Site-Specific Self-Catalyzed DNA Depurination: A Biological Mechanism That Leads to Mutations and Creates Sequence Diversity.

Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.

Assembly and comparative analysis of the complete mitochondrial genome of Vaccinium carlesii Dunn.

Vaccinium L. is an important fruit tree with nutritional, medicinal, and ornamental values. However, the mitochondrial (mt) genome of Vaccinium L. remains largely unexplored. Vaccinium carlesii Dunn is an endemic wild resource in China, which is crucial for blueberry breeding. The V. carlesii mt genomes were sequenced using Illumina and Nanopore, which total length was 636,904 bp with 37 protein coding genes, 20 tRNA genes, and three rRNA genes. We found four pairs of long repeat fragments homologous recombination mediated the generation of substructures in the V. carlesii mt genome. We predicted 383 RNA editing sites, all converting cytosine (C) to uracil (U). According to the phylogenetic analysis, V. carlesii and V. macrocarpon of the Ericaceae exhibited the closest genetic relationship. This study provides a theoretical basis for understanding the evolution of higher plants, species classification and identification, and will also be useful for further utilization of Vaccinium germplasm resources.

Mitochondrial genome features and systematic evolution of diospyros kaki thunb 'Taishuu'.

BACKGROUND: 'Taishuu' has a crisp texture, abundant juice, and sweet flavor with hints of cantaloupe. The availability of mitochondrial genome data of Diospyros species is far from the known number of species. RESULTS: The sequencing data were assembled into a closed circular mitochondrial chromosome with a 421,308 bp length and a 45.79% GC content. The mitochondrial genome comprised 40 protein-coding, 24 tRNA, and three rRNA genes. The most common codons for arginine (Arg), proline (Pro), glycine (Gly), tryptophan (Trp), valine (Val), alanine (Ala), and leucine (Leu) were AGA, CCA, GGA, UGG, GUA, GCA, and CUA, respectively. The start codon for cox1 and nad4L protein-coding genes was ACG (ATG), whereas the remaining protein-coding genes started with ATG. There are four types of stop codons: CGA, TAA, TAG, and TGA, with TAA being the most frequently used stop codon (45.24%). In the D. kaki Thunb. 'Taishuu' mitochondrial genome, a total of 645 repeat sequences were identified, including 125 SSRs, 7 tandem repeats, and 513 dispersed repeats. Collinearity analysis revealed a close relationship between D. kaki Thunb. 'Taishuu' and Diospyros oleifera, with conserved homologous gene fragments shared among these species in large regions of the mitochondrial genome. The protein-coding genes ccmB and nad4L were observed to undergo positive selection. Analysis of homologous sequences between chloroplasts and mitochondria identified 28 homologous segments, with a total length of 24,075 bp, accounting for 5.71% of the mitochondrial genome. These homologous segments contain 8 annotated genes, including 6 tRNA genes and 2 protein-coding genes (rrn18 and ccmC). There are 23 homologous genes between chloroplasts and nuclei. Mitochondria, chloroplasts, and nuclei share two homologous genes, which are trnV-GAC and trnW-CCA. CONCLUSION: In conclusion, a high-quality chromosome-level draft genome for D. kaki was generated in this study, which will contribute to further studies of major economic traits in the genus Diospyros.

Assembly of continuous high-resolution draft genome sequence of Hemicentrotus pulcherrimus using long-read sequencing.

The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided ~600-Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5% and 77.8%, respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcherrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of ~100 Mb, N50 = 143 kb, BUSCO completeness score = 86.1%, and TMMR = 55.4%. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using H. pulcherrimus.

Comparative genomics reveals insights into anuran genome size evolution.

BACKGROUND: Amphibians, particularly anurans, display an enormous variation in genome size. Due to the unavailability of whole genome datasets in the past, the genomic elements and evolutionary causes of anuran genome size variation are poorly understood. To address this, we analyzed whole-genome sequences of 14 anuran species ranging in size from 1.1 to 6.8 Gb. By annotating multiple genomic elements, we investigated the genomic correlates of anuran genome size variation and further examined whether the genome size relates to habitat types. RESULTS: Our results showed that intron expansions or contraction and Transposable Elements (TEs) diversity do not contribute significantly to genome size variation. However, the recent accumulation of transposable elements (TEs) and the lack of deletion of ancient TEs primarily accounted for the evolution of anuran genome sizes. Our study showed that the abundance and density of simple repeat sequences positively correlate with genome size. Ancestral state reconstruction revealed that genome size exhibits a taxon-specific pattern of evolution, with families Bufonidae and Pipidae experiencing extreme genome expansion and contraction events, respectively. Our result showed no relationship between genome size and habitat types, although large genome-sized species are predominantly found in humid habitats. CONCLUSIONS: Overall, our study identified the genomic element and their evolutionary dynamics accounting for anuran genome size variation, thus paving a path to a greater understanding of the size evolution of the genome in amphibians.

The complete chloroplast genome of two Firmiana species and comparative analysis with other related species.

Firmiana is a small genus within the subfamily Sterculioideae of the Malvaceae. There are nine Firmiana species distributed in South and South-west China, most of which are endangered. Due to the shortage of plastid genomes data, the phylogenetic relationships and the evolutionary history of this genus remain unclear. Therefore, the complete chloroplast genomes of F. calcarean and F. hainanensis were sequenced using high-throughput sequencing and then compared with the chloroplast genomes of other reported Firmiana species. The genome size of F. calcarean and F. hainanensis is 161,263 and 160,031 bp long, respectively, containing a total of 131 genes (including 85 protein coding genes, 37 tRNAs, 8 rRNAs, and one pseudogene). Comparative analysis revealed that the genome structure, GC content, gene content and order, as well as the RNA editing sites within the chloroplast genomes of F. calcarean and F. hainanensis were similar to previously reported Firmiana species. ML phylogenetic analysis revealed that F. danxiaensis, F. hainanensis, F. calcarean, F. simplex, and F. major form a sister group to F. colorata, F. pulcherrima, and F. kwangsiensis. The SSRs, long repeats, and 21 highly divergent regions (Pi > 0.01) identified in this study might provide potential DNA markers for further population genetics and phylogenetic studies of Firmiana. Our findings can help design new species-specific molecular markers and the general framework to further explore the evolutionary history of Firmiana and to address their conservation challenges.

Systematic identification and characterization of repeat sequences in African swine fever virus genomes.

African swine fever virus (ASFV) is a large DNA virus that infects domestic pigs with high morbidity and mortality rates. Repeat sequences, which are DNA sequence elements that are repeated more than twice in the genome, play an important role in the ASFV genome. The majority of repeat sequences, however, have not been identified and characterized in a systematic manner. In this study, three types of repeat sequences, including microsatellites, minisatellites and short interspersed nuclear elements (SINEs), were identified in the ASFV genome, and their distribution, structure, function, and evolutionary history were investigated. Most repeat sequences were observed in noncoding regions and at the 5' end of the genome. Noncoding repeat sequences tended to form enhancers, whereas coding repeat sequences had a lower ratio of alpha-helix and beta-sheet and a higher ratio of loop structure and surface amino acids than nonrepeat sequences. In addition, the repeat sequences tended to encode penetrating and antimicrobial peptides. Further analysis of the evolution of repeat sequences revealed that the pan-repeat sequences presented an open state, showing the diversity of repeat sequences. Finally, CpG islands were observed to be negatively correlated with repeat sequence occurrences, suggesting that they may affect the generation of repeat sequences. Overall, this study emphasizes the importance of repeat sequences in ASFVs, and these results can aid in understanding the virus's function and evolution.

The influence of regulatory elements on Mycoplasma hyopneumoniae 7448 transcriptional response during oxidative stress and heat shock.

BACKGROUND: The comprehension of genome organization and gene modulation is essential for understanding pathogens' infection mechanisms. Mycoplasma hyopneumoniae 7448 genome is organized in transcriptional units (TUs), which are flanked by regulatory elements such as putative promoters, terminators and repetitive sequences. Yet the relationship between the presence of these elements and bacterial responses during stress conditions remains unclear. Thus, in this study, in silico and RT-qPCR analyses were associated to determine the effect of regulatory elements in gene expression regulation upon heat shock and oxidative stress conditions. METHODS AND RESULTS: Thirteen TU's organizational profiles were found based on promoters and terminators distribution. Differential expression in genes sharing the same TUs was observed, suggesting the activity of internal regulatory elements. Moreover, 88.8% of tested genes were differentially expressed under oxidative stress in comparison to the control condition, being 81.3% of them surrounded by their own regulatory elements. Similarly, under heat shock, 44.4% of the genes showed regulation when compared to control condition, being 75.0% of them surrounded by their own regulatory elements. CONCLUSIONS: Altogether, this data suggests the activity of internal regulatory elements in gene modulation of M. hyopneumoniae 7448 transcription.

PlantRep: a database of plant repetitive elements.

KEY MESSAGE: We re-annotated repeats of 459 plant genomes and released a new database: PlantRep ( http://www.plantrep.cn/ ). PlantRep sheds lights of repeat evolution and provides fundamental data for deep exploration of genome.

Analysis of CRISPR-Cas system and antimicrobial resistance in Staphylococcus coagulans isolates.

CRISPR-Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR-Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR-Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR-Cas system. Three isolates from Antarctica seals neither contain CRISPR-Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR-Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR-Cas system, and this Type IIIA CRISPR-Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR-Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR-Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.

A highly mutable GST is essential for bract colouration in Euphorbia pulcherrima Willd. Ex Klotsch.

BACKGROUND: Mutation breeding is an extraordinary tool in plant breeding to increase the genetic variability, where mutations in anthocyanin biosynthesis are targets to generate distinctive phenotypes in ornamental species. In poinsettia, ionizing radiation is routinely applied in breeding programs to obtaining a range of colours, with nearly all pink and white varieties being obtained after γ- or X-ray mutagenesis of red varieties. In the present study we performed a thorough characterization of a potential mutagenesis target gene as the main responsible for the 'white paradox' in poinsettia. RESULTS: We identified a GST gene in poinsettia (Bract1) as an essential factor for the expression of anthocyanin-based red colouration of bracts, which presents a high phylogenetic similarity to known anthocyanin-related GSTs. Red poinsettia varieties and white mutants generated from these varieties by X-ray were analysed for polymorphisms related to the 'white paradox' in the species. A 4 bp mutation in a short repeat within the coding region of Bract1 is most likely responsible for the appearance of white phenotypes upon irradiation treatment. The polymorphism between wild-type and mutant alleles co-segregates with the phenotype in progeny from heterozygous red and white parents. Moreover, overexpression of Bract1 wild-type allele in Arabidopsis tt19 mutants restored the anthocyanin phenotype, while the Bract1 mutated allele showed to be non-functional. CONCLUSIONS: The identified repeat seems to be highly unstable, since mutated plants can be easily detected among fewer than 200 shoots derived from 10 mutated plants. Our data indicate that particular short repeat sequences, similar to microsatellite sequences or so-called dynamic mutations, might be hot spots for genetic variability. Moreover, the identification of the Bract1 mutation fills a gap on the understanding on the molecular mechanism of colour formation in poinsettia.

Sequence-dependent nucleosome formation in trinucleotide repeats evaluated by in vivo chemical mapping.

Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4-S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT)12 and (ACT)12 act as strong nucleosome-promoting sequences, while (AGG)12 and (CCG)12 act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG)12, is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.

Molecular Cytogenetic Analysis of Karyotype and Y Chromosome Conservation in Species of the Genus Talpa (Insectivora).

The Talpidae family has a highly stable karyotype. Most of the chromosome studies in this mammal group, however, employed classical cytogenetic techniques. Molecular cytogenetic analyses are still scarce and, for example, no repeated DNA sequences have been described to date. In this work, we used sequence analysis, chromosomal mapping of a LINE1 retroelement sequence, as well as chromosome painting with a whole Y chromosome probe of T. occidentalis to compare the karyotypes of 3 species of the genus Talpa (T. occidentalis, T. romana, and T. aquitania). Our results demonstrate that in Talpa genomes LINE1 sequences are widely distributed on all chromosomes but are enriched in pericentromeric C-band-positive regions. In addition, these LINE1 accumulate on the Y chromosomes of the 3 Talpa species regardless of their euchromatic or heterochromatic condition. Chromosome painting shows that the Y chromosomes in these 3 species are highly conserved. Interestingly, they share sequences with heterochromatic blocks on chromosome pairs 14 and 16 and, to a lesser degree, with the pericentromeric regions of other autosomes. Together, our analyses demonstrate that the repetitive DNA content of chromosomes from Talpa species is highly conserved.

Urine cell-free DNA as a promising biomarker for early detection of non-small cell lung cancer.

BACKGROUND: While blood-derived cell-free DNA has been shown to be a candidate biomarker able to provide diagnostic and prognostic insight in cancer patients, little is known regarding the potential application of urine cell-free DNA (ucfDNA) in diagnosis of cancer. Thus, the aim of this study was to investigate ucfDNA concentration and integrity index as potential biomarkers for early detection of non-small-cell lung cancer (NSCLC). METHODS: Urine samples were collected from 35 healthy controls and 55 NSCLC patients at various tumor node metastasis (TNM) stages. Two long interspersed nuclear element 1 (LINE1) fragments (LINE1-97 and 266 bp) were quantified via quantitative real-time PCR (qPCR). DNA integrity index was calculated as the ratio of LINE1-266/LINE-97. RESULTS: LINE1 fragments concentrations of ucfDNA (LINE1-97, 266 bp) were significantly higher in NSCLC patients with stage III/IV than in stage I/II and in healthy controls. The receiver operating characteristic (ROC) curves for discriminating patients with stage III/IV from healthy controls had areas under the curves (AUC) of 0.84 and 0.886, respectively. Moreover, ucfDNA integrity LINE1-266/97 was significantly higher in patients with stage III/IV than in stage I/II and in healthy controls. The AUC of ROC curve for discriminating patients with stage III/IV from healthy controls was 0.800. Furthermore, LINE1-266 fragment concentration was significantly higher in lymph node metastasis (LNM)-positive patients relative to LNM-negative patients. The ROC curve for discriminating LNM-positive from LNM-negative patients had an AUC of 0.822. CONCLUSION: UcfDNA could serve as a promising biomarker for early detection of NSCLC.

G6PD A- is the major cause of G6PD deficiency among the Siddis of Karnataka, India.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human erythroenzymopathy affecting more than 400 million people worldwide. G6PD deficiency was reported in India more than 50 years ago and the prevalence rate varies from 5.7% to 27.9% in different caste and tribal groups.Aim: To study the prevalence of, and the mutations causing, G6PD deficiency among the Siddis of Karnataka.Subjects and methods: A total of 755 individuals were screened using the DPIP dye decolorisation method and the deficiency was further confirmed by quantitative assay. Molecular characterisation was performed by PCR-RFLP method and DNA sequencing. Biochemical characterisation was performed as per WHO criteria.Results: Of the 755 individuals, 71 individuals (9.4%) were found to be G6PD deficient with an enzyme activity ranging from 0.02 to 3.83 IU/gm Hb. Mutational analysis could be performed on 49 G6PD deficient individuals and 45 (91.8%) of them showed the presence of the G6PD A- variant while the remaining 4 (8.2%) had the G6PD Kerala-Kalyan mutation. Microsatellite analysis in G6PD A- individuals showed the presence of 166/195 bp, AC/CTT alleles.Conclusions: G6PD deficiencies among the Siddis are predominantly due to G6PD A- mutation. Furthermore, biochemical parameters and the microsatellite repeat markers in the Siddi A- chromosome confirmed they are African descendants with Indian admixture.

Intergeneric Relationships within the Family Salicaceae s.l. based on Plastid Phylogenomics.

Many Salicaceae s.l. plants are recognized for their important role in the production of products such as wood, oils, and medicines, and as a model organism in life studies. However, the difference in plastid sequence, phylogenetic relationships, and lineage diversification of the family Salicaceae s.l. remain poorly understood. In this study, we compare 24 species representing 18 genera of the family. Simple sequence repeats (SSRs) are considered effective molecular markers for plant species identification and population genetics. Among them, a total of 1798 SSRs were identified, among which mononucleotide repeat was the most common with 1455 accounts representing 80.92% of the total. Most of the SSRs are located in the non-coding region. We also identified five other types of repeats, including 1750 tandems, 434 forward, 407 palindromic, 86 reverse, and 30 complementary repeats. The species in Salicaceae s.l. have a conserved plastid genome. Each plastome presented a typical quadripartite structure and varied in size due to the expansion and contraction of the inverted repeat (IR) boundary, lacking major structural variations, but we identified six divergence hotspot regions. We obtained phylogenetic relationships of 18 genera in Salicaceae s.l. and the 24 species formed a highly supported lineage. Casearia was identified as the basal clade. The divergence time between Salicaceae s.l. and the outgroup was estimated as ~93 Mya; Salix, and Populus diverged around 34 Mya, consistent with the previously reported time. Our research will contribute to a better understanding of the phylogenetic relationships among the members of the Salicaceae s.l.

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent "a hypothetical ancestral super-sex chromosome" or random distribution?

BACKGROUND: Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. RESULTS: We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. CONCLUSIONS: The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes.

Fluorescence in situ hybridization karyotyping reveals the presence of two distinct genomes in the taxon Aegilops tauschii.

BACKGROUND: Aegilops tauschii is the donor of the bread wheat D genome. Based on spike morphology, the taxon has conventionally been subdivided into ssp. tauschii and ssp. strangulata. The present study was intended to address the poor match between this whole plant morphology-based subdivision and genetic relationships inferred from genotyping by fluorescence in situ hybridization karyotyping a set of 31 Ae. tauschii accessions. RESULTS: The distribution of sites hybridizing to the two probes oligo-pTa-535 and (CTT)10 split the Ae. tauschii accessions into two clades, designated Dt and Ds, which corresponded perfectly with a previously assembled phylogeny based on marker genotype. The Dt cluster was populated exclusively by ssp. tauschii accessions, while the Ds cluster harbored both ssp. strangulata and morphologically intermediate accessions. As a result, it is proposed that Ae. tauschii ssp. tauschii is restricted to carriers of the Dt karyotype: their spikelets are regularly spaced along the rachis, at least in the central portion of their spike. Accessions classified as Ae. tauschii ssp. strangulata carry the Ds karyotype; their spikelets are irregularly spaced. Based on this criterion, forms formerly classified as ssp. tauschii var. meyeri have been re-designated ssp. strangulata var. meyeri. CONCLUSIONS: According to the reworking of the taxon, the bread wheat D genome was most probably donated by ssp. strangulata var. meyeri. Chromosomal differentiation reveals intra-species taxon of Ae. tauschii. Ae. tauschii ssp. tauschii has more distant relationship with breed wheat than ssp. strangulata and can be used for breeding improving effectively.

An expanded global inventory of allelic variation in the most extremely polymorphic region of Plasmodium falciparum merozoite surface protein 1 provided by short read sequence data.

BACKGROUND: Within Plasmodium falciparum merozoite surface protein 1 (MSP1), the N-terminal block 2 region is a highly polymorphic target of naturally acquired antibody responses. The antigenic diversity is determined by complex repeat sequences as well as non-repeat sequences, grouping into three major allelic types that appear to be maintained within populations by natural selection. Within these major types, many distinct allelic sequences have been described in different studies, but the extent and significance of the diversity remains unresolved. METHODS: To survey the diversity more extensively, block 2 allelic sequences in the msp1 gene were characterized in 2400 P. falciparum infection isolates with whole genome short read sequence data available from the Pf3K project, and compared with the data from previous studies. RESULTS: Mapping the short read sequence data in the 2400 isolates to a reference library of msp1 block 2 allelic sequences yielded 3815 allele scores at the level of major allelic family types, with 46% of isolates containing two or more of these major types. Overall frequencies were similar to those previously reported in other samples with different methods, the K1-like allelic type being most common in Africa, MAD20-like most common in Southeast Asia, and RO33-like being the third most abundant type in each continent. The rare MR type, formed by recombination between MAD20-like and RO33-like alleles, was only seen in Africa and very rarely in the Indian subcontinent but not in Southeast Asia. A combination of mapped short read assembly approaches enabled 1522 complete msp1 block 2 sequences to be determined, among which there were 363 different allele sequences, of which 246 have not been described previously. In these data, the K1-like msp1 block 2 alleles are most diverse and encode 225 distinct amino acid sequences, compared with 123 different MAD20-like, 9 RO33-like and 6 MR type sequences. Within each of the major types, the different allelic sequences show highly skewed geographical distributions, with most of the more common sequences being detected in either Africa or Asia, but not in both. CONCLUSIONS: Allelic sequences of this extremely polymorphic locus have been derived from whole genome short read sequence data by mapping to a reference library followed by assembly of mapped reads. The catalogue of sequence variation has been greatly expanded, so that there are now more than 500 different msp1 block 2 allelic sequences described. This provides an extensive reference for molecular epidemiological genotyping and sequencing studies, and potentially for design of a multi-allelic vaccine.

Evolutionary Analysis of Plastid Genomes of Seven Lonicera L. Species: Implications for Sequence Divergence and Phylogenetic Relationships.

Plant plastomes play crucial roles in species evolution and phylogenetic reconstruction studies due to being maternally inherited and due to the moderate evolutionary rate of genomes. However, patterns of sequence divergence and molecular evolution of the plastid genomes in the horticulturally- and economically-important Lonicera L. species are poorly understood. In this study, we collected the complete plastomes of seven Lonicera species and determined the various repeat sequence variations and protein sequence evolution by comparative genomic analysis. A total of 498 repeats were identified in plastid genomes, which included tandem (130), dispersed (277), and palindromic (91) types of repeat variations. Simple sequence repeat (SSR) elements analysis indicated the enriched SSRs in seven genomes to be mononucleotides, followed by tetra-nucleotides, dinucleotides, tri-nucleotides, hex-nucleotides, and penta-nucleotides. We identified 18 divergence hotspot regions (rps15, rps16, rps18, rpl23, psaJ, infA, ycf1, trnN-GUU-ndhF, rpoC2-rpoC1, rbcL-psaI, trnI-CAU-ycf2, psbZ-trnG-UCC, trnK-UUU-rps16, infA-rps8, rpl14-rpl16, trnV-GAC-rrn16, trnL-UAA intron, and rps12-clpP) that could be used as the potential molecular genetic markers for the further study of population genetics and phylogenetic evolution of Lonicera species. We found that a large number of repeat sequences were distributed in the divergence hotspots of plastid genomes. Interestingly, 16 genes were determined under positive selection, which included four genes for the subunits of ribosome proteins (rps7, rpl2, rpl16, and rpl22), three genes for the subunits of photosystem proteins (psaJ, psbC, and ycf4), three NADH oxidoreductase genes (ndhB, ndhH, and ndhK), two subunits of ATP genes (atpA and atpB), and four other genes (infA, rbcL, ycf1, and ycf2). Phylogenetic analysis based on the whole plastome demonstrated that the seven Lonicera species form a highly-supported monophyletic clade. The availability of these plastid genomes provides important genetic information for further species identification and biological research on Lonicera.