Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts
We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated “Edit Plasmids,” were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.
Yoo et al. (2020) Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts. PeerJ 8:e8362 https://doi.org/10.7717/peerj.8362
Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp
There are four species of mud crabs within the genus Scylla, and most of them live sympatrically in the equatorial region. Apart from a report in Japan about the finding of a natural Scylla hybrid more than a decade ago after the division of genus Scylla into four species by Keenan, Davie & Mann (1998), no subsequent sighting was found. Thus, this study investigates the possible natural occurrence of potential hybridization among Scylla species in the wild. A total of 76,211 individuals from mud crab landing sites around the Malacca Straits, South China Sea and Sulu Sea were screened. In addition to the four-purebred species, four groups (SH 1, n = 2, 627; SH 2, n = 136; SH 3, n = 1; SH 4, n = 2) with intermediate characteristics were found, mostly at Sulu Sea. Discriminant Function Analysis revealed that all Scylla species, including SH 1 - 4, are distinguishable via their morphometric ratios. The most powerful discriminant ratios for each character and the top five discriminant ratios of males and females were suggested. The carapace width of SH 1 males and females were significantly smaller than pure species. Based on the discriminant ratios and the description of morphological characters, we hypothesize that the additional four groups of Scylla with intermediate characteristics could be presumed hybrids. Future work at the molecular level is urgently needed to validate this postulate.
Fazhan et al. (2020) Morphological descriptions and morphometric discriminant function analysis reveal an additional four groups of Scylla spp. PeerJ 8:e8066 https://doi.org/10.7717/peerj.8066
OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes
Background OLA1 is a member of the GTPase protein family; unlike other members, it possess both GTPase and ATPase activities, and can bind and hydrolyze ATP more efficiently than GTP. OLA1 participates in cell proliferation, oxidative response, protein synthesis and tumorigenesis. However, whether OLA1 is also required for oocyte meiosis is still unknown. Methods In this study, the localization, expression, and functions of OLA1 in the mouse oocyte meiosis were examined. Immunofluorescent and confocal microscopy were used to explore the location pattern of OLA1 in the mouse oocyte. Moreover, nocodazole treatment was used to confirm the spindle-like location of OLA1 during mouse meiosis. Western blot was used to explore the expression pattern of OLA1 in the mouse oocyte. Microinjection of siRNA was used to explore the OLA1 functions in the mouse oocyte meiosis. In addition, chromosome spreading was used to investigate the spindle assembly checkpoint (SAC) activity. Results Immunofluorescent staining showed that OLA1 evenly distributed in the cytoplasm at germinal vesicle (GV) stage. After meiosis resumption (GVBD), OLA1 co-localized with spindles, which was further identified by nocodazole treatment experiments. Knockdown of OLA1 impaired the germinal vesicle breakdown progression and finally resulted in a lower polar body extrusion rate. Immunofluorescence analysis indicated that knockdown of OLA1 led to abnormal spindle assembly, which was evidenced by multipolar spindles in OLA1-RNAi-oocytes. After 6 h post-GVBD in culture, an increased proportion of oocyte which has precociously entered into anaphase/telephase I (A/TI) was observed in OLA1-knockdown oocytes, suggesting that loss of OLA1 resulted in the premature segregation of homologous chromosomes. In addition, the chromosome spread analysis suggested that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. Taken together, we concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis.
Xie et al. (2020) OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes. PeerJ 8:e8180 https://doi.org/10.7717/peerj.8180
Estimation of regional irrigation water requirements and water balance in Xinjiang, China during 1995–2017
Estimating water requirements and water balance for irrigated agricultural areas are important and will facilitate the efficient allocation of water resources for agriculture while minimizing the impact on natural ecosystems in arid regions. Based on the Penman-Monteith formula and GIS technology, the irrigation water requirements (IWR) of three main crops (cotton, corn and wheat) during the growing season were estimated and their spatio-temporal changes over the past 23 years (1995–2017) were analyzed in Xinjiang province, China. Our results indicated a dramatic increase in IWR from 14.12 billion m3 in 1995 to 38.99 billion m3 in 2017 due to the rapid cropland expansion of approximately 2.58 × 104 km2 in this period. Monthly IWR usually peaked in summer from May to July and varied in different basins. From the perspective of crops, cotton was identified to have consumed the largest amount of water, reaching 26.39 billion m3 in 2017, accounting for 67.68% of total water consumption. Spatially, the fastest increasing rate of IWR was Tarim Basin, which was attributable to the increase in water requirement of cotton. By comparing IWR and actual irrigation of Xinjiang in 2014, the amount of water scarcity had reached −15.01 billion m3 (−9.80 billion m3 in Tarim Basin and −6.58 billion m3 in Junggar Basin). The planting areas of three main crops (wheat, corn and cotton) were more sensitive to IWR than rising temperature indicated by our model. This study is of great significance for the scientific allocation of water resources in the irrigated areas of the different prefectures of Xinjiang.
Li et al. (2020) Estimation of regional irrigation water requirements and water balance in Xinjiang, China during 1995–2017. PeerJ 8:e8243 https://doi.org/10.7717/peerj.8243
Phylogeography of the rare and endangered lycophyte Isoetes yunguiensis
Background Isoetes yunguiensis Q. F. Wang & W. C. Taylor is a lycophyte of an ancient genus, and it is endemic to China. It is a first-class protected plant in China. This living fossil is used in paleoecology and studies on the evolution of Lycophytes in the Yunnan-Guizhou Plateau. In recent years, human activities have caused the disappearance of several wild populations, and the number of plants in the existing populations is low. Study of the genetic structure, distribution pattern, and historical dynamics of I. yunguiensis in all areas of its distribution is of guiding significance for its rational and effective protection. Methods Expressed sequence tag-simple sequence repeat (EST-SSR) markers were used to study the genetic diversity and structure of I. yunguiensis, and noncoding chloroplast DNA (cpDNA) sequences were used to study the pedigree, population dynamics history, and glacial shelter of I. yunguiensis. A maximum entropy model was used to predict the past, present, and future distribution patterns of I. yunguiensis. Results Analysis with EST-SSR markers revealed that I. yunguiensis showed high genetic diversity and that genetic variation was significantly higher within populations than between populations. Based on cpDNA data, it was concluded that there was no significant geographic pedigree in the whole area of I. yunguiensis distribution (NST = 0.344 > GST = 0.183, p > 0.05); 21 haplotypes were detected using DnaSP v5. Neutral test and LAMARC simulation showed that I. yunguiensis has experienced rapid expansion in recent years. The maximum entropy model predicted that the potential distribution area of I. yunguiensis in the last glacial maximum period has increased significantly compared with the present distribution area, but the future distribution area did not show substantial changes.
Zheng et al. (2020) Phylogeography of the rare and endangered lycophyte Isoetes yunguiensis. PeerJ 8:e8270 https://doi.org/10.7717/peerj.8270
The inflated mitochondrial genomes of siphonous green algae reflect processes driving expansion of noncoding DNA and proliferation of introns
Within the siphonous green algal order Bryopsidales, the size and gene arrangement of chloroplast genomes has been examined extensively, while mitochondrial genomes have been mostly overlooked. The recently published mitochondrial genome of Caulerpa lentillifera is large with expanded noncoding DNA, but it remains unclear if this is characteristic of the entire order. Our study aims to evaluate the evolutionary forces shaping organelle genome dynamics in the Bryopsidales based on the C. lentillifera and Ostreobium quekettii mitochondrial genomes. In this study, the mitochondrial genome of O. quekettii was characterised using a combination of long and short read sequencing, and bioinformatic tools for annotation and sequence analyses. We compared the mitochondrial and chloroplast genomes of O. quekettii and C. lentillifera to examine hypotheses related to genome evolution. The O. quekettii mitochondrial genome is the largest green algal mitochondrial genome sequenced (241,739 bp), considerably larger than its chloroplast genome. As with the mtDNA of C. lentillifera, most of this excess size is from the expansion of intergenic DNA and proliferation of introns. Inflated mitochondrial genomes in the Bryopsidales suggest effective population size, recombination and/or mutation rate, influenced by nuclear-encoded proteins, differ between the genomes of mitochondria and chloroplasts, reducing the strength of selection to influence evolution of their mitochondrial genomes.
Repetti et al. (2020) The inflated mitochondrial genomes of siphonous green algae reflect processes driving expansion of noncoding DNA and proliferation of introns. PeerJ 8:e8273 https://doi.org/10.7717/peerj.8273
Identification of biological pathways and genes associated with neurogenic heterotopic ossification by text mining
Background Neurogenic heterotopic ossification is a disorder of aberrant bone formation affecting one in five patients sustaining a spinal cord injury or traumatic brain injury (SCI-TBI-HO). However, the underlying mechanisms of SCI-TBI-HO have proven difficult to elucidate. The aim of the present study is to identify the most promising candidate genes and biological pathways for SCI-TBI-HO. Methods In this study, we used text mining to generate potential explanations for SCI-TBI-HO. Moreover, we employed several additional datasets, including gene expression profile data, drug data and tissue-specific gene expression data, to explore promising genes that associated with SCI-TBI-HO. Results We identified four SCI-TBI-HO-associated genes, including GDF15, LDLR, CCL2, and CLU. Finally, using enrichment analysis, we identified several pathways, including integrin signaling, insulin pathway, internalization of ErbB1, urokinase-type plasminogen activator and uPAR-mediated signaling, PDGFR-beta signaling pathway, EGF receptor (ErbB1) signaling pathway, and class I PI3K signaling events, which may be associated with SCI-TBI-HO. Conclusions These results enhance our understanding of the molecular mechanisms of SCI-TBI-HO and offer new leads for researchers and innovative therapeutic strategies.
Zhang et al. (2020) Identification of biological pathways and genes associated with neurogenic heterotopic ossification by text mining. PeerJ 8:e8276 https://doi.org/10.7717/peerj.8276
Genetic diversity and population structure of two subspecies of western honey bees (Apis mellifera L.) in the Republic of South Africa as revealed by microsatellite genotyping
Apis mellifera scutellata and Apis mellifera capensis, two native subspecies of western honey bees in the Republic of South Africa (RSA), are important to beekeepers in their native region because beekeepers use these bees for honey production and pollination purposes. Additionally, both bees are important invasive pests outside of their native ranges. Recently, whole mitogenome sequencing and single nucleotide polymorphisms were used to study their genetic diversity. To add to our knowledge of the molecular ecology of both bees, we tested the ability of microsatellites to be used as a tool to discriminate between A.m. capensis and A.m. scutellata. We analyzed the genetic variability and overall population structure of both bee subspecies and hybrids of the two by genotyping individuals collected from RSA (N = 813 bees from 75 apiaries) at 19 microsatellite DNA loci. Overall, populations averaged between 9.2 and 11.3 alleles per locus, with unbiased heterozygosity values ranging from 0.81 to 0.86 per population. Bayesian clustering analyses revealed two distinct evolutionary units, though the results did not match those of earlier morphometric and molecular analyses. This suggests that the microsatellites we tested were not sufficient for subspecies identification purposes, especially for Cape and hybrid bees. Nevertheless, the microsatellite data highlight the considerable genetic diversity within both populations and a larger-than-expected hybridization zone between the natural distributions of A.m. capensis and A.m. scutellata.
Eimanifar et al. (2020) Genetic diversity and population structure of two subspecies of western honey bees (Apis mellifera L.) in the Republic of South Africa as revealed by microsatellite genotyping. PeerJ 8:e8280 https://doi.org/10.7717/peerj.8280
Temporal and spatial variability of terrestrial diatoms at the catchment scale: controls on communities
Diatoms are generally regarded as inhabitants of water bodies. However, numerous taxa are able to survive and reproduce in a variety of non-aquatic ecosystems. Although terrestrial diatoms are discussed extensively in the literature, most of those studies covered floristic aspects and few information exists on their ecology. This lack of knowledge thwarts their potential use as environmental markers in various applications. As a way forward, we investigated the seasonal patterns and the role of different disturbances on the community composition. We collected soil diatom samples in 16 sites across the Attert River basin (Luxembourg) every 4 weeks for a period of 14 months. Our results indicate that forests create a stable microhabitat for diatoms and that temporal variation of the diatom communities is mainly controlled by farming practices rather than seasonal changes in environmental variables. We also found out that communities need one to 2 months to reestablish a new, stable community after a significant change in the environment. We were able to confirm the applicability of the Pollution-Sensitivity Index (IPS) to identify anthropic disturbances.
Foets et al. (2020) Temporal and spatial variability of terrestrial diatoms at the catchment scale: controls on communities. PeerJ 8:e8296 https://doi.org/10.7717/peerj.8296
Population genetic structure and variability in Lindera glauca (Lauraceae) indicates low levels of genetic diversity and skewed sex ratios in natural populations in mainland China
Lindera glauca (Lauraceae) is a tree of economic and ecological significance that reproduces sexually and asexually via apomictic seeds. It is widely distributed in the low-altitude montane forests of East Asia. Despite the potential implications of a mixed reproductive system in terms of genetic diversity, few studies have focused on this aspect. In this study, the genetic structure of wild populations of L. glauca was investigated via genetic analyses. Overall, 13 nuclear microsatellites (nSSRs) and five chloroplast microsatellites (cpSSRs) were used to genotype 300 individual plants, taken from 20 wild populations (a small sample size in some wild populations is due to the limitation of its specific reproduction, leading to certain limitations in the results of this study) and two cultivated populations ranging across nearly the entire natural distribution of mainland China. The populations exhibited low levels of genetic diversity (nSSR: AR = 1.75, Ho = 0.32, He = 0.36; cpSSR: Nb = 2.01, Hrs = 0.40), and no significant effect of isolation by distance between populations existed, regardless of marker type (nSSR: R2 = 0.0401, P = 0.068; cpSSR: R2 = 0.033, P = 0.091). Haplotype networks showed complex relationships among populations, and the H12 haplotype was predominant in most populations. Analyses of molecular variance obtained with nuclear markers (Fsc = 0.293, FST = 0.362) and chloroplast markers (Fsc = 0.299, FST = 0.312) were similar. The migration ratio of pollen flow versus seed flow in this study was negative (r = −1.149). Results suggest that weak barriers of dispersal between populations and/or the similarity of founders shared between neighbors and distant populations are indicative of the gene flow between populations more likely involving seeds. Wild L. glauca in mainland China was inferred to have highly skewed sex ratios with predominant females. In addition, some populations experienced a recent bottleneck effect, especially in Gujianshan, Chongqing, and southwest China (population GJS). It is suggested that few wild male individuals should be conserved in order to maintain overall genetic diversity in the wild populations of this species. These findings provide important information for the sustainable utilization and preservation of the overall genetic diversity of L. glauca.
Xiong et al. (2020) Population genetic structure and variability in Lindera glauca (Lauraceae) indicates low levels of genetic diversity and skewed sex ratios in natural populations in mainland China. PeerJ 8:e8304 https://doi.org/10.7717/peerj.8304