Next-generation sequencing-based bulked segregant analysis without sequencing the parental genomes

Genomic regions that control traits of interest can be rapidly identified using BSA-Seq, a technology in which next-generation sequencing (NGS) is applied to bulked segregant analysis (BSA). We recently developed the significant structural variant method for BSA-Seq data analysis that exhibits higher detection power than standard BSA-Seq analysis methods. Our original algorithm was developed to analyze BSA-Seq data in which genome sequences of one parent served as the reference sequences in genotype calling, and thus required the availability of high-quality assembled parental genome sequences. Here we modified the original script to effectively detect the genomic region-trait associations using only bulk genome sequences. We analyzed two public BSA-Seq datasets using our modified method and the standard allele frequency and G-statistic methods with and without the aid of the parental genome sequences. Our results demonstrate that the genomic region(s) associated with the trait of interest could be reliably identified via the significant structural variant method without using the parental genome sequences.

Identification of Parental Genome Construction and Inherited Morphological Characteristics in Triploid and AneuploidIntergeneric Hybrids from a Diploid−Diploid Cross between Citrus and Fortunella

We previously obtained two intergeneric hybrids with different ploidies, i.e., aneuploid (2n = 28) and eutriploid, from diploid−diploid crosses between ‘Kiyomi’ tangor (Citrus unshiu Marcow. × C. sinensis (L.) Osbeck) and Meiwa kumquat (Fortunella crassifolia Swingle) as novel breeding materials for a seedless kumquat. In this study, we attempted to clarify the construction of the parental genomes of these hybrids by SSR genotyping and genomic in situ hybridization (GISH)−chromomycin A3 (CMA) analysis. SSR genotyping in NSX43 (LG5) and CiBE2227 (LG8) loci revealed that both hybrids inherited one allele from ‘Kiyomi’ tangor and two heterozygous alleles from Meiwa kumquat. The GISH analysis failed due to the high genomic homology between Citrus and Fortunella. At the same time, the CMA karyotype compositions of the two intergeneric hybrids (H15-701: 2A + 1B + 3C + 13D + 7E + 1F + 1Dst; H15-702: 3A + 1B + 2C + 15D + 4E +1F + 1Dst) and both parents (‘Kiyomi’ tangor: 1A + 2B + 2C + 6D + 7E; Meiwa kumquat: 2A + 2C + 12D + 1F + 1Dst) were completely revealed. We identified the parental genome construction and polyploidization processes in both intergeneric hybrids on the basis of these SSR genotypes and CMA karyotype compositions according to the following theory: the SSR genotypes and chromosome compositions were the same as those of the somatic chromosome and two-fold after the first division (even number) in unreduced gametes caused by first-division restitution (FDR) and second-division restitution (SDR), respectively. Consequently, we determined that both intergeneric hybrids may have had two genomes derived from the 2n male unreduced gamete as a result of the FDR of the Meiwa kumquat. In addition, most horticultural traits of the leaves, flowers, and fruits of both hybrids showed intermediate traits of the parents, but the fruit sizes and flowering habits were more like those of the two inherited genomes of Meiwa kumquat.

Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.

Dynamics of Verticillium dahliae race 1 population under managed agricultural ecosystems

Background Plant pathogens and their hosts undergo adaptive changes in managed agricultural ecosystems, by overcoming host resistance, but the underlying genetic adaptations are difficult to determine in natural settings. Verticillium dahliae is a fungal pathogen that causes Verticillium wilt on many economically important crops including lettuce. We assessed the dynamics of changes in the V. dahliae genome under selection in a long-term field experiment. Results In this study, a field was fumigated before the Verticillium dahliae race 1 strain (VdLs.16) was introduced. A derivative 145-strain population was collected over a 6-year period from this field in which a seggregating population of lettuce derived from Vr1/vr1 parents were evaluated. We de novo sequenced the parental genome of VdLs.16 strain and resequenced the derivative strains to analyze the genetic variations that accumulate over time in the field cropped with lettuce. Population genomics analyses identified 2769 single-nucleotide polymorphisms (SNPs) and 750 insertion/deletions (In-Dels) in the 145 isolates compared with the parental genome. Sequence divergence was identified in the coding sequence regions of 378 genes and in the putative promoter regions of 604 genes. Five-hundred and nine SNPs/In-Dels were identified as fixed. The SNPs and In-Dels were significantly enriched in the transposon-rich, gene-sparse regions, and in those genes with functional roles in signaling and transcriptional regulation. Conclusions Under the managed ecosystem continuously cropped to lettuce, the local adaptation of V. dahliae evolves at a whole genome scale to accumulate SNPs/In-Dels nonrandomly in hypervariable regions that encode components of signal transduction and transcriptional regulation.

Uniparental genome elimination in Australian carp gudgeons

Metazoans usually reproduce sexually, blending the unique identity of parental genomes for the next generation through functional crossing-over and recombination in meiosis. However, some metazoan lineages have evolved reproductive systems where offspring are either full (clonal) or partial (hemiclonal) genetic replicas. In the latter group, the process of uniparental genome elimination selectively eliminates either the maternal or paternal genome from germ cells, and only one parental genome is selected for transmission. Although fairly common in plants, hybridogenesis (i.e. clonal haploidization via chromosome elimination) remains a poorly understood process in animals. Here, we explore the proximal cytogenomic mechanisms of somatic and germ cell chromosomes in sexual and hybrid genotypes of Australian carp gudgeons (Hypseleotris) by tracing the fate of each set during mitosis (in somatic tissues) and meiosis (in gonads). Our comparative study of diploid hybrid and sexual individuals revealed visually functional gonads in male and female hybrid genotypes and generally high karyotype variability, although the number of chromosome arms remains constant. Our results delivered direct evidence for classic hybridogenesis as a reproductive mode in carp gudgeons. Two parental sets with integral structure in the hybrid soma (the F1 constitution) contrasted with uniparental chromosomal inheritance detected in gonads. The inheritance mode happens through pre-meiotic genome duplication of the parental genome to be transmitted, while the second parental genome is likely gradually eliminated already in juvenile individuals. The role of metacentric chromosomes in hybrid evolution is also discussed.

Next-generation sequencing-based bulked segregant analysis without sequencing the parental genomes

The genomic region(s) that controls a trait of interest can be rapidly identified using BSA-Seq, a technology in which next-generation se-quencing (NGS) is applied to bulked segregant analysis (BSA). We recently developed the significant structural variant method for BSA-Seq data analysis that exhibits higher detection power than standard BSA-Seq analysis methods. Our original algorithm was developed to analyze BSA-Seq data in which genome sequences of one parent served as the reference sequences in genotype calling, and thus required the availability of high-quality assembled parental genome sequences. Here we modified the original script to allow for the effective detection of the genomic region-trait associations using only bulk genome sequences. We analyzed a public BSA-Seq dataset using our modified method and the standard allele frequency and G-statistic methods with and without the aid of the parental genome sequences. Our results demonstrate that the genomic region(s) associated with the trait of interest could be reliably identified only via the significant structural variant method without using the parental genome sequences.Significance StatementBSA-Seq can be utilized to rapidly identify structural varianttrait associations, and our modified significant structural variant method allows the detection of such associations without sequencing the parental genomes, leading to further lower the sequencing cost and making BSA-Seq more accessible to the research community and more applicable to the species with a large genome.

Marker Assisted Gene Pyramiding for Bacterial Blight Resistance in Some Improved Rice Genotypes

Bacterial blight is one of the major diseases of rice, causing huge economic loss to the rice farmers around the world. In the present study, marker assisted selection (MAS) was used to pyramid broad spectrum resistance genes (Xa33 and Xa38) into two improved rice genotypes (ADT 47 and ASD 16) which already have some bacterial blight resistance genes (xa5, xa13 and Xa21). The rice genotypes FBRI-15 and PR114 were used as donors of Xa33 and Xa38 genes respectively. The marker RMWR7.1 linked to Xa33 and the sequenced-tagged site marker Os04g53050-1 specific to Xa38 were used for foreground selection. True F1 hybrids were selected using the polymorphic markers. Out of one hundred and thirty-seven SSR markers, forty-two were polymorphic for improved ADT 47 x FBRI-16 and forty-six were polymorphic for Improved ASD 16 x PR114. The polymorphic markers were used for background selection. Foreground selection revealed that a single F1 plant was heterozygote in Improved ADT 47 X FBRI-15 whereas two plants were heterozygotes in Improved ASD 16 x PR114. At BC1F1, one and three plants were found to be heterozygous respectively for Improved ADT 47 x FBRI-15 and Improved ASD 16 x PR114. At BC2F1, single plant each was found heterozygous from the two crosses. In the final BC3F1 population, one heterozygote each was obtained from the two crosses. In BC3F1, plant number 11 had 92.80% parental genome recovery in the Improved ADT 47 x FBRI-15, whereas plant no 16 had 91.33% parental genome recovery in the Improved ASD 16 x PR114

Non-rodent mammalian zygotes assemble dual spindles despite the presence of paternal centrosomes

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded and, instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In non-rodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether non-rodent zygotes assemble a single centrosomal spindle around both genomes, or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form around the parental genomes despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in non-rodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.SummaryThis study investigates spindle assembly during the first embryonic division in bovine zygotes that, like human, inherit centrosomes from the sperm. It shows that two independent microtubule arrays form by self-organization around parental genomes with only loosely connected centrosomes.