Diversity of ecotypes of five species of ryegrass from Northwestern Spain by phenotypic traits and microsatellites.

BACKGROUND: The Agricultural Research Centre of Mabegondo (Xunta de Galicia, A Coruña, Spain) conserves one of the most important collections of phytogenetic resources of ecotypes and natural populations of grassland species from northwestern Spain, among them populations of ryegrass (Lolium spp.), one of the most cultivated forage grasses in the world. The objective of the present study was to evaluate the diversity among commercial cultivars and natural ryegrass populations with phenotypic traits and molecular markers. RESULTS: Eleven polymorphic microsatellites loci were used to analyze 58 ecotypes and 10 cultivars (680 DNA samples in total) differentiating 673 genotypes. Two main groups were detected by the Structure analysis, one related to Lolium perenne and a second to Lolium multiflorum. The first group showed two subgroups and the second three. The cluster of L. multiflorum showed two subgroups not related with the third cluster including commercial varieties, one from the Canary Islands (with Lolium rigidum included) and a second one from northwestern Spain, which presented specific agromorphological characteristics, such as lower FES (number of days from 1 January, when three heads per plant were flowering per plot), CRE (growth in flowering, in g of dry matter), and AIN (number of inflorescences per plant). CONCLUSIONS: This is the first time that a large amount of data on ryegrass from the Iberian Peninsula has been analyzed, obtaining a clear genetic differentiation of the autochthonous varieties from the commercial varieties analyzed. In addition, the genetic structure found in the ecotypes was related to the phenotypic variation analyzed. Being of interest in the conservation of biodiversity and in obtaining better adapted varieties of ryegrasses, due to their specific phenotypic traits, such as a lower FES, CRE and AIN.

Development of EST-SSR markers in Bergenia ciliata using de novo transcriptome sequencing.

Bergenia ciliata (Haw.) Sternb. is an important herb predominantly found in the Indian Himalayan Region. It is widely used in medicines, healthcare systems, cosmetics, fodder, and ornamental purposes. The Illumina sequencing and de novo transcriptome assembly were carried out in B. ciliata to develop and identify simple sequence repeat markers. A total of 18 226 simple sequence repeats (SSRs) were identified wherein di-nucleotides were found to be abundant (47.88%), followed by mono-nucleotide (35.03%) and tri-nucleotide (15.88%) repeats. A total of 11 839 EST-SSR primers were designed, of which 96 primer pairs were commercially synthesized. Finally, 17 primer pairs revealed clear, distinct polymorphic bands, and these primers were validated with 40 diverse B. ciliata accessions. The present study revealed moderate level of genetic diversity (Ho = 0.389, He = 0.542, and PIC = 0.513). Furthermore, the transcriptome data and EST-SSR markers generated during the present investigation could be an important genetic resource for functional genomics, population studies, and conservation genetics of the genus Bergenia.

Mental health outcomes in patients with moderate to severe psoriasis treated with bimekizumab: Analysis of phase 2/3 randomized trials.

BACKGROUND: Patients with psoriasis have increased risk of suicidal ideation and behavior (SIB) and depression. Bimekizumab, a biologic that inhibits interleukin (IL)-17A and IL-17F, received Food and Drug Administration approval in 2023 for moderate to severe plaque psoriasis, following 2021 European Medicines Agency approval. OBJECTIVE: To report SIB and depression in patients with moderate to severe psoriasis treated in bimekizumab clinical trials. METHODS: Mental health changes, including neuropsychiatric events, were actively monitored across 9 bimekizumab clinical trials in psoriasis phase 2/3 trials. The patient-reported electronic Columbia-Suicide Severity Rating Scale (measuring SIB) and Patient Health Questionnaire-9 (measuring depression) were administered, monitored by an independent Neuropsychiatric Adjudication Committee. RESULTS: Throughout 7166 patient-years (PY) of bimekizumab exposure, the adjudicated SIB rate was 0.13/100PY; SIB ranges for the general psoriasis population and patients receiving anti-IL-17A/anti-IL-23 therapies are 0.09 to 0.54/100PY and 0.09 to 0.19/100PY, respectively. At week 16, 92.9% vs 81.1% of bimekizumab- vs placebo-treated patients had no/minimal depression. Newonset positive electronic Columbia-Suicide Severity Rating Scale responses and mean Patient Health Questionnaire-9 scores were low for bimekizumab-treated patients. LIMITATIONS: Patient exclusion for significant/severe prespecified SIB/depression history. CONCLUSION: The long-term adjudicated SIB rate with bimekizumab was low and within ranges reported in the general psoriasis patient population and psoriasis patients treated with anti-IL-17A/anti-IL-23 biologics. Screening/monitoring questionnaires reported low SIB and depression levels.

Development of eighteen novel microsatellite loci for Masked Bobwhite, Colinus virginianus ridgwayi.

BACKGROUND: Masked Bobwhite (Colinus virginianus ridgwayi) is a critically-endangered New World quail species endemic to Sonoran Desert grasslands of North America. It suffered severe population declines during the nineteenth and twentieth centuries, with its persistence now reliant upon a captive breeding program that requires careful genetic management to maintain extant genetic diversity. Although nuclear microsatellite DNA markers existed for the closely related Northern Bobwhite (C. virginianus), none were available for Masked Bobwhite to inform necessary management decisions. METHODS AND RESULTS: Paired-end Illumina© sequencing was conducted to screen the Masked Bobwhite genome for microsatellite loci. We identified 18 loci exhibiting high polymorphism and limited deviations from genetic equilibrium expectations. These loci were amplified in 78 individuals. Familial relationships were reconstructed via sibship methods and compared to manually-curated pedigree data. Thirteen of fifteen full-sibling groups in the pedigree were exactly reconstructed (86.6%). Three other full-sibling groups partially matched pedigree relationships with high statistical confidence, and likely represented pedigree inaccuracies. Four additional full-sibling pairs were identified with low statistical confidence and likely resulted from analytical artifacts. CONCLUSIONS: The novel microsatellite loci accurately reconstructed parent-offspring and sibling relationships. These loci will be useful for guiding genetic management decisions and identifying pedigree inaccuracies in the captive breeding program.

Repetitive Sequences, Codon Usage Bias and Phylogenetic Analysis of the Plastome of Miliusa glochidioides.

Annonaceae is the largest family in Magnoliales, exhibiting the greatest diversity among and within genera. In this study, we conducted an analysis of repetitive sequences and codon usage bias in the previously acquired plastome of Miliusa glochidioides. Using a concatenated dataset of shared genes, we constructed the phylogenetic relationships among 27 Annonaceae species. The results showed that the size of the plastomes in the Annonaceae ranged from 159 to 202 kb, with the size of the inverted repeat region ranging from 40 to 65 kb. Within the plastome of M. glochidioides, we identified 42 SSRs, 36 tandem repeats, and 9 dispersed repeats. These SSRs consist of three nucleotide types and eight motif types, with a preference for A/T bases, primarily located in the large single-copy regions and intergenic spacers. Tandem and dispersed repeat sequences were predominantly detected in the IR region. Through codon usage bias analysis, we identified 30 high-frequency codons and 11 optimal codons. The plastome of M. glochidioides demonstrated relatively weak codon usage bias, favoring codons with A/T endings, primarily influenced by natural selection. Phylogenetic analysis revealed that all four subfamilies formed monophyletic groups, with Cananga odorata (Ambavioideae) and Anaxagorea javanica (Anaxagoreoideae) successively nested outside Annonoideae + Malmeoideae. These findings improve our understanding of the plastome of M. glochidioides and provide additional insights for studying plastome evolution in Annonaceae.

Dig-up Primers: A Pipeline for Identification of Polymorphic Microsatellites Loci within Assemblies of Related Species.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers and are used in numerous fields, including conservation genetics, population genetic studies, and genetic mapping. Advances in next-generation sequencing technology and the growing amount of genomic data are driving the development of bioinformatics tools for SSR marker design. These tools work with different combinations of input data, which can be raw reads or assemblies, and with one or more input datasets. We present here a new strategy and implementation of a simple standalone pipeline that utilizes more than one assembly for the in silico design of PCR primers for microsatellite loci in more than one species. Primers are tested in silico to determine if they are polymorphic, eliminating the need to test time-consuming cross-species amplification in the laboratory. The end result is a set of markers that are in silico polymorphic in all analyzed species and have great potential for the identification of interspecies hybrids. The efficiency of the tool is demonstrated using two examples at different taxonomic levels and with different numbers of input assemblies to generate promising, high-quality SSR markers.

Chloroplast Genomes Evolution and Phylogenetic Relationships of Caragana species.

Caragana sensu lato (s.l.) includes approximately 100 species that are mainly distributed in arid and semi-arid regions. Caragana species are ecologically valuable for their roles in windbreaking and sand fixation. However, the taxonomy and phylogenetic relationships of the genus Caragana are still unclear. In this study, we sequenced and assembled the chloroplast genomes of representative species of Caragana and reconstructed robust phylogenetic relationships at the section level. The Caragana chloroplast genome has lost the inverted repeat region and wascategorized in the inverted repeat loss clade (IRLC). The chloroplast genomes of the eight species ranged from 128,458 bp to 135,401 bp and contained 110 unique genes. All the Caragana chloroplast genomes have a highly conserved structure and gene order. The number of long repeats and simple sequence repeats (SSRs) showed significant variation among the eight species, indicating heterogeneous evolution in Caragana. Selective pressure analysis of the genes revealed that most of the protein-coding genes evolved under purifying selection. The phylogenetic analyses indicated that each section forms a clade, except the section Spinosae, which was divided into two clades. This study elucidated the evolution of the chloroplast genome within the widely distributed genus Caragana. The detailed information obtained from this study can serve as a valuable resource for understanding the molecular dynamics and phylogenetic relationships within Caragana.

Factors associated with self-harm behaviors during military service in the Israel Defense Forces, rates of such behaviors and their outcomes.

The military environment involves stressful situations that may trigger or aggravate suicidal behaviors, such as suicide attempts (SAs), which significantly increase the likelihood of future suicide. This cross-sectional study aims to assess risk factors for severe SAs and non-suicidal self-injury (NSSI) among Israel Defense Forces (IDF) soldiers. Data were retrieved from an IDF computerized self-harm surveillance database and were based on the criteria of the Columbia Suicide Severity Rating Scale (C-SSRS) and the Suicide Attempt Self-Injury Interview (SASII). The cohort included all 1,238 occurrences of self-harm behavior, during 2017-2021. Other investigated variables included adjustment difficulty (AD, as per IDF definition) and psychiatric diagnosis (PD) as reported by mental health officers (MHOs) during recruitment. Higher rates of adjustment difficulties were found among soldiers who had conducted NSSIs. Higher rates of previous psychiatric diagnoses were found among individuals with SAs, and their risk of dying by suicide during military service was twice as high (OR = 2.356; p < .001). If the latter also served in a combat unit, the risk was almost fourfold (OR = .3.860; p < .001). The current study demonstrates a clear difference between IDF soldiers who conduct NSSI vs. those conducting SA with regard to adjustment difficulty (as per IDF definition) and PD.

The complete chloroplast genome of Ulmus mianzhuensis with insights into structural variations, adaptive evolution, and phylogenetic relationships of Ulmus (Ulmaceae).

BACKGROUND: Ulmus mianzhuensis is an endemic tree species in China with high ornamental and economic value. Currently, little is known regarding its genomic architecture, phylogenetic position, or adaptive evolution. Here, we sequenced the complete chloroplast genome (cp genome) of U. mianzhuensis and further compared the variations in gene organization and structure within Ulmus species to define their genomic evolution, then reconstructed the phylogenomic relationship of 31 related Ulmus species to explore the systematic position of U. mianzhuensis and the utility of cp genome for resolving phylogenetics among Ulmus species. RESULTS: Our results revealed that all the Ulmus species exhibited a typical quadripartite structure, with a large single copy (LSC) region of 87,170 - 88,408 bp, a small single copy (SSC) region of 18,650 - 19,038 bp and an inverted repeat (IR) region of 26,288 - 26,546 bp. Within Ulmus species, gene structure and content of cp genomes were highly conserved, although slight variations were found in the boundary of SC/IR regions. Moreover, genome-wide sliding window analysis uncovered the variability of ndhC-trnV-UAC, ndhF-rpl32, and psbI-trnS-GCU were higher among 31 Ulmus that may be useful for the population genetics and potential DNA barcodes. Two genes (rps15 and atpF) were further detected under a positive selection of Ulmus species. Comparative phylogenetic analysis based on the cp genome and protein-coding genes revealed consistent topology that U. mianzhuensis is a sister group to U. parvifolia (sect. Microptelea) with a relatively low-level nucleotide variation of the cp genome. Additionally, our analyses also found that the traditional taxonomic system of five sections in Ulmus is not supported by the current phylogenomic topology with a nested evolutionary relationship between sections. CONCLUSIONS: Features of the cp genome length, GC content, organization, and gene order were highly conserved within Ulmus. Furthermore, molecular evidence from the low variation of the cp genome suggested that U. mianzhuensis should be merged into U. parvifolia and regarded as a subspecies of U. parvifolia. Overall, we demonstrated that the cp genome provides valuable information for understanding the genetic variation and phylogenetic relationship in Ulmus.

The complete chloroplast genome of Diplodiscus trichospermus and phylogenetic position of Brownlowioideae within Malvaceae.

BACKGROUND: Malvaceae is an economically important plant family of 4,225 species in nine subfamilies. Phylogenetic relationships among the nine subfamilies have always been controversial, especially for Brownlowioideae, whose phylogenetic position remains largely unknown due to the lack of samples in previous analysis datasets. To greatly clarify the phylogenetic relationship of Malvaceae, we newly sequenced and assembled the plastome of Diplodiscus trichospermus taxonomically located in Brownlowioideae, and downloaded the allied genomes from public database to build a dataset covering all subfamily members of Malvaceae. RESULTS: The annotation results showed that the plastome of Diplodiscus trichospermus has a typical quadripartite structure, comprising 112 unique genes, namely 78 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The total length was 158,570 bp with 37.2% GC content. Based on the maximum likelihood method and Bayesian inference, a robust phylogenetic backbone of Malvaceae was reconstructed. The topology showed that Malvaceae was divided distinctly into two major branches which were previously recognized as Byttneriina and Malvadendrina. In the Malvadendrina clade, Malvoideae and Bombacoideae formed, as always, a close sister clade named as Malvatheca. Subfamily Helicteroideae occupied the most basal position and was followed by Sterculioideae which was sister to the alliance of Malvatheca, Brownlowioideae, Dombeyoideae, and Tilioideae. Brownlowioideae together with the clade comprising Dombeyoideae and Tilioideae formed a sister clade to Malvatheca. In addition, one specific conservation SSR and three specific palindrome sequences were observed in Brownlowioideae. CONCLUSIONS: In this study, the phylogenetic framework of subfamilies in Malvaceae has been resolved clearly based on plastomes, which may contribute to a better understanding of the classification and plastome evolution for Malvaceae.

De novo full-length transcriptome analysis of two ecotypes of Phragmites australis (swamp reed and dune reed) provides new insights into the transcriptomic complexity of dune reed and its long-term adaptation to desert environments.

BACKGROUND: The extremely harsh environment of the desert is changing dramatically every moment, and the rapid adaptive stress response in the short term requires enormous energy expenditure to mobilize widespread regulatory networks, which is all the more detrimental to the survival of the desert plants themselves. The dune reed, which has adapted to desert environments with complex and variable ecological factors, is an ideal type of plant for studying the molecular mechanisms by which Gramineae plants respond to combinatorial stress of the desert in their natural state. But so far, the data on the genetic resources of reeds is still scarce, therefore most of their research has focused on ecological and physiological studies. RESULTS: In this study, we obtained the first De novo non-redundant Full-Length Non-Chimeric (FLNC) transcriptome databases for swamp reeds (SR), dune reeds (DR) and the All of Phragmites australis (merged of iso-seq data from SR and DR), using PacBio Iso-Seq technology and combining tools such as Iso-Seq3 and Cogent. We then identified and described long non-coding RNAs (LncRNA), transcription factor (TF) and alternative splicing (AS) events in reeds based on a transcriptome database. Meanwhile, we have identified and developed for the first time a large number of candidates expressed sequence tag-SSR (EST-SSRs) markers in reeds based on UniTransModels. In addition, through differential gene expression analysis of wild-type and homogenous cultures, we found a large number of transcription factors that may be associated with desert stress tolerance in the dune reed, and revealed that members of the Lhc family have an important role in the long-term adaptation of dune reeds to desert environments. CONCLUSIONS: Our results provide a positive and usable genetic resource for Phragmites australis with a widespread adaptability and resistance, and provide a genetic database for subsequent reeds genome annotation and functional genomic studies.

Mapping QTLs for Super-Earliness and Agro-Morphological Traits in RILs Population Derived from Interspecific Crosses between Pisum sativum × P. fulvum.

Earliness in crop plants has a vital role in prevention of heat-induced drought stress and in combating global warming, which is predicted to exacerbate in the near future. Furthermore, earliness may expand production into northern areas or higher altitudes, having relatively shorter growing season and may also expand arable lands to meet global food demands. The primary objective of the present study was to investigate quantitative trait loci (QTLs) for super-earliness and important agro-morphological traits in a recombinant inbred line (RIL) population derived from an interspecific cross. A population of 114 RILs developed through single-seed descent from an interspecific cross involving Pisum sativum L. and P. fulvum Sibth. et Sm. was evaluated to identify QTLs for super-earliness and important agro-morphological traits. A genetic map was constructed with 44 SSRs markers representing seven chromosomes with a total length of 262.6 cM. Of the 14 QTLs identified, two were for super-earliness on LG2, one for plant height on LG3, six for number of pods per plant on LG2, LG4, LG5 and LG6, one for number of seeds per pod on LG6, one for pod length on LG4 and three for harvest index on LG3, LG5, and LG6. AA205 and AA372-1 flanking markers for super-earliness QTLs were suggested for marker-assisted selection (MAS) in pea breeding programs due to high heritability of the trait. This is the first study to map QTLs originating from P. sativum and P. fulvum recently identified species with super-earliness character and the markers (AA205 and AA372-1) linked to QTLs were valuable molecular tools for pea breeding.

Detailed assessment of suicidal ideation in youth with bipolar disorder versus major depressive disorder.

OBJECTIVES: There is a critical need to better understand the factors underlying the increased suicide risk for youth with bipolar disorder (BD) in order to develop targeted prevention efforts. This study aimed to examine differences in characteristics of suicide ideation (SI) in youth with BD compared to youth with major depressive disorder (MDD) that may be associated with increased suicide risk. METHODS: One hundred and fifty-one participants (92 MDD and 59 BD), ages 13-21, completed a diagnostic interview and clinical assessments. Lifetime symptoms of SI and SA were assessed using the Columbia Suicide Severity Rating Scale. Ordinal logistic regression models were used to investigate whether the diagnostic group predicted the severity and intensity of the most severe or most common SI with the age of onset, age, and gender as covariates. RESULTS: Compared to MDD youth, BD youth were more likely to report experiencing more severe SI, p = 0.039, experiencing the most severe SI more frequently, p = 0.002, having less control of the most severe SI, p = 0.012, and that deterrents were less likely to stop them from acting on the most severe SI, p = 0.006. CONCLUSION: This study highlights differences in the severity and intensity of SI in youth with BD and suggests that youth with BD have greater difficulty inhibiting thoughts of SI which may lead to less resistance to suicide action. Findings underscore the need for a more detailed assessment of SI in youth with BD to better understand SI as a proximal risk factor for future SA and a potential target for intervention.

Development and characterization of microsatellite loci in Ulex parviflorus Pourr. And its cross-transferability to other Genisteae.

BACKGROUND: The genus Ulex is composed by 15 species distributed in Europe and Africa, but the majority of them are restricted to the Iberian Peninsula and Northwest Africa. Some of these species are common elements at the landscape level, and others contribute to global biodiversity as narrow endemics. Assayed nuclear and plastid Sanger-sequenced regions do not provide enough resolution to perform evolutionary studies on the genus, neither at the intraspecific population level nor at the interspecific phylogenetic level. Thus, we have developed and characterized a set of nuclear microsatellite loci in U. parviflorus to provide new highly polymorphic molecular markers for the genus Ulex. METHODS AND RESULTS: Genomic DNA enriched in microsatellite motifs using streptavidin-coated M-280 magnetic beads attached to 5'-biotinylated oligonucleotides was sequenced in a 454GS Junior System. After primer design, fluorescent-dyed amplicons were analyzed through capillary sequencing (ABI3730XL). Here we present twelve new high polimorphic SSRs markers developed in U. parviflorus specimens and tested in 120 individuals. The 12 SSR loci amplified a total of 152 alleles, and detected expected heterozygosities that ranged from 0.674 to 0.725 in the genotyped populations. Successful cross-species transferability of the 12 SSR loci to the rest of species included in the genus Ulex and three other representative Genisteae was achieved. CONCLUSIONS: The 12 novel proposed SSRs loci will contribute to perform evolutionary studies and genetic research on the genus Ulex and in other Genisteae.

Identification and validation of the first EST-SSR markers based on transcriptome of Anopheles darlingi, the primary transmitter of malaria in Brazil.

BACKGROUND: Anopheles darlingi is a monotypic species in terms of its morphological, genetic, and behavioral aspects and is the primary transmitter of human malaria (99%) in Brazil, especially in the Brazilian Amazon. In this pioneering study, 15 expressed sequence tag (EST)-simple sequence repeat (SSR) markers were obtained and characterized in samples from the municipality of São Gabriel da Cachoeira, Amazonas state, Brazil, with polymorphisms that can be used for further genetic research. METHODS AND RESULTS: The specimens (from egg to larval stage) collected were bred in the insectary at INPA (National Institute for Amazonian Research). The SSR repeats within the contigs of the A. darlingi EST banks were confirmed on the Vector Base site. DNA was extracted and amplified using polymerase chain reaction and then genotyped. Fifteen polymorphic SSR loci were identified and characterized. The number of alleles totaled 76 and ranged from 2 to 9. The observed heterozygosity varied between 0.026 and 0.769, the expected heterozygosity between 0.025 and 0.776, and the mean polymorphism information content was 0.468. Eight loci showed Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (P: (5%) ≤ 0.0033). No linkage disequilibrium was found among the loci. CONCLUSIONS: The polymorphic SSRs of the loci have been shown to be efficient for investigation of the variability and genetic population structure of A. darlingi.

Screening and validation of salt-stress responsive cg-SSR markers in wheat (Triticum aestivum L.) germplasm of Pakistan.

BACKGROUND: Soil salinity has been affecting wheat production worldwide over past few decades. Evaluation of wheat genotypes for salinity tolerance at germination and vegetative growth level is crucial. Marker assisted selection is a technique used extensively for choosing salt-tolerant genotypes from breeding populations to introduce novel genes. METHODS AND MATERIALS: The current study's main goal was to discover salt-stress resistant genes; genetic divergence and genome-wide connection by using recently designed candidate gene-based simple-sequence-repeat markers (cg-SSRs). The phenotypic connection of morphological features during the germination growth stage i.e., germination period, root length/weight and shoot length/weight, and vegetative growth stages i.e., root length/weight and shoot length/weight were tested in a group of 50 wheat genotypes. Significant difference was observed in germination rate, root length and weight among control and saline treatments. CONCLUSION: Total 30 SSR markers were utilized to test salinity resistance genes in wheat genotypes. Three (10%) of which were monomorphic, one (3.34%) showed no result, and the other 26 (86%) were polymorphic. Using 30 polymorphic markers discovered total 37 alleles. The polymorphic information content (PIC), quantifies each SSR locus capacity to discriminate between wheat, varied from 0.00 to 0.38 with an average of 0.19. Association analysis revealed that 26 primers were associated with morphological features, 03 with root length and the remaining 23 with germination. Utilizing morphological data, stress tolerance index (STI) was designed concluding that Auqab-2000, Margala-99 and Ufaq showed better resistance against salinity among other wheat genotypes. Cluster analysis demonstrated that wheat genotypes have vast genetic variability.

Genomic-based microsatellite development for Ternstroemia (Pentaphylacaceae) and transferability to other Ericales.

BACKGROUND: The genus Ternstroemia is associated with the vulnerable tropical montane cloud forest in Mexico and with other relevant vegetation types worldwide. It contains threatened and pharmacologically important species and has taxonomic issues regarding its species limits. This study describes 38 microsatellite markers generated using a genomic-based approach. METHODS AND RESULTS: We tested 23 of these markers in a natural population of Ternstroemia lineata. These markers are highly polymorphic (all loci polymorphic with 3-14 alleles per locus and expected heterozygosity between 0.202 and 0.908), most of them (19 out of 23) are in Hardy-Weinberg Equilibrium and free of null alleles (18 out of 23). Also we found no evidence of linkage among them. Finally, we tested the transferability to six other American species of Ternstroemia, two other Pentaphylacaceae species, and four species from different families within the order Ericales. CONCLUSIONS: These molecular resources are promising tools to investigate genetic diversity loss and as barcodes for ethnopharmacological applications and species delimitation in the family Pentaphylacaceae and some Ericales, among other applications.

Development of twenty-one novel microsatellite loci for Gila topminnow, Poeciliopsis occidentalis occidentalis.

BACKGROUND: Gila topminnow (Poeciliopsis occidentalis occidentalis) was once highly abundant throughout the Lower Colorado River Basin of the southwestern United States. However, this Sonoran Desert endemic suffered extreme population declines over the past century because of habitat degradation and nonnative species introductions. Much of the prior conservation genetic work conducted on the species relied upon a small number of microsatellite loci; many exhibiting low variability in extant populations. Consequently, there was a need for additional microsatellite loci to provide high-resolution delimitation of populations for conservation purposes. METHODS AND RESULTS: Paired-end Illumina sequencing was utilized to screen the Gila topminnow genome for novel microsatellite loci. We identified 21 novel loci that exhibited no deviations from expectations of genetic equilibrium, and cross-amplified in Yaqui topminnow (P. o. sonoriensis). These loci were amplified from 401 samples representing eight populations of Gila topminnow and Yaqui topminnow. Although diversity was low for all populations (observed heterozygosity = 0.12 to 0.45), these novel markers provided ample power to identify population of origin for each individual in Bayesian assignment tests. CONCLUSIONS: This novel set of microsatellite loci provide a useful genetic tool to assess population genetic parameters of the endangered Gila topminnow and delineate populations for identifying conservation priorities. The cross-amplification of these loci in Yaqui topminnow shows promise for application to other Poeciliopsis species of Mexico and Central America.

De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.

CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.