QTL mapping of shoot and seed traits impacted by Drought in Barley using a recombinant inbred line Population.

BACKGROUND: With ongoing climate change, drought events are severely limiting barley production worldwide and pose a significant risk to the malting, brewing and food industry. The genetic diversity inherent in the barley germplasm offers an important resource to develop stress resiliency. The purpose of this study was to identify novel, stable, and adaptive Quantitative Trait Loci (QTL), and candidate genes associated with drought tolerance. A recombinant inbred line (RIL) population (n = 192) developed from a cross between the drought tolerant 'Otis' barley variety, and susceptible 'Golden Promise'(GP) was subjected to short-term progressive drought during heading in the biotron. This population was also evaluated under irrigated and rainfed conditions in the field for yields and seed protein content. RESULTS: Barley 50k iSelect SNP Array was used to genotype the RIL population to elucidate drought-adaptive QTL. Twenty-three QTL (eleven for seed weight, eight for shoot dry weight and four for protein content) were identified across several barley chromosomes. QTL analysis identified genomic regions on chromosome 2 and 5 H that appear to be stable across both environments and accounted for nearly 60% variation in shoot weight and 17.6% variation in seed protein content. QTL at approximately 29 Mbp on chromosome 2 H and 488 Mbp on chromosome 5 H are in very close proximity to ascorbate peroxidase (APX) and in the coding sequence of the Dirigent (DIR) gene, respectively. Both APX and DIR are well-known key players in abiotic stress tolerance in several plants. In the quest to identify key recombinants with improved tolerance to drought (like Otis) and good malting profiles (like GP), five drought tolerant RILs were selected for malt quality analysis. The selected drought tolerant RILs exhibited one or more traits that were outside the realms of the suggested limits for acceptable commercial malting quality. CONCLUSIONS: The candidate genes can be used for marker assisted selection and/or genetic manipulation to develop barley cultivars with improved tolerance to drought. RILs with genetic network reshuffling necessary to generate drought tolerance of Otis and favorable malting quality attributes of GP may be realized by screening a larger population.

Malting quality and preharvest sprouting traits are genetically correlated in spring malting barley.

KEY MESSAGE: Malt for craft "all-malt" brewing can have high quality, PHS resistance, and malted in normal timeframes. Canadian style adjunct malt is associated with PHS susceptibility. Expansion of malting barley production into non-traditional growing regions and erratic weather has increased the demand for preharvest sprouting (PHS) resistant, high quality malting barley cultivars. This is hindered by the relatively unknown relationships between PHS resistance and malting quality. Here we present a three-year study of malting quality and germination at different after-ripening durations post physiological maturity. Malting quality traits alpha amylase (AA) and free amino nitrogen (FAN) and germination rate at six days post PM shared a common association with a SNP in HvMKK3 on chromosome 5H in the Seed Dormancy 2 (SD2) region responsible for PHS susceptibility. Soluble protein (SP) and soluble over total protein (S/T) both shared a common association with a marker in the SD2 region. Significant genetic correlations between PHS resistance and the malting quality traits AA, FAN, SP, S/T were detected across and within HvMKK3 allele groups. High adjunct malt quality was related to PHS susceptibility. Selection for PHS resistance led to a correlated response in malting quality traits. Results strongly suggest pleiotropy of HvMKK3 on malting quality traits and that the classic "Canadian-style" malt is caused by a PHS susceptible allele of HvMKK3. PHS susceptibility appears to benefit the production of malt intended for adjunct brewing, while PHS resistance is compatible with all-malt brewing specifications. Here we present our analysis on the effect of combining complexly inherited and correlated traits with contrasting goals to inform breeding practice in malting barley, the general principles of which can be extended to other breeding programs.

The Barley (Hordeum vulgare ssp. vulgare) Respiratory Burst Oxidase Homolog (HvRBOH) Gene Family and Their Plausible Role on Malting Quality.

Controlled generation of reactive oxygen species (ROS) is pivotal for normal plant development and adaptation to changes in the external milieu. One of the major enzymatic sources of ROS in plants are the plasma-membrane localized NADPH oxidases, also called as Respiratory Burst Oxidase Homologs (RBOH). In addition to the six previously reported, seven new members of RBOH gene family were identified in barley using in silico analysis. Conservation of genomic structure and key residues important for catalytic activity and co-factor binding was observed in barley RBOH genes. Phylogenetic analysis of plant RBOHs revealed distinct clades for monocot and dicot RBOH proteins. Hence, we propose to use the rice nomenclature for naming barley RBOH genes. Temporal changes in ROS profiles were observed during barley malting and was accompanied by changes in protein carbonylation, lipid peroxidation, and antioxidant capacity. Among the nine differentially expressed HvRBOHs during various malting stages, HvRBOHA and HvRBOHC showed most significant sustained changes in expression. RNAi knockdown lines with reduced expression of HvRBOHA/C gene exhibited genetic compensation via inducible expression of other gene family members during malting. However, the physiological consequence of reduced expression of HvRBOHA/C manifested as a poor malting quality profile attributable to low alpha-amylase activity and high levels of beta-glucan. We propose that the HvRBOHs play a critical role in modulating the redox milieu during the early stages of malting, which in turn can significantly impact carbohydrate metabolism.

Genome-wide association analysis of natural variation in seed tocochromanols of barley.

Tocochromanols (tocols for short), commonly called Vitamin E, are lipid-soluble plant antioxidants vital for regulating lipid peroxidation in chloroplasts and seeds. Barley (Hordeum vulgare L.) seeds contain all eight different isoforms of tocols; however, the extent of natural variation in their composition and their underlying genetic basis is not known. Tocol levels in barley seeds were quantified in diverse H. vulgare panels comprising 297 wild lines from a diversity panel and 160 cultivated spring-type accessions from the mini-core panel representing the genetic diversity of the USDA barley germplasm collection. Significant differences were observed in the concentration of tocols between the two panels. To identify the genes associated with tocols, genome-wide association analysis was conducted with single nucleotide polymorphisms (SNPs) from Illumina arrays for the mini-core panel and genotyping-by-sequencing for the wild barley panel. Forty unique SNPs in the wild barley and 27 SNPs in the mini-core panel were significantly associated with various tocols. Marker-trait associations (MTAs) were identified on chromosomes 1, 6, and 7 for key genes in the tocol biosynthesis pathway, which have also been reported in other studies. Several novel MTAs were identified on chromosomes 2, 3, 4 and 5 and were found to be in proximity to genes involved in the generation of precursor metabolites required for tocol biosynthesis. This study provides a valuable resource for barley breeding programs targeting specific isoforms of seed tocols and for investigating the physiological roles of these metabolites in seed longevity, dormancy, and germination.

Genomic survey of RNA recognition motif (RRM) containing RNA binding proteins from barley (Hordeum vulgare ssp. vulgare).

One of the major mechanisms of post-transcriptional gene regulation is achieved by proteins bearing well-defined sequence motifs involved in 'RNA binding'. In eukaryotes, RNA binding proteins (RBPs) are key players of RNA metabolism that includes synthesis, processing, editing, modifying, transport, storage and stability of RNA. In plants, the family of RBPs is vastly expanded compared to other eukaryotes including humans. In this study we identified 363 RBPs in the barley genome. Gene ontology enrichment analysis of barley RBPs indicated these proteins were in all the major cellular compartments and associated with key biological processes including translation, splicing, seed development and stress signaling. Members with the classical RNA binding motifs such as the RNA recognition motif (RRM), KH domain, Helicase, CRM, dsRNA and Pumilio were identified in the repertoire of barley RBPs. Similar to Arabidopsis, the RRM containing RBPs were the most abundant in barley genome. In-depth analysis of the RRM containing proteins - polyA binding proteins, Ser/Arg rich proteins and Glycine-rich RBPs were undertaken. Reanalysis of the proteome dataset of various stages during barley malting identified 38 RBPs suggesting an important role for these proteins during the malting process. This survey provides a systematic analysis of barley RBPs and serves as the basis for the further functional characterization of this important family of proteins.

Author Correction: Transcriptomics analysis of host liver and meta-transcriptome analysis of rumen epimural microbial community in young calves treated with artificial dosing of rumen content from adult donor cow.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Transcriptomics analysis of host liver and meta-transcriptome analysis of rumen epimural microbial community in young calves treated with artificial dosing of rumen content from adult donor cow.

In mammals, microbial colonization of the digestive tract (GIT) occurs right after birth by several bacterial phyla. Numerous human and mouse studies have reported the importance of early gut microbial inhabitants on host health. However, few attempts have been undertaken to directly interrogate the role of early gut/rumen microbial colonization on GIT development or host health in neonatal ruminants through artificial manipulation of the rumen microbiome. Thus, the molecular changes associated with bacterial colonization are largely unknown in cattle. In this study, we dosed young calves with exogenous rumen fluid obtained from an adult donor cow, starting at birth, and repeated every other week until six weeks of age. Eight Holstein bull calves were included in this study and were separated into two groups of four: the first group was treated with rumen content freshly extracted from an adult cow, and the second group was treated with sterilized rumen content. Using whole-transcriptome RNA-sequencing, we investigated the transcriptional changes in the host liver, which is a major metabolic organ and vital to the calf's growth performance. Additionally, the comparison of rumen epimural microbial communities between the treatment groups was performed using the rRNA reads generated by sequencing. Liver transcriptome changes were enriched with genes involved in cell signaling and protein phosphorylation. Specifically, up-regulation of SGPL1 suggests a potential increase in the metabolism of sphingolipids, an essential molecular signal for bacterial survival in digestive tracts. Notably, eight genera, belonging to four phyla, had significant increases in abundance in treated calves. Our study provides insight into host liver transcriptome changes associated with early colonization of the microbial communities in neonatal calves. Such knowledge provides a foundation for future probiotics-based research in microbial organism mediated rumen development and nutrition in ruminants.

Comparative gene expression analysis of the ß-amylase and hordein gene families in the developing barley grain.

Expression of hordeins and ß-amylase during barley grain development is important in determining malting quality parameters that are controlled by protein and malt enzyme levels. The relationship between protein and enzyme levels is confounding because, in general, protein and malt enzyme activity are positively correlated and the malting and brewing industries demand relatively low levels of protein and relatively high levels of enzymes. Separation of these traits is desirable because high protein levels are one of the primary causes of barley not meeting malt quality standards. Studies on barley grain development have not resulted in a consensus on the temporal accumulation of hordein and endosperm-specific ß-amylase (Bmy1) and thus, it is unclear whether hordeins and Bmy1 are under control of the same temporal regulator (s). Therefore, temporal expression patterns of hordeins (B- [Hor2], C- [Hor1], D- [Hor3], and γ-hordein [Hor5]) were compared to Bmy1 throughout grain development (5 to 35 days after anthesis (DAA)). Transcript accumulation between hordeins and Bmy1 occurred simultaneously beginning during the pre-storage phase of grain development whereas the B1-hordein protein appeared two days before Bmy1 most likely due to variations in gene copy number. Interestingly, the largest increase in hordein and Bmy1 transcript levels occurred between 5 and 9 (Hor2, Hor2-B1, Hor2-B3, Hor3, Hor5-γ1, and Hor5-γ3) or 9 and 13 DAA (Hor1 and Bmy1). Additionally, ubiquitous ß-amylase (Bmy2) has a novel expression pattern and was the predominant ß-amylase present between 5 and 15 DAA whereas Bmy1 was the predominant ß-amylase present between 17 and 35 DAA.

Comparison of Oryza sativa and Oryza brachyantha Genomes Reveals Selection-Driven Gene Escape from the Centromeric Regions.

Centromeres are dynamic chromosomal regions, and the genetic and epigenetic environment of the centromere is often regarded as oppressive to protein-coding genes. Here, we used comparative genomic and phylogenomic approaches to study the evolution of centromeres and centromere-linked genes in the genus Oryza We report a 12.4-Mb high-quality BAC-based pericentromeric assembly for Oryza brachyantha, which diverged from cultivated rice (Oryza sativa) ∼15 million years ago. The synteny analyses reveal seven medium (>50 kb) pericentric inversions in O. sativa and 10 in O. brachyantha Of these inversions, three resulted in centromere movement (Chr1, Chr7, and Chr9). Additionally, we identified a potential centromere-repositioning event, in which the ancestral centromere on chromosome 12 in O. brachyantha jumped ∼400 kb away, possibly mediated by a duplicated transposition event (>28 kb). More strikingly, we observed an excess of syntenic gene loss at and near the centromeric regions (P < 2.2 × 10-16). Most (33/47) of the missing genes moved to other genomic regions; therefore such excess could be explained by the selective loss of the copy in or near centromeric regions after gene duplication. The pattern of gene loss immediately adjacent to centromeric regions suggests centromere chromatin dynamics (e.g., spreading or microrepositioning) may drive such gene loss.

Evaluation and selection of internal reference genes from two- and six-row U.S. malting barley varieties throughout micromalting for use in RT-qPCR.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a popular method for measuring transcript abundance. The most commonly used method of interpretation is relative quantification and thus necessitates the use of normalization controls (i.e. reference genes) to standardize transcript abundance. The most popular gene targets for RT-qPCR are housekeeping genes because they are thought to maintain a static transcript level among a variety of samples. However, more recent studies have shown, several housekeeping genes are not reliably stable. This is the first study to examine the potential of several reference genes for use in RT-qPCR normalization during barley malting. The process of malting barley mechanizes the imbibition and subsequent germination of barley seeds under controlled conditions. Malt quality is controlled by many pleiotropic genes that are determined by examining the result of physiological changes the barley seed undergoes during the malting process. We compared the stability of 13 reference genes across both two-and six-row malting barleys (Conrad and Legacy, respectfully) throughout the entirety of the malting process. Initially, primer target specificity, amplification efficiency and average Ct values were determined for each of the selected primer pairs. Three statistical programs (geNorm, NormFinder, and BestKeeper) were used to rank the stability of each reference gene. Rankings were similar between the two- and six-row with the exception of BestKeeper's ranking of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A consensus ranking among programs was determined using RefFinder. Our results show that Actin (ACT) and Heat Shock Protein 70 (HSP70) were the most stable throughout micromalting, while GAPDH and Cyclophilin (CYP) were the least stable. Two reference genes are necessary for stable transcript normalization according to geNorm and the best two reference genes (ACT and HSP70) provided a sufficient level of stability.

Fluorescence in situ hybridization techniques for cytogenetic and genomic analyses.

Fluorescent in situ hybridization (FISH) is a powerful method to visualize DNA sequences in the context of the whole chromosome. Yet despite the value of FISH analysis for cytogenetic studies, there are surprisingly few labs that are able to adapt the technique for their experiments in chromosomal and genome biology. Here we present a comprehensive FISH protocol acquired from over 20 years of collective experience using different plant species. Our description uses rice as a model for performing a complete FISH procedure, but the protocol can be readily adapted for other plant species. We have provided more specialized instruction beyond routine FISH, which includes the preparation of meiotic and mitotic samples suitable for FISH analysis, procedures for direct and indirect labeling of DNA probes, and techniques for increasing signal strength using layers of antibodies.

Conservation and purifying selection of transcribed genes located in a rice centromere.

Recombination is strongly suppressed in centromeric regions. In chromosomal regions with suppressed recombination, deleterious mutations can easily accumulate and cause degeneration of genes and genomes. Surprisingly, the centromere of chromosome8 (Cen8) of rice (Oryza sativa) contains several transcribed genes. However, it remains unclear as to what selective forces drive the evolution and existence of transcribed genes in Cen8. Sequencing of orthologous Cen8 regions from two additional Oryza species, Oryza glaberrima and Oryza brachyantha, which diverged from O. sativa 1 and 10 million years ago, respectively, revealed a set of seven transcribed Cen8 genes conserved across all three species. Chromatin immunoprecipitation analysis with the centromere-specific histone CENH3 confirmed that the sequenced orthologous regions are part of the functional centromere. All seven Cen8 genes have undergone purifying selection, representing a striking phenomenon of active gene survival within a recombination-free zone over a long evolutionary time. The coding sequences of the Cen8 genes showed sequence divergence and mutation rates that were significantly reduced from those of genes located on the chromosome arms. This suggests that Oryza has a mechanism to maintain the fidelity and functionality of Cen8 genes, even when embedded in a sea of repetitive sequences and transposable elements.

A neo-sex chromosome that drives postzygotic sex determination in the hessian fly (Mayetiola destructor).

Two nonoverlapping autosomal inversions defined unusual neo-sex chromosomes in the Hessian fly (Mayetiola destructor). Like other neo-sex chromosomes, these were normally heterozygous, present only in one sex, and suppressed recombination around a sex-determining master switch. Their unusual properties originated from the anomalous Hessian fly sex determination system in which postzygotic chromosome elimination is used to establish the sex-determining karyotypes. This system permitted the evolution of a master switch (Chromosome maintenance, Cm) that acts maternally. All of the offspring of females that carry Cm-associated neo-sex chromosomes attain a female-determining somatic karyotype and develop as females. Thus, the chromosomes act as maternal effect neo-W's, or W-prime (W') chromosomes, where ZW' females mate with ZZ males to engender female-producing (ZW') and male-producing (ZZ) females in equal numbers. Genetic mapping and physical mapping identified the inversions. Their distribution was determined in nine populations. Experimental matings established the association of the inversions with Cm and measured their recombination suppression. The inversions are the functional equivalent of the sciarid X-prime chromosomes. We speculate that W' chromosomes exist in a variety of species that produce unisexual broods.

A lineage-specific centromere retrotransposon in Oryza brachyantha.

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3-gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo-LTRs to full-length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3-associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.

Molecular and chromosomal evidence for allopolyploidy in soybean.

Recent studies have documented that the soybean (Glycine max) genome has undergone two rounds of large-scale genome and/or segmental duplication. To shed light on the timing and nature of these duplication events, we characterized and analyzed two subfamilies of high-copy centromeric satellite repeats, CentGm-1 and CentGm-2, using a combination of computational and molecular cytogenetic approaches. These two subfamilies of satellite repeats mark distinct subsets of soybean centromeres and, in at least one case, a pair of homologs, suggesting their origins from an allopolyploid event. The satellite monomers of each subfamily are arranged in large tandem arrays, and intermingled monomers of the two subfamilies were not detected by fluorescence in situ hybridization on extended DNA fibers nor at the sequence level. This indicates that there has been little recombination and homogenization of satellite DNA between these two sets of centromeres. These satellite repeats are also present in Glycine soja, the proposed wild progenitor of soybean, but could not be detected in any other relatives of soybean examined in this study, suggesting the rapid divergence of the centromeric satellite DNA within the Glycine genus. Together, these observations provide direct evidence, at molecular and chromosomal levels, in support of the hypothesis that the soybean genome has experienced a recent allopolyploidization event.

Structure and evolution of plant centromeres.

Investigations of centromeric DNA and proteins and centromere structures in plants have lagged behind those conducted with yeasts and animals; however, many attractive results have been obtained from plants during this decade. In particular, intensive investigations have been conducted in Arabidopsis and Gramineae species. We will review our understanding of centromeric components, centromere structures, and the evolution of these attributes of centromeres among plants using data mainly from Arabidopsis and Gramineae species.

The position of repetitive DNA sequence in the southern cattle tick genome permits chromosome identification.

Fluorescent in-situ hybridization (FISH) using meiotic chromosome preparations and highly repetitive DNA from the southern cattle tick, Rhipicephalus microplus, was undertaken to investigate genome organization. Several classes of highly repetitive DNA elements were identified by screening a R. microplus bacterial artificial chromosome (BAC) library. A repeat unit of approximately 149 bp, RMR-1 was localized to the subtelomeric regions of R. microplus autosomes 1-6 and 8-10. A second repeat unit, RMR-2 was localized to the subtelomeric regions of all autosomes and the X chromosome. RMR-2 was composed of three distinct repeat populations, RMR-2a, RMR-2b and RMR-2c of 178, 177 and 216 bp in length, respectively. Localization of an rDNA probe identified a single nucleolar organizing region on one autosome. Using a combination of labeled probes, we developed a preliminary karyotype for R. microplus. We present evidence that R. microplus has holocentric chromosomes and explore the implications of these findings for tick chromosome biology and genomic research.

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean.

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.

Evolutionary dynamics of an ancient retrotransposon family provides insights into evolution of genome size in the genus Oryza.

Long terminal repeat (LTR) retrotransposons constitute a significant portion of most eukaryote genomes and can dramatically change genome size and organization. Although LTR retrotransposon content variation is well documented, the dynamics of genomic flux caused by their activity are poorly understood on an evolutionary time scale. This is primarily because of the lack of an experimental system composed of closely related species whose divergence times are within the limits of the ability to detect ancestrally related retrotransposons. The genus Oryza, with 24 species, ten genome types, different ploidy levels and over threefold genome size variation, constitutes an ideal experimental system to explore genus-level transposon dynamics. Here we present data on the discovery and characterization of an LTR retrotransposon family named RWG in the genus Oryza. Comparative analysis of transposon content (approximately 20 to 27,000 copies) and transpositional history of this family across the genus revealed a broad spectrum of independent and lineage-specific changes that have implications for the evolution of genome size and organization. In particular, we provide evidence that the basal GG genome of Oryza (O. granulata) has expanded by nearly 25% by a burst of the RWG lineage Gran3 subsequent to speciation. Finally we describe the recent evolutionary origin of Dasheng, a large retrotransposon derivative of the RWG family, specifically found in the A, B and C genome lineages of Oryza.

Comparative physical mapping between Oryza sativa (AA genome type) and O. punctata (BB genome type).

A comparative physical map of the AA genome (Oryza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. sativa genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. sativa with individual chromosome differences of 1.5-16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged approximately 2 million years ago.