Plant 3D Chromatin Organization: Important Insights from Chromosome Conformation Capture Analyses of the Last 10 Years.

Over the past few decades, eukaryotic linear genomes and epigenomes have been widely and extensively studied for understanding gene expression regulation. More recently, the three-dimensional (3D) chromatin organization was found to be important for determining genome functionality, finely tuning physiological processes for appropriate cellular responses. With the development of visualization techniques and chromatin conformation capture (3C)-based techniques, increasing evidence indicates that chromosomal architecture characteristics and chromatin domains with different epigenetic modifications in the nucleus are correlated with transcriptional activities. Subsequent studies have further explored the intricate interplay between 3D genome organization and the function of interacting regions. In this review, we summarize spatial distribution patterns of chromatin, including chromatin positioning, configurations and domains, with a particular focus on the effect of a unique form of interaction between varieties of factors that shape the 3D genome conformation in plants. We further discuss the methods, advantages and limitations of various 3C-based techniques, highlighting the applications of these technologies in plants to identify chromatin domains, and address their dynamic changes and functional implications in evolution, and adaptation to development and changing environmental conditions. Moreover, the future implications and emerging research directions of 3D genome organization are discussed.

FISH-Based Karyotype Analyses of Four Dracaena Species.

The genus Dracaena is the main source of dragon's blood, which is a plant resin and has been used as traditional medicine since ancient times in different civilizations. However, the chromosome numbers and karyotypes present in this genus remain poorly understood. In this study, fluorescence in situ hybridization (FISH) using oligonucleotide probes for ribosomal DNAs (5S and 45S rDNA) and telomeric repeats (TTTAGGG)3 was applied to analyze 4 related species: Dracaena terniflora Roxb., Dracaena cambodiana Pierre ex Gagnep., Aizong (Dracaena sp.), and Dracaena cochinchinensis (Lour.) S.C. Chen. In all 4 species, both 5S and 45S rDNA showed hybridization signals in the paracentromeric region of a pair of chromosomes; the sizes of the 45S rDNA signals were larger than those of the 5S rDNA. Importantly, the telomeric repeat signals were located in the telomeric regions of almost all chromosomes. The results indicated that the chromosome number of all 4 Dracaena species is 2n = 40, and the lengths of the mitotic metaphase chromosomes range from 0.99 to 2.98 µm. Our results provide useful cytogenetic information, which will be beneficial to future studies in genome structure of the genus Dracaena.

A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

Optical maps refine the bread wheat Triticum aestivum cv. Chinese Spring genome assembly.

Until recently, achieving a reference-quality genome sequence for bread wheat was long thought beyond the limits of genome sequencing and assembly technology, primarily due to the large genome size and > 80% repetitive sequence content. The release of the chromosome scale 14.5-Gb IWGSC RefSeq v1.0 genome sequence of bread wheat cv. Chinese Spring (CS) was, therefore, a milestone. Here, we used a direct label and stain (DLS) optical map of the CS genome together with a prior nick, label, repair and stain (NLRS) optical map, and sequence contigs assembled with Pacific Biosciences long reads, to refine the v1.0 assembly. Inconsistencies between the sequence and maps were reconciled and gaps were closed. Gap filling and anchoring of 279 unplaced scaffolds increased the total length of pseudomolecules by 168 Mb (excluding Ns). Positions and orientations were corrected for 233 and 354 scaffolds, respectively, representing 10% of the genome sequence. The accuracy of the remaining 90% of the assembly was validated. As a result of the increased contiguity, the numbers of transposable elements (TEs) and intact TEs have increased in IWGSC RefSeq v2.1 compared with v1.0. In total, 98% of the gene models identified in v1.0 were mapped onto this new assembly through development of a dedicated approach implemented in the MAGAAT pipeline. The numbers of high-confidence genes on pseudomolecules have increased from 105 319 to 105 534. The reconciled assembly enhances the utility of the sequence for genetic mapping, comparative genomics, gene annotation and isolation, and more general studies on the biology of wheat.

Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes.

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

The genetic basis of cytoplasmic male sterility and fertility restoration in wheat.

Hybrid wheat varieties give higher yields than conventional lines but are difficult to produce due to a lack of effective control of male fertility in breeding lines. One promising system involves the Rf1 and Rf3 genes that restore fertility of wheat plants carrying Triticum timopheevii-type cytoplasmic male sterility (T-CMS). Here, by genetic mapping and comparative sequence analyses, we identify Rf1 and Rf3 candidates that can restore normal pollen production in transgenic wheat plants carrying T-CMS. We show that Rf1 and Rf3 bind to the mitochondrial orf279 transcript and induce cleavage, preventing expression of the CMS trait. The identification of restorer genes in wheat is an important step towards the development of hybrid wheat varieties based on a CMS-Rf system. The characterisation of their mode of action brings insights into the molecular basis of CMS and fertility restoration in plants.

Ph2 encodes the mismatch repair protein MSH7-3D that inhibits wheat homoeologous recombination.

Meiotic recombination is a critical process for plant breeding, as it creates novel allele combinations that can be exploited for crop improvement. In wheat, a complex allohexaploid that has a diploid-like behaviour, meiotic recombination between homoeologous or alien chromosomes is suppressed through the action of several loci. Here, we report positional cloning of Pairing homoeologous 2 (Ph2) and functional validation of the wheat DNA mismatch repair protein MSH7-3D as a key inhibitor of homoeologous recombination, thus solving a half-century-old question. Similar to ph2 mutant phenotype, we show that mutating MSH7-3D induces a substantial increase in homoeologous recombination (up to 5.5 fold) in wheat-wild relative hybrids, which is also associated with a reduction in homologous recombination. These data reveal a role for MSH7-3D in meiotic stabilisation of allopolyploidy and provides an opportunity to improve wheat's genetic diversity through alien gene introgression, a major bottleneck facing crop improvement.

Molecular breeding for rust resistance in wheat genotypes.

Rusts are a group of major diseases that have an adverse effect on crop production. Those targeting wheat are found in three principal forms: leaf, stripe, and stem rust. Leaf rust causes foliar disease in wheat; in Egypt, this causes a significant annual yield loss. The deployment of resistant genotypes has proved to be a relatively economical and environmentally sustainable method of controlling the disease. Gene pyramiding can be performed using traditional breeding techniques. Additionally, pathotypes can be introduced to examine specific leaf rust genes, or the breeder may conduct more complex breeding methods. Indirect selection via DNA markers linked to resistance genes may facilitate the transfer of targeted genes, either individually or in combination, even in a disease-free environment. The use of selective crosses to counter virulent races of leaf, stripe, and stem rust has resulted in the transfer of several resistance genes into new wheat germplasm from cultivated or wild species. Quantitative trait locus (QTL) technology has been adopted in a wide variety of novel approaches and is becoming increasingly recognized in wheat breeding. Moreover, several researchers have reported the transference of leaf and stripe rust resistance genes into susceptible wheat cultivars.

GWAS reveals consistent QTL for drought and salt tolerance in a MAGIC population of 550 lines derived from intermating of 11 Upland cotton (Gossypium hirsutum) parents.

Cotton is grown in arid and semi-arid regions where abiotic stresses such as drought and salt are prevalent. There is a lack of studies that simultaneously address the genetic and genomic basis of tolerance to drought and salt stress. In this study, a multi-parent advanced generation inter-cross (MAGIC) population of 550 recombinant inbred lines (RILs) together with their 11 Upland cotton parents with a total of 473,516 polymorphic SNP markers was used to identify quantitative trait loci (QTL) for drought tolerance (DT) and salt tolerance (ST) at the seedling stage based on two replicated greenhouse tests. Transgressive segregation occurred in the MAGIC-RILs, indicating that tolerant and sensitive alleles recombined for tolerance to the abiotic stress during the intermating process for the population development. A total of 20 QTL were detected for DT including 13 and 7 QTL based on plant height (PH) and dry shoot weight (DSW), respectively; and 23 QTL were detected for ST including 12 and 11 QTL for PH and DSW, respectively. There were several chromosomes with QTL clusters for abiotic stress tolerance including four QTL on chromosome A13 and three QTL on A01 for DT, and four QTL on D08 and three QTL on A11 for ST. Nine QTL (21% of the 43 QTL) detected were in common between DT and ST, indicating a common genetic basis for DT and ST. The narrow chromosomal regions for most of the QTL detected in this study allowed identification of 53 candidate genes associated with responses to salt and drought stress and abiotic stimulus. The QTL identified for both DT and ST have significantly augmented the repertoire of QTL for abiotic stress tolerance that can be used for marker-assisted selection to develop cultivars with resilience to drought and/or salt and further genomic studies towards the identification of drought and salt tolerance genes in cotton.

Construction of high-density genetic maps defined sex determination region of the Y chromosome in spinach.

Spinach (Spinacia olracea L.) is a dioecious leafy vegetable with a highly repetitive genome of around 990 Mb, which is challenging for de-novo genome assembly. In our study, a segregating F1 (double pseudo-testcross) population from 'Viroflay' × 'Cornell-NO. 9' was used for genetic mapping by resequencing genotyping. In the paternal 'Cornell-NO. 9' map, 212,414 SNPs were mapped, and the total linkage distance was 476.83 cM; the maternal 'Viroflay' map included 29,282 SNPs with 401.28 cM total genetic distance. Both paternal and maternal maps have the expected number of six linkage groups (LGs). A non-recombining region with 5678 SNPs (39 bin markers) co-segregates with sex type which located at 45.2 cM of LG1 in the 'Cornell-NO. 9' map while indicates the sex determination region (SDR). Integration of two maps into a consensus map guided us to anchor additional 1242 contigs to six pseudomolecules from the published reference genome, which improved additional 233 Mb (23.4%) assembly based on spinach estimated genome size. Particularly, the X counterpart of SDR in our assembly is estimated around 18.4 Mb which locates at the largest chromosome, as consensus with sex-biased FISH signals from previous cytogenetics studies. The region is featured by reduced gene density, higher percentage of repetitive sequences, and no recombination. Our linkage maps provide the resource for improving spinach genome de-novo assembly and identification of sex-determining genes in spinach.

Elucidation of sequence polymorphism in fuzzless-seed cotton lines.

Most commercially produced cotton cultivars have two types of fibers on the seed coat, short fuzz and long lint. Lint fiber is used in the textile industry, while fuzz is considered an undesirable trait. Both types of fibers are believed to be controlled by the same regulators; however, their mechanisms of actions are still obscure. Cotton fiber mutants provide an excellent system to study the genes that regulate fiber development. Here we described four uncharacterized and three previously reported cotton mutants with fuzzless seed phenotypes. To evaluate whether or not the genes previously associated with fuzzless seed phenotypes have mutations we sequenced whole genomic DNA of seven mutants and wild type varieties. We identified multiple polymorphic changes among the tested genes. Non-synonymous SNPs in the coding region of the MML3-A gene was common in the six mutant lines tested in this study, showing both dominant and recessive fuzzless phenotypes. We have mapped the locus of the causative mutation for one of the uncharacterized fuzzless lines using an F2 population that originated from a cross between the dominant fuzzless mutant and a wild type. Further, we have clarified the current knowledge about the causative n2 mutations by analyzing the sequence data and previously reported mapping data. The key genes and possible mechanisms of fiber differentiation are discussed in this study.

Wheat Breeding through Genetic and Physical Mapping.

The Special Issue of "Wheat breeding through genetic and physical mapping" aimed to collect recent advances in research on the genetic and physical mapping of quantitative trait loci (QTLs), candidate genes and regulatory sequences involved in the control of wheat's important agronomic traits, such as grain yield and quality, biotic and abiotic stress resistance [...].

Deciphering the Impact of a Bacterial Infection on Meiotic Recombination in Arabidopsis with Fluorescence Tagged Lines.

Plants are under strong evolutionary pressure to maintain surveillance against pathogens. One major disease resistance mechanism is based on NB-LRR (NLR) proteins that specifically recognize pathogen effectors. The cluster organization of the NLR gene family could favor sequence exchange between NLR genes via recombination, favoring their evolutionary dynamics. Increasing data, based on progeny analysis, suggest the existence of a link between the perception of biotic stress and the production of genetic diversity in the offspring. This could be driven by an increased rate of meiotic recombination in infected plants, but this has never been strictly demonstrated. In order to test if pathogen infection can increase DNA recombination in pollen meiotic cells, we infected Arabidopsis Fluorescent Tagged Lines (FTL) with the virulent bacteria Pseudomonas syringae. We measured the meiotic recombination rate in two regions of chromosome 5, containing or not an NLR gene cluster. In all tested intervals, no significant difference in genetic recombination frequency between infected and control plants was observed. Although it has been reported that pathogen exposure can sometimes increase the frequency of recombinant progeny in plants, our findings suggest that meiotic recombination rate in Arabidopsis may be resilient to at least some pathogen attack. Alternative mechanisms are discussed.

Identification of QTL for perenniality and floral scent in cowpea (Vigna unguiculata [L.] Walp.).

Perennial habit and floral scent are major traits that distinguish domesticated cowpeas from their wild relatives. However, the genetic basis of these two important traits remains largely unknown in cowpea. Plant longevity, a perenniality-related trait, and floral scent, an outcrossing trait, were investigated using a RIL population derived from a cross between a domesticated and a wild cowpea. QTL analysis revealed three significant loci, one on chromosome 8 associated with plant longevity and two, on chromosomes 1 and 11, for floral scent. Genes within the QTL regions were identified. Genes encoding an F-box protein (Vigun08g215300) and two kinases (Vigun08g217000, Vigun08g217800), and involved in physiological processes including regulation of flowering time and plant longevity, were identified within the perenniality QTL region. A cluster of O-methyltransferase genes (Vigun11g096800, Vigun11g096900, Vigun11g097000, Vigun11g097600, and Vigun11g097800) was identified within the floral scent QTL region. These O-methyltransferase cowpea genes are orthologs of the Arabidopsis N-acetylserotonin O-methyltransferase (ASMT) gene, which is involved in the biosynthesis of melatonin. Melatonin is an indole derivative, which is an essential molecule for plant interactions with pollinators. These findings lay the foundation for further exploration of the genetic mechanisms of perenniality and floral scent in cowpea. Knowledge from this study can help in the development of new extended-growth cycle lines with increased yield or lines with increased outcrossing for population breeding.

Improved Genetic Map Identified Major QTLs for Drought Tolerance- and Iron Deficiency Tolerance-Related Traits in Groundnut.

A deep understanding of the genetic control of drought tolerance and iron deficiency tolerance is essential to hasten the process of developing improved varieties with higher tolerance through genomics-assisted breeding. In this context, an improved genetic map with 1205 loci was developed spanning 2598.3 cM with an average 2.2 cM distance between loci in the recombinant inbred line (TAG 24 × ICGV 86031) population using high-density 58K single nucleotide polymorphism (SNP) "Axiom_Arachis" array. Quantitative trait locus (QTL) analysis was performed using extensive phenotyping data generated for 20 drought tolerance- and two iron deficiency tolerance-related traits from eight seasons (2004-2015) at two locations in India, one in Niger, and one in Senegal. The genome-wide QTL discovery analysis identified 19 major main-effect QTLs with 10.0-33.9% phenotypic variation explained (PVE) for drought tolerance- and iron deficiency tolerance- related traits. Major main-effect QTLs were detected for haulm weight (20.1% PVE), SCMR (soil plant analytical development (SPAD) chlorophyll meter reading, 22.4% PVE), and visual chlorosis rate (33.9% PVE). Several important candidate genes encoding glycosyl hydrolases; malate dehydrogenases; microtubule-associated proteins; and transcription factors such as MADS-box, basic helix-loop-helix (bHLH), NAM, ATAF, and CUC (NAC), and myeloblastosis (MYB) were identified underlying these QTL regions. The putative function of these genes indicated their possible involvement in plant growth, development of seed and pod, and photosynthesis under drought or iron deficiency conditions in groundnut. These genomic regions and candidate genes, after validation, may be useful to develop molecular markers for deploying genomics-assisted breeding for enhancing groundnut yield under drought stress and iron-deficient soil conditions.

Molecular marker dissection of stem rust resistance in Nebraska bread wheat germplasm.

Stem rust (caused by Puccinia graminis f. sp. tritici) is a major disease of wheat. To understand the genetic basis of stem rust resistance in Nebraska winter wheat, a set of 330 genotypes representing two nurseries (DUP2015 and TRP2015) were evaluated for resistance to a Nebraska stem rust race (QFCSC) in two replications. The TRP2015 nursery was also evaluated for its resistance to an additional 13 stem rust races. The analysis of variance revealed significant variation among genotypes in both populations for stem rust resistance. Nine stem rust genes, Sr6, Sr31, Sr1RSAmigo, Sr24, Sr36, SrTmp, Sr7b, Sr9b, and Sr38, were expected and genotyped using gene-specific markers. The results of genetic analysis confirmed the presence of seven stem rust resistance genes. One genotype (NE15680) contained target alleles for five stem rust resistance genes and had a high level of stem rust resistance against different races. Single marker analysis indicated that Sr24 and Sr38 were highly significantly associated with stem rust resistance in the DUP2015 and TRP2015 nurseries, respectively. Linkage disequilibrium analysis identified the presence of 17 SNPs in high linkage with the Sr38-specific marker. These SNPs potentially tagging the Sr38 gene could be used in marker-assisted selection after validating them in additional genetic backgrounds.

A SWI/SNF subunit regulates chromosomal dissociation of structural maintenance complex 5 during DNA repair in plant cells.

DNA damage decreases genome stability and alters genetic information in all organisms. Conserved protein complexes have been evolved for DNA repair in eukaryotes, such as the structural maintenance complex 5/6 (SMC5/6), a chromosomal ATPase involved in DNA double-strand break (DSB) repair. Several factors have been identified for recruitment of SMC5/6 to DSBs, but this complex is also associated with chromosomes under normal conditions; how SMC5/6 dissociates from its original location and moves to DSB sites is completely unknown. In this study, we determined that SWI3B, a subunit of the SWI/SNF complex, is an SMC5-interacting protein in Arabidopsis thialiana Knockdown of SWI3B or SMC5 results in increased DNA damage accumulation. During DNA damage, SWI3B expression is induced, but the SWI3B protein is not localized at DSBs. Notably, either knockdown or overexpression of SWI3B disrupts the DSB recruitment of SMC5 in response to DNA damage. Overexpression of a cotranscriptional activator ADA2b rescues the DSB localization of SMC5 dramatically in the SWI3B-overexpressing cells but only weakly in the SWI3B knockdown cells. Biochemical data confirmed that ADA2b attenuates the interaction between SWI3B and SMC5 and that SWI3B promotes the dissociation of SMC5 from chromosomes. In addition, overexpression of SMC5 reduces DNA damage accumulation in the SWI3B knockdown plants. Collectively, these results indicate that the presence of an appropriate level of SWI3B enhances dissociation of SMC5 from chromosomes for its further recruitment at DSBs during DNA damage in plant cells.

The genome of broomcorn millet.

Broomcorn millet (Panicum miliaceum L.) is the most water-efficient cereal and one of the earliest domesticated plants. Here we report its high-quality, chromosome-scale genome assembly using a combination of short-read sequencing, single-molecule real-time sequencing, Hi-C, and a high-density genetic map. Phylogenetic analyses reveal two sets of homologous chromosomes that may have merged ~5.6 million years ago, both of which exhibit strong synteny with other grass species. Broomcorn millet contains 55,930 protein-coding genes and 339 microRNA genes. We find Paniceae-specific expansion in several subfamilies of the BTB (broad complex/tramtrack/bric-a-brac) subunit of ubiquitin E3 ligases, suggesting enhanced regulation of protein dynamics may have contributed to the evolution of broomcorn millet. In addition, we identify the coexistence of all three C4 subtypes of carbon fixation candidate genes. The genome sequence is a valuable resource for breeders and will provide the foundation for studying the exceptional stress tolerance as well as C4 biology.

Chromosome conformation capture resolved near complete genome assembly of broomcorn millet.

Broomcorn millet (Panicum miliaceum L.) has strong tolerance to abiotic stresses, and is probably one of the oldest crops, with its earliest cultivation that dated back to ca. ~10,000 years. We report here its genome assembly through a combination of PacBio sequencing, BioNano, and Hi-C (in vivo) mapping. The 18 super scaffolds cover ~95.6% of the estimated genome (~887.8 Mb). There are 63,671 protein-coding genes annotated in this tetraploid genome. About ~86.2% of the syntenic genes in foxtail millet have two homologous copies in broomcorn millet, indicating rare gene loss after tetraploidization in broomcorn millet. Phylogenetic analysis reveals that broomcorn millet and foxtail millet diverged around ~13.1 Million years ago (Mya), while the lineage specific tetraploidization of broomcorn millet may be happened within ~5.91 million years. The genome is not only beneficial for the genome assisted breeding of broomcorn millet, but also an important resource for other Panicum species.

A cytogenetic analysis of male meiosis in Asparagus officinalis.

Asparagus (Asparagus officinalis) has several traits that make it a useful model for cytogenetic studies, however, few studies of the meiosis process have been made in asparagus. Here, we present in detail an atlas of male meiosis in asparagus, from preleptotene to telophase II. The meiosis process in asparagus is largely similar to those of the well-characterized model plants Arabidopsis thaliana, Zea mays, and Oryza sativa. However, most asparagus prophase I meiotic chromosomes show a strongly aggregated morphology, and this phenotype persists through the pachytene stage, highlighting a property in the control of chromosome migration and distribution in asparagus. Further, we observed no obvious banding of autofluorescent dots between divided nuclei of asparagus meiocytes, as one would expect in Arabidopsis. This description of wild-type asparagus meiosis will serve as a reference for the analyses of meiotic mutants, as well as for comparative studies among difference species. Abbreviations: DAPI: 4',6-diamidino-2-phenylindole; FISH: fluorescence in situ hybridization; PBS: phosphate-buffered saline; PMC: pollen mother cell; SEM: Scanning Electron Microscope.