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1.
Front Genet ; 15: 1373028, 2024.
Article in English | MEDLINE | ID: mdl-38784030

ABSTRACT

Hippophae rhamnoides subsp. sinensis Rousi is a cold- and drought-tolerant pioneer species with significant economic and ecological value. Evaluating its genetic diversity and population structure is of great importance for guiding the development and utilization of resources. In this study, a total of 41,804 SSRs were generated by transcriptome sequencing of Hippophae rhamnoides subsp. sinensis Rousi. Among the different SSR motif types, mononucleotide repeats (26,972) were the most abundant, followed by trinucleotides, tetranucleotides, and pentanucleotides. 200 pairs of SSR primers were selected to detect polymorphisms, of which 15 pairs primers were selected as validated polymorphic SSRs used for genetic diversity and population structure analysis. A total of 63 alleles were identified with 15 pairs primers, with Nei's genetic diversity index ranged from 0.27 to 0.83 (average: 0.54), and the expected heterozygosity ranged from 0.16 to 0.73 (average: 0.46). The polymorphism information content ranged from 0.23 to 0.81 (average: 0.48). Genetic structure analyses showed that the 10 populations could be broadly categorized into two groups. AMOVA denoted that genetic variations primarily originated from within the populations, with minimal differences observed between the groups, accounting for only 7% of the total genetic variation. This implies that mutation in H. rhamnoides subsp. sinensis Rousi mainly occurred within the populations. The results showed that the 10 populations of H. rhamnoides subsp. sinensis Rousi are rich in genetic diversity, with low levels of population differentiation and a high degree of gene exchange, which should be taken into consideration for the future work of germplasm resource preservation and seedling breeding.

2.
J Fungi (Basel) ; 10(5)2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38786670

ABSTRACT

The greater yam (Dioscorea alata), a widely cultivated and nutritious food crop, suffers from widespread yield reduction due to anthracnose caused by Colletotrichum gloeosporioides. Latent infection often occurs before anthracnose phenotypes can be detected, making early prevention difficult and causing significant harm to agricultural production. Through comparative genomic analysis of 60 genomes of 38 species from the Colletotrichum genus, this study identified 17 orthologous gene groups (orthogroups) that were shared by all investigated C. gloeosporioides strains but absent from all other Colletotrichum species. Four of the 17 C. gloeosporioides-specific orthogroups were used as molecular markers for PCR primer designation and C. gloeosporioides detection. All of them can specifically detect C. gloeosporioides out of microbes within and beyond the Colletotrichum genus with different sensitivities. To establish a rapid, portable, and operable anthracnose diagnostic method suitable for field use, specific recombinase polymerase amplification (RPA) primer probe combinations were designed, and a lateral flow (LF)-RPA detection kit for C. gloeosporioides was developed, with the sensitivity reaching the picogram (pg) level. In conclusion, this study identified C. gloeosporioides-specific molecular markers and developed an efficient method for C. gloeosporioides detection, which can be applied to the prevention and control of yam anthracnose as well as anthracnose caused by C. gloeosporioides in other crops. The strategy adopted by this study also serves as a reference for the identification of molecular markers and diagnosis of other plant pathogens.

3.
BMC Ecol Evol ; 24(1): 73, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38822255

ABSTRACT

Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.


Subject(s)
DNA Barcoding, Taxonomic , DNA Primers , DNA, Environmental , Mollusca , Mollusca/genetics , Animals , DNA Barcoding, Taxonomic/methods , DNA, Environmental/analysis , DNA, Environmental/genetics , DNA Primers/genetics , Biodiversity
4.
Mol Biol Rep ; 51(1): 87, 2024 Jan 06.
Article in English | MEDLINE | ID: mdl-38183556

ABSTRACT

BACKGROUND: The Eastern Tropical Pacific (ETP) harbors a great diversity of Porifera. In particular, the Aplysina genus has acquired biotechnological and pharmacological importance. Nevertheless, the ecological aspects of their species and populations have been poorly studied. Aplysina gerardogreeni is the most conspicuous verongid sponge from the ETP, where it is usually found on rocky-coralline ecosystems. We evaluated the polymorphism levels of 18 microsatellites obtained from next-generation sequencing technologies. Furthermore, we tested the null hypothesis of panmixia in A. gerardogreeni population from two Mexican-Pacific localities. METHODS AND RESULTS: A total of 6,128,000 paired reads were processed of which primer sets of 18 microsatellites were designed. The loci were tested in 64 specimens from Mazatlan, Sinaloa (N = 32) and Isabel Island, Nayarit (N = 32). The microsatellites developed were moderately polymorphic with a range of alleles between 2 and 11, and Ho between 0.069 and 0.785. Fifteen loci displayed significant deviation from the Hardy-Weinberg equilibrium. No linkage disequilibrium was detected. A strong genetic structure was confirmed between localities using hierarchical Bayesian analyses, principal coordinates analyses, and fixation indices (FST = 0.108*). All the samples were assigned to their locality; however, there was a small sign of mixing between localities. CONCLUSIONS: Despite the moderate values of diversity in microsatellites, they showed a strong signal of genetic structure between populations. We suggest that these molecular markers can be a relevant tool to evaluate all populations across the ETP. In addition, 17 of these microsatellites were successfully amplified in the species A. fistularis and A. lacunosa, meaning they could also be applied in congeneric sponges from the Caribbean Sea. The use of these molecular markers in population genetic studies will allow assessment of the connectivity patterns in species of the Aplysina genus.


Subject(s)
Biotechnology , Ecosystem , Bayes Theorem , Alleles , Microsatellite Repeats/genetics
5.
Front Plant Sci ; 14: 1259736, 2023.
Article in English | MEDLINE | ID: mdl-38259948

ABSTRACT

Introduction: Simple sequence repeats (SSR), also known as microsatellites, are crucial molecular markers in both animals and plants. Despite extensive previous research on SSRs, the development of microsatellite markers in Brassica crops remains limited and inefficient. Methods: Krait software was used to identify microsatellites by genome-wide and marker development based on three recently sequenced basic species of Brassica crops in the triangle of U (Brassica rapa, B. nigra and B. oleracea), as well as three allotetraploids (B. juncea, B. napus and B. carinata) using public databases. Subsequently, the primers and the characteristics of microsatellites for most of them were accordingly designed on each chromosome of each of the six Brassica species, and their physical locations were identified,and the cross-transferability of primers have been carried out. In addition, a B-genome specific SSR marker was screened out. Results: A total of 79341, 92089, 125443, 173964, 173604, and 222160 SSR loci have been identified from the whole genome sequences of Brassica crops within the triangle of U crops, B. rapa (AA), B. nigra (BB), B. oleracea (CC), B. napus (AACC), B. juncea (AABB) and B. carinata (BBCC), respectively. Comparing the number distribution of the three allotetraploid SSR loci in the three subgenomes AA, BB and CC, results indicate that the allotetraploid species have significant reduction in the number of SSR loci in the genome compared with their basic diploid counterparts. Moreover, we compared the basic species with their corresponding varieties, and found that the microsatellite characters between the allotetraploids and their corresponding basic species were very similar or almost identical. Subsequently, each of the 40 SSR primers was employed to investigate the polymorphism potential of B. rapa (85.27%), B. nigra (81.33%) and B. oleracea (73.45%), and B. rapa was found to have a higher cross-transfer rate among the basic species in the triangle of U. Meanwhile, a B-genome specific SSR marker, BniSSR23228 possessing the (AAGGA)3 sequence characteristics was obtained, and it located in chromosome B3 with a total length of 97 bp. Discussion: In this study, results suggest that the pattern of distribution may be highly conserved during the differentiation of basic Brassica species and their allotetraploid counterparts. Our data indicated that the allotetraploidization process resulted in a significant reduction in SSR loci in the three subgenomes AA, BB and CC. The reasons may be partial gene dominated chromosomal homologous recombination and rearrangement during the evolution of basic diploid species into allotetraploids. This study provides a basis for future genomics and genetic research on the relatedness of Brassica species.

6.
Mol Biol Rep ; 49(10): 10121-10125, 2022 Oct.
Article in English | MEDLINE | ID: mdl-36057875

ABSTRACT

BACKGROUND: Next-generation sequencing technology has allowed for the rapid development of microsatellites, neutral polymorphic markers that can be used for the analysis of population structure. METHODS AND RESULTS: In this study, we performed whole-genome sequencing using the Illumina MiSeq system and de novo assembly to design microsatellite primers for Triops granarius populations in Qatar. The developed microsatellites are suitable for future studies of genetic structuring among geographically isolated freshwater pools. A total of 23 different primer pairs produced typical microsatellite results, with each pair successfully amplified in up to 40 individuals. Only five of the loci produced a significant departure from Hardy-Weinberg equilibrium. CONCLUSIONS: Some of the underlying mechanisms regarding the few loci that deviated from HWE may be further investigated to determine the source of deviation. As T. granarius is the most widely distributed species of the family, the development of these molecular markers would be useful for conducting population genetics and biogeographical studies broadly.


Subject(s)
Genetics, Population , Microsatellite Repeats , Animals , Crustacea/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Microsatellite Repeats/genetics , Technology
7.
Front Microbiol ; 11: 541554, 2020.
Article in English | MEDLINE | ID: mdl-33123100

ABSTRACT

The gene pair hgcAB is essential for microbial mercury methylation. Our understanding of its abundance and diversity in nature is rapidly evolving. In this study we developed a new broad-range primer set for hgcAB, plus an expanded hgcAB reference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy to hgcA sequences from clone, amplicon, and metagenomic datasets. We evaluated potential biases introduced in primer design, sequence length, and classification, and suggest best practices for studying Hg-methylator diversity. Our study confirms the emerging picture of an expanded diversity of HgcAB-encoding microbes in many types of ecosystems, with abundant putative mercury methylators Nitrospirae and Chloroflexi in several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB included Phycisphaerae, Aminicenantes, Spirochaetes, and Elusimicrobia. Combined with high-throughput amplicon specific sequencing, the new primer set also indentified novel hgcAB sequences similar to Lentisphaerae, Bacteroidetes, Atribacteria, and candidate phyla WOR-3 and KSB1 bacteria. Gene abundance data also corroborate the important role of two "classic" groups of methylators (Deltaproteobacteria and Methanomicrobia) in many environments, but generally show a scarcity of hgcAB+ Firmicutes. The new primer set was developed to specifically target hgcAB sequences found in nature, reducing degeneracy and providing increased sensitivity while maintaining broad diversity capture. We evaluated mock communities to confirm primer improvements, including culture spikes to environmental samples with variable DNA extraction and PCR amplification efficiencies. For select sites, this new workflow was combined with direct high-throughput hgcAB sequencing. The hgcAB diversity generated by direct amplicon sequencing confirmed the potential for novel Hg-methylators previously identified using metagenomic screens. A new phylogenetic analysis using sequences from freshwater, saline, and terrestrial environments showed Deltaproteobacteria HgcA sequences generally clustered among themselves, while metagenome-resolved HgcA sequences in other phyla tended to cluster by environment, suggesting horizontal gene transfer into many clades. HgcA from marine metagenomes often formed distinct subtrees from those sequenced from freshwater ecosystems. Overall the majority of HgcA sequences branch from a cluster of HgcAB fused proteins related to Thermococci, Atribacteria (candidate division OP9), Aminicenantes (OP8), and Chloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity of hgcAB+ microbes in nature.

8.
Mol Biol Rep ; 47(10): 8305-8310, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32974841

ABSTRACT

Xenocypris davidi is one of the most economically important freshwater fish in China. However, few molecular markers have been reported for this species, impeding in-depth population genetic, dispersal, and gene flow studies. In the present study, a batch of novel polymorphic microsatellites from the genome of X. davidi were isolated and characterized using high-throughput sequencing. A total of 20 microsatellite markers were isolated. Analysis of 33 individuals revealed an average of 7.35 alleles per locus, ranging from 3 to 18. The observed and expected heterozygosities ranged from 0.3 to 1 and from 0.426 to 0.93, respectively. Only one tested locus significantly deviated from Hardy-Weinberg equilibrium. 18 microsatellite loci were highly polymorphic (PIC > 0.5). These newly isolated microsatellite markers would be useful to study the population genetics and stock management of X. davidi.


Subject(s)
Alleles , Cyprinidae/genetics , Genetic Loci , Heterozygote , Microsatellite Repeats , Polymorphism, Genetic , Animals
9.
Mol Biol Rep ; 47(8): 6407-6415, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32617956

ABSTRACT

This study was conducted to develop the first species-specific microsatellite markers in Betula costata. A total of 178 primers were designed from 95,755 contigs and screened in two B. costata populations sampled from Mt. Hwaaksan and Mt. Gyebangsan. A total of 16 polymorphic microsatellite loci were selected and used for population genetic characterization. The average values of observed heterozygosity (HO) and expected heterozygosity (HE) of the Mt. Hwaaksan population were 0.488 and 0.493, respectively. The average values of HO and HE in the Mt. Gyebangsan population were 0.492 and 0.481, respectively. The null allele frequency was less than 0.2 in all loci. No significant linkage disequilibrium was detected in all combinations of loci. In addition, 26 polymorphic markers were selected by cross-species transferability test to B. costata using the microsatellite markers developed in four other Betula species. The cross-species transferability of the microsatellite markers developed in B. costata was conducted in two other Betula species. The transferability was 75% in B. ermanii and 100% in B. davurica. Therefore, the microsatellite markers developed and characterized in this study were expected to be useful for further genetic studies in B. costata and related species in the genus Betula.


Subject(s)
Betula/genetics , Microsatellite Repeats , DNA, Plant/genetics , Gene Frequency , Genome, Plant , Heterozygote , High-Throughput Nucleotide Sequencing , Polymorphism, Genetic
10.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-851457

ABSTRACT

Objective To explore genetic diversity of and genetic relationships among 18 Picria felterrae populations to provide references for the resource assessment and utilization. Methods The genetic diversity of 18 P. felterrae populations were analyzed using the EST-SSR primer development technology and SSR molecular markers, and cluster analysis was performed based on genetic distances to determine the relationships among those populations. Results A total of 48 pairs of polymorphic primers were selected from 100 pairs of EST-SSR markers, of which 20 pairs were randomly selected and used for amplification of 18 populations. A total of 71 alleles were amplified, 3.55 alleles per primer. Among the primers, the percentage of polymorphic loci (P) varied from 0 to 40.7%, with an average of 19.9%; The polymorphism information content (PIC) varied from 0 to 0.794 1, 0.397 7 on average; The Shannon diversity information index (I) varied from 0 to 1.814 3, with an average of 0.808 4; Obs_Het varied from 0 to 0.442 3, with an average of 0.212 7; And the Exp_Het varied from 0 to 0.826 9, with an average of 0.455 8. For the 18 populations, the Inbreeding Coefficient (Fis) varied from -0.095 3 to 0.663 9, with an average of 0.159 2; The inbreeding coefficient of subgroups (Fit) varied from 0.062 6 to 0.858 7, with an average of 0.537 2; The genetic differentiation coefficient (Fst) varied from 0 to 0.686, with an average of 0.449 6; The gene flow (Nm) varied from 0.114 4 to 0.759 4, with an average of 0.306 1. For the 18 samples tested, the gene diversity index (Nei) varied from 0 to 0.401 6, the I varied from 0 to 0.620 9, Wuzhou Guangxi having the maximum value and Longtan Yunnan the minimum value. Menglong and Jingha, two towns in Yunnan, had the shortest genetic distance (0.031 9), whereas Longzhou Guangxi and Menghai Yunnan had the maximum genetic distance (0.963 8). The 18 populations could be divided into four groups at the location where genetic distance was 0.321 3. The three populations in Guangxi belonged to the same group, populations from Menglong, Menglun and Mengzhe of Yunnan belonged in the same group, populations from Mengsong Yunnan became an independent group, and the rest belonged in the fourth group. Conclusion The genetic differentiation levels of 18 populations were not consistent, and the heterogeneity difference was significant. The gene flow among populations was small, which indicated that the population gene exchange was low. A certain inbreeding rate exists among the populations. The relationship among populations was influenced by geographical isolation and environmental factors. Key words:

11.
Appl Microbiol Biotechnol ; 101(3): 1267-1287, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28032194

ABSTRACT

PCR primers targeting genes encoding the two proteins of anammox bacteria, hydrazine synthase and cytochrome c biogenesis protein, were designed and tested in this study. Three different ecotypes of samples, namely ocean sediments, coastal wetland sediments, and wastewater treatment plant (WWTP) samples, were used to assess the primer efficiency and the community structures of anammox bacteria retrieved by 16S ribosomal RNA (rRNA) and the functional genes. Abundances of hzsB gene of anammox bacteria in South China Sea (SCS) samples were significantly correlated with 16S rRNA gene by qPCR method. And hzsB and hzsC gene primer pair hzsB364f-hzsB640r and hzsC745f-hzsC862r in combination with anammox bacterial 16S rRNA gene primers were recommended for quantifying anammox bacteria. Congruent with 16S rRNA gene-based community study, functional gene hzsB could also delineate the coastal-ocean distributing pattern, and seawater depth was positively associated with the diversity and abundance of anammox bacteria from shallow- to deep-sea. Both hzsC and ccsA genes could differentiate marine samples between deep and shallow groups of the Scalindua sp. clades. As for WWTP samples, non-Scalindua anammox bacteria reflected by hzsB, hzsC, ccsA, and ccsB gene-based libraries showed a similar distribution pattern with that by 16S rRNA gene. NH4+ and NH4+/Σ(NO3- + NO2-) positively correlated with anammox bacteria gene diversity, but organic matter contents correlated negatively with anammox bacteria gene diversity in SCS. Salinity was positively associated with diversity indices of hzsC and ccsB gene-harboring anammox bacteria communities and could potentially differentiate the distribution patterns between shallow- and deep-sea sediment samples. SCS surface sediments harbored considerably diverse community of Scalindua. A new Mai Po clade representing coastal estuary wetland anammox bacteria group based on 16S rRNA gene phylogeny is proposed. Existence of anammox bacteria within wider coverage of genera in Mai Po wetland indicates this unique niche is very complex, and species of anammox bacteria are niche-specific with different physiological properties towards substrates competing and chemical tolerance capability.


Subject(s)
Ammonium Compounds/metabolism , Bacteria/enzymology , Bacteria/genetics , Cytochromes c/biosynthesis , DNA Primers , Hydrazines/metabolism , Polymerase Chain Reaction , Biodiversity , China , Cytochromes c/genetics , DNA, Bacterial/genetics , Genetic Variation , Microbial Consortia/genetics , Microbial Consortia/physiology , Oxidation-Reduction , Phylogeny , RNA, Ribosomal, 16S , Seawater/microbiology , Sequence Analysis, DNA , Wetlands
12.
J Biotechnol ; 244: 4-15, 2017 Feb 20.
Article in English | MEDLINE | ID: mdl-28011128

ABSTRACT

Direct molecular approaches provide hints that lactic acid bacteria play an important role in the degradation process of organic material to methanogenetic substrates in biogas plants. However, their diversity in biogas fermenter samples has not been analyzed in detail yet. For that reason, five different biogas fermenters, which were fed mainly with maize silage and manure from cattle or pigs, were examined for the occurrence of lactic acid-forming bacteria. A total of 197 lactic acid-forming bacterial strains were isolated, which we assigned to 21 species, belonging to the genera Bacillus, Clostridium, Lactobacillus, Pediococcus, Streptococcus and Pseudoramibacter-related. A qualitative multiplex system and a real-time quantitative PCR could be developed for most isolates, realized by the selection of specific primers. Their role in biogas plants was discussed on the basis of the quantitative results and on physiological data of the isolates.


Subject(s)
Bacteria/classification , Bacteria/isolation & purification , Lactic Acid/biosynthesis , Plants/microbiology , Bacillus/classification , Bacillus/isolation & purification , Bacteria/genetics , Biodiversity , Biofuels , Clostridium/classification , Clostridium/isolation & purification , Fermentation , Lactobacillus/classification , Lactobacillus/isolation & purification , Manure/microbiology , Multiplex Polymerase Chain Reaction , Pediococcus/classification , Pediococcus/isolation & purification , Real-Time Polymerase Chain Reaction , Streptococcus/classification , Streptococcus/isolation & purification
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