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The rhesus macaque (Macaca mulatta) is the most widely studied nonhuman primate (NHP) in biomedical research. We present an updated reference genome assembly (Mmul_10, contig N50 = 46 Mbp) that increases the sequence contiguity 120-fold and annotate it using 6.5 million full-length transcripts, thus improving our understanding of gene content, isoform diversity, and repeat organization. With the improved assembly of segmental duplications, we discovered new lineage-specific genes and expanded gene families that are potentially informative in studies of evolution and disease susceptibility. Whole-genome sequencing (WGS) data from 853 rhesus macaques identified 85.7 million single-nucleotide variants (SNVs) and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay, providing a framework for developing noninvasive NHP models of human disease.

Rhesus macaque is an Old World monkey that shared a common ancestor with human ∼25 Myr ago and is an important animal model for human disease studies. A deep understanding of its genetics is therefore required for both biomedical and evolutionary studies. Among structural variants, inversions represent a driving force in speciation and play an important role in disease predisposition. Here we generated a genome-wide map of inversions between human and macaque, combining single-cell strand sequencing with cytogenetics. We identified 375 total inversions between 859 bp and 92 Mbp, increasing by eightfold the number of previously reported inversions. Among these, 19 inversions flanked by segmental duplications overlap with recurrent copy number variants associated with neurocognitive disorders. Evolutionary analyses show that in 17 out of 19 cases, the Hominidae orientation of these disease-associated regions is always derived. This suggests that duplicated sequences likely played a fundamental role in generating inversions in humans and great apes, creating architectures that nowadays predispose these regions to disease-associated genetic instability. Finally, we identified 861 genes mapping at 156 inversions breakpoints, with some showing evidence of differential expression in human and macaque cell lines, thus highlighting candidates that might have contributed to the evolution of species-specific features. This study depicts the most accurate fine-scale map of inversions between human and macaque using a two-pronged integrative approach, such as single-cell strand sequencing and cytogenetics, and represents a valuable resource toward understanding of the biology and evolution of primate species.

Inversions play an important role in disease and evolution but are difficult to characterize because their breakpoints map to large repeats. We increased by sixfold the number (n = 1,069) of previously reported great ape inversions by using single-cell DNA template strand and long-read sequencing. We find that the X chromosome is most enriched (2.5-fold) for inversions, on the basis of its size and duplication content. There is an excess of differentially expressed primate genes near the breakpoints of large (>100 kilobases (kb)) inversions but not smaller events. We show that when great ape lineage-specific duplications emerge, they preferentially (approximately 75%) occur in an inverted orientation compared to that at their ancestral locus. We construct megabase-pair scale haplotypes for individual chromosomes and identify 23 genomic regions that have recurrently toggled between a direct and an inverted state over 15 million years. The direct orientation is most frequently the derived state for human polymorphisms that predispose to recurrent copy number variants associated with neurodevelopmental disease.

Disorders of sexual development (DSDs) in dogs, similar to humans, arise from genetic mutations, gonadal differentiation, or phenotypic sex development. The French Bulldog, a breed that has seen a surge in popularity and demand, has also shown a marked increase in DSD incidence. This study aims to characterize the genetic underpinnings of DSDs in a French Bulldog named Brutus, exhibiting ambiguous genitalia and internal sexual anatomy, and to explore the impact of breeding practices on genetic diversity within the breed. We utilized a comprehensive approach combining conventional cytogenetics, molecular techniques, and deep sequencing to investigate the genetic profile of Brutus. The sequence data were compared to three other male French Bulldogs' genome sequences with typical reproductive anatomy, including Brutus's father and the canine reference genome (CanFam6). We found a Robertsonian fusion involving chromosome 23 previously reported in dogs as a causative mutation responsible for sex reversal syndrome. Our findings revealed a 22% mosaicism (78,XX/77,XX), the absence of the sex-determining region (SRY) gene, and the presence of 43 unique Single Nucleotide Variants (SNVs) not inherited from the father. Notably, the run of homozygosity (ROH) analysis showed Brutus has a higher number of homozygous segments compared to other Bulldogs, with a total length of these fragments 50% greater than the average, strongly suggesting this dog is the product of the mating between siblings. Although no direct causative genes for the DSD phenotype were identified, four candidate loci warrant further investigation. Our study highlighted the need for a better annotated and curated reference dog genome to define genes causative of any specific phenotype, suggests a potential genetic basis for the DSD phenotype in dogs, and underscores the consequences of uncontrolled breeding practices in French Bulldogs. These findings highlight the importance of implementing strategic genetic management to preserve genetic health and diversity in canine populations. [ABSTRACT FROM AUTHOR]

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

BackgroundGene innovation by duplication is a fundamental evolutionary process but is difficult to study in humans due to the large size, high sequence identity, and mosaic nature of segmental duplication blocks. The human-specific gene hydrocephalus-inducing 2, HYDIN2, was generated by a 364 kbp duplication of 79 internal exons of the large ciliary gene HYDIN from chromosome 16q22.2 to chromosome 1q21.1. Because the HYDIN2 locus lacks the ancestral promoter and seven terminal exons of the progenitor gene, we sought to characterize transcription at this locus by coupling reverse transcription polymerase chain reaction and long-read sequencing.Results5' RACE indicates a transcription start site for HYDIN2 outside of the duplication and we observe fusion transcripts spanning both the 5' and 3' breakpoints. We observe extensive splicing diversity leading to the formation of altered open reading frames (ORFs) that appear to be under relaxed selection. We show that HYDIN2 adopted a new promoter that drives an altered pattern of expression, with highest levels in neural tissues. We estimate that the HYDIN duplication occurred ~3.2 million years ago and find that it is nearly fixed (99.9%) for diploid copy number in contemporary humans. Examination of 73 chromosome 1q21 rearrangement patients reveals that HYDIN2 is deleted or duplicated in most cases.ConclusionsTogether, these data support a model of rapid gene innovation by fusion of incomplete segmental duplications, altered tissue expression, and potential subfunctionalization or neofunctionalization of HYDIN2 early in the evolution of the Homo lineage.

BackgroundAlthough many algorithms are now available that aim to characterize different classes of structural variation, discovery of balanced rearrangements such as inversions remains an open problem. This is mainly due to the fact that breakpoints of such events typically lie within segmental duplications or common repeats, which reduces the mappability of short reads. The algorithms developed within the 1000 Genomes Project to identify inversions are limited to relatively short inversions, and there are currently no available algorithms to discover large inversions using high throughput sequencing technologies.ResultsHere we propose a novel algorithm, VALOR, to discover large inversions using new sequencing methods that provide long range information such as 10X Genomics linked-read sequencing, pooled clone sequencing, or other similar technologies that we commonly refer to as long range sequencing. We demonstrate the utility of VALOR using both pooled clone sequencing and 10X Genomics linked-read sequencing generated from the genome of an individual from the HapMap project (NA12878). We also provide a comprehensive comparison of VALOR against several state-of-the-art structural variation discovery algorithms that use whole genome shotgun sequencing data.ConclusionsIn this paper, we show that VALOR is able to accurately discover all previously identified and experimentally validated large inversions in the same genome with a low false discovery rate. Using VALOR, we also predicted a novel inversion, which we validated using fluorescent in situ hybridization. VALOR is available at https://github.com/BilkentCompGen/VALOR.

Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates, including more recently the genomes of archaic hominins. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for approximately 1% of cases of autism and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. Here we reconstruct the evolutionary history of the locus and identify bolA family member 2 (BOLA2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95-kilobase-pair segment containing BOLA2 duplicated across the critical region approximately 282 thousand years ago (ka), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carried one or more copies of the duplication, which nearly fixed early in the human lineage--a pattern unlikely to have arisen so rapidly in the absence of selection (P

The unique inheritance pattern of the X chromosome exposes it to natural selection in a way that is different from that of the autosomes, potentially resulting in accelerated evolution. We perform a comparative analysis of X chromosome polymorphism in 10 great ape species, including humans. In most species, we identify striking megabase-wide regions, where nucleotide diversity is less than 20% of the chromosomal average. Such regions are found exclusively on the X chromosome. The regions overlap partially among species, suggesting that the underlying targets are partly shared among species. The regions have higher proportions of singleton SNPs, higher levels of population differentiation, and a higher nonsynonymous-to-synonymous substitution ratio than the rest of the X chromosome. We show that the extent to which diversity is reduced is incompatible with direct selection or the action of background selection and soft selective sweeps alone, and therefore, we suggest that very strong selective sweeps have independently targeted these specific regions in several species. The only genomic feature that we can identify as strongly associated with loss of diversity is the location of testis-expressed ampliconic genes, which also have reduced diversity around them. We hypothesize that these genes may be responsible for selective sweeps in the form of meiotic drive caused by an intragenomic conflict in male meiosis.

We analyzed 83 fully sequenced great ape genomes for mobile element insertions, predicting a total of 49,452 fixed and polymorphic Alu and long interspersed element 1 (L1) insertions not present in the human reference assembly and assigning each retrotransposition event to a different time point during great ape evolution. We used these homoplasy-free markers to construct a mobile element insertions-based phylogeny of humans and great apes and demonstrate their differential power to discern ape subspecies and populations. Within this context, we find a good correlation between L1 diversity and single-nucleotide polymorphism heterozygosity (r(2) = 0.65) in contrast to Alu repeats, which show little correlation (r(2) = 0.07). We estimate that the "rate" of Alu retrotransposition has differed by a factor of 15-fold in these lineages. Humans, chimpanzees, and bonobos show the highest rates of Alu accumulation--the latter two since divergence 1.5 Mya. The L1 insertion rate, in contrast, has remained relatively constant, with rates differing by less than a factor of three. We conclude that Alu retrotransposition has been the most variable form of genetic variation during recent human-great ape evolution, with increases and decreases occurring over very short periods of evolutionary time.

Most great ape genetic variation remains uncharacterized; however, its study is critical for understanding population history, recombination, selection and susceptibility to disease. Here we sequence to high coverage a total of 79 wild- and captive-born individuals representing all six great ape species and seven subspecies and report 88.8 million single nucleotide polymorphisms. Our analysis provides support for genetically distinct populations within each species, signals of gene flow, and the split of common chimpanzees into two distinct groups: Nigeria-Cameroon/western and central/eastern populations. We find extensive inbreeding in almost all wild populations, with eastern gorillas being the most extreme. Inferred effective population sizes have varied radically over time in different lineages and this appears to have a profound effect on the genetic diversity at, or close to, genes in almost all species. We discover and assign 1,982 loss-of-function variants throughout the human and great ape lineages, determining that the rate of gene loss has not been different in the human branch compared to other internal branches in the great ape phylogeny. This comprehensive catalogue of great ape genome diversity provides a framework for understanding evolution and a resource for more effective management of wild and captive great ape populations.

Chronic myeloid leukemia (CML) is a rare myeloproliferative disorder caused by the reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem cells (HSCs). This chromosomal translocation results in the formation of an extra-short chromosome 22, called a Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene responsible for the expression of a constitutively active tyrosine kinase that causes uncontrolled growth and replication of leukemic cells. Mechanisms behind the formation of this chromosomal rearrangement are not well known, even if, as observed in tumors, repetitive DNA may be involved as core elements in chromosomal rearrangements. We have participated in the explorative investigations of the PhilosoPhi34 study to evaluate residual Ph+ cells in patients with negative FISH analysis on CD34+/lin- cells with gDNA qPCR. Using targeted next-generation deep sequencing strategies, we analyzed the genomic region around the t(9;22) translocations of 82 CML patients and one CML cell line and assessed the relevance of interspersed repeat elements at breakpoints (BP). We found a statistically higher presence of LINE elements, in particular belonging to the subfamily L1M, in BP cluster regions of both chromosome 22 and 9 compared to the whole human genome. These data suggest that L1M elements could be potential drivers of t(9;22) translocation leading to the generation of the BCR-ABL1 chimeric gene and the expression of the active BCR-ABL1-controlled tyrosine kinase chimeric protein responsible for CML. [ABSTRACT FROM AUTHOR]

In recent years the wine sector has been characterized by an increased focus on the diversification of wine products. This change has been welcomed by consumers. In fact, today's consumers are willing to experiment with new products and unusual combinationswhich has stimulated research aimed at the discovery of native grape varieties suitable for for winemaking. In this context, particular attention are receiving vines for the production of dessert wines. Many regions have made efforts in the search for varieties and techniques able to better promote the drying process. At CREA-VE Turi-BA, there is an ongoing recovery and exploitation of indigenous grape varieties in the regions of Southern Italy and today more than 3,000 accessions of wine grapes are preserved. In the present work we have investigate the possibility of the use of certain accessions of Vitis vinifera varieties (collection CREA-VE Turi-BA) deemed attractive for the production of dessert wines. Accessions presenting a good sugar content and a good titratable acidity were selected; namely, Greco bianco b. (named Q19) and Mantonico bianco b. (named R8) were compared to the cv of Malvasia di Lipari b. Ampelographic and ampelometric analyses, followed by molecular analysis, were carried out for varietal assessment. A rate of the grapes were fermented immediately after harvest with standard procedure, in comparison to rates vinified after withering. The latter was made in a greenhouse type structure with parts of natural and unforced air, without any additional energy expense. At the end of the process, a loss in weight of the grapes equal to 30–35% was realized. The grapes are then stemmed, crushed, pressed and fermented by selected yeast under controlled temperature. Upon completion of the fermentation and subsequent stabilization, the wines were analyzed for the most important enological parameters and subjected to evaluation by a panel of expert tasters. Chemical analysis of wines showed that an alcohol content ranging to 11–12% volume was reached with in the standard wine-making; whilst, for the vinification of dried grapes the alcohol content was equal to 16% vol for Mantonico bianco b. and 18% vol. for and Greco bianco. Organoleptic analysis of the wines showed a greater appreciation for both of dessert wines. The best ratings were given for the olfactory characteristics (floral and fruity aromas) and taste characteristics. In particular, the Greco bianco b. had excellent scores slightly lower than Malvasia di Lipari b. Therefore, the tested varieties have shown a good potential for the production of dessert wines related to typicality of the grape and the region. Dessert wines produced, have a complex flavor profile and interesting for the high acidic component, which improved the taste perception of wine and attenuated the sensation of sweetness. That feature could allow a good wine aging, too.

Grapevine is one of the most important crop plants in the world. Recently there was great expansion of genomics resources about grapevine genome, thus providing increasing efforts for molecular breeding. Current cultivars display a great level of inter-specific differentiation that needs to be investigated to reach a comprehensive understanding of the genetic basis of phenotypic differences, and to find responsible genes selected by cross breeding programs. While there have been significant advances in resolving the pattern and nature of single nucleotide polymorphisms (SNPs) on plant genomes, few data are available on copy number variation (CNV). Furthermore association between structural variations and phenotypes has been described in only a few cases. We combined high throughput biotechnologies and bioinformatics tools, to reveal the first inter-varietal atlas of structural variation (SV) for the grapevine genome. We sequenced and compared four table grape cultivars with the Pinot noir inbred line PN40024 genome as the reference. We detected roughly 8% of the grapevine genome affected by genomic variations. Taken into account phenotypic differences existing among the studied varieties we performed comparison of SVs among them and the reference and next we performed an in-depth analysis of gene content of polymorphic regions. This allowed us to identify genes showing differences in copy number as putative functional candidates for important traits in grapevine cultivation.