Chromosome-level genomes of three key Allium crops and their trait evolution.

Allium crop breeding remains severely hindered due to the lack of high-quality reference genomes. Here we report high-quality chromosome-level genome assemblies for three key Allium crops (Welsh onion, garlic and onion), which are 11.17 Gb, 15.52 Gb and 15.78 Gb in size with the highest recorded contig N50 of 507.27 Mb, 109.82 Mb and 81.66 Mb, respectively. Beyond revealing the genome evolutionary process of Allium species, our pathogen infection experiments and comparative metabolomic and genomic analyses showed that genes encoding enzymes involved in the metabolic pathway of Allium-specific flavor compounds may have evolved from an ancient uncharacterized plant defense system widely existing in many plant lineages but extensively boosted in alliums. Using in situ hybridization and spatial RNA sequencing, we obtained an overview of cell-type categorization and gene expression changes associated with spongy mesophyll cell expansion during onion bulb formation, thus indicating the functional roles of bulb formation genes.

Thirteen Dipterocarpoideae genomes provide insights into their evolution and borneol biosynthesis.

Dipterocarpoideae, the largest subfamily of the Dipterocarpaceae, is a dominant component of Southeast Asian rainforests and is widely used as a source of wood, damar resin, medicine, and essential oil. However, many Dipterocarpoideae species are currently on the IUCN Red List owing to severe degradation of their habitats under global climate change and human disturbance. Genetic information regarding these taxa has only recently been reported with the sequencing of four Dipterocarp genomes, providing clues to the function and evolution of these species. Here, we report on 13 high-quality Dipterocarpoideae genome assemblies, ranging in size from 302.6 to 494.8 Mb and representing the five most species-rich genera in Dipterocarpoideae. Molecular dating analyses support the Western Gondwanaland origin of Dipterocarpaceae. Based on evolutionary analysis, we propose a three-step chromosome evolution scenario to describe the karyotypic evolution from an ancestor with six chromosomes to present-day species with 11 and 7 chromosomes. We discovered an expansion of genes encoding cellulose synthase (CesA), which is essential for cellulose biosynthesis and secondary cell-wall formation. We functionally identified five bornyl diphosphate synthase (BPPS) genes, which specifically catalyze the biosynthesis of borneol, a natural medicinal compound extracted from damar resin and oils, thus providing a basis for large-scale production of natural borneol in vitro.

Comparison of ONT and CCS sequencing technologies on the polyploid genome of a medicinal plant showed that high error rate of ONT reads are not suitable for self-correction.

BACKGROUND: Many medicinal plants are known for their complex genomes with high ploidy, heterozygosity, and repetitive content which pose severe challenges for genome sequencing of those species. Long reads from Oxford nanopore sequencing technology (ONT) or Pacific Biosciences Single Molecule, Real-Time (SMRT) sequencing offer great advantages in de novo genome assembly, especially for complex genomes with high heterozygosity and repetitive content. Currently, multiple allotetraploid species have sequenced their genomes by long-read sequencing. However, we found that a considerable proportion of these genomes (7.9% on average, maximum 23.7%) could not be covered by NGS (Next Generation Sequencing) reads (uncovered region by NGS reads, UCR) suggesting the questionable and low-quality of those area or genomic areas that can't be sequenced by NGS due to sequencing bias. The underlying causes of those UCR in the genome assembly and solutions to this problem have never been studied. METHODS: In the study, we sequenced the tetraploid genome of Veratrum dahuricum (Turcz.) O. Loes (VDL), a Chinese medicinal plant, with ONT platform and assembled the genome with three strategies in parallel. We compared the qualities, coverage, and heterozygosity of the three ONT assemblies with another released assembly of the same individual using reads from PacBio circular consensus sequencing (CCS) technology, to explore the cause of the UCR. RESULTS: By mapping the NGS reads against the three ONT assemblies and the CCS assembly, we found that the coverage of those ONT assemblies by NGS reads ranged from 49.15 to 76.31%, much smaller than that of the CCS assembly (99.53%). And alignment between ONT assemblies and CCS assembly showed that most UCR can be aligned with CCS assembly. So, we conclude that the UCRs in ONT assembly are low-quality sequences with a high error rate that can't be aligned with short reads, rather than genomic regions that can't be sequenced by NGS. Further comparison among the intermediate versions of ONT assemblies showed that the most probable origin of those errors is a combination of artificial errors introduced by "self-correction" and initial sequencing error in long reads. We also found that polishing the ONT assembly with CCS reads can correct those errors efficiently. CONCLUSIONS: Through analyzing genome features and reads alignment, we have found the causes for the high proportion of UCR in ONT assembly of VDL are sequencing errors and additional errors introduced by self-correction. The high error rates of ONT-raw reads make them not suitable for self-correction prior to allotetraploid genome assembly, as the self-correction will introduce artificial errors to > 5% of the UCR sequences. We suggest high-precision CCS reads be used to polish the assembly to correct those errors effectively for polyploid genomes.

Comparative and phylogenetic analyses of eleven complete chloroplast genomes of Dipterocarpoideae.

BACKGROUND: In South-east Asia, Dipterocarpoideae is predominant in most mature forest communities, comprising around 20% of all trees. As large quantity and high quality wood are produced in many species, Dipterocarpoideae plants are the most important and valuable source in the timber market. The d-borneol is one of the essential oil components from Dipterocarpoideae (for example, Dryobalanops aromatica or Dipterocarpus turbinatus) and it is also an important traditional Chinese medicine (TCM) formulation known as "Bingpian" in Chinese, with antibacterial, analgesic and anti-inflammatory effects and can enhance anticancer efficiency. METHODS: In this study, we analyzed 20 chloroplast (cp) genomes characteristics of Dipterocarpoideae, including eleven newly reported genomes and nine cp genomes previously published elsewhere, then we explored the chloroplast genomic features, inverted repeats contraction and expansion, codon usage, amino acid frequency, the repeat sequences and selective pressure analyses. At last, we constructed phylogenetic relationships of Dipterocarpoideae and found the potential barcoding loci. RESULTS: The cp genome of this subfamily has a typical quadripartite structure and maintains a high degree of consistency among species. There were slightly more tandem repeats in cp genomes of Dipterocarpus and Vatica, and the psbH gene was subjected to positive selection in the common ancestor of all the 20 species of Dipterocarpoideae compared with three outgroups. Phylogenetic tree showed that genus Shorea was not a monophyletic group, some Shorea species and genus Parashorea are placed in one clade. In addition, the rpoC2 gene can be used as a potential marker to achieve accurate and rapid species identification in subfamily Dipterocarpoideae. CONCLUSIONS: Dipterocarpoideae had similar cp genomic features and psbM, rbcL, psbH may function in the growth of Dipterocarpoideae. Phylogenetic analysis suggested new taxon treatment is needed for this subfamily indentification. In addition, rpoC2 is potential to be a barcoding gene to TCM distinguish.

Molecular mechanisms and topological consequences of drastic chromosomal rearrangements of muntjac deer.

Muntjac deer have experienced drastic karyotype changes during their speciation, making it an ideal model for studying mechanisms and functional consequences of mammalian chromosome evolution. Here we generated chromosome-level genomes for Hydropotes inermis (2n = 70), Muntiacus reevesi (2n = 46), female and male M. crinifrons (2n = 8/9) and a contig-level genome for M. gongshanensis (2n = 8/9). These high-quality genomes combined with Hi-C data allowed us to reveal the evolution of 3D chromatin architectures during mammalian chromosome evolution. We find that the chromosome fusion events of muntjac species did not alter the A/B compartment structure and topologically associated domains near the fusion sites, but new chromatin interactions were gradually established across the fusion sites. The recently borne neo-Y chromosome of M. crinifrons, which underwent male-specific inversions, has dramatically restructured chromatin compartments, recapitulating the early evolution of canonical mammalian Y chromosomes. We also reveal that a complex structure containing unique centromeric satellite, truncated telomeric and palindrome repeats might have mediated muntjacs' recurrent chromosome fusions. These results provide insights into the recurrent chromosome tandem fusion in muntjacs, early evolution of mammalian sex chromosomes, and reveal how chromosome rearrangements can reshape the 3D chromatin regulatory conformations during species evolution.

A Genetic Mechanism for Convergent Skin Lightening during Recent Human Evolution.

Skin lightening among Eurasians is thought to have been a convergence occurring independently in Europe and East Asia as an adaptation to high latitude environments. Among Europeans, several genes responsible for such lightening have been found, but the information available for East Asians is much more limited. Here, a genome-wide comparison between dark-skinned Africans and Austro-Asiatic speaking aborigines and light-skinned northern Han Chinese identified the pigmentation gene OCA2, showing unusually deep allelic divergence between these groups. An amino acid substitution (His615Arg) of OCA2 prevalent in most East Asian populations-but absent in Africans and Europeans-was significantly associated with skin lightening among northern Han Chinese. Further transgenic and targeted gene modification analyses of zebrafish and mouse both exhibited the phenotypic effect of the OCA2 variant manifesting decreased melanin production. These results indicate that OCA2 plays an important role in the convergent skin lightening of East Asians during recent human evolution.

Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

The interleukin 3 gene (IL3) contributes to human brain volume variation by regulating proliferation and survival of neural progenitors.

One of the most significant evolutionary changes underlying the highly developed cognitive abilities of humans is the greatly enlarged brain volume. In addition to being far greater than in most other species, the volume of the human brain exhibits extensive variation and distinct sexual dimorphism in the general population. However, little is known about the genetic mechanisms underlying normal variation as well as the observed sex difference in human brain volume. Here we show that interleukin-3 (IL3) is strongly associated with brain volume variation in four genetically divergent populations. We identified a sequence polymorphism (rs31480) in the IL3 promoter which alters the expression of IL3 by affecting the binding affinity of transcription factor SP1. Further analysis indicated that IL3 and its receptors are continuously expressed in the developing mouse brain, reaching highest levels at postnatal day 1-4. Furthermore, we found IL3 receptor alpha (IL3RA) was mainly expressed in neural progenitors and neurons, and IL3 could promote proliferation and survival of the neural progenitors. The expression level of IL3 thus played pivotal roles in the expansion and maintenance of the neural progenitor pool and the number of surviving neurons. Moreover, we found that IL3 activated both estrogen receptors, but estrogen didn't directly regulate the expression of IL3. Our results demonstrate that genetic variation in the IL3 promoter regulates human brain volume and reveals novel roles of IL3 in regulating brain development.

Genetic association and identification of a functional SNP at GSK3ß for schizophrenia susceptibility.

OBJECTIVE: GSK3ß is a key gene in neurodevelopment, and also an important target of antipsychotics. Several lines of evidence including association and gene expression studies have suggested GSK3ß as a susceptibility gene for schizophrenia, but the underlying genetic mechanism is still unknown. In this study, we test whether the genetic variants in GSK3ß contribute to the risk of schizophrenia in Chinese population. METHODS: We first conducted an association analysis of 9 representative SNPs spanning the entire genomic region of GSK3ß in two independent Han Chinese case-control samples from southwestern China (the Kunming sample and the Yuxi sample, a total of 2550 subjects).Then using EMSA and reporter gene assays, we tested the functional impact of the identified risk SNP on transcriptional factor binding affinity and promoter activity. RESULTS: We observed weak allelic associations of three GSK3ß SNPs (rs3755557, rs7431209 and rs13320980) with schizophrenia in the combined Han Chinese samples. Further analysis using genotypes (under recessive genetic model) supported the association of rs3755557 (p = 0.01, corrected), which is located in the GSK3ß promoter region. The functional assays demonstrated that the risk SNP (rs3755557) could influence the transcription factor binding affinities, resulting in a higher promoter activity of the risk allele. CONCLUSION: Our findings suggest that GSK3ß is likely a risk gene for schizophrenia, and its expression alteration caused by the risk SNP in the promoter region may contribute to the etiology of schizophrenia.

Changes in histomorphometric and mechanical properties of femurs induced by acupuncture at the Shenshu point in the SAMP6 mouse model of senile osteoporosis.

BACKGROUND/OBJECTIVE: The effect of acupuncture on the changes in the histomorphometric and mechanical properties of femurs in senescence-accelerated mice strain P6 (SAMP6) was evaluated in this work. METHODS: Six-month-old male SAMP6 and SAMR1 mice were allocated to 1 of 4 groups: SAMP6 control group (Pc), SAMP6 non-acupoint control group (Pn), SAMP6 acupuncture group (Pa) and SAMR1 control group (Rc). The Pa group was acupunctured at the Shenshu point (BL23) once daily for 8 weeks. Two non-acupoints at the hypochondria were needled for the Pn group. Mice in the other 2 groups were grasped using the same method as for the Pa group. The serum testosterone and osteocalcin (OC) levels were determined by radioimmunoassay. The histomorphometric data were obtained from undecalcified specimens, and the mechanical properties of the femur were assessed by the 3-point bending test. RESULTS: After acupuncture treatment, the decreased serum testosterone level in SAMP6 mice increased markedly, whereas the increased OC concentration declined sharply. The bone histomorphometric and mechanical indexes of SAMP6 mice also improved significantly. The values of trabecular thickness, trabecular bone volume, osteoid volume, mineral apposition rate and bone formation rate in Pa mice increased by 20.4, 18.1, 14.1, 9.9 and 14.7%, respectively, compared with Pc mice. The scores for ultimate force, yield force, elastic stress, ultimate stress and energy to yield force for Pa mice were significantly higher than those of Pc and Pn mice. CONCLUSION: Therefore, acupuncture at BL23 was effective in promoting bone formation, restoring the amount of bone volume, improving bone architecture and reversing osteoporosis in SAMP6 mice to some degree by enhancing the secretion of testosterone and declining bone turnover.

[Effect of puncturing shenshu point on the femoral biomechanical properties in senescence accelerated mouse prone 6].

OBJECTIVE: To explore the mechanisms of puncturing Shenshu point in improving osteoporosis. METHODS: Serum levels of testosterone (T) and osteocalcin (BGP) in senescence accelerated mouse prone 6 (SAMP6, test animals) and senescence accelerated mouse resistant 1 (SAMR1, for control) were determined by radioimmunoassay and their femoral biomechanical properties were determined with three-point bending test before and after puncturing to observe the effect of puncturing on the femoral biomechanical properties and bone mineral contents. RESULTS: Compared with the SAMR1 control group, the serum level of T (20.91 +/- 3.41 nmol/L) decreased (11.09 +/- 1.48 nmol/L in SAMP6 mouse), BGP (6.7 +/- 2.07 microg/L) increased (12.29 +/- 2.29 microg/L in SAMP6 mouse), femoral bending strength lowered and fragility increased. These changes were all improved to some extent or normalized, serum T level 15.05 +/- 2.63 nmol/L and BGP 8.88 +/- 1.85 microg/L after needling at Shenshu point showed significant difference when compared with those in SAMP6. CONCLUSION: Puncturing Shenshu point could effectively prevent the bone loss in SAMP6 mice, increase their bone strength, the therapeutic effect is partly by way of promoting the secretion of sex hormone, improving bone metabolism, suppressing bone transformation rate and increasing bone minerals.

Acupuncture improves cognitive deficits and regulates the brain cell proliferation of SAMP8 mice.

Senescence-accelerated mouse prone 8 (SAMP8) is an autogenic senile strain characterized by early cognitive impairment and age-related deterioration of learning and memory. To investigate the effect of acupuncture on behavioral changes and brain cell events, male 4-month-old SAMP8 and age-matched homologous normal aging SAMR1 mice were divided into four groups: SAMP8 acupuncture group (Pa), SAMP8 non-acupoint control group (Pn), SAMP8 control group (Pc) and SAMR1 normal control group (Rc). By Morris water maze test, the cognitive deficit of SAMP8 was revealed and significantly improved by "Yiqitiaoxue and Fubenpeiyuan" acupuncture. Meanwhile, by 5'-bromo-2'-deoxyuridine (BrdU) specific immunodetection, the decreased cell proliferation in dentate gyrus (DG) of SAMP8 was greatly enhanced by the therapeutic acupuncture, suggesting acupoint-related specificity. Even though no significant differences were found in ventricular/subventricular zones (VZ/SVZ) of the third ventricle (V3) and lateral ventricle (LV) between groups, we obtained interesting results: a stream-like distribution of newly proliferated cells presented along the dorsum of alveus hippocampi (Alv), extending from LV to corpus callosum (CC), and the therapeutic acupuncture showed a marked effect on this region. Our research suggests that acupuncture can induce different cell proliferation in different brain regions of SAMP8, which brings forth the need to explore further for the mechanism of cognitive deficits and acupuncture intervention in this field.