Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali (Coleoptera: Buprestidae), a serious wood-boring beetle of the western Tianshan Mountains in China.

Agrilus mali stands as a significant wood-boring pest prevalent in Northeast Asia. Identifying this pest beetle is often hindered by insufficient efficient, rapid, on-site discrimination methods beyond examining adult morphological features. As a result, an urgent need arises for developing and implementing a rapid and accurate molecular technique to distinguish and manage the beetle. This study presents a straightforward, swift, highly specific, and sensitive method built upon recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD). This method demonstrates the capability to promptly identify the beetle, even during its larval stage. RPA primers and probes were designed using the internal transcribed spacer 1 region. Through probe optimization, false-positive signals were successfully eliminated, with an accompanying discussion on the underlying causes of such signals. The RPA-LFD assays exhibited remarkable specificity and sensitivity, requiring as little as 10-3 ng of purified DNA. Furthermore, the extraction of crude DNA was achieved through immersion in sterile distilled water, thus streamlining the assay process. Achievable at temperatures ranging from 30 to 50 °C, the RPA-LFD assay can be executed manually without specialized equipment. By merging the RPA-LFD assay with DNA coarse extraction, A. mali can be detected within just 30 min. This current study effectively demonstrates the immense potential of RPA-LFD in quarantine and pest management. Additionally, it presents a universal technique for the rapid on-site diagnosis of insects, showcasing the wide applicability of this method.

Characterization and expression analysis of the SPL gene family during floral development and abiotic stress in pecan (Carya illinoinensis).

SQUAMOSA promoter binding protein-like (SPL) genes are a type of plant-specific transcription factors that play crucial roles in the regulation of phase transition, floral transformation, fruit development, and various stresses. Although SPLs have been characterized in several model species, no systematic analysis has been studied in pecans, an important woody oil tree species. In this study, a total of 32 SPL genes (CiSPLs) were identified in the pecan genome. After conducting phylogenetic analysis of the conserved SBP proteins from Arabidopsis, rice, and poplar, the CiSPLs were separated into eight subgroups. The CiSPL genes within the same subgroup contained very similar exon-intron structures and conserved motifs. Nine segmentally duplicated gene pairs in the pecan genome and 16 collinear gene pairs between the CiSPL and AtSPL genes were identified. Cis-element analysis showed that CiSPL genes may regulate plant meristem differentiation and seed development, participate in various biological processes, and respond to plant hormones and environmental stresses. Therefore, we focused our study on the expression profiles of CiSPL genes during flower and fruit development. Most of the CiSPL genes were predominantly expressed in buds and/or female flowers. Additionally, quantitative real time PCR (qRT-PCR) analyses confirmed that CiSPL genes showed distinct spatiotemporal expression patterns in response to drought and salt treatments. The study provides foundation for the further exploration of the function and evolution of SPL genes in pecan.

Genetic Variation May Have Promoted the Successful Colonization of the Invasive Gall Midge, Obolodiplosis robiniae, in China.

Invasive species often cause serious economic and ecological damage. Despite decades of extensive impacts of invasives on bio-diversity and agroforestry, the mechanisms underlying the genetic adaptation and rapid evolution of invading populations remain poorly understood. The black locust gall midge, Obolodiplosis robiniae, a highly invasive species that originated in North America, spread widely throughout Asia and Europe in the past decade. Here, we used 11 microsatellite DNA markers to analyze the genetic variation of 22 O. robiniae populations in China (the introduced region) and two additional US populations (the native region). A relatively high level of genetic diversity was detected among the introduced populations, even though they exhibited lower diversity than the native US populations. Evidence for genetic differentiation among the introduced Chinese populations was also found based on the high Fst value compared to the relatively low among the native US populations. Phylogenetic trees, structure graphical output, and principal coordinate analysis plots suggested that the Chinese O. robiniae populations (separated by up to 2,540 km) cluster into two main groups independent of geographical distance. Genetic variation has been observed to increase rapidly during adaptation to a new environment, possibly contributing to population establishment and spread. Our results provide insights into the genetic mechanisms underlying successful invasion, and identify factors that have contributed to colonization by an economically important pest species in China. In addition, the findings improve our understanding of the role that genetic structure plays during invasion by O. robiniae.

The conserved mitochondrial genome of the jewel beetle (Coleoptera: Buprestidae) and its phylogenetic implications for the suborder Polyphaga.

In this study, we sequenced the mitochondrial (mt) genome of Agrilus mali (Coleoptera: Buprestidae) using next-generation sequencing, and accordingly annotated 13 protein-coding, 22 tRNA, and 2 rRNA genes and a 1458-bp non-coding region. Comparative analysis indicated that the mt genome of A. mali is relatively conserved, with a typical gene content and order identical to those of other coleopterans. However, the newly sequenced mt genome is characterized by a relatively higher A + T content compared with that of other species within the family Buprestidae. Phylogenetic analysis based on Bayesian inference revealed that the evolutionary relationship among the six infraorders of the suborder Polyphaga is (Scirtiformia + (Elateriformia + ((Scarabaeiformia + Staphyliniformia) + (Bostrichiformia + (Cucujiformia))))). However, the topology indicated that the family Buprestidae is a sister group to other Polyphaga infraorders, excluding Scirtiformia as a monophyly, and thus the monophyly of Elateriformia was not supported. This study not only presents the mt genome of a species in the family Buprestidae and a comparative analysis of jewel beetles but also examines the contribution of mt genomes in elucidating phylogenetic relationships within the suborder Polyphaga of Coleoptera.

Evaluation of four commonly used DNA barcoding Loci for chinese medicinal plants of the family schisandraceae.

Many species of Schisandraceae are used in traditional Chinese medicine and are faced with contamination and substitution risks due to inaccurate identification. Here, we investigated the discriminatory power of four commonly used DNA barcoding loci (ITS, trnH-psbA, matK, and rbcL) and corresponding multi-locus combinations for 135 individuals from 33 species of Schisandraceae, using distance-, tree-, similarity-, and character-based methods, at both the family level and the genus level. Our results showed that the two spacer regions (ITS and trnH-psbA) possess higher species-resolving power than the two coding regions (matK and rbcL). The degree of species resolution increased with most of the multi-locus combinations. Furthermore, our results implied that the best DNA barcode for the species discrimination at the family level might not always be the most suitable one at the genus level. Here we propose the combination of ITS+trnH-psbA+matK+rbcL as the most ideal DNA barcode for discriminating the medicinal plants of Schisandra and Kadsura, and the combination of ITS+trnH-psbA as the most suitable barcode for Illicium species. In addition, the closely related species Schisandra rubriflora Rehder & E. H. Wilson and Schisandra grandiflora Hook.f. & Thomson, were paraphyletic with each other on phylogenetic trees, suggesting that they should not be distinct species. Furthermore, the samples of these two species from the southern Hengduan Mountains region formed a distinct cluster that was separated from the samples of other regions, implying the presence of cryptic diversity. The feasibility of DNA barcodes for identification of geographical authenticity was also verified here. The database and paradigm that we provide in this study could be used as reference for the authentication of traditional Chinese medicinal plants utilizing DNA barcoding.