Genomic variation of 363 diverse tea accessions unveils the genetic diversity, domestication, and structural variations associated with tea adaptation.

Domestication has shaped the population structure and agronomic traits of tea plants, yet the complexity of tea population structure and genetic variation that determines these traits remains unclear. We here investigated the resequencing data of 363 diverse tea accessions collected extensively from almost all tea distributions and found that the population structure of tea plants was divided into eight subgroups, which were basically consistent with their geographical distributions. The genetic diversity of tea plants in China decreased from southwest to east as latitude increased. Results also indicated that Camellia sinensis var. assamica (CSA) illustrated divergent selection signatures with Camellia sinensis var. sinensis (CSS). The domesticated genes of CSA were mainly involved in leaf development, flavonoid and alkaloid biosynthesis, while the domesticated genes in CSS mainly participated in amino acid metabolism, aroma compounds biosynthesis, and cold stress. Comparative population genomics further identified ~730 Mb novel sequences, generating 6,058 full-length protein-encoding genes, significantly expanding the gene pool of tea plants. We also discovered 217,376 large-scale structural variations and 56,583 presence and absence variations (PAVs) across diverse tea accessions, some of which were associated with tea quality and stress resistance. Functional experiments demonstrated that two PAV genes (CSS0049975 and CSS0006599) were likely to drive trait diversification in cold tolerance between CSA and CSS tea plants. The overall findings not only revealed the genetic diversity and domestication of tea plants, but also underscored the vital role of structural variations in the diversification of tea plant traits.

Pangenomics Comes of Age: From Bacteria to Plant and Animal Applications.

The pangenome refers to a collection of genomic sequence found in the entire species or population rather than in a single individual; the sequence can be core, present in all individuals, or accessory (variable or dispensable), found in a subset of individuals only. While pangenomic studies were first undertaken in bacterial species, developments in genome sequencing and assembly approaches have allowed construction of pangenomes for eukaryotic organisms, fungi, plants, and animals, including two large-scale human pangenome projects. Analysis of the these pangenomes revealed key differences, most likely stemming from divergent evolutionary histories, but also surprising similarities.

Comparative pangenome analyses provide insights into the evolution of Brassica rapa resistance gene analogues (RGAs).

Brassica rapa is grown worldwide as economically important vegetable and oilseed crop. However, its production is challenged by yield-limiting pathogens. The sustainable control of these pathogens mainly relies on the deployment of genetic resistance primarily driven by resistance gene analogues (RGAs). While several studies have identified RGAs in B. rapa, these were mainly based on a single genome reference and do not represent the full range of RGA diversity in B. rapa. In this study, we utilized the B. rapa pangenome, constructed from 71 lines encompassing 12 morphotypes, to describe a comprehensive repertoire of RGAs in B. rapa. We show that 309 RGAs were affected by presence-absence variation (PAV) and 223 RGAs were missing from the reference genome. The transmembrane leucine-rich repeat (TM-LRR) RGA class had more core gene types than variable genes, while the opposite was observed for nucleotide-binding site leucine-rich repeats (NLRs). Comparative analysis with the B. napus pangenome revealed significant RGA conservation (93%) between the two species. We identified 138 candidate RGAs located within known B. rapa disease resistance QTL, of which the majority were under negative selection. Using blackleg gene homologues, we demonstrated how these genes in B. napus were derived from B. rapa. This further clarifies the genetic relationship of these loci, which may be useful in narrowing-down candidate blackleg resistance genes. This study provides a novel genomic resource towards the identification of candidate genes for breeding disease resistance in B. rapa and its relatives.

The role of gene presence-absence variations on genetic incompatibility in Asian rice.

Genetic incompatibilities are widespread between species. However, it remains unclear whether they all originated after population divergence as suggested by the Bateson-Dobzhansky-Muller model, and if not, what is their prevalence and distribution within populations. The gene presence-absence variations (PAVs) provide an opportunity for investigating gene-gene incompatibility. Here, we searched for the repulsion of coexistence between gene PAVs to identify the negative interaction of gene functions separately in two Oryza sativa subspecies. Many PAVs are involved in subspecies-specific negative epistasis and segregate at low-to-intermediate frequencies in focal subspecies but at low or high frequencies in the other subspecies. Incompatible PAVs are enriched in two functional groups, defense response and protein phosphorylation, which are associated with plant immunity and consistent with autoimmunity being a known mechanism of hybrid incompatibility in plants. Genes in the two enriched functional groups are older and seldom directly interact with each other. Instead, they interact with other younger gene PAVs with diverse functions. Our results illustrate the landscape of genetic incompatibility at gene PAVs in rice, where many incompatible pairs have already segregated as polymorphisms within subspecies, and many are novel negative interactions between older defense-related genes and younger genes with diverse functions.

Single-parent expression drives dynamic gene expression complementation in maize hybrids.

Single-parent expression (SPE) is defined as gene expression in only one of the two parents. SPE can arise from differential expression between parental alleles, termed non-presence/absence (non-PAV) SPE, or from the physical absence of a gene in one parent, termed PAV SPE. We used transcriptome data of diverse Zea mays (maize) inbreds and hybrids, including 401 samples from five different tissues, to test for differences between these types of SPE genes. Although commonly observed, SPE is highly genotype and tissue specific. A positive correlation was observed between the genetic distance of the two inbred parents and the number of SPE genes identified. Regulatory analysis showed that PAV SPE and non-PAV SPE genes are mainly regulated by cis effects, with a small fraction under trans regulation. Polymorphic transposable element insertions in promoter sequences contributed to the high level of cis regulation for PAV SPE and non-PAV SPE genes. PAV SPE genes were more frequently expressed in hybrids than non-PAV SPE genes. The expression of parentally silent alleles in hybrids of non-PAV SPE genes was relatively rare but occurred in most hybrids. Non-PAV SPE genes with expression of the silent allele in hybrids are more likely to exhibit above high parent expression level than hybrids that do not express the silent allele, leading to non-additive expression. This study provides a comprehensive understanding of the nature of non-PAV SPE and PAV SPE genes and their roles in gene expression complementation in maize hybrids.

A New Catalog of Structural Variants in 1,301 A. thaliana Lines from Africa, Eurasia, and North America Reveals a Signature of Balancing Selection at Defense Response Genes.

Genomic variation in the model plant Arabidopsis thaliana has been extensively used to understand evolutionary processes in natural populations, mainly focusing on single-nucleotide polymorphisms. Conversely, structural variation has been largely ignored in spite of its potential to dramatically affect phenotype. Here, we identify 155,440 indels and structural variants ranging in size from 1 bp to 10 kb, including presence/absence variants (PAVs), inversions, and tandem duplications in 1,301 A. thaliana natural accessions from Morocco, Madeira, Europe, Asia, and North America. We show evidence for strong purifying selection on PAVs in genes, in particular for housekeeping genes and homeobox genes, and we find that PAVs are concentrated in defense-related genes (R-genes, secondary metabolites) and F-box genes. This implies the presence of a "core" genome underlying basic cellular processes and a "flexible" genome that includes genes that may be important in spatially or temporally varying selection. Further, we find an excess of intermediate frequency PAVs in defense response genes in nearly all populations studied, consistent with a history of balancing selection on this class of genes. Finally, we find that PAVs in genes involved in the cold requirement for flowering (vernalization) and drought response are strongly associated with temperature at the sites of origin.

Presence and absence of type VI secretion systems in bacteria.

The type VI secretion system (T6SS) is a molecular puncturing device that enables Gram-negative bacteria to kill competitors, manipulate host cells and take up nutrients. Who would want to miss such superpowers? Indeed, many studies show how widespread the secretion apparatus is among microbes. However, it is becoming evident that, on multiple taxonomic levels, from phyla to species and strains, some bacteria lack a T6SS. Here, we review who does and does not have a type VI secretion apparatus and speculate on the dynamic process of gaining and losing the secretion system to better understand its spread and distribution across the microbial world.

Isolation and Sequencing of Chromosome Arm 7RS of Rye, Secale cereale.

Rye (Secale cereale) is a climate-resilient cereal grown extensively as grain or forage crop in Northern and Eastern Europe. In addition to being an important crop, it has been used to improve wheat through introgression of genomic regions for improved yield and disease resistance. Understanding the genomic diversity of rye will assist both the improvement of this crop and facilitate the introgression of more valuable traits into wheat. Here, we isolated and sequenced the short arm of rye chromosome 7 (7RS) from Triticale 380SD using flow cytometry and compared it to the public Lo7 rye whole genome reference assembly. We identify 2747 Lo7 genes present on the isolated chromosome arm and two clusters containing seven and sixty-five genes that are present on Triticale 380SD 7RS, but absent from Lo7 7RS. We identified 29 genes that are not assigned to chromosomal locations in the Lo7 assembly but are present on Triticale 380SD 7RS, suggesting a chromosome arm location for these genes. Our study supports the Lo7 reference assembly and provides a repertoire of genes on Triticale 7RS.

Pangenomes as a Resource to Accelerate Breeding of Under-Utilised Crop Species.

Pangenomes are a rich resource to examine the genomic variation observed within a species or genera, supporting population genetics studies, with applications for the improvement of crop traits. Major crop species such as maize (Zea mays), rice (Oryza sativa), Brassica (Brassica spp.), and soybean (Glycine max) have had pangenomes constructed and released, and this has led to the discovery of valuable genes associated with disease resistance and yield components. However, pangenome data are not available for many less prominent crop species that are currently under-utilised. Despite many under-utilised species being important food sources in regional populations, the scarcity of genomic data for these species hinders their improvement. Here, we assess several under-utilised crops and review the pangenome approaches that could be used to build resources for their improvement. Many of these under-utilised crops are cultivated in arid or semi-arid environments, suggesting that novel genes related to drought tolerance may be identified and used for introgression into related major crop species. In addition, we discuss how previously collected data could be used to enrich pangenome functional analysis in genome-wide association studies (GWAS) based on studies in major crops. Considering the technological advances in genome sequencing, pangenome references for under-utilised species are becoming more obtainable, offering the opportunity to identify novel genes related to agro-morphological traits in these species.

Myb10-D confers PHS-3D resistance to pre-harvest sprouting by regulating NCED in ABA biosynthesis pathway of wheat.

Pre-harvest sprouting (PHS), the germination of grain before harvest, is a serious problem resulting in wheat yield and quality losses. Here, we mapped the PHS resistance gene PHS-3D from synthetic hexaploid wheat to a 2.4 Mb presence-absence variation (PAV) region and found that its resistance effect was attributed to the pleiotropic Myb10-D by integrated omics and functional analyses. Three haplotypes were detected in this PAV region among 262 worldwide wheat lines and 16 Aegilops tauschii, and the germination percentages of wheat lines containing Myb10-D was approximately 40% lower than that of the other lines. Transcriptome and metabolome profiling indicated that Myb10-D affected the transcription of genes in both the flavonoid and abscisic acid (ABA) biosynthesis pathways, which resulted in increases in flavonoids and ABA in transgenic wheat lines. Myb10-D activates 9-cis-epoxycarotenoid dioxygenase (NCED) by biding the secondary wall MYB-responsive element (SMRE) to promote ABA biosynthesis in early wheat seed development stages. We revealed that the newly discovered function of Myb10-D confers PHS resistance by enhancing ABA biosynthesis to delay germination in wheat. The PAV harboring Myb10-D associated with grain color and PHS will be useful for understanding and selecting white grained PHS resistant wheat cultivars.

Characterization of disease resistance genes in the Brassica napus pangenome reveals significant structural variation.

Methods based on single nucleotide polymorphism (SNP), copy number variation (CNV) and presence/absence variation (PAV) discovery provide a valuable resource to study gene structure and evolution. However, as a result of these structural variations, a single reference genome is unable to cover the entire gene content of a species. Therefore, pangenomics analysis is needed to ensure that the genomic diversity within a species is fully represented. Brassica napus is one of the most important oilseed crops in the world and exhibits variability in its resistance genes across different cultivars. Here, we characterized resistance gene distribution across 50 B. napus lines. We identified a total of 1749 resistance gene analogs (RGAs), of which 996 are core and 753 are variable, 368 of which are not present in the reference genome (cv. Darmor-bzh). In addition, a total of 15 318 SNPs were predicted within 1030 of the RGAs. The results showed that core R-genes harbour more SNPs than variable genes. More nucleotide binding site-leucine-rich repeat (NBS-LRR) genes were located in clusters than as singletons, with variable genes more likely to be found in clusters. We identified 106 RGA candidates linked to blackleg resistance quantitative trait locus (QTL). This study provides a better understanding of resistance genes to target for genomics-based improvement and improved disease resistance.

The limited role of differential fractionation in genome content variation and function in maize (Zea mays L.) inbred lines.

Maize is a diverse paleotetraploid species with considerable presence/absence variation and copy number variation. One mechanism through which presence/absence variation can arise is differential fractionation. Fractionation refers to the loss of duplicate gene pairs from one of the maize subgenomes during diploidization. Differential fractionation refers to non-shared gene loss events between individuals following a whole-genome duplication event. We investigated the prevalence of presence/absence variation resulting from differential fractionation in the syntenic portion of the genome using two whole-genome de novo assemblies of the inbred lines B73 and PH207. Between these two genomes, syntenic genes were highly conserved with less than 1% of syntenic genes being subject to differential fractionation. The few variably fractionated syntenic genes that were identified are unlikely to contribute to functional phenotypic variation, as there is a significant depletion of these genes in annotated gene sets. In further comparisons of 60 diverse inbred lines, non-syntenic genes were six times more likely to be variable than syntenic genes, suggesting that comparisons among additional genome assemblies are not likely to result in the discovery of large-scale presence/absence variation among syntenic genes.

Transcriptomic and presence/absence variation in the barley genome assessed from multi-tissue mRNA sequencing and their power to predict phenotypic traits.

BACKGROUND: Barley is the world's fourth most cultivated cereal and is an important crop model for genetic studies. One layer of genomic information that remains poorly explored in barley is presence/absence variation (PAV), which has been suggested to contribute to phenotypic variation of agronomic importance in various crops. RESULTS: An mRNA sequencing approach was used to study genomic PAV and transcriptomic variation in 23 spring barley inbreds. 1502 new genes identified here were physically absent from the Morex reference sequence, and 11,523 previously unannotated genes were not expressed in Morex. The procedure applied to detect expression PAV revealed that more than 50% of all genes of our data set are not expressed in all inbreds. Interestingly, expression PAV were not in strong linkage disequilibrium with neighboring sequence variants (SV), and therefore provided an additional layer of genetic information. Optimal combinations of expression PAV, SV, and gene abundance data could enhance the prediction accuracy of predicting three different agronomic traits. CONCLUSIONS: Our results highlight the advantage of mRNA sequencing for genomic prediction over other technologies, as it allows extracting multiple layers of genomic data from a single sequencing experiment. Finally, we propose low coverage mRNA sequencing based characterization of breeding material harvested as seedlings in petri dishes as a powerful and cost efficient approach to replace current single nucleotide polymorphism (SNP) based characterizations.

Sequence analysis of European maize inbred line F2 provides new insights into molecular and chromosomal characteristics of presence/absence variants.

BACKGROUND: Maize is well known for its exceptional structural diversity, including copy number variants (CNVs) and presence/absence variants (PAVs), and there is growing evidence for the role of structural variation in maize adaptation. While PAVs have been described in this important crop species, they have been only scarcely characterized at the sequence level and the extent of presence/absence variation and relative chromosomal landscape of inbred-specific regions remain to be elucidated. RESULTS: De novo genome sequencing of the French F2 maize inbred line revealed 10,044 novel genomic regions larger than 1 kb, making up 88 Mb of DNA, that are present in F2 but not in B73 (PAV). This set of maize PAV sequences allowed us to annotate PAV content and to analyze sequence breakpoints. Using PAV genotyping on a collection of 25 temperate lines, we also analyzed Linkage Disequilibrium in PAVs and flanking regions, and PAV frequencies within maize genetic groups. CONCLUSIONS: We highlight the possible role of MMEJ-type double strand break repair in maize PAV formation and discover 395 new genes with transcriptional support. Pattern of linkage disequilibrium within PAVs strikingly differs from this of flanking regions and is in accordance with the intuition that PAVs may recombine less than other genomic regions. We show that most PAVs are ancient, while some are found only in European Flint material, thus pinpointing structural features that may be at the origin of adaptive traits involved in the success of this material. Characterization of such PAVs will provide useful material for further association genetic studies in European and temperate maize.

Finding invisible quantitative trait loci with missing data.

Evolutionary processes during plant polyploidization and speciation have led to extensive presence-absence variation (PAV) in crop genomes, and there is increasing evidence that PAV associates with important traits. Today, high-resolution genetic analysis in major crops frequently implements simple, cost-effective, high-throughput genotyping from single nucleotide polymorphism (SNP) hybridization arrays; however, these are normally not designed to distinguish PAV from failed SNP calls caused by hybridization artefacts. Here, we describe a strategy to recover valuable information from single nucleotide absence polymorphisms (SNaPs) by population-based quality filtering of SNP hybridization data to distinguish patterns associated with genuine deletions from those caused by technical failures. We reveal that including SNaPs in genetic analyses elucidate segregation of small to large-scale structural variants in nested association mapping populations of oilseed rape (Brassica napus), a recent polyploid crop with widespread structural variation. Including SNaP markers in genomewide association studies identified numerous quantitative trait loci, invisible using SNP markers alone, for resistance to two major fungal diseases of oilseed rape, Sclerotinia stem rot and blackleg disease. Our results indicate that PAV has a strong influence on quantitative disease resistance in B. napus and that SNaP analysis using cost-effective SNP array data can provide extensive added value from 'missing data'. This strategy might also be applicable for improving the precision of genetic mapping in many important crop species.

Long-term balancing selection at the Phosphorus Starvation Tolerance 1 (PSTOL1) locus in wild, domesticated and weedy rice (Oryza).

BACKGROUND: The ability to grow in phosphorus-depleted soils is an important trait for rice cultivation in many world regions, especially in the tropics. The Phosphorus Starvation Tolerance 1 (PSTOL1) gene has been identified as underlying the ability of some cultivated rice varieties to grow under low-phosphorus conditions; however, the gene is absent from other varieties. We assessed PSTOL1 presence/absence in a geographically diverse sample of wild, domesticated and weedy rice and sequenced the gene in samples where it is present. RESULTS: We find that the presence/absence polymorphism spans cultivated, weedy and wild Asian rice groups. For the subset of samples that carry PSTOL1, haplotype sequences suggest long-term selective maintenance of functional alleles, but with repeated evolution of loss-of-function alleles through premature stops and frameshift mutations. The loss-of-function alleles have evolved convergently in multiple rice species and cultivated rice varieties. Greenhouse assessments of plant growth under low- and high-phosphorus conditions did not reveal significant associations with PSTOL1 genotype variation; however, the striking signature of balancing selection at this locus suggests that further phenotypic characterizations of PSTOL1 allelic variants is warranted and may be useful for crop improvement. CONCLUSIONS: These findings suggest balancing selection for both functional and non-functional PSTOL1 alleles that predates and transcends Asian rice domestication, a pattern that may reflect fitness tradeoffs associated with geographical variation in soil phosphorus content.

Towards plant pangenomics.

As an increasing number of genome sequences become available for a wide range of species, there is a growing understanding that the genome of a single individual is insufficient to represent the gene diversity within a whole species. Many studies examine the sequence diversity within genes, and this allelic variation is an important source of phenotypic variation which can be selected for by man or nature. However, the significant gene presence/absence variation that has been observed within species and the impact of this variation on traits is only now being studied in detail. The sum of the genes for a species is termed the pangenome, and the determination and characterization of the pangenome is a requirement to understand variation within a species. In this review, we explore the current progress in pangenomics as well as methods and approaches for the characterization of pangenomes for a wide range of plant species.

An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly.

BACKGROUND AND AIMS: High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing. METHODS: The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40 kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach. KEY RESULTS: The assembly comprises 424 750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212 bp and a contig N50 of 3790 bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200 Mb of the reference assembly. CONCLUSIONS: The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.

Presence-absence variation in A. thaliana is primarily associated with genomic signatures consistent with relaxed selective constraints.

The sequencing of multiple genomes of the same plant species has revealed polymorphic gene and exon loss. Genes associated with disease resistance are overrepresented among those showing structural variations, suggesting an adaptive role for gene and exon presence-absence variation (PAV). To shed light on the possible functional relevance of polymorphic coding region loss and the mechanisms driving this process, we characterized genes that have lost entire exons or their whole coding regions in 17 fully sequenced Arabidopsis thaliana accessions. We found that although a significant enrichment in genes associated with certain functional categories is observed, PAV events are largely restricted to genes with signatures of reduced essentiality: PAV genes tend to be newer additions to the genome, tissue specific, and lowly expressed. In addition, PAV genes are located in regions of lower gene density and higher transposable element density. Partial coding region PAV events were associated with only a marginal reduction in gene expression level in the affected accession and occurred in genes with higher levels of alternative splicing in the Col-0 accession. Together, these results suggest that although adaptive scenarios cannot be ruled out, PAV events can be explained without invoking them.

Next-generation re-sequencing as a tool for rapid bioinformatic screening of presence and absence of genes and accessory chromosomes across isolates of Zymoseptoria tritici.

The wheat pathogen Zymoseptoria tritici possesses a large number of accessory chromosomes that may be present or absent in its genome. The genome of the reference isolate IPO323 has been assembled to a very high standard and contains 21 full length chromosome sequences, 8 of which represent accessory chromosomes. The IPO323 reference, when combined with low-cost next-generation sequencing and bioinformatics, can be used as a powerful tool to assess the presence or absence of accessory chromosomes. We present an outline of a range of bioinformatics techniques that can be applied to the analysis of presence-absence variation among accessory chromosomes across 13 novel isolates of Z. tritici.