Development and Application of EST-SSR Markers in Cephalotaxus oliveri From Transcriptome Sequences

Cephalotaxus oliveri is an endemic conifer of China, which has medicinal and ornamental value. However, the limited molecular markers and genetic information are insufficient for further genetic studies of this species. In this study, we characterized and developed the EST-SSRs from transcriptome sequences for the first time. The results showed that a total of 5089 SSRs were identified from 36446 unigenes with a density of one SSR per 11.1 kb. The most common type was trinucleotide repeats, excluding mononucleotide repeats, followed by dinucleotide repeats. AAG/CTT and AT/AT exhibited the highest frequency in the trinucleotide and dinucleotide repeats, respectively. Of the identified SSRs, 671, 1125, and 1958 SSRs were located in CDS, 3′UTR, and 5′UTR, respectively. Functional annotation showed that the SSR-containing unigenes were involved in growth and development with various biological functions. Among successfully designed primer pairs, 238 primer pairs were randomly selected for amplification and validation of EST-SSR markers and 47 primer pairs were identified as polymorphic. Finally, 28 high-polymorphic primers were used for genetic analysis and revealed a moderate level of genetic diversity. Seven natural C. oliveri sampling sites were divided into two genetic groups. Furthermore, the 28 EST-SSRs had 96.43, 71.43, and 78.57% of transferability rate in Cephalotaxus fortune, Ametotaxus argotaenia, and Pseudotaxus chienii, respectively. These markers developed in this study lay the foundation for further genetic and adaptive evolution studies in C. oliveri and related species.

Common Threads: Aphidicolin-Inducible and Folate-Sensitive Fragile Sites in the Human Genome

The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed via some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome.

Genetic Variation and Association Mapping in the F2 Population of the Perilla Crop (Perilla Frutescens L.) using new Developed Perilla SSR Markers

The transcriptome sequencing approach RNA-seq represents a powerful tool for transcriptional analysis and development of SSR markers for nonmodel crop. In the Perilla crop, analysis of the distribution of different repeat motifs showed that dinucleotide repeats were the most abundant (62.0%) type, followed by trinucleotide repeats (35.3%), with the two together comprising 97.3% of the eSSR repeats. In this study, we developed 39 new SSR primer sets by the transcriptome sequencing approach RNA-sEq. A total of 130 alleles were detected segregating in nine Perilla accessions with an average of 3.3 alleles per locus, ranging from 125 to 360 bp. The number of alleles per locus ranged from two to six. To detect SSR markers associated with morphological characteristics of Perilla crop, a total of 40 individuals from the F2 population of Perilla were selected for association analysis based on their leaf- and plant-related characteristics. In an association analysis of 37 SSR markers and 9 leaf- and plant-related traits in the 40 individuals of the F2 population, we identified 12 and 11 SSR markers associated with leaf-related traits and plant-related traits, respectively. Therefore, the new Perilla SSR primers described in this study could be helpful in identifying genetic diversity and genetic mapping, designating important genes/QTLs for Perilla crop breeding programs, and allowing Perilla breeders to improve leaf and plant quality through marker-assisted selection (MAS) breeding programs.

Dinucleotide (AC)n Repeat Polymorphism (rs36213840) in the Promoter Region of IL18R1 Gene and Genetic Susceptibility to Severe Malaria

Background: Cytokines are key regulator s of human immune response to malaria but polymorphisms within the regulatory or coding regions of their genes may lead to differences in expression levels which may consequently influence disease susceptibility. In this study, we characterized an adeninecytosine (AC)n dinucleotide repeat polymorphism (rs36213840) at the promoter region of the Interleukin 18 Receptor 1 (IL18R1) gene and investigated its association with severe malaria. Methods: We utilised the case-control study design to enrol a total of 207 children including 87 severe malaria cases and 120 asymptomatic controls. DNA was extracted from blood spot on filter paper using QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany). Genotyping for dinucleotide repeat polymorphisms was done by PCR and capillary electrophoresis of sequenced products on ABI PRISM® 3100 DNA sequencer (PE Applied Biosystems). Results: The genotype frequencies of the dinucleotide repeats differed significantly between the two groups (χ2 = 8.69, P=0.026). We found a significantly higher frequency of the 14bp (AC)7 allele in severe malaria patients than in asymptomatic controls (odds ratio 1.945, 95% CI: 1.23 – 3.09, P = 0.005) while the frequency of the 16bp (AC)8 allele was significant higher in the asymptomatic controls than in severe cases (odds ratio 0.431, 95% CI: 0.244 – 0.761, P = 0.004). Conclusion: Results of this suggest that the 14bp (AC)7 dinucleotide repeats might be a genetic risk factor for susceptibility to severe malaria while the 16bp (AC)8 dinucleotide repeats might be a protective factor against severe malaria

A Potential Role for Mitochondrial DNA in the Activation of Oxidative Stress and Inflammation in Liver Disease

Mitochondria are organelles that are essential for cellular homeostasis including energy harvesting through oxidative phosphorylation. Mitochondrial dysfunction plays a vital role in liver diseases as it produces a large amount of reactive oxygen species (ROS), in turn leading to further oxidative damage to the structure and function of mitochondria and other cellular components. More severe oxidative damage occurred in mitochondrial DNA (mtDNA) than in nuclear DNA. mtDNA dysfunction results in further oxidative damage as it participates in encoding respiratory chain polypeptides. In addition, mtDNA can leave the mitochondria and enter the cytoplasm and extracellular environment. mtDNA is derived from ancient bacteria, contains many unmethylated CpG dinucleotide repeats similar to bacterial DNA, and thus can induce inflammation to exacerbate damage to liver cells and distal organs by activating toll-like receptor 9, inflammatory bodies, and stimulator of interferon genes (STING). In this review, we focus on the mechanism by which mtDNA alterations cause liver injuries, including nonalcoholic fatty liver, alcoholic liver disease, drug-induced liver injury, viral hepatitis, and liver cancer.

Genomic characteristics and profile of microsatellite primers for Acanthogobius ommaturus by genome survey sequencing

Acanthogobius ommaturus is one of the suitable species to study the genetic mechanism of adaptive evolution, but there are few reports on its genetics. In the present study, the genomic survey sequencing method was used to analyze the genome characters of A. ommaturus. A total of 50.50 G high-quality sequence data were obtained in the present study. From the 19-mer distribution frequency, the estimated genome size was 928.01 Mb. The calculated sequence repeat rate was about 38.31%, the heterozygosity was approximately 0.17%, and the GC% content was approximately 40.88%. Moreover, 475,724 simple sequence repeats (SSRs) were identified. Among them, dinucleotide repeats were the most (53.70% of the total SSRs), followed by tri- (35.36%), hexa- (4.59%), tetra- (4.57%) and penta- (1.77%) nucleotide repeats type. This is the first genome-wide feature of this species to be reported.

Genome survey of Zanthoxylum bungeanum and development of genomic-SSR markers in congeneric species

Zanthoxylum bungeanum, a spice and medicinal plant, is cultivated in many parts of China and some countries in Southeast Asia; however, data on its genome are lacking. In the present study, we performed a whole-genome survey and developed novel genomic-SSR markers of Z. bungeanum. Clean data (∼197.16 Gb) were obtained and assembled into 11185221 scaffolds with an N50 of 183 bp. K-mer analysis revealed that Z. bungeanum has an estimated genome size of 3971.92 Mb, and the GC content, heterozygous rate, and repeat sequence rate are 37.21%, 1.73%, and 86.04%, respectively. These results indicate that the genome of Z. bungeanum is complex. Furthermore, 27153 simple sequence repeat (SSR) loci were identified from 57288 scaffolds with a minimum length > 1 kb. Mononucleotide repeats (19706) were the most abundant type, followed by dinucleotide repeats (5154). The most common motifs were A/T, followed by AT/AT; these SSRs accounted for 71.42% and 11.84% of all repeats, respectively. A total of 21243 non-repeating primer pairs were designed, and 100 were randomly selected and validated by PCR analysis using DNA from 10 Z. bungeanum individuals and 5 Zanthoxylum armatum individuals. Finally, 36 polymorphic SSR markers were developed with polymorphism information content (PIC) values ranging from 0.16 to 0.75. Cluster analysis revealed that Z. bungeanum and Z. armatum could be divided into two major clusters, suggesting that these newly developed SSR markers are useful for genetic diversity and germplasm resource identification in Z. bungeanum and Z. armatum.

The Increase of Simple Sequence Repeats during Diversification of Marchantiidae, An Early Land Plant Lineage, Leads to the First Known Expansion of Inverted Repeats in the Evolutionarily-Stable Structure of Liverwort Plastomes

The chloroplast genomes of liverworts, an early land plant lineage, exhibit stable structure and gene content, however the known resources are very limited. The newly sequenced plastomes of Conocephalum, Riccia and Sphaerocarpos species revealed an increase of simple sequence repeats during the diversification of complex thalloid liverwort lineage. The presence of long TA motifs forced applying the long-read nanopore sequencing method for proper and dependable plastome assembly, since the length of dinucleotide repeats overcome the length of Illumina short reads. The accumulation of SSRs (simple sequence repeats) enabled the expansion of inverted repeats by the incorporation of rps12 and rps7 genes, which were part of large single copy (LSC) regions in the previously sequenced plastomes. The expansion of inverted repeat (IR) at the genus level is reported for the first time for non-flowering plants. Moreover, comparative analyses with remaining liverwort lineages revealed that the presence of SSR in plastomes is specific for simple thalloid species. Phylogenomic analysis resulted in trees confirming monophyly of Marchantiidae and partially congruent with previous studies, due to dataset-dependent results of Dumortiera-Reboulia relationships. Despite the lower evolutionary rate of Marchantiales plastomes, significant barcoding gap was detected, even for recently divergent holarctic Conocephalum species. The sliding window analyses revealed the presence of 18 optimal (500 bp long) barcodes that enable the molecular identification of all studied species.

Genome Survey of Male and Female Spotted Scat (Scatophagus argus)

The spotted scat, Scatophagus argus, is a species of fish that is widely propagated within the Chinese aquaculture industry and therefore has significant economic value. Despite this, studies of its genome are severely lacking. In the present study, a genomic survey of S. argus was conducted using next-generation sequencing (NGS). In total, 55.699 GB (female) and 51.047 GB (male) of high-quality sequence data were obtained. Genome sizes were estimated to be 598.73 (female) and 597.60 (male) Mbp. The sequence repeat ratios were calculated to be 27.06% (female) and 26.99% (male). Heterozygosity ratios were 0.37% for females and 0.38% for males. Reads were assembled into 444,961 (female) and 453,459 (male) contigs with N50 lengths of 5,747 and 5,745 bp for females and males, respectively. The average guanine-cytosine (GC) content of the female genome was 41.78%, and 41.82% for the male. A total of 42,869 (female) and 43,283 (male) genes were annotated to the non-redundant (NR) and SwissProt databases. The female and male genomes contained 66.6% and 67.8% BUSCO core genes, respectively. Dinucleotide repeats were the dominant form of simple sequence repeats (SSR) observed in females (68.69%) and males (68.56%). Additionally, gene fragments of Dmrt1 were only observed in the male genome. This is the first report of a genome-wide characterization of S. argus.

A First Insight into a Draft Genome of Silver Sillago (Sillago sihama) via Genome Survey Sequencing

Sillago sihama has high economic value and is one of the most attractive aquaculture species in China. Despite its economic importance, studies of its genome have barely been performed. In this study, we conducted a first genomic survey of S. sihama using next-generation sequencing (NGS). In total, 45.063 Gb of high-quality sequence data were obtained. For the 17-mer frequency distribution, the genome size was estimated to be 508.50 Mb. The sequence repeat ratio was calculated to be 21.25%, and the heterozygosity ratio was 0.92%. Reads were assembled into 1,009,363 contigs, with a N50 length of 1362 bp, and then into 814,219 scaffolds, with a N50 length of 2173 bp. The average Guanine and Cytosine (GC) content was 45.04%. Dinucleotide repeats (56.55%) were the dominant form of simple sequence repeats (SSR).