic separation between all rootstocks and all scions. However, the two remaining populations, both from northern Yunnan, showed genetic similarity between scions and rootstocks, possibly indicating that wild populations here are derived from feralized local cultivars. Moreover, our data indicated probable crop-to-wild gene flow between scions and rootstocks, across all populations. CONCLUSIONS: Our results indicate that large-scale grafting has been causing genetic diversity erosion and genetic structure breakdown in the wild material of J. sigillata within Yunnan. To mitigate these effects, we caution against the overuse of grafting in wild populations of iron walnut and other crop species and recommend the preservation of natural genotypes through in situ  and ex situ conservation.

Insights into the mechanisms involved in the fungal degradation of plastics.

Fungi are considered among the most efficient microbial degraders of plastics, as they produce salient enzymes and can survive on recalcitrant compounds with limited nutrients. In recent years, studies have reported numerous species of fungi that can degrade different types of plastics, yet there remain many gaps in our understanding of the processes involved in biodegradation. In addition, many unknowns need to be resolved regarding the fungal enzymes responsible for plastic fragmentation and the regulatory mechanisms which fungi use to hydrolyse, assimilate and mineralize synthetic plastics. This review aims to detail the main methods used in plastic hydrolysis by fungi, key enzymatic and molecular mechanisms, chemical agents that enhance the enzymatic breakdown of plastics, and viable industrial applications. Considering that polymers such as lignin, bioplastics, phenolics, and other petroleum-based compounds exhibit closely related characteristics in terms of hydrophobicity and structure, and are degraded by similar fungal enzymes as plastics, we have reasoned that genes that have been reported to regulate the biodegradation of these compounds or their homologs could equally be involved in the regulation of plastic degrading enzymes in fungi. Thus, this review highlights and provides insight into some of the most likely regulatory mechanisms by which fungi degrade plastics, target enzymes, genes, and transcription factors involved in the process, as well as key limitations to industrial upscaling of plastic biodegradation and biological approaches that can be employed to overcome these challenges.

Genetic Diversity and Structure of Persian Walnut (Juglans regia L.) in Pakistan: Implications for Conservation.

Persian (Common) walnut (Juglans regia L.) is a famous fruit tree species valued for its nutritious nuts and high-quality wood. Although walnut is widely distributed and plays an important role in the economy and culture of Pakistan, the genetic diversity and structure of its populations in the country remains poorly understood. Therefore, using 31 nuclear microsatellites, we assessed the genetic diversity and population structure of 12 walnut populations sampled across Pakistan. We also implemented the geostatistical IDW technique in ArcGIS to reveal "hotspots" of genetic diversity. Generally, the studied populations registered relatively low indices of genetic diversity (NA = 3.839, HO = 0.558, UHE = 0.580), and eight populations had positive inbreeding coefficient (FIS) values. Low among-population differentiation was indicated by AMOVA, pairwise FST and DC. STRUCTURE, PCoA and neighbor joining (NJ) analysis revealed a general lack of clear clustering in the populations except that one population in Upper Dir was clearly genetically distinct from the rest. Furthermore, the Mantel test showed no correlation between the geographic and genetic distance (r = 0.14, p = 0.22), while barrier analysis suggested three statistically significant genetic barriers. Finally, the spatial interpolation results indicated that populations in Ziarat, Kashmir, Dir, Swat, Chitral, and upper Dir had high intrapopulation genetic diversity, suggesting the need to conserve populations in those areas. The results from this study will be important for future breeding improvement and conservation of walnuts in Pakistan.