The Power of QTL Mapping with RILs

QTL (quantitative trait loci) mapping is commonly used to identify genetic regions responsible to important phenotype variation. A common strategy of QTL mapping is to use recombinant inbred lines (RILs), which are usually established by several generations of inbreeding of an F1 population (usually up to F6 or F7 populations). As this inbreeding process involves a large amount of labor, we are particularly interested in the effect of the number of inbreeding generations on the power of QTL mapping; a part of the labor could be saved if a smaller number of inbreeding provides sufficient power. By using simulations, we investigated the performance of QTL mapping with recombinant inbred lines (RILs). As expected, we found that the power of F4 population could be almost comparable to that of F6 and F7 populations. A potential problem in using F4 population is that a large proportion of RILs are heterozygotes. We here introduced a new method to partly relax this problem. The performance of this method was verified by simulations with a wide range of parameters including the size of the segregation population, recombination rate, genome size and the density of markers. We found our method works better than the commonly used standard method especially when there are a number of heterozygous markers. Our results imply that in most cases, QTL mapping does not necessarily require RILs at F6 or F7 generations; rather, F4 (or even F3) populations would be almost as useful as F6 or F7 populations. Because the cost to establish a number of RILs for many generations is enormous, this finding will cause a reduction in the cost of QTL mapping, thereby accelerating gene mapping in many species

Identical sets of methylated and nonmethylated genes in Ciona intestinalis sperm and muscle cells

BackgroundThe discovery of gene body methylation, which refers to DNA methylation within gene coding region, suggests an as yet unknown role of DNA methylation at actively transcribed genes. In invertebrates, gene bodies are the primary targets of DNA methylation, and only a subset of expressed genes is modified. ResultsHere we investigate the tissue variability of both the global levels and distribution of 5-methylcytosine (5mC) in the sea squirt Ciona intestinalis. We find that global 5mC content of early developmental embryos is high, but is strikingly reduced in body wall tissues. We chose sperm and adult muscle cells, with high and reduced levels of global 5mC respectively, for genome-wide analysis of 5mC targets. By means of CXXC-affinity purification followed by deep sequencing (CAP-seq), and genome-wide bisulfite sequencing (BS-seq), we designated body-methylated and unmethylated genes in each tissue. Surprisingly, body-methylated and unmethylated gene groups are identical in the sperm and muscle cells. Our analysis of microarray expression data shows that gene body methylation is associated with broad expression throughout development. Moreover, transgenic analysis reveals contrasting gene body methylation at an identical gene-promoter combination when integrated at different genomic sites. ConclusionsWe conclude that gene body methylation is not a direct regulator of tissue specific gene expression in C. intestinalis. Our findings reveal constant targeting of gene body methylation irrespective of cell type, and they emphasize a correlation between gene body methylation and ubiquitously expressed genes. Our transgenic experiments suggest that the promoter does not determine the methylation status of the associated gene body

From many, one: Genetic control of prolificacy during maize domestication

A reduction in number and an increase in size of inflorescences is a common aspect of plant domestication. When maize was domesticated from teosinte, the number and arrangement of ears changed dramatically. Teosinte has long lateral branches that bear multiple small ears at their nodes and tassels at their tips. Maize has much shorter lateral branches that are tipped by a single large ear with no additional ears at the branch nodes. To investigate the genetic basis of this difference in prolificacy (the number of ears on a plant), we performed a genome-wide QTL scan. A large effect QTL for prolificacy (prol1.1) was detected on the short arm of chromosome 1 in a location that has previously been shown to influence multiple domestication traits. We fine-mapped prol1.1 to a 2.7 kb “causative region” upstream of the grassy tillers1 (gt1) gene, which encodes a homeodomain leucine zipper transcription factor. Tissue in situ hybridizations reveal that the maize allele of prol1.1 is associated with up-regulation of gt1 expression in the nodal plexus. Given that maize does not initiate secondary ear buds, the expression of gt1 in the nodal plexus in maize may suppress their initiation. Population genetic analyses indicate positive selection on the maize allele of prol1.1, causing a partial sweep that fixed the maize allele throughout most of domesticated maize. This work shows how a subtle cis-regulatory change in tissue specific gene expression altered plant architecture in a way that improved the harvestability of maize

QTL Map Meets Population Genomics: An Application to Rice

Genes involved in the transition from wild to cultivated crop species should be of great agronomic importance. Population genomic approaches utilizing genome resequencing data have been recently applied for this purpose, although it only reports a large list of candidate genes with no biological information. Here, by resequencing more than 30 genomes altogether of wild rice Oryza rufipogon and cultivated rice O. sativa, we identified a number of regions with clear footprints of selection during the domestication process. We then focused on identifying candidate domestication genes in these regions by utilizing the wealth of QTL information in rice. We were able to identify a number of interesting candidates such as transcription factors that should control key domestication traits such as shattering, awn length, and seed dormancy. Other candidates include those that might have been related to the improvement of grain quality and those that might have been involved in the local adaptation to dry conditions and colder environments. Our study shows that population genomic approaches and QTL mapping information can be used together to identify genes that might be of agronomic importance

A role for palindromic structures in the cis-region of maize Sirevirus LTRs in transposable element evolution and host epigenetic response

Transposable elements (TEs) proliferate within the genome of their host, which responds by silencing them epigenetically. Much is known about the mechanisms of silencing in plants, particularly the role of siRNAs in guiding DNA methylation. In contrast, little is known about siRNA targeting patterns along the length of TEs, yet this information may provide crucial insights into the dynamics between hosts and TEs. By focusing on 6456 carefully annotated, full-length Sirevirus LTR retrotransposons in maize, we show that their silencing associates with underlying characteristics of the TE sequence and also uncover three features of the host–TE interaction. First, siRNA mapping varies among families and among elements, but particularly along the length of elements. Within the cis-regulatory portion of the LTRs, a complex palindrome-rich region acts as a hotspot of both siRNA matching and sequence evolution. These patterns are consistent across leaf, tassel, and immature ear libraries, but particularly emphasized for floral tissues and 21- to 22-nt siRNAs. Second, this region has the ability to form hairpins, making it a potential template for the production of miRNA-like, hairpin-derived small RNAs. Third, Sireviruses are targeted by siRNAs as a decreasing function of their age, but the oldest elements remain highly targeted, partially by siRNAs that cross-map to the youngest elements. We show that the targeting of older Sireviruses reflects their conserved palindromes. Altogether, we hypothesize that the palindromes aid the silencing of active elements and influence transposition potential, siRNA targeting levels, and ultimately the fate of an element within the genome

Population genomics of the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has been widely used as a model eukaryote to study a diverse range of biological processes. However, population genetic studies of this species have been limited to date, and we know very little about the evolutionary processes and selective pressures that are shaping its genome. Here, we sequenced the genomes of 32 worldwide S. pombe strains and examined the pattern of polymorphisms across their genomes. In addition to introns and untranslated regions (UTRs), intergenic regions also exhibited lower levels of nucleotide diversity than synonymous sites, suggesting that a considerable amount of noncoding DNA is under selective constraint and thus likely to be functional. A number of genomic regions showed a reduction of nucleotide diversity probably caused by selective sweeps. We also identified a region close to the end of chromosome 3 where an extremely high level of divergence was observed between 5 of the 32 strains and the remain 27, possibly due to introgression, strong positive selection, or that region being responsible for reproductive isolation. Our study should serve as an important starting point in using a population genomics approach to further elucidate the biology of this important model organism

Identical sets of methylated and nonmethylated genes in Ciona intestinalis sperm and muscle cells

BACKGROUND: The discovery of gene body methylation, which refers to DNA methylation within gene coding region, suggests an as yet unknown role of DNA methylation at actively transcribed genes. In invertebrates, gene bodies are the primary targets of DNA methylation, and only a subset of expressed genes is modified. RESULTS: Here we investigate the tissue variability of both the global levels and distribution of 5-methylcytosine (5mC) in the sea squirt Ciona intestinalis. We find that global 5mC content of early developmental embryos is high, but is strikingly reduced in body wall tissues. We chose sperm and adult muscle cells, with high and reduced levels of global 5mC respectively, for genome-wide analysis of 5mC targets. By means of CXXC-affinity purification followed by deep sequencing (CAP-seq), and genome-wide bisulfite sequencing (BS-seq), we designated body-methylated and unmethylated genes in each tissue. Surprisingly, body-methylated and unmethylated gene groups are identical in the sperm and muscle cells. Our analysis of microarray expression data shows that gene body methylation is associated with broad expression throughout development. Moreover, transgenic analysis reveals contrasting gene body methylation at an identical gene-promoter combination when integrated at different genomic sites. CONCLUSIONS: We conclude that gene body methylation is not a direct regulator of tissue specific gene expression in C. intestinalis. Our findings reveal constant targeting of gene body methylation irrespective of cell type, and they emphasize a correlation between gene body methylation and ubiquitously expressed genes. Our transgenic experiments suggest that the promoter does not determine the methylation status of the associated gene body

エピジェネティック修飾に関わる自然選択の検出

2015～2017研究成果の概要（和文）：ゲノム進化学にエピジェネティックスを取り入れ、ゲノム-エピゲノム間の進化的インタラクションと新たな自然選択の検出を目指して研究を行った。エピゲノムのうち、DNAメチル化に着目して研究を行った。陸上植物のDNAメチル化、特に遺伝子的メチル化に焦点を当てて、解析を行った。遺伝子内メチル化は自然選択により、長期間植物種のオーソログで保存されていた。遺伝子内メチル化レベルは、トランスポゾン増加によるDNAメチル化レベルの影響を受け進化していた。さらに、遺伝子内メチル化を保護するため、そのメチル化を持つ遺伝子のDNA配列への自然選択が検出された。研究成果の概要（英文）：DNA methylation is an important epigenetic modification that affects both chromatin packing and transcription. DNA methylation occurs in three sequence contexts, that is, CG,CHG, and CHH(where H is A,C,orT) in plants. All three contexts are methylated within repetitive elements. The major role of DNA methylation within repetitive elements is to silence transcription and functions as a host defense against transposons. On the other hand, only the CG context is mainly methylated within coding regions in plants that is called gene body methylation. Gene body methylation is considered a byproduct of the process of removing heterochromatic marks within active genes; gene body methylation might not be functional. However, I found that gene body methylation was observed in a biased subset of genes, tended to be conserved between plant orthologs, and the drastic changes of gene body methylation significantly affected expression levels.　科学研究費補助金研究成果報告書研究代表者：宅野将平[総合研究大学院大学・先導科学研究科・助教