PSXII-8 Mitochondrial DNA diversity within domestic reindeer in Russia

Domestic reindeer in Russia are a valuable resource of vital importance to the physical and cultural survival of the Northern indigenous minority. During the last decades, the mitochondrial (mt) genetic markers have been widely used as a molecular tool to investigate genetic structure and diversity of livestock species. Here we aimed at the assessing the mtDNA diversity of the domestic reindeer inhabiting the area from the Kola Peninsula in the west to the Chukotka region in the east. A complete cytochrome b (cytb) sequences (1,140 bp) from representatives of six populations, including Nenets (NEN, n = 16), Evenk (EVK, n = 12), Even (EVN, n = 6), Chukotka (CHU, n = 6), Chukotka-Khargin (CHUKH, n = 6) and Tuva (TUVA, n = 6) were obtained. Sequences’ alignment was conducted using MUSCLE algorithm in R package msa. In total, 34 haplotypes were identified. Median-joining network, constructed in PopART 1.7, revealed three major groups of haplotypes: the first one joined the samples of all the populations, the second one included NEN, EVN and CHUKH, and the third group was presented by the one sample of CHU. AMOVA, calculated in Arlequin 3.5.2.2, showed that only 9.58% of molecular variance could be explained by the differences between populations and 90.42% - within populations. Genetic diversity parameters calculated in DnaSP 6.12.03, demonstrated that average number of nucleotide differences (K) was highest in CHUKH (28.333) and EVN (27.409) and lowest in TUVA (4.533) and EVK (5.400). Nucleotide diversity (Pi) was 0.01238±0.00559, 0.00474±0.00091, 0.02404±0.00453, 0.01281±0.00464, 0.02485±0.00744, and 0.00398±0.00110 for NEN, EVK, EVN, CHU, CHUKH and TUVA, respectively. Our study demonstrated the lack of clear genetic structure of the studied reindeer populations in relation to cytb sequence. The level of genetic diversity was associated with census size and was lowest in the smallest Tuva population. This study was supported by RSF-21-16-00071 and Russian Ministry of Science and Higher Education-0445-2019-0024.

Application of DNA Molecular Identification Method to Distinguish Ejiao and its Adulterants

To identify Ejiao and its adulterants at the DNA level by using DNA molecula marker. Ejiao (Asini Corii Colla) is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the morphological characteristics of Asini Corii Colla and its adulterants, traditional identification techniques often complex and professional, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Asini Corii Colla and its adulterants accurately, this study identified and its adulterant samples based on the CytB sequence. Sequence characteristics, Basic Local Alignment Search Tool (BLAST) application, genetic distance, construction of phylogenetic tree showed the CytB sequence to accurately identify Asini Corii Colla from its adulterants. Furthermore, in this study, we designed a specific primer, based on the CytB sequence, and established a PCR detection system for rapid, sensitive, and specific identification of Asini Corii Colla. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Asini Corii Colla.

A re-examination of the molecular systematics and phylogeography of taxa of the Peromyscus aztecus species group, with comments on the distribution of P. winkelmanni

The objectives of this study are to examine the available molecular data from the mitochondrial cytochrome-b gene (Cytb) and a concatenated dataset with this gene and two nuclear introns (Adh-1-I2 and Fgb-I7) to reexamine the systematic and phylogeographic conclusions reached by Sullivan et al. (1997) concerning the Peromyscus aztecus species group. The divergence of samples of P. aztecus oaxacensis across the Isthmus of Tehuantepec are further examined and taxonomic revisions are suggested. In addition, this study reviews the sources of data that lead to the conclusion that P. winkelmanni occurred in the Sierra Madre del Sur in Guerrero including a morphometric examination of a reported voucher. Bayesian and maximum likelihood analyses were conducted on a dataset of 31 Cytb sequences of all taxa in the P. aztecus group except for P. a. cordillerae and a concatenated dataset including five individuals of this group. Representative taxa of the P. boylii, P. mexicanus, and P. truei groups were included in both analyses. Body and cranial measurements of the voucher of the P. winkelmanni from Guerrero from which a Cytb sequence is reported to have been obtained was compared with measurements from specimens taken from the vicinity of Dos Aguas, Michoacán, including the type locality. We identified seven instances involving problematic identifications in GenBank. Once these issues were addressed, well-supported monophyletic sister clades of the P. aztecus and P. boylii species groups were recovered from phylogenetic analyses of Cytb sequences (Fig 1). Phylogenetic analyses of the Cytb and the concatenated datasets recover similar topologies that support the relationships of taxa of the aztecus group proposed by an earlier molecular study. Populations of P. a. oaxacensis southeast of the Isthmus of Tehuantepec represent a distinct species. Measurements of the voucher from Guerrero identified as the source of a P. winkelmanni Cytb sequence are smaller than P. winkelmanni for several characters. The divergent populations of P. a. oaxacensis from southeast of the Isthmus of Tehuantepec are recognized as two subspecies of P. cordillerae, P. c. cordillerae and P. c. hondurensis, whereas those northwest of the Isthmus are retained as P. a. oaxacensis. The lack of genetic divergence observed between P. a. evides and P. a. oaxacensis questions whether these two taxa should continue to be recognized as separate subspecies. Northern and southern populations of P. spicilegus demonstrate moderate divergence and additional examination of morphological and molecular differentiation within this taxon is warranted. The distribution of P. winkelmanni should be restricted to the vicinity of Dos Aguas, Michoacán, due to the lack of a voucher specimen that would confirm its reported occurrence in Guerrero.

Molecular Identification of Mutations Conferring Resistance to Azoxystrobin in Cercospora nicotianae

Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for over a decade through azoxystrobin applications targeting other major foliar diseases. From 2016 to 2018, a total of 124 isolates were collected from tobacco fields throughout Kentucky. Sensitivity of these isolates to azoxystrobin was previously characterized by determining the effective concentration to inhibit 50% conidial germination (EC50). Based on azoxystrobin EC50, isolates were categorized into three discrete groups: high azoxystrobin sensitivity (< 0.08 µg/ml), moderate azoxystrobin sensitivity (0.14 to 0.64 µg/ml) and low azoxystrobin sensitivity (> 1.18 µg/ml). Variability in sensitivity in a limited number of C. nicotianae isolates was previously shown to be a result of resistance mutations in the fungicide target gene. To improve understanding of C. nicotianae cytochrome b (cytb) structure, the gene was cloned from three isolates representing each EC50group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1161 nucleotides encoding 386 amino acids. Cytb sequence among the cloned isolates was identical with the exception of the F129L and G143A point mutations. To more rapidly determine the resistance status of C. nicotianae isolates to azoxystrobin, a PCR assay was developed to screen for mutations. Using this assay, 80% (n=99) of testedC. nicotianae isolates carried an F129L mutation and were moderately resistant to azoxystrobin, and 7% (n=9) carried the G143A mutation and were highly resistant. A receiver operator characteristic curve analysis suggested the PCR assay is a robust diagnostic tool to identify C. nicotianae isolates with different sensitivity to azoxystrobin in Kentucky tobacco production. The prevalence of both the F129L and G143A mutations in C. nicotianae from Kentucky differs from other pathosystems where resistance to QoI fungicides has been identified, in which the majority of sampled isolates of the pathogen species have a broadly-occurring cytb mutation.

Heteroplasmy for the Cytochrome b Gene in Podosphaera xanthii and its Role in Resistance to QoI Fungicides in Spain

In Spain, management of the cucurbit powdery mildew pathogen Podosphaera xanthii is strongly dependent on chemicals such as quinone outside inhibitor (QoI) fungicides. In a previous report, widespread resistance to QoI fungicides in populations of P. xanthii in south-central Spain was documented, but the molecular mechanisms of resistance remained unclear. In this work, the role of the Rieske-FeS (risp) and the cytochrome b (cytb) gene mutations in QoI resistance of P. xanthii were examined. No point mutations in the risp gene were found in the three QoI-resistant isolates analyzed. For cytb, sequence analysis revealed the presence of a G143A substitution that occurs in many QoI-resistant fungi. This mutation was always detected in QoI-resistant isolates of P. xanthii; however, it was also detected in sensitive isolates. To better understand the role of heteroplasmy for cytb in QoI resistance of P. xanthii, an allele-specific quantitative PCR was developed to quantify the relative abundance of the G143 (sensitive) and A143 (resistant) alleles. High relative abundance of A143 allele (70%) was associated with isolates resistant to QoI fungicides; however, QoI-sensitive isolates also carried the mutated allele in frequencies ranged from 10 to 60%. Our data suggest that G143A mutation in cytb is the primary factor involved in QoI resistance of P. xanthii but the proportion of G143 and A143 alleles in an isolate may determine its QoI resistance level.

First record of Myotis flavus (Chiroptera: Vespertilionidae) from mainland China and a reassessment of its taxonomic status

Myotis flavus, a synonym of M. formosus found in Taiwan, is reported for the first time for mainland China. We captured one bat in Jiangxi province in Southeast China and identified the taxon as M. flavus based upon its external, cranial and baculum morphology. The uncorrected cytb sequence divergence between M. flavus from Jiangxi and specimen from Taiwan was only 0.61%, indicating intraspecific divergence. Both, morphological and genetic evidence indicated that the specimen from mainland China was a new locality record of M. flavus. In contrast, M. flavus from Jiangxi and from Taiwan exhibited 15.53–16.67% sequence divergence from samples of M. formosus obtained from locations outside Taiwan, and they were not sister lineages. Therefore M. flavus should be recognized as a species from M. formosus. In addition, the sequence similarity between M. watasei from Taiwan and M. formosus from mainland China and their phylogenetic grouping strongly suggests that M. watasei is a synonym of M. formosus.

Mitochondrial DNA variation in water shrews (Sorex palustris, Sorex bendirii) from western North America: implications for taxonomy and phylogeography

Inter- and intra-specific variations in cytochrome b (Cytb) sequence were assessed in 22 specimens of Sorex palustris Richardson, 1828 and 6 specimens of Sorex bendirii (Merriam, 1884) from 20 locations in western North America. Phylogenetic analyses revealed three distinct clades: Boreal (S. p. palustris), Cordilleran (S. p. brooksi, S. p. navigator), and Coastal (S. b. palmeri, S. b. bendirii). Sequence divergence between the Boreal and the Coastal–Cordilleran lineages was 6.9%, while the divergence between the Coastal and the Cordilleran clades was 3.1%. Sorex palustris brooksi, a subspecies endemic to Vancouver Island, showed minor divergence from mainland samples of S. p. navigator. The results suggest that S. palustris may consist of two species: a boreal eastern form (S. palustris) and a Cordilleran form (S. navigator). The taxonomic validity of S. p. brooksi is unresolved. Distribution of the three clades are consistent with vicariance and isolation in coastal, Cordilleran, and eastern refugia in the Late Pliocene or Pleistocene. The Vancouver Island subspecies S. p. brooksi is probably derived from postglacial colonization in the Late Pleistocene.