Cytonuclear interplay in auto- and allopolyploids: a multifaceted perspective from the Festuca-Lolium complex.

Restoring cytonuclear stoichiometry is necessary after whole-genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto- and allopolyploids of the Festuca-Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well-established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post-transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.

Reciprocal allopolyploid grasses (Festuca × Lolium) display stable patterns of genome dominance.

Allopolyploidization entailing the merger of two distinct genomes in a single hybrid organism, is an important process in plant evolution and a valuable tool in breeding programs. Newly established hybrids often experience massive genomic perturbations, including karyotype reshuffling and gene expression modifications. These phenomena may be asymmetric with respect to the two progenitors, with one of the parental genomes being "dominant." Such "genome dominance" can manifest in several ways, including biased homoeolog gene expression and expression level dominance. Here we employed a k-mer-based approach to study gene expression in reciprocal Festuca pratensis Huds. × Lolium multiflorum Lam. allopolyploid grasses. Our study revealed significantly more genes where expression mimicked that of the Lolium parent compared with the Festuca parent. This genome dominance was heritable to successive generation and its direction was only slightly modified by environmental conditions and plant age. Our results suggest that Lolium genome dominance was at least partially caused by its more efficient trans-acting gene expression regulatory factors. Unraveling the mechanisms responsible for propagation of parent-specific traits in hybrid crops contributes to our understanding of allopolyploid genome evolution and opens a way to targeted breeding strategies.

Chromosome Pairing in Polyploid Grasses.

Polyploids are species in which three or more sets of chromosomes coexist. Polyploidy frequently occurs in plants and plays a major role in their evolution. Based on their origin, polyploid species can be divided into two groups: autopolyploids and allopolyploids. The autopolyploids arise by multiplication of the chromosome sets from a single species, whereas allopolyploids emerge from the hybridization between distinct species followed or preceded by whole genome duplication, leading to the combination of divergent genomes. Having a polyploid constitution offers some fitness advantages, which could become evolutionarily successful. Nevertheless, polyploid species must develop mechanism(s) that control proper segregation of genetic material during meiosis, and hence, genome stability. Otherwise, the coexistence of more than two copies of the same or similar chromosome sets may lead to multivalent formation during the first meiotic division and subsequent production of aneuploid gametes. In this review, we aim to discuss the pathways leading to the formation of polyploids, the occurrence of polyploidy in the grass family (Poaceae), and mechanisms controlling chromosome associations during meiosis, with special emphasis on wheat.

One Major Challenge of Sequencing Large Plant Genomes Is to Know How Big They Really Are.

Any project seeking to deliver a plant or animal reference genome sequence must address the question as to the completeness of the assembly. Given the complexity introduced particularly by the presence of sequence redundancy, a problem which is especially acute in polyploid genomes, this question is not an easy one to answer. One approach is to use the sequence data, along with the appropriate computational tools, the other is to compare the estimate of genome size with an experimentally measured mass of nuclear DNA. The latter requires a reference standard in order to provide a robust relationship between the two independent measurements of genome size. Here, the proposal is to choose the human male leucocyte genome for this standard: its 1C DNA amount (the amount of DNA contained within unreplicated haploid chromosome set) of 3.50 pg is equivalent to a genome length of 3.423 Gbp, a size which is just 5% longer than predicted by the most current human genome assembly. Adopting this standard, this paper assesses the completeness of the reference genome assemblies of the leading cereal crops species wheat, barley and rye.

Physical Map of the Short Arm of Bread Wheat Chromosome 3D.

Bread wheat ( L.) is one of the most important crops worldwide. Although a reference genome sequence would represent a valuable resource for wheat improvement through genomics-assisted breeding and gene cloning, its generation has long been hampered by its allohexaploidy, high repeat content, and large size. As a part of a project coordinated by the International Wheat Genome Sequencing Consortium (IWGSC), a physical map of the short arm of wheat chromosome 3D (3DS) was prepared to facilitate reference genome assembly and positional gene cloning. It comprises 869 contigs with a cumulative length of 274.5 Mbp and represents 85.5% of the estimated chromosome arm size. Eighty-six Mbp of survey sequences from chromosome arm 3DS were assigned in silico to physical map contigs via next-generation sequencing of bacterial artificial chromosome pools, thus providing a high-density framework for physical map ordering along the chromosome arm. About 60% of the physical map was anchored in this single experiment. Finally, 1393 high-confidence genes were anchored to the physical map. Comparisons of gene space of the chromosome arm 3DS with genomes of closely related species [ (L.) P.Beauv., rice ( L.), and sorghum [ (L.) Moench] and homeologous wheat chromosomes provided information about gene movement on the chromosome arm.

Variation in genome composition of blue-aleurone wheat.

KEY MESSAGE: Different blue-aleurone wheats display major differences in chromosome composition, ranging from disomic chromosome additions, substitutions, single chromosome arm introgressions and chromosome translocation of Thinopyrum ponticum. Anthocyanins are of great importance for human health due to their antioxidant, anti-inflammatory, anti-microbial and anti-cancerogenic potential. In common wheat (Triticum aestivum L.) their content is low. However, elite lines with blue aleurone exhibit significantly increased levels of anthocyanins. These lines carry introgressed chromatin from wild relatives of wheat such as Thinopyrum ponticum and Triticum monococcum. The aim of our study was to characterize genomic constitutions of wheat lines with blue aleurone using genomic and fluorescence in situ hybridization. We used total genomic DNA of Th. ponticum and two repetitive DNA sequences (GAA repeat and the Afa family) as probes to identify individual chromosomes. This enabled precise localization of introgressed Th. ponticum chromatin. Our results revealed large variation in chromosome constitutions of the blue-aleurone wheats. Of 26 analyzed lines, 17 carried an introgression from Th. ponticum; the remaining nine lines presumably carry T. monococcum chromatin undetectable by the methods employed. Of the Th. ponticum introgressions, six different types were present, ranging from a ditelosomic addition (cv. Blue Norco) to a disomic substitution (cv. Blue Baart), substitution of complete (homologous) chromosome arms (line UC66049) and various translocations of distal parts of a chromosome arm(s). Different types of introgressions present support a hypothesis that the introgressions activate the blue aleurone trait present, but inactivated, in common wheat germplasm.

Intraspecific sequence comparisons reveal similar rates of non-collinear gene insertion in the B and D genomes of bread wheat.

BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.

Chromosome-based genomics in the cereals.

The cereals are of enormous importance to mankind. Many of the major cereal species - specifically, wheat, barley, oat, rye, and maize - have large genomes. Early cytogenetics, genome analysis and genetic mapping in the cereals benefited greatly from their large chromosomes, and the allopolyploidy of wheat and oats that has allowed for the development of many precise cytogenetic stocks. In the genomics era, however, large genomes are disadvantageous. Sequencing large and complex genomes is expensive, and the assembly of genome sequence is hampered by a significant content of repetitive DNA and, in allopolyploids, by the presence of homoeologous genomes. Dissection of the genome into its component chromosomes and chromosome arms provides an elegant solution to these problems. In this review we illustrate how this can be achieved by flow cytometric sorting. We describe the development of methods for the preparation of intact chromosome suspensions from the major cereals, and their analysis and sorting using flow cytometry. We explain how difficulties in the discrimination of specific chromosomes and their arms can be overcome by exploiting extant cytogenetic stocks of polyploid wheat and oats, in particular chromosome deletion and alien addition lines. Finally, we discuss some of the applications of flow-sorted chromosomes, and present some examples demonstrating that a chromosome-based approach is advantageous for the analysis of the complex genomes of cereals, and that it can offer significant potential for the delivery of genome sequencing and gene cloning in these crops.

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B.

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.

Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat.

The analysis of the hexaploid wheat genome (Triticum aestivum L., 2 n=6 x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 10(7) of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.

Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome.

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24,960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11,904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.