EST-SSR marker development based on transcriptome sequencing and genetic analyses of Phoebe bournei (Lauraceae).

This study aimed to compare and assess the genetic diversity and trends among the introduced family provenance, first-cycle superior trees breeding provenance, and improved-generation superior trees breeding provenance of Pinus elliottii using EST-SSR markers. The goal was to provide a foundation for advanced genetic improvement and sustainable utilization of P. elliottii in Jiangxi Province. A total of 417 individuals were analyzed for their genetic diversity and population structure using 19 pairs of SSR markers. The analysis identified 103 alleles across all the samples, with an average of 5.421 alleles per locus. Compared to other coniferous species, P. elliottii exhibited a moderate to high level of genetic diversity (I = 0.862, He = 0.457). Analysis of the molecular variance (AMOVA) revealed that 97.90% of the genetic variation occurred within provenances, consistent with a low genetic differentiation coefficient (Fst = 0.016 < 0.05) and high gene flow (Nm = 15.715) among provenances. In addition, analysis using STRUCTURE v. 2.3.4 software divided the 417 germplasm samples into two distinct groups, corroborating the results of the principal coordinates analysis (PCoA) and the unweighted pair group method with arithmetic (UPGMA) clustering analysis. Overall, the germplasm resources of P. elliottii exhibited rich genetic diversity, with the majority of the genetic variation occurring within provenances. For the genetic improvement of high-resin-yielding slash pines, breeding programs should prioritize populations with high genetic diversity while carefully selecting superior individuals from within those populations. These findings provide a solid foundation for breeding high-resin-yielding varieties and for future research on the sustainable utilization of these valuable resources.

Blumea balsamifera is a widely used ancient medicinal herb in tropical and subtropical Asia. Studying the genetic diversity of B. balsamifera germplasm resources and developing molecular markers related to the content of active constituents in B. balsamifera are of great significance for breeding varieties with high active ingredient content. In this study, the contents of 6 key active constituents were determined in 51 B. balsamifera accessions. EST-SSR markers were used to study their genetic diversity. At the same time, genetic structure analysis and linkage disequilibrium analysis were performed. On this basis, association analysis between EST-SSR markers and active ingredient content was performed. Quantitative analysis revealed substantial variation in six bioactive compounds, with coefficients of variation (CV) exceeding 50% across all metabolites. This result revealed its suitability for association analysis. Genetic diversity analysis revealed a total of 102 alleles amplified from 22 pairs of primers, with effective alleles accounting for 53.52%. The average polymorphism information content was 0.488, with 9 pairs of primers exhibiting high polymorphism (PIC > 0.5), 11 pairs of primers showing moderate polymorphism (0.25 < PIC < 0.5). The proposed primers demonstrated strong effectiveness and good polymorphism. The average Nei diversity index and Shannon information index were 0.542 and 1.023, respectively, indicating a high level of genetic diversity within the population. UPGMA clustering and population structure analyses classified the 51 accessions into four distinct groups correlated with geographical origins. LD analysis indicated over 50% of marker pairs showed substantial linkage (D' > 0.5), validating the suitability of this germplasm set for association mapping. Association analysis between quality traits and EST-SSR markers showed that, in both GLM and MLM models, Bbf137 and Bbf377 were found to be associated with 3,3',5,7-tetrahydroxy-4'- methoxyflavanone (TMF, P < 0.05) and Blumeatin (P < 0.01) respectively. These findings lay a robust foundation for future breeding strategies and genetic enhancement of B. balsamifera.

Spatial distribution and risk assessment of heavy metal pollution from enterprises in China

Tartary buckwheat (Fagopyrum tataricum) has been cultivated for over one thousand years in the Liangshan Prefecture of Sichuan, China. Growing population pressures, economic modernization pressures, and the erosion of traditional culture have led to the rapid loss of area covered by Tartary buckwheat landraces. Morphological and molecular characterization of 112 Tartary buckwheat accessions from 29 populations were assessed based on 10 morphological traits of seeds and 10 SSR markers, respectively. The coefficient of variation and Shannon index showed diversity within the morphological characteristics of the seeds. All accessions were divided into three categories according to phylogenetic dendrogram analysis, which was consistent with folk nomenclature and taxonomy. Genetic analysis using SSR markers identified 45 alleles with a mean value of 4.5 alleles per locus. The high average PIC value (0.459) indicated polymorphism of the SSR markers. The genetic similarity coefficient of the 112 Tartary buckwheat accessions showed a high level of genetic diversity ranging from 0.130 to 0.978. The genetic structure analysis revealed high genetic differentiation (Nei = 0.255). The folk nomenclature, folk taxonomy, and sociocultural norms may also contribute to a significant influence on the diversity of folk nomenclature and taxonomy. The assessment of the genetic diversity of Tartary buckwheat landraces and detection of SSR loci associated with traits could be used as scientific guidance for selecting Tartary buckwheat seed for improved production relative to local farmers and consumer preferences. Local traditional knowledge (seed exchange network) and culture also contribute to breeding and the maintenance of the genetic diversity of Tartary buckwheat.

Genetic diversity is the basis for adaptability of any organism against environmental changes through natural selection. Microsatellites markers are mostly used for studying population genetic diversity because of their locus-specific and hyper-variability. Availability of sequences (DNA) paved a way for the development of EST-SSRs. The ISSR markers were identified based on the amplification regions; usually 100–3000 bp present between inversely oriented closely spaced microsatellites. 40 SSR markers, 33 EST-SSR markers, 10 ISSR markers were used in the current study to check diversity among 30 diverse finger millet accessions from different geographical regions. 29 SSR markers, 21 EST-SSR markers and 10 ISSR markers exhibited polymorphism in which 19 SSR markers, 13 EST SSR markers and 8 ISSR markers have a PIC value above 0.5. 30 accessions are categorized into four major groups basing on the distribution of their polymorphic alleles. This current study demonstrates the use of different primers in studying diverse finger millet accessions. These identified primers will be valuable sources for any further genetic studies on Finger millet.

Geographical genetic diversity and divergence of common wild rice (O. rufipogon Griff.) in China

Triadica sebifera is an important landscaping tree species because of its colorful autumn leaves. In recent years, some cultivars have been bred and licensed, but it can be difficult to identify them from their morphological traits due to their similar phenotypes. To explore the genetic relationships and construct a fingerprint of the cultivars, the licensed T. sebifera cultivars were analyzed using SSR markers. A total of 179 alleles were identified among the 21 cultivars at 16 SSR loci, and these alleles exhibited a high level of genetic diversity (He = 0.86). The genetic variations mainly occurred among cultivars based on an analysis of molecular variance (AMOVA). According to phylogenetic analysis, principal coordinate analysis (PCoA), and Bayesian clustering analysis, the genetic relationships were independent of geographic distances, which may be mainly due to transplantations between regions. Some cultivars with different leaf colors showed obvious genetic differentiation and may be preliminary candidates for cross-breeding. Finally, the fingerprint for the licensed cultivars was constructed with two SSR markers. The results of this study can provide technical support for the application and legal protection of licensed Triadica sebifera cultivars.

The co-existence of several ploidy types in natural populations makes the cyprinid loach Misgurnus anguillicaudatus an exciting model system to study the genetic and phenotypic consequences of ploidy variations. A first step in such effort is to identify the specific ploidy of an individual. Currently popular methods of karyotyping via cytological preparation or flow cytometry require a large amount of tissue (such as blood) samples, which can be damaging or fatal to the fishes. Here, we developed novel microsatellite markers (SSR markers) from M. anguillicaudatus and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple M. anguillicaudatus using the Illumina paired-end sequencing. Approximately 150 million raw reads were assembled into 76,544 non-redundant unigenes. A total of 8,194 potential SSR markers were identified. We selected 98 pairs with more than five tandem repeats for further assays. Out of 45 putative EST-SSR markers that successfully amplified and harbored polymorphism in diploids, 11 markers displayed high variability in tetraploids. We further demonstrate that a set of five EST-SSR markers selected from these are sufficient to distinguish ploidy levels, by first validating them on 69 reference specimens with known ploidy levels and then subsequently using fresh-collected 96 ploidy-unknown specimens. The results from EST-SSR markers are highly concordant with those from independent flow cytometry analysis. The novel EST-SSR markers developed here should facilitate genetic studies of polyploidy in the emerging model system M. anguillicaudatus.

BackgroundTheileriosis, caused by a number of species within the genus Theileria, is a common disease of livestock in Oman. It is a major constraint to the development of the livestock industry due to a high rate of morbidity and mortality in both cattle and sheep. Since little is currently known about the genetic diversity of the parasites causing theileriosis in Oman, the present study was designed to address this issue with specific regard to T. annulata in cattle.MethodsBlood samples were collected from cattle from four geographically distinct regions in Oman for genetic analysis of the Theileria annulata population. Ten genetic markers (micro- and mini-satellites) representing all four chromosomes of T. annulata were applied to these samples using a combination of PCR amplification and fragment analysis. The resultant genetic data was analysed to provide a first insight into the structure of the T. annulata population in Oman.ResultsWe applied ten micro- and mini-satellite markers to a total of 310 samples obtained from different regions (174 [56%] from Dhofar, 68 [22%] from Dhira, 44 [14.5%] from Batinah and 24 [8%] from Sharqia). A high degree of allelic diversity was observed among the four parasite populations. Expected heterozygosity for each site ranged from 0.816 to 0.854. A high multiplicity of infection was observed in individual hosts, with an average of 3.3 to 3.4 alleles per locus, in samples derived from Batinah, Dhofar and Sharqia regions. In samples from Dhira region, an average of 2.9 alleles per locus was observed. Mild but statistically significant linkage disequilibrium between pairs of markers was observed in populations from three of the four regions. In contrast, when the analysis was performed at farm level, no significant linkage disequilibrium was observed. Finally, no significant genetic differentiation was seen between the four populations, with most pair-wise FST values being less than 0.03. Slightly higher FST values (GST’ = 0.075, θ = 0.07) were detected when the data for T. annulata parasites in Oman was compared with that previously generated for Turkey and Tunisia.ConclusionGenetic analyses of T. annulata samples representing four geographical regions in Oman revealed a high level of genetic diversity in the parasite population. There was little evidence of genetic differentiation between parasites from different regions, and a high level of genetic diversity was maintained within each sub-population. These findings are consistent with a high parasite transmission rate and frequent movement of animals between different regions in Oman.

Re-emergence of Severe Acute Diarrhea Syndrome Coronavirus (SADS-CoV) in Guangxi, China, 2021

The ascomycete pathogen Ramularia collo-cygni causes Ramularia leaf spot (RLS) on barley. Although R. collo-cygni is considerd an emerging disease of barley, little is known about genetic diversity or population genetic structure of this pathogen. We applied a set of polymorphic AFLP (Amplified Fragment Length Polymorphism) markers to investigate population genetic structure in two Northern European populations of R. collo-cygni. The distribution of AFLP alleles revealed low levels of population subdivision and high levels of genetic diversity at both locations. Our analyses included 87 isolates and of these 84 showed a unique genotype pattern. The genetic structure of populations in Scotland and Denmark is highly similar and we find no evidence of population sub-division. An analysis of molecular variance was used to show that 86 % of the variance is attributable to within field genetic variance. In spite of the high levels of genetic and genotypic diversity in the R. collo-cygni populations, we find significant evidence of linkage disequilibrium among the AFLP alleles using a multilocus analysis. We propose that the high levels of genotypic diversity and the lack of population differentiation result from considerable levels of gene flow between populations most likely mediated by seed borne dispersal of inoculum.

BackgroundAmorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of West Africa and South Asia. Its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. Genetic studies of Amorphophallus have been hindered by a lack of genetic markers. A large number of molecular markers are required for genetic diversity study and improving disease resistance in Amorphophallus. Here, we report large scale of transcriptome sequencing of two species: Amorphophallus konjac and Amorphophallus bulbifer using deep sequencing technology, and microsatellite (SSR) markers were identified based on these transcriptome sequences.ResultscDNAs of A. konjac and A. bulbifer were sequenced using Illumina HiSeq™ 2000 sequencing technology. A total of 135,822 non-redundant unigenes were assembled from about 9.66 gigabases, and 19,596 SSRs were identified in 16,027 non-redundant unigenes. Di-nucleotide SSRs were the most abundant motif (61.6%), followed by tri- (30.3%), tetra- (5.6%), penta- (1.5%), and hexa-nucleotides (1%) repeats. The top di- and tri-nucleotide repeat motifs included AG/CT (45.2%) and AGG/CCT (7.1%), respectively. A total of 10,754 primer pairs were designed for marker development. Of these, 320 primers were synthesized and used for validation of amplification and assessment of polymorphisms in 25 individual plants. The total of 275 primer pairs yielded PCR amplification products, of which 205 were polymorphic. The number of alleles ranged from 2 to 14 and the polymorphism information content valued ranged from 0.10 to 0.90. Genetic diversity analysis was done using 177 highly polymorphic SSR markers. A phenogram based on Jaccard’s similarity coefficients was constructed, which showed a distinct cluster of 25 Amorphophallus individuals.ConclusionA total of 10,754 SSR markers have been identified in Amorphophallus using transcriptome sequencing. One hundred and seventy-seven polymorphic markers were successfully validated in 25 individuals. The large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in Amorphophallus.

Aim of study: To evaluate genetic variability and population structure of C. lindemuthianum isolates in Turkey and to record the reactions of some common bean cultivars to the pathogen isolates representing different genetic groups.Area of study: The study was performed in seven provinces of Turkey.Material and methods: Genetic diversity of 91 C. lindemuthianum isolates obtained from different provinces of Turkey was characterized by 27 iPBS and 30 ISSR primers. Also, the resistance of 40 common bean cultivars was scored against three isolates representing different genetic groups.Main results: The dendrogram based on the combined dataset of iPBS and ISSR markers classified the isolates into two main groups with a genetic similarity of 72%, which closely associated with the geographic distribution of the isolates. The dendrogram of Nei’s genetic distances and Structure analysis supported the clustering of C. lindemuthianum isolates according to the geographical provinces. The results indicated that high level of genetic diversity (GST= 0.4) and low level of gene flow (NM=0.748) exist among the populations. AMOVA analysis showed that 58.7% of total genetic variability resulted from genetic differences between the isolates within populations, while 41.29% was among populations. Four cultivars showed resistant reaction to three isolates, while the other cultivars were susceptible to at least one isolate.Research highlights: The results indicated that iPBS and ISSR markers were reliable and effective tools for analyzing population structure of C. lindemuthianum and revealed high level of genetic and pathogenic diversity among pathogen populations in Turkey.

The Burmese python (Python bivittatus) has recently suffered large population declines in the wild in China due to illegal capture, overexploitation, deforestation and the loss of its natural habitat. Greater knowledge of the genetic diversity and structure of wild P. bivittatus populations is needed to help ensure its effective management. In this study, we investigated the genetic diversity and population genetic structure of wild P. bivittatus in China in detail. 109 P. bivittatus individuals from five distribution areas in Guangdong (GD), Guangxi (GX), Hainan (HN), Fujian (FJ) and Yunnan (YN) province of China were collected, and their genetic structure and diversity were analyzed. Eight highly polymorphic microsatellite loci were utilized to reveal high levels of genetic diversity in the P. bivittatus population. Genetic diversity was highest in GX, and lowest in GD. All geographic populations demonstrated a bottleneck effect indicating recent population decline. Fst and AMOVA analyses revealed that there was moderate genetic differentiation among the five populations, and that only 10.59 % of total genetic diversity occurred among populations. Fst values between populations were positively correlated with their geographical distances. Genetic structure analyses revealed that the HN, GX and GD populations, which were geographically closest, were assigned to a genetic cluster, while the YN and FJ populations constituted a single cluster, respectively.

The fast development of aquaculture over the past decades has made it the main source of fish protein and led to its integration into the global food system. Mostly originating from inland production systems, aquaculture has emerged as strategy to decrease malnutrition in low-income countries. The Nile tilapia (Oreochromis niloticus) was introduced to Madagascar in the 1950s, and is now produced nationally at various scales. Aquaculture mostly relies on fry harvested from wild populations and grow-out in ponds for decades. It has recently been diversified by the introduction of several fast-growing strains. Little is known how local genetic diversity compares to recently introduced strains, although high and comparable levels of genetic diversity have previously been observed for both wild populations and local stocks. Our study compares DNA barcode genetic diversity among eight farms and several strains belonging to three species sampled. DNA-based lineage delimitation methods were applied and resulted in the detection of six well differentiated and highly divergent lineages. A comparison of DNA barcode records to sequences on the Barcode of Life Data System (BOLD) helped to trace the origin of several of them. Both haplotype and nucleotide diversity indices highlight high levels of mitochondrial genetic diversity, with several local strains displaying higher diversity than recently introduced strains. This allows for multiple options to maintain high levels of genetic diversity in broodstock and provides more options for selective breeding programs.