Problem-Elephant Translocation: Translocating the Problem and the Elephant?

Human-elephant conflict (HEC) threatens the survival of endangered Asian elephants (Elephas maximus). Translocating “problem-elephants” is an important HEC mitigation and elephant conservation strategy across elephant range, with hundreds translocated annually. In the first comprehensive assessment of elephant translocation, we monitored 16 translocations in Sri Lanka with GPS collars. All translocated elephants were released into national parks. Two were killed within the parks where they were released, while all the others left those parks. Translocated elephants showed variable responses: “homers” returned to the capture site, “wanderers” ranged widely, and “settlers” established home ranges in new areas soon after release. Translocation caused wider propagation and intensification of HEC, and increased elephant mortality. We conclude that translocation defeats both HEC mitigation and elephant conservation goals.     

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N₅₀ of 10,384 bp and scaffold N₅₀ of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.     

Linkage Disequilibrium and Demographic History of Wild and Domestic Canids

Assessing the extent of linkage disequilibrium (LD) in natural populations of a nonmodel species has been difficult due to the lack of available genomic markers. However, with advances in genotyping and genome sequencing, genomic characterization of natural populations has become feasible. Using sequence data and SNP genotypes, we measured LD and modeled the demographic history of wild canid populations and domestic dog breeds. In 11 gray wolf populations and one coyote population, we find that the extent of LD as measured by the distance at which r² = 0.2 extends <10 kb in outbred populations to >1.7 Mb in populations that have experienced significant founder events and bottlenecks. This large range in the extent of LD parallels that observed in 18 dog breeds where the r² value varies from ~20 kb to >5 Mb. Furthermore, in modeling demographic history under a composite-likelihood framework, we find that two of five wild canid populations exhibit evidence of a historical population contraction. Five domestic dog breeds display evidence for a minor population contraction during domestication and a more severe contraction during breed formation. Only a 5% reduction in nucleotide diversity was observed as a result of domestication, whereas the loss of nucleotide diversity with breed formation averaged 35%.     

Three-Dimensional Genome Architecture Influences Partner Selection for Chromosomal Translocations in Human Disease

Chromosomal translocations are frequent features of cancer genomes that contribute to disease progression. These rearrangements result from formation and illegitimate repair of DNA double-strand breaks (DSBs), a process that requires spatial colocalization of chromosomal breakpoints. The “contact first” hypothesis suggests that translocation partners colocalize in the nuclei of normal cells, prior to rearrangement. It is unclear, however, the extent to which spatial interactions based on three-dimensional genome architecture contribute to chromosomal rearrangements in human disease. Here we intersect Hi-C maps of three-dimensional chromosome conformation with collections of 1,533 chromosomal translocations from cancer and germline genomes. We show that many translocation-prone pairs of regions genome-wide, including the cancer translocation partners BCR-ABL and MYC-IGH, display elevated Hi-C contact frequencies in normal human cells. Considering tissue specificity, we find that translocation breakpoints reported in human hematologic malignancies have higher Hi-C contact frequencies in lymphoid cells than those reported in sarcomas and epithelial tumors. However, translocations from multiple tissue types show significant correlation with Hi-C contact frequencies, suggesting that both tissue-specific and universal features of chromatin structure contribute to chromosomal alterations. Our results demonstrate that three-dimensional genome architecture shapes the landscape of rearrangements directly observed in human disease and establish Hi-C as a key method for dissecting these effects.     

Targeted Resequencing of the Pericentromere of Chromosome 2 Linked to Constitutional Delay of Growth and Puberty

Constitutional delay of growth and puberty (CDGP) is the most common cause of pubertal delay. CDGP is defined as the proportion of the normal population who experience pubertal onset at least 2 SD later than the population mean, representing 2.3% of all adolescents. While adolescents with CDGP spontaneously enter puberty, they are at risk for short stature, decreased bone mineral density, and psychosocial problems. Genetic factors contribute heavily to the timing of puberty, but the vast majority of CDGP cases remain biologically unexplained, and there is no definitive test to distinguish CDGP from pathological absence of puberty during adolescence. Recently, we published a study identifying significant linkage between a locus at the pericentromeric region of chromosome 2 (chr 2) and CDGP in Finnish families. To investigate this region for causal variation, we sequenced chr 2 between the genomic coordinates of 79–124 Mb (genome build GRCh37) in the proband and affected parent of the 13 families contributing most to this linkage signal. One gene, DNAH6, harbored 6 protein-altering low-frequency variants (< 6% in the Finnish population) in 10 of the CDGP probands. We sequenced an additional 135 unrelated Finnish CDGP subjects and utilized the unique Sequencing Initiative Suomi (SISu) population reference exome set to show that while 5 of these variants were present in the CDGP set, they were also present in the Finnish population at similar frequencies. Additional variants in the targeted region could not be prioritized for follow-up, possibly due to gaps in sequencing coverage or lack of functional knowledge of non-genic genomic regions. Thus, despite having a well-characterized sample collection from a genetically homogeneous population with a large population-based reference sequence dataset, we were unable to pinpoint variation in the linked region predisposing delayed puberty. This study highlights the difficulties of detecting genetic variants under linkage regions for complex traits and suggests that advancements in annotation of gene function and regulatory regions of the genome will be critical for solving the genetic background of complex phenotypes like CDGP.     

Evaluation of Linkage Disequilibrium Pattern and Association Study on Seed Oil Content in Brassica napus Using ddRAD Sequencing

High-density genetic markers are the prerequisite for understanding linkage disequilibrium (LD) and genome-wide association studies (GWASs) of complex traits in crops. To evaluate the LD pattern in oilseed rape, we sequenced a previous association panel containing 189 B. napus inbred lines using double-digested restriction-site associated DNA (ddRAD) and genotyped 19,327 RAD tags. A total of 15,921 RAD tags were assigned to a published genetic linkage map and the majority (71.1%) of these tags was uniquely mapped to the draft reference genome “Darmor-bzh.” The distance of LD decay was 1,214 kb across the genome at the background level (r² = 0.26), with the distances of LD decay being 405 kb and 2,111 kb in the A and C subgenomes, respectively. A total of 361 haplotype blocks with length > 100 kb were identified in the entire genome. The association panel could be classified into two groups, P1 and P2, which are essentially consistent with the geographical origins of varieties. A large number of group-specific haplotypes were identified, reflecting that varieties in the P1 and P2 groups experienced distinct selection in breeding programs to adapt their different growth habitats. GWAS repeatedly detected two loci significantly associated with oil content of seeds based on the developed SNPs, suggesting that the high-density SNPs were useful for understanding the genetic determinants of complex traits in GWAS.     

Chromosome segmental dosage analysis of maize morphogenesis using B-A-A translocations

The B–A–A translocations have enabled us to simultaneously assess the possible dosage-sensitive interactions of two nonhomologous chromosome segments in affecting maize plant development. Maize B–A–A translocations contain segments of two nonhomologous essential A chromosomes in tandem arrangement attached to a segment of the long arm of a supernumerary B chromosome. By utilizing the frequent nondisjunction of the B centromere at the second pollen mitosis we produced plants containing an extra copy of the two A chromosome segments. We compared these hyperploid plants with nonhyperploid plants by measuring leaf width, plant height, ear height, internode length, stalk circumference, leaf length, and tassel-branch number in 20 paired families that involved one of the chromosome arms 1S, 1L, 4L, 5S, and 10L. One or more of the seven measured traits displayed dosage sensitivity among 17 of the 20 B–A–A translocations, which included the involvement of chromosome arms 2L, 3L, 5L, 6L, and 7L. The most obvious effect of an increased dosage of the B–A–A translocation was a significant decrease in the traits in the hyperploid plants. These effects may be either the additive effects of hyperploidy for the two chromosome segments or a result of gene interaction between them.     

Evaluation of LD decay and various LD-decay estimators in simulated and SNP-array data of tetraploid potato

Key message

The number of SNPs required for QTL discovery is justified by the distance at which linkage disequilibrium has decayed. Simulations and real potato SNP data showed how to estimate and interpret LD decay.

Abstract

The magnitude of linkage disequilibrium (LD) and its decay with genetic distance determine the resolution of association mapping, and are useful for assessing the desired numbers of SNPs on arrays. To study LD and LD decay in tetraploid potato, we simulated autotetraploid genotypes and used it to explore the dependence on: (1) the number of haplotypes in the population (the amount of genetic variation) and (2) the percentage of haplotype specific SNPs (hs-SNPs). Several estimators for short-range LD were explored, such as the average r ², median r ², and other percentiles of r ² (80, 90, and 95 %). For LD decay, we looked at LD_½,90, the distance at which the short-range LD is halved when using the 90 % percentile of r ² at short range, as estimator for LD. Simulations showed that the performance of various estimators for LD decay strongly depended on the number of haplotypes, although the real value of LD decay was not influenced very much by this number. The estimator LD_½,90 was chosen to evaluate LD decay in 537 tetraploid varieties. LD_½,90 values were 1.5 Mb for varieties released before 1945 and 0.6 Mb in varieties released after 2005. LD_½,90 values within three different subpopulations ranged from 0.7 to 0.9 Mb. LD_½,90 was 2.5 Mb for introgressed regions, indicating large haplotype blocks. In pericentromeric heterochromatin, LD decay was negligible. This study demonstrates that several related factors influencing LD decay could be disentangled, that no universal approach can be suggested, and that the estimation of LD decay has to be performed with great care and knowledge of the sampled material.

     

Genome-Wide Association Studies Using Single Nucleotide Polymorphism Markers Developed by Re-Sequencing of the Genomes of Cultivated Tomato

With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ∼13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ∼1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.     

Punctuated Distribution of Recombination Hotspots and Demarcation of Pericentromeric Regions in Phaseolus vulgaris L.

High density genetic maps are a reliable tool for genetic dissection of complex plant traits. Mapping resolution is often hampered by the variable crossover and non-crossover events occurring across the genome, with pericentromeric regions (pCENR) showing highly suppressed recombination rates. The efficiency of linkage mapping can further be improved by characterizing and understanding the distribution of recombinational activity along individual chromosomes. In order to evaluate the genome wide recombination rate in common beans (Phaseolus vulgaris L.) we developed a SNP-based linkage map using the genotype-by-sequencing approach with a 188 recombinant inbred line family generated from an inter gene pool cross (Andean x Mesoamerican). We identified 1,112 SNPs that were subsequently used to construct a robust linkage map with 11 groups, comprising 513 recombinationally unique marker loci spanning 943 cM (LOD 3.0). Comparative analysis showed that the linkage map spanned >95% of the physical map, indicating that the map is almost saturated. Evaluation of genome-wide recombination rate indicated that at least 45% of the genome is highly recombinationally suppressed, and allowed us to estimate locations of pCENRs. We observed an average recombination rate of 0.25 cM/Mb in pCENRs as compared to the rest of genome that showed 3.72 cM/Mb. However, several hot spots of recombination were also detected with recombination rates reaching as high as 34 cM/Mb. Hotspots were mostly found towards the end of chromosomes, which also happened to be gene-rich regions. Analyzing relationships between linkage and physical map indicated a punctuated distribution of recombinational hot spots across the genome.     

An S-Locus Independent Pollen Factor Confers Self-Compatibility in ‘Katy’ Apricot

Loss of pollen-S function in Prunus self-compatible cultivars has been mostly associated with deletions or insertions in the S-haplotype-specific F-box (SFB) genes. However, self-compatible pollen-part mutants defective for non-S-locus factors have also been found, for instance, in the apricot (Prunus armeniaca) cv. ‘Canino’. In the present study, we report the genetic and molecular analysis of another self-compatible apricot cv. termed ‘Katy’. S-genotype of ‘Katy’ was determined as S ₁ S ₂ and S-RNase PCR-typing of selfing and outcrossing populations from ‘Katy’ showed that pollen gametes bearing either the S ₁- or the S ₂-haplotype were able to overcome self-incompatibility (SI) barriers. Sequence analyses showed no SNP or indel affecting the SFB ₁ and SFB ₂ alleles from ‘Katy’ and, moreover, no evidence of pollen-S duplication was found. As a whole, the obtained results are compatible with the hypothesis that the loss-of-function of a S-locus unlinked factor gametophytically expressed in pollen (M’-locus) leads to SI breakdown in ‘Katy’. A mapping strategy based on segregation distortion loci mapped the M’-locus within an interval of 9.4 cM at the distal end of chr.3 corresponding to ∼1.29 Mb in the peach (Prunus persica) genome. Interestingly, pollen-part mutations (PPMs) causing self-compatibility (SC) in the apricot cvs. ‘Canino’ and ‘Katy’ are located within an overlapping region of ∼273 Kb in chr.3. No evidence is yet available to discern if they affect the same gene or not, but molecular markers seem to indicate that both cultivars are genetically unrelated suggesting that every PPM may have arisen independently. Further research will be necessary to reveal the precise nature of ‘Katy’ PPM, but fine-mapping already enables SC marker-assisted selection and paves the way for future positional cloning of the underlying gene.     

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the “hemoglobin (Hb) superfamily.” The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His¹¹⁷ and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His¹¹⁷ is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.     

The Antibiotic Dosage of Fastest Resistance Evolution: Gene Amplifications Underpinning the Inverted-U

To determine the dosage at which antibiotic resistance evolution is most rapid, we treated Escherichia coli in vitro, deploying the antibiotic erythromycin at dosages ranging from zero to high. Adaptation was fastest just below erythromycin’s minimal inhibitory concentration (MIC) and genotype-phenotype correlations determined from whole genome sequencing revealed the molecular basis: simultaneous selection for copy number variation in three resistance mechanisms which exhibited an “inverted-U” pattern of dose-dependence, as did several insertion sequences and an integron. Many genes did not conform to this pattern, however, reflecting changes in selection as dose increased: putative media adaptation polymorphisms at zero antibiotic dosage gave way to drug target (ribosomal RNA operon) amplification at mid dosages whereas prophage-mediated drug efflux amplifications dominated at the highest dosages. All treatments exhibited E. coli increases in the copy number of efflux operons acrAB and emrE at rates that correlated with increases in population density. For strains where the inverted-U was no longer observed following the genetic manipulation of acrAB, it could be recovered by prolonging the antibiotic treatment at subMIC dosages.     

Insights into chromosomal evolution and sex determination of Pseudobagrus ussuriensis (Bagridae,Siluriformes) based on a chromosome-level genome

Pseudobagrus ussuriensis is an aquaculture catfish with significant sexual dimorphism. In this study, a chromosome-level genome with a size of 741.97 Mb was assembled for female P. ussuriensis. A total of 26 chromosome-level contigs covering 97.34% of the whole-genome assembly were obtained with an N50 of 28.53 Mb and an L50 of 11. A total of 24,075 protein-coding genes were identified, with 91.54% (22,039) genes being functionally annotated. Based on the genome assembly, four chromosome evolution clusters of catfishes were identified and the formation process of P. ussuriensis chromosomes was predicted. A total of 55 sex-related quantitative trait loci (QTLs) with a phenotypic variance explained value of 100% were located on chromosome 8 (chr08). The QTLs and other previously identified sex-specific markers were located in a sex-determining region of 16.83 Mb (from 6.90 to 23.73 Mb) on chr08, which was predicted as the X chromosome. The sex-determining region comprised 554 genes, with 135 of which being differently expressed between males and females/pseudofemales, and 16 candidate sex-determining genes were screened out. The results of this study provided a useful chromosome-level genome for genetic, genomic and evolutionary studies of P. ussuriensis, and also be useful for further studies on sex-determination mechanism analysis and sex-control breeding of this fish.     

A Mutant with Expression Deletion of Gene Sec-1 in a 1RS.1BL Line and Its Effect on Production Quality of Wheat

The chromosome arm 1RS of rye (Secale cereal L.) has been used worldwide as a source of genes for agronomic and resistant improvement. However, the 1RS arm in wheat has end-use quality defects that are partially attributable to the presence of ω-secalins, which are encoded by genes at the Sec-1 locus. Various attempts in removing the Sec-1 genes from the 1RS.1BL translocation chromosome have been made. In the present study, two new primary 1RS.1BL translocation lines, T917-26 and T917-15, were developed from a cross between wheat variety “{"type":"entrez-nucleotide","attrs":{"text":"A42912","term_id":"2298361"}}A42912” and Chinese local rye “Weining.” The lines T917-15 and T917-26 carried a pair of intact and homogeneous 1RS.1BL chromosomes. The line T917-26 also harbored an expression deletion of some genes at the Sec-1 locus, which originated from a mutation that occurred simultaneously with wheat-rye chromosome translocations. These results suggest that the accompanying mutations of the evolutionarily significant translocations are remarkable resources for plant improvement. Comparison of translocation lines with its wheat parent showed improvements in the end-use quality parameters, which included protein content (PC), water absorption (WA), sodium dodecyl sulfate sedimentation (SDSS), wet gluten (WG), dry gluten (DG) and dough stickiness (DS), whereas significant reduction in gluten index (GI) and stability time (ST) were observed. These findings indicate that 1RS in wheat has produced a higher amount of protein, although these comprised worse compositions. However, in the T917-26 line that harbored an expression deletion mutation in the Sec-1 genes, the quality parameters were markedly improved relative to its sister line, T917-15, especially for GI and DS (P < 0.05). These results indicated that expression deletion of Sec-1 genes significantly improves the end-use quality of wheat cultivars harboring the 1RS.1BL translocation. Strategies to remove the Sec-1 genes from the 1RS.1BL translocation in wheat improvement are discussed.     

KIAA1462, A Coronary Artery Disease Associated Gene,Is a Candidate Gene for Late Onset Alzheimer Disease in APOE Carriers

Alzheimer disease (AD) is a devastating neurodegenerative disease affecting more than five million Americans. In this study, we have used updated genetic linkage data from chromosome 10 in combination with expression data from serial analysis of gene expression to choose a new set of thirteen candidate genes for genetic analysis in late onset Alzheimer disease (LOAD). Results in this study identify the KIAA1462 locus as a candidate locus for LOAD in APOE4 carriers. Two genes exist at this locus, KIAA1462, a gene associated with coronary artery disease, and “rokimi”, encoding an untranslated spliced RNA The genetic architecture at this locus suggests that the gene product important in this association is either “rokimi”, or a different isoform of KIAA1462 than the isoform that is important in cardiovascular disease. Expression data suggests that isoform f of KIAA1462 is a more attractive candidate for association with LOAD in APOE4 carriers than “rokimi” which had no detectable expression in brain.     

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.