BAC Sequences Research Articles

High-resolution mapping of SrTm4, a recessive resistance gene to wheat stem rust

Key messageThe diploid wheat recessive stem rust resistance gene SrTm4 was fine-mapped to a 754-kb region on chromosome arm 2AmL and potential candidate genes were identified.Race Ug99 of Puccinia graminis f. sp. tritici (Pgt), the causal agent of wheat stem (or black) rust is one of the most serious threats to global wheat production. The identification, mapping, and deployment of effective stem rust resistance (Sr) genes are critical to reduce this threat. In this study, we generated SrTm4 monogenic lines and found that this gene confers resistance to North American and Chinese Pgt races. Using a large mapping population (9522 gametes), we mapped SrTm4 within a 0.06 cM interval flanked by marker loci CS4211 and 130K1519, which corresponds to a 1.0-Mb region in the Chinese Spring reference genome v2.1. A physical map of the SrTm4 region was constructed with 11 overlapping BACs from the resistant Triticum monococcum PI 306540. Comparison of the 754-kb physical map with the genomic sequence of Chinese Spring and a discontinuous BAC sequence of DV92 revealed a 593-kb chromosomal inversion in PI 306540. Within the candidate region, we identified an L-type lectin-domain containing receptor kinase (LLK1), which was disrupted by the proximal inversion breakpoint, as a potential candidate gene. Two diagnostic dominant markers were developed to detect the inversion breakpoints. In a survey of T. monococcum accessions, we identified 10 domesticated T. monococcum subsp. monococcum genotypes, mainly from the Balkans, carrying the inversion and showing similar mesothetic resistant infection types against Pgt races. The high-density map and tightly linked molecular markers developed in this study are useful tools to accelerate the deployment of SrTm4-mediated resistance in wheat breeding programs.

37 Genomic improvement of the horse X chromosome and characterization of the pseudoautosomal boundary

The horse reference genome has been improved with the release of EquCab3 and the first Y chromosome reference. The most complex sequences, such as those in the sex chromosomes, are still unresolved. These include large amplicons, repeats, and the pseudoautosomal boundary (PAB). We initiated a comprehensive study specifically to improve the assembly of the horse sex chromosomes. To refine the assembly of complex regions, we are utilizing 3 new technologies: trio-binning, Hi-C and Bionano optical mapping. Trio-binning uses long read sequences from F1 interspecific hybrids and short reads from parent species. High molecular weight blood DNA was extracted from a female hinny and sequenced on 2 PacBio Sequel cells. Paired-end Illumina reads (150bp) for horse ( Twilight ) and donkey ( Willy ) were obtained from SRA. These sequences as well as the hinny long reads were assembled with trio-binning function of the Canu assembler program. The initial assembly is 2.5 Gb separated into 1,757 contigs with an N50 of 41.5 Mb. We have completed Hi-C sequencing and Bionano optical maps for one thoroughbred stallion ( Bravo ). Both technologies are needed to scaffold the trio-binning Canu assembly. Our initial goal was to use this assembly to better define the PAB in the horse. The PAB demarcates the end of the pseudoautosomal region (PAR) where X-Y recombination stops. Despite the evolutionary and biological importance of the PAB, the region has been characterized at molecular level in only a few species. The PAB of the horse is presently not well defined. Previously, we identified and Sanger sequenced 4 BAC clones - 2 spanning PAB-X and PAB-Y. To identify the PAB of the horse, BAC sequences were aligned to the Y assembly, EquCab3 and a 42 Mb-size contig from trio-binning assembly which corresponds to the short arm of the X. We identified a region on both X and Y where X-Y homology drops from over 97% (PAR) to almost zero, indicative of the PAB. This region corresponds to the location of the XKR3Y gene in the Y but is not well-annotated in the X. We also identified a duplication and an inversion in EquCab3 which was not consistent with the corresponding region in the X-BACs, or the new 42 Mb Xp contig, suggesting a mis-assembly in EquCab3. We believe that these approaches combined will also resolve other complex and repetitive portions in the horse sex chromosomes.

Genome evolution in Oryza allopolyploids of various ages: Insights into the process of diploidization.

The prevalence and recurrence of whole-genome duplication in plants and its major role in evolution have been well recognized. Despite great efforts, many aspects of genome evolution, particularly the temporal progression of genomic responses to allopolyploidy and the underlying mechanisms, remain poorly understood. The rice genus Oryza consists of both recently formed and older allopolyploid species, representing an attractive system for studying the genome evolution after allopolyploidy. In this study, through screening BAC libraries and sequencing and annotating the targeted BAC clones, we generated orthologous genomic sequences surrounding the DEP1 locus, a major grain yield QTL in cultivated rice, from four Oryza polyploids of various ages and their likely diploid genome donors or close relatives. Based on sequenced DEP1 region and published data from three other genomic regions, we investigated the temporal evolutionary dynamics of four polyploid genomes at both genetic and expression levels. In the recently formed BBCC polyploid, Oryza minuta, genome dominance was not observed and its short-term responses to allopolyploidy are mainly manifested as a high proportion of homoeologous gene pairs showing unequal expression. This could partly be explained by parental legacy, rewiring of divergent regulatory networks and epigenetic modulation. Moreover, we detected an ongoing diploidization process in this genus, and suggest that the expression divergence driven by changes of selective constraint probably plays a big role in the long-term diploidization. These findings add novel insights into our understanding of genome evolution after allopolyploidy, and could facilitate crop improvements through hybridization and polyploidization.

Lampreys, the jawless vertebrates, contain three Pax6 genes with distinct expression in eye, brain and pancreas

The transcription factor Pax6 is crucial for the development of the central nervous system, eye, olfactory system and pancreas, and is implicated in human disease. While a single Pax6 gene exists in human and chicken, Pax6 occurs as a gene family in other vertebrates, with two members in elephant shark, Xenopus tropicalis and Anolis lizard and three members in teleost fish such as stickleback and medaka. However, the complement of Pax6 genes in jawless vertebrates (cyclostomes), the sister group of jawed vertebrates (gnathostomes), is unknown. Using a combination of BAC sequencing and genome analysis, we discovered three Pax6 genes in lampreys. Unlike the paired-less Pax6 present in some gnathostomes, all three lamprey Pax6 have a highly conserved full-length paired domain. All three Pax6 genes are expressed in the eye and brain, with variable expression in other tissues. Notably, lamprey Pax6α transcripts are found in the pancreas, a vertebrate-specific organ, indicating the involvement of Pax6 in development of the pancreas in the vertebrate ancestor. Multi-species sequence comparisons revealed only a single conserved non-coding element, in the lamprey Pax6β locus, with similarity to the PAX6 neuroretina enhancer. Using a transgenic zebrafish enhancer assay we demonstrate functional conservation of this element over 500 million years of vertebrate evolution.

Multicolored Fluorescent In Situ Hybridization to Assess Pairing Configurations at Metaphase I in Brassica Hybrids.

Genetic diversity can be introduced into polyploid crop species through meiotic recombination by exchanges between homologous or homoeologous chromosomes. Fluorescent in situ hybridization (FISH) enables the characterization of these homologous and homoeologous chromosome pairs during meiosis and identification of structural rearrangements during mitosis in metaphase I. In this chapter, we describe a protocol for the multicolored fluorescent labeling of chromosome spreads. This protocol allows the characterization of each A and C homoeologous subgenomes in a polyploid species using a genome-specific BAC combined with specific chromosome labeling BAC sequences.

Physical mapping of an adult plant stripe rust resistance gene from Triticum monococcum

Stripe rust (Puccinia striiformis f. sp. tritici) is one of the major devastating disease which causes large reduction in wheat yield. T. monococcum is an attractive diploid species for gene discovery in wheat with smaller genome size of 5700 Mb compared to 17,300 Mb of bread wheat. An adult plant stripe rust resistance QTL QYrtm.pau-2A was mapped on chromosome 2A flanked by two SSR markers Xwmc170 and Xwmc407. In the present study, two gene based markers Pau_Ta2AL_Gene45 and Pau_Ta2AL_Gene54 developed from 2A specific ESTs were found to map close to QYrtmpau-2A to narrow down the region for candidate gene identification. Utilizing sequence information of these two markers, four BAC clones were identified from the Minimum Tiling Path of 2AL assembly and were sequenced. SSR markers were designed from these BAC sequences and mapped to chromosome 2A. A 50 Mb region of wheat chromomse 2A was identified to harbor stripe rust resistance gene of T. monococcum. Gene based markers identified in the present investigation can be used for marker assisted introgression of QYrtm.pau-2A from T. monococcum to cultivated wheat.

High-quality genome assembly of the silkworm, Bombyx mori

In 2008, the genome assembly and gene models for the domestic silkworm, Bombyx mori, were published by a Japanese and Chinese collaboration group. However, the genome assembly contains a non-negligible number of misassembled and gap regions due to the presence of many repetitive sequences within the silkworm genome. The erroneous genome assembly occasionally causes incorrect gene prediction. Here we performed hybrid assembly based on 140 × deep sequencing of long (PacBio) and short (Illumina) reads. The remaining gaps in the initial genome assembly were closed using BAC and Fosmid sequences, giving a new total length of 460.3 Mb, with 30 gap regions and an N50 comprising 16.8 Mb in scaffolds and 12.2 Mb in contigs. More RNA-seq and piRNA-seq reads were mapped on the new genome assembly compared with the previous version, indicating that the new genome assembly covers more transcribed regions, including repetitive elements. We performed gene prediction based on the new genome assembly using available mRNA and protein sequence data. The number of gene models was 16,880 with an N50 of 2154 bp. The new gene models reflected more accurate coding sequences and gene sets than old ones. The proportion of repetitive elements was also reestimated using the new genome assembly, and was calculated to be 46.8% in the silkworm genome. The new genome assembly and gene models are provided in SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp).

A preliminary integrated genetic map distinguishes every chromosome pair and locates essential genes related to abiotic adaptation of Crassostrea angulata/gigas

BackgroundThe re-sequencing of C. angulata has revealed many polymorphisms in candidate genes related to adaptation to abiotic stress that are not present in C. gigas; these genes, therefore, are probably related to the ability of this oyster to retain high concentrations of toxic heavy metals. There is, in addition, an unresolved controversy as to whether or not C. angulata and C. gigas are the same species or subspecies. Both oysters have 20 metacentric chromosomes of similar size that are morphologically indistinguishable. From a genomic perspective, as a result of the great variation and selection for heterozygotes in C. gigas, the assembly of its draft genome was difficult: it is fragmented in more than seven thousand scaffolds.ResultsIn this work sixty BAC sequences of C. gigas downloaded from NCBI were assembled in BAC-contigs and assigned to BACs that were used as probes for mFISH in C. angulata and C. gigas. In addition, probes of H3, H4 histone, 18S and 5S rDNA genes were also used. Hence we obtained markers identifying 8 out the 10 chromosomes constituting the karyotype. Chromosomes 1 and 9 can be distinguished morphologically. The bioinformatic analysis carried out with the BAC-contigs annotated 88 genes. As a result, genes associated with abiotic adaptation, such as metallothioneins, have been positioned in the genome. The gene ontology analysis has also shown many molecular functions related to metal ion binding, a phenomenon associated with detoxification processes that are characteristic in oysters. Hence the provisional integrated map obtained in this study is a useful complementary tool for the study of oyster genomes.ConclusionsIn this study 8 out of 10 chromosome pairs of Crassostrea angulata/gigas were identified using BAC clones as probes. As a result all chromosomes can now be distinguished. Moreover, FISH showed that H3 and H4 co-localized in two pairs of chromosomes different that those previously escribed. 88 genes were annotated in the BAC-contigs most of them related with Molecular Functions of protein binding, related to the resistance of the species to abiotic stress. An integrated genetic map anchored to the genome has been obtained in which the BAC-contigs structure were not concordant with the gene structure of the C. gigas scaffolds displayed in the Genomicus database.

Micro- and macroevolutionary adaptation through repeated loss of a complete metabolic pathway.

There is growing evidence for the convergent evolution of physically linked gene clusters encoding chemical defense pathways. Metabolic clusters are proposed to evolve because they ensure co-inheritance of all required genes where the defense is favored, and prevent inheritance of toxic partial pathways where it is not. This hypothesis rests on the assumption that clusters evolve in species where selection favors intraspecific polymorphism for the defense; however, they have not been examined in polymorphic species. We examined metabolic cluster evolution in relation to an adaptive polymorphism for cyanogenic glucoside (CNglc) production in clover. Using 163 accessions, we performed CNglc assays, BAC sequencing, Southern hybridizations and molecular evolutionary analyses. We find that the CNglc pathway forms a 138-kb cluster in white clover, and that the adaptive polymorphism occurs through presence/absence of the complete cluster. Component genes are orthologous to those in the distantly related legume Lotus japonicus. These findings provide empirical support for the co-inheritance hypothesis, and they indicate that adaptive CNglc variation in white clover evolves through recurrent deletions of the entire pathway. They further indicate that the shared ancestor of many important legume crops was likely cyanogenic and that this defense was lost repeatedly over the last 50Myr.

High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell

The handheld Oxford Nanopore MinION sequencer generates ultra-long reads with minimal cost and time requirements, which makes sequencing genomes at the bench feasible. Here, we sequence the gold standard Arabidopsis thaliana genome (KBS-Mac-74 accession) on the bench with the MinION sequencer, and assemble the genome using typical consumer computing hardware (4 Cores, 16 Gb RAM) into chromosome arms (62 contigs with an N50 length of 12.3 Mb). We validate the contiguity and quality of the assembly with two independent single-molecule technologies, Bionano optical genome maps and Pacific Biosciences Sequel sequencing. The new A. thaliana KBS-Mac-74 genome enables resolution of a quantitative trait locus that had previously been recalcitrant to a Sanger-based BAC sequencing approach. In summary, we demonstrate that even when the purpose is to understand complex structural variation at a single region of the genome, complete genome assembly is becoming the simplest way to achieve this goal.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Legume isoflavone synthase genes have evolved by whole-genome and local duplications yielding transcriptionally active paralogs

Isoflavone synthase (IFS) is the key enzyme of isoflavonoid biosynthesis. IFS genes were identified in numerous species, although their evolutionary patterns have not yet been reconstructed. To address this issue, we performed structural and functional genomic analysis.Narrow leafed lupin, Lupinus angustifolius L., was used as a reference species for the genus, because it has the most developed molecular tools available. Nuclear genome BAC library clones carrying IFS homologs were localized by linkage mapping and fluorescence in situ hybridization in three chromosome pairs. Annotation of BAC, scaffold and transcriptome sequences confirmed the presence of three full-length IFS genes in the genome.Microsynteny analysis and Bayesian inference provided clear evidence that IFS genes in legumes have evolved by lineage-specific whole-genome and tandem duplications. Gene expression profiling and RNA-seq data mining showed that the vast majority of legume IFS copies have maintained their transcriptional activity. L. angustifolius IFS homologs exhibited organ-specific expression patterns similar to those observed in other Papilionoideae. Duplicated lupin IFS homologs retained non-negligible levels of substitutions in conserved motifs, putatively due to positive selection acting during early evolution of the genus, before the whole-genome duplication. Strong purifying selection preserved newly arisen IFS duplicates from further nonsynonymous changes.

The landscape and structural diversity of LTR retrotransposons in Musa genome.

Long terminal repeat retrotransposons represent a major component of plant genomes and act as drivers of genome evolution and diversity. Musa is an important fruit crop and also used as a starchy vegetable in many countries. BAC sequence analysis by dot plot was employed to investigate the LTR retrotransposons from Musa genomes. Fifty intact LTR retrotransposons from selected Musa BACs were identified by dot plot analysis and further BLASTN searches retrieved 153 intact copies, 61 truncated, and a great number of partial copies/remnants from GenBank database. LARD-like elements were also identified with several copies dispersed among the Musa genotypes. The predominant elements were the LTR retrotransposons Copia and Gypsy, while Caulimoviridae (pararetrovirus) were rare in the Musa genome. PCR amplification of reverse transcriptase (RT) sequences revealed their abundance in almost all tested Musa accessions and their ancient nature before the divergence of Musa species. The phylogenetic analysis based on RT sequences of Musa and other retrotransposons clustered them into Gypsy, Caulimoviridae, and Copia lineages. Most of the Musa-related elements clustered in their respective groups, while some grouped with other elements indicating homologous sequences. The present work will be helpful to understand the LTR retrotransposons landscape, giving a complete picture of the nature of the elements, their structural features, annotation, and evolutionary dynamics in the Musa genome.

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions

BackgroundTilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.ResultsA homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recent vasa gene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus.ConclusionsThis study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species.

Patterns of Polymorphism at the Self-Incompatibility Locus in 1,083 Arabidopsis thaliana Genomes

Although the transition to selfing in the model plant Arabidopsis thaliana involved the loss of the self-incompatibility (SI) system, it clearly did not occur due to the fixation of a single inactivating mutation at the locus determining the specificities of SI (the S-locus). At least three groups of divergent haplotypes (haplogroups), corresponding to ancient functional S-alleles, have been maintained at this locus, and extensive functional studies have shown that all three carry distinct inactivating mutations. However, the historical process of loss of SI is not well understood, in particular its relation with the last glaciation. Here, we took advantage of recently published genomic resequencing data in 1,083 Arabidopsis thaliana accessions that we combined with BAC sequencing to obtain polymorphism information for the whole S-locus region at a species-wide scale. The accessions differed by several major rearrangements including large deletions and interhaplogroup recombinations, forming a set of haplogroups that are widely distributed throughout the native range and largely overlap geographically. “Relict” A. thaliana accessions that directly derive from glacial refugia are polymorphic at the S-locus, suggesting that the three haplogroups were already present when glacial refugia from the last Ice Age became isolated. Interhaplogroup recombinant haplotypes were highly frequent, and detailed analysis of recombination breakpoints suggested multiple independent origins. These findings suggest that the complete loss of SI in A. thaliana involved independent self-compatible mutants that arose prior to the last Ice Age, and experienced further rearrangements during postglacial colonization.

Expression map of a complete set of gustatory receptor genes in chemosensory organs of Bombyx mori

Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27–BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO2 receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for finding genes involved in plant-insect interactions in Lepidoptera and establishing correlations between these genes and vital insect behaviors like host plant selection and courtship for mating.

Identification of microsatellite loci based on BAC sequencing data and their physical mapping into the soft wheat 5B chromosome

The shortage of polymorphic markers for the regions of the wheat chromosomes that encode commercially valuable traits determines the need for studying the wheat microsatellite SSR loci. In this work, SSR markers for individual regions of the short arm of soft wheat chromosome 5B (5BS) were designed based on the sequence data obtained from BAC clones, and regions of the corresponding chromosome were saturated with these markers. Totally, 130 randomly selected BAC clones from 5BS library were sequenced using the IonTorrent platform and assembled in contigs using MIRA software. The assembly characteristics (N50 = 4136 bp) are comparable to the recently obtained data for wheat and related species and are acceptable for the identification of the microsatellite loci. The algorithm utilizing the properties of complex decompositions in the sliding-window mode was used to detect DNA sequences with a repeat unit of 2–4 bp. Analysis of 17770 contigs with a total length of 25879921 bp allowed for the design of 113, 79, and 67 microsatellite SSR loci with a repeat unit of 2, 3, and 4 bp, respectively. SSR markers with a motif of 3 bp were tested using nullitetrasomic lines of Chinese Spring wheat homoeologous group 5. In total, 21 markers specific for chromosome 5B were identified. Eight of these markers were mapped into the distal region of this chromosome (bin 5BS6) using a set of Chinese Spring deletion lines for 5BS. Eight and four markers were mapped to the interstitial region (bins 5BS5 and 5BS4, respectively). One marker was mapped to a pericentromeric bin. Comparative analysis of the distribution of trinucleotide microsatellites over wheat chromosome 5B, and in different cereal species, suggests that the (AAG) n repeat proliferates and is conserved during the evolution of cereals.

English

Cassava is an economically important crop in sub-Saharan Africa; however, its yield potential is constrained by cassava mosaic disease (CMD) infection. Classical genetics and biotechnology are being harnessed to overcome the disease and secure yields for farmers. The CMD2 resistance locus flanked by three simple sequence repeats (SSR) markers and one sequence characterized amplified region (SCAR) marker were mapped in West African genotypes and shown to impart qualitative resistant to all species of CMGs. However, gene(s) associated with the CMD2 locus and their mode of actions remains unknown. In an effort to discover gene(s) located in CMD2 locus region, TME3 BAC collections were screened for the presence of CMD2 flanking markers. CMD susceptible and resistant cassava genotypes were found to contain 100% of the markers flanking CMD2 locus. SNPs and nucleotide deletions were identified within the marker sequences but there was no evidence of trait and marker association. All the SSR markers flanking CMD2, and the more recently characterized CMD3 loci were to be located on chromosome 12. Through BAC pools library hybridization with marker probes, 130 BACs were identified, but only 23 BACs contained at least CMD2 specific two markers. Whole BAC sequencing identified five clones that mapped to the marker regions. BAC29 assembled into a 100 kb contig and encoded tandem repeats of three full length R genes (3.5 kb) and two partial repeats. These R genes were conserved and highly expressed in CMD susceptible and CMD resistant cassava genotypes. Promoter sequences derived from R genes showed similar transient expression of GUS as 35S promoter. On cassava genome V6.1 BAC29 sequences were mapped to chromosome 16, eliminating their potential role in CMD resistant. Key words: Bacteria artificial chromosome, CMD2, cassava, cassava mosaic disease.

Variant discovery and breakpoint region prediction for studying the human 22q11.2 deletion using BAC clone and whole genome sequencing analysis.

Velo-cardio-facial syndrome/DiGeorge syndrome/22q11.2 deletion syndrome (22q11.2DS) is caused by meiotic non-allelic homologous recombination events between flanking low copy repeats termed LCR22A and LCR22D, resulting in a 3 million base pair (Mb) deletion. Due to their complex structure, large size and high sequence identity, genetic variation within LCR22s among different individuals has not been well characterized. In this study, we sequenced 13 BAC clones derived from LCR22A/D and aligned them with 15 previously available BAC sequences to create a new genetic variation map. The thousands of variants identified by this analysis were not uniformly distributed in the two LCR22s. Moreover, shared single nucleotide variants between LCR22A and LCR22D were enriched in the Breakpoint Cluster Region pseudogene (BCRP) block, suggesting the existence of a possible recombination hotspot there. Interestingly, breakpoints for atypical 22q11.2 rearrangements have previously been located to BCRPs To further explore this finding, we carried out in-depth analyses of whole genome sequence (WGS) data from two unrelated probands harbouring a de novo 3Mb 22q11.2 deletion and their normal parents. By focusing primarily on WGS reads uniquely mapped to LCR22A, using the variation map from our BAC analysis to help resolve allele ambiguity, and by performing PCR analysis, we infer that the deletion breakpoints were most likely located near or within the BCRP module. In summary, we found a high degree of sequence variation in LCR22A and LCR22D and a potential recombination breakpoint near or within the BCRP block, providing a starting point for future breakpoint mapping using additional trios.

Ancestral Y-linked genes were maintained by translocation to the X and Y chromosomes fused to an autosomal pair in the Okinawa spiny rat Tokudaia muenninki.

Two species of the genus Tokudaia lack the Y chromosome and SRY, but several Y-linked genes have been rescued by translocation or transposition to other chromosomes. Tokudaia muenninki is the only species in the genus that maintains the Y owing to sex chromosome-autosome fusions. According to previous studies, many SRY pseudocopies and other Y-linked genes have evolved by excess duplication in this species. Using RNA-seq and RT-PCR, we found that ZFY, EIF2S3Y, TSPY, UTY, DDX3Y, USP9Y, and RBMY, but not UBA1Y, had high deduced amino acid sequence similarity and similar expression patterns with other rodents, suggesting that these genes were functional. Based on FISH and quantitative real-time PCR, all of the genes except for UTY and DDX3Y were amplified on the X and Y chromosomes with approximately 10-66 copies in the male genome. In a comparative analysis of the 372.4-kb BAC sequence and Y-linked gene transcripts from T. muenninki with the mouse Y genomic sequence, we observed that multiple-copy genes in the ancestral Y genome were nonfunctional, indicating that the gene functions were assumed by amplified copies. We also found a LTR sequence at the distal end of a SRY duplication unit, suggesting that unequal sister chromatid exchange mediated by retrotransposable elements could have been involved in SRY amplification. Our results revealed that the Y-linked genes were rescued from degeneration via translocations to other sex chromosomal regions and amplification events in T. muenninki.