A k-mer-based bulked segregant analysis approach to map seed traits in unphased heterozygous potato genomes.

High-throughput sequencing-based methods for bulked segregant analysis (BSA) allow for the rapid identification of genetic markers associated with traits of interest. BSA studies have successfully identified qualitative (binary) and quantitative trait loci (QTLs) using QTL mapping. However, most require population structures that fit the models available and a reference genome. Instead, high-throughput short-read sequencing can be combined with BSA of k-mers (BSA-k-mer) to map traits that appear refractory to standard approaches. This method can be applied to any organism and is particularly useful for species with genomes diverged from the closest sequenced genome. It is also instrumental when dealing with highly heterozygous and potentially polyploid genomes without phased haplotype assemblies and for which a single haplotype can control a trait. Finally, it is flexible in terms of population structure. Here, we apply the BSA-k-mer method for the rapid identification of candidate regions related to seed spot and seed size in diploid potato. Using a mixture of F1 and F2 individuals from a cross between 2 highly heterozygous parents, candidate sequences were identified for each trait using the BSA-k-mer approach. Using parental reads, we were able to determine the parental origin of the loci. Finally, we mapped the identified k-mers to a closely related potato genome to validate the method and determine the genomic loci underlying these sequences. The location identified for the seed spot matches with previously identified loci associated with pigmentation in potato. The loci associated with seed size are novel. Both loci are relevant in future breeding toward true seeds in potato.

Unlikely heroes on the long and winding road to potato inbreeding.

Conversion of potato from a tetraploid, heterozygous, vegetatively propagated crop to a diploid F1 hybrid, propagated via botanical seed, would constitute a considerable advance for global agriculture, but faces multiple challenges. One such challenge is the difficulty in inbreeding potato, which involves purging deleterious alleles from its genome. This commentary discusses possible reasons for this difficulty and highlights a recent sequence-based effort to classify SNP variation, in potato germplasm, according to its deleterious potential. Tools and strategies connected to this database may facilitate development of F1 hybrids.

OsBTBZ1 Confers Salt Stress Tolerance in Arabidopsis thaliana.

Rice (Oryza sativa L.), one of the most important commodities and a primary food source worldwide, can be affected by adverse environmental factors. The chromosome segment substitution line 16 (CSSL16) of rice is considered salt-tolerant. A comparison of the transcriptomic data of the CSSL16 line under normal and salt stress conditions revealed 511 differentially expressed sequence (DEseq) genes at the seedling stage, 520 DEseq genes in the secondary leaves, and 584 DEseq genes in the flag leaves at the booting stage. Four BTB genes, OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7, were differentially expressed under salt stress. Interestingly, only OsBTBZ1 was differentially expressed at the seedling stage, whereas the other genes were differentially expressed at the booting stage. Based on the STRING database, OsBTBZ1 was more closely associated with other abiotic stress-related proteins than other BTB genes. The highest expression of OsBTBZ1 was observed in the sheaths of young leaves. The OsBTBZ1-GFP fusion protein was localized to the nucleus, supporting the hypothesis of a transcriptionally regulatory role for this protein. The bt3 Arabidopsis mutant line exhibited susceptibility to NaCl and abscisic acid (ABA) but not to mannitol. NaCl and ABA decreased the germination rate and growth of the mutant lines. Moreover, the ectopic expression of OsBTBZ1 rescued the phenotypes of the bt3 mutant line and enhanced the growth of wild-type Arabidopsis under stress conditions. These results suggest that OsBTBZ1 is a salt-tolerant gene functioning in ABA-dependent pathways.

Genome-Wide Association Study of Starch Properties in Local Thai Rice.

Rice (Oryza sativa L.) is the main source of energy for humans and a staple food of high cultural significance for much of the world's population. Rice with highly resistant starch (RS) is beneficial for health and can reduce the risk of disease, especially type II diabetes. The identification of loci affecting starch properties will facilitate breeding of high-quality and health-supportive rice. A genome-wide association study (GWAS) of 230 rice cultivars was used to identify candidate loci affecting starch properties. The apparent amylose content (AAC) among rice cultivars ranged from 7.04 to 33.06%, and the AAC was positively correlated with RS (R2 = 0.94) and negatively correlated with rapidly available glucose (RAG) (R2 = -0.73). Three loci responsible for starch properties were detected on chromosomes 1, 6, and 11. On chromosome 6, the most significant SNP corresponded to LOC_Os06g04200 which encodes granule-bound starch synthase I (GBSSI) or starch synthase. Two novel loci associated with starch traits were LOC_Os01g65810 and LOC_Os11g01580, which encode an unknown protein and a sodium/calcium exchanger, respectively. The markers associated with GBSSI and LOC_Os11g01580 were tested in two independent sets of rice populations to confirm their effect on starch properties. The identification of genes associated with starch traits will further the understanding of the molecular mechanisms affecting starch in rice and may be useful in the selection of rice varieties with improved starch.

Gene expression profile during seed development of Bixa orellana accessions varying in bixin pigment.

Diverse morphological, cellular and physiological changes occur during seed maturation in Bixa orellana when the seed tissues form specialized cell glands that produce reddish latex with high bixin amounts. Transcriptomic profiling during seed development in three B. orellana accessions (P12, N4 and N5) with contrasting morphologic characteristics showed enrichment in pathways of triterpenes, sesquiterpenes, and cuticular wax biosynthesis. WGCNA allows groups of all identified genes in six modules the module turquoise, the largest and highly correlated with the bixin content. The high number of genes in this module suggests a diversification of regulatory mechanisms for bixin accumulation with the genes belonging to isoprene, triterpenes and carotene pathways, being more highly correlated with the bixin content. Analysis of key genes of the mevalonate (MVA) and the 2C-methyl-D-erythritol-4-phosphate (MEP) pathways revealed specific activities of orthologs of BoHMGR, BoFFP, BoDXS, and BoHDR. This suggests that isoprenoid production is necessary for compounds included in the reddish latex of developing seeds. The carotenoid-related genes BoPSY2, BoPDS1 and BoZDS displayed a high correlation with bixin production, consistent with the requirement for carotene precursors for apocarotenoid biosynthesis. The BoCCD gene member (BoCCD4-4) and some BoALDH (ALDH2B7.2 and ALDH3I1) and BoMET (BoSABATH1 and BoSABATH8) gene members were highly correlated to bixin in the final seed development stage. This suggested a contributing role for several genes in apocarotenoid production. The results revealed high genetic complexity in the biosynthesis of reddish latex and bixin in specialized seed cell glands in different accessions of B. orellana suggesting gene expression coordination between both metabolite biosynthesis processes.

The Arabidopsis Hop1 homolog ASY1 mediates cross-over assurance and interference.

The chromosome axis plays a crucial role in meiotic recombination. Here, we study the function of ASY1, the Arabidopsis homolog of the yeast chromosome axis-associated component Hop1. Specifically, we characterized cross-over (CO) distribution in female and male meiosis by deep sequencing of the progeny of an allelic series of asy1 mutants. Combining data from nearly 1,000 individual plants, we find that reduced ASY1 functionality leads to genomic instability and sometimes drastic genomic rearrangements. We further observed that COs are less frequent and appear in more distal chromosomal regions in plants with no or reduced ASY1 functionality, consistent with previous analyses. However, our sequencing approach revealed that the reduction in CO number is not as dramatic as suggested by cytological analyses. Analysis of double mutants of asy1 with mutants with three other CO factors, MUS81, MSH4, and MSH5, as well as the determination of foci number of the CO regulator MLH1 demonstrates that the majority of the COs in asy1, similar to the situation in the wildtype (WT), largely belong to the class I, which are subject to interference. However, these COs are redistributed in asy1 mutants and typically appear much closer than in the WT. Hence, ASY1 plays a key role in CO interference that spaces COs along a chromosome. Conversely, since a large proportion of chromosomes do not receive any CO, we conclude that CO assurance, the process that ensures the obligatory assignment of one CO per chromosome, is also affected in asy1 mutants.

Establishment and inheritance of minichromosomes from Arabidopsis haploid induction.

Minichromosomes are small, sometimes circular, rearranged chromosomes consisting of one centromere and short chromosomal arms formed by treatments that break DNA, including plant transformation. Minichromosomes have the potential to serve as vectors to quickly move valuable genes across a wide range of germplasm, including into adapted crop varieties. To realize this potential, minichromosomes must be reliably generated, easily manipulated, and stably inherited. Here we show a reliable method for minichromosome formation in haploids resulting from CENH3-mediated genome elimination, a process that generates genome instability and karyotypic novelty specifically on one parental genome. First, we identified 2 out of 260 haploids, each containing a single-copy minichromosome originating from centromeric regions of chromosomes 1 and 3, respectively. The chromosome 1 minichromosome we characterized did not pair at meiosis but displayed consistent transmission over nine selfing generations. Next, we demonstrated that CENH3-based haploid induction can produce minichromosomes in a targeted manner. Haploid inducers carrying a selectable pericentromeric marker were used to isolate additional chromosome-specific minichromosomes, which occurred in 3 out of 163 haploids. Our findings document the formation of heritable, rearranged chromosomes, and we provide a method for convenient minichromosome production.

Chromoanagenesis in plants: triggers, mechanisms, and potential impact.

Chromoanagenesis is a single catastrophic event that involves, in most cases, localized chromosomal shattering and reorganization, resulting in a dramatically restructured chromosome. First discovered in cancer cells, it has since been observed in various other systems, including plants. In this review, we discuss the origin, characteristics, and potential mechanisms underlying chromoanagenesis in plants. We report that multiple processes, including mutagenesis and genetic engineering, can trigger chromoanagenesis via a variety of mechanisms such as micronucleation, breakage-fusion-bridge (BFB) cycles, or chain-like translocations. The resulting rearranged chromosomes can be preserved during subsequent plant growth, and sometimes inherited to the next generation. Because of their high tolerance to genome restructuring, plants offer a unique system for investigating the evolutionary consequences and potential practical applications of chromoanagenesis.

Chromoanagenesis in the asy1 meiotic mutant of Arabidopsis.

Chromoanagenesis is a catastrophic event that involves localized chromosomal shattering and reorganization. In this study, we report a case of chromoanagenesis resulting from defective meiosis in the MEIOTIC ASYNAPTIC MUTANT 1 (asy1) background in Arabidopsis thaliana. We provide a detailed characterization of the genomic structure of this individual with a severely shattered segment of chromosome 1. We identified 260 novel DNA junctions in the affected region, most of which affect gene sequence on 1 or both sides of the junction. Our results confirm that asy1-related defective meiosis is a potential trigger for chromoanagenesis. This is the first example of chromoanagenesis associated with female meiosis and indicates the potential for genome evolution during oogenesis. PLAIN LANGUAGE SUMMARY: Chromoanagenesis is a complex and catastrophic event that results in severely restructured chromosomes. It has been identified in cancer cells and in some plant samples, after specific triggering events. Here, we identified this kind of genome restructuring in a mutant that exhibits defective meiosis in the model plant system Arabidopsis thaliana.

Identification of a Negative Regulator for Salt Tolerance at Seedling Stage via a Genome-Wide Association Study of Thai Rice Populations.

Salt stress is a major limiting factor in crop production and yield in many regions of the world. The objective of this study was to identify the genes responsible for salt tolerance in Thai rice populations. We performed a genome-wide association study with growth traits, relative water content, and cell membrane stability at the seedling stage, and predicted 25 putative genes. Eleven of them were located within previously reported salt-tolerant QTLs (ST-QTLs). OsCRN, located outside the ST-QTLs, was selected for gene characterization using the Arabidopsis mutant line with T-DNA insertion in the orthologous gene. Mutations in the AtCRN gene led to the enhancement of salt tolerance by increasing the ability to maintain photosynthetic pigment content and relative water content, while the complemented lines with ectopic expression of OsCRN showed more susceptibility to salt stress detected by photosynthesis performance. Moreover, the salt-tolerant rice varieties showed lower expression of this gene than the susceptible rice varieties under salt stress conditions. The study concludes that by acting as a negative regulator, OsCRN plays an important role in salt tolerance in rice.

Salt stress responses and SNP-based phylogenetic analysis of Thai rice cultivars.

Genetic diversity is important for developing salt-tolerant rice (Oryza sativa L.) cultivars. Certain Thai rice accessions display salt tolerance at the adult or reproductive stage, but their response to salinity at the seedling stage is unknown. In this study, a total of 10 rice cultivars/line, including eight Thai cultivars and standard salt-tolerant cultivar and susceptible line, were screened using a hydroponic system to identify salt-tolerant genotypes at the seedling stage. Different morphophysiological indicators were used to classify tolerant and susceptible genotypes. Phylogenetic analyses were performed to determine the evolutionary relationships between the cultivars. Results showed that 'Lai Mahk', 'Jao Khao', 'Luang Pratahn', and 'Ma Gawk' exhibited salt stress tolerance comparable with the standard salt-tolerance check 'Pokkali'. Whole-exome single-nucleotide polymorphism (SNP)-based phylogenetic analysis showed that the Thai rice cultivars were monophyletic and distantly related to Pokkali and IR29. Lai Mahk and Luang Pratahn were found closely related when using the whole-exome SNPs for the analysis. This is also the case for the analysis of SNPs from 164 salt-tolerance genes and transcription regulatory genes. The tolerant cultivars shared the same haplotype for 16 genes. Overall, the findings of this study identified four salt-stress-tolerant Thai rice cultivars, which could be used in rice breeding programs for salinity tolerance.

Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential.

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (-)-carvone and (-)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

Identification of Key Genes in 'Luang Pratahn', Thai Salt-Tolerant Rice, Based on Time-Course Data and Weighted Co-expression Networks.

Salinity is an important environmental factor causing a negative effect on rice production. To prevent salinity effects on rice yields, genetic diversity concerning salt tolerance must be evaluated. In this study, we investigated the salinity responses of rice (Oryza sativa) to determine the critical genes. The transcriptomes of 'Luang Pratahn' rice, a local Thai rice variety with high salt tolerance, were used as a model for analyzing and identifying the key genes responsible for salt-stress tolerance. Based on 3' Tag-Seq data from the time course of salt-stress treatment, weighted gene co-expression network analysis was used to identify key genes in gene modules. We obtained 1,386 significantly differentially expressed genes in eight modules. Among them, six modules indicated a significant correlation within 6, 12, or 48h after salt stress. Functional and pathway enrichment analysis was performed on the co-expressed genes of interesting modules to reveal which genes were mainly enriched within important functions for salt-stress responses. To identify the key genes in salt-stress responses, we considered the two-state co-expression networks, normal growth conditions, and salt stress to investigate which genes were less important in a normal situation but gained more impact under stress. We identified key genes for the response to biotic and abiotic stimuli and tolerance to salt stress. Thus, these novel genes may play important roles in salinity tolerance and serve as potential biomarkers to improve salt tolerance cultivars.

LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci.

Large-scale structural variations, such as chromosomal translocations, can have profound effects on fitness and phenotype, but are difficult to identify and characterize. Here, we describe a simple and effective method aimed at identifying translocations using only the dosage of sequence reads mapped on the reference genome. We binned reads on genomic segments sized according to sequencing coverage and identified instances when copy number segregated in populations. For each dosage-polymorphic 1 Mb bin, we tested independence, effectively an apparent linkage disequilibrium (LD), with other variable bins. In nine potato (Solanum tuberosum) dihaploid families translocations affecting pericentromeric regions were common and in two cases were due to genomic misassembly. In two populations, we found evidence for translocation affecting euchromatic arms. In cv. PI 310467, a nonreciprocal translocation between chromosomes (chr.) 7 and 8 resulted in a 5-3 copy number change affecting several Mb at the respective chromosome tips. In cv. "Alca Tarma," the terminal arm of chr. 4 translocated to the tip of chr. 1. Using oligonucleotide-based fluorescent in situ hybridization painting probes (oligo-FISH), we tested and confirmed the predicted arrangement in PI 310467. In 192 natural accessions of Arabidopsis thaliana, dosage haplotypes tended to vary continuously and resulted in higher noise, while apparent LD between pericentromeric regions suggested the effect of repeats. This method, LD-CNV, should be useful in species where translocations are suspected because it tests linkage without the need for genotyping.

Epigenetically mismatched parental centromeres trigger genome elimination in hybrids.

Wide crosses result in postzygotic elimination of one parental chromosome set, but the mechanisms that result in such differential fate are poorly understood. Here, we show that alterations of centromeric histone H3 (CENH3) lead to its selective removal from centromeres of mature Arabidopsis eggs and early zygotes, while wild-type CENH3 persists. In the hybrid zygotes and embryos, CENH3 and essential centromere proteins load preferentially on the CENH3-rich centromeres of the wild-type parent, while CENH3-depleted centromeres fail to reconstitute new CENH3-chromatin and the kinetochore and are frequently lost. Genome elimination is opposed by E3 ubiquitin ligase VIM1. We propose a model based on cooperative binding of CENH3 to chromatin to explain the differential CENH3 loading rates. Thus, parental CENH3 polymorphisms result in epigenetically distinct centromeres that instantiate a strong mating barrier and produce haploids.

Efficient construction of a linkage map and haplotypes for Mentha suaveolens using sequence capture.

The sustainability of many crops is hindered by the lack of genomic resources and a poor understanding of natural genetic diversity. Particularly, application of modern breeding requires high-density linkage maps that are integrated into a highly contiguous reference genome. Here, we present a rapid method for deriving haplotypes and developing linkage maps, and its application to Mentha suaveolens, one of the diploid progenitors of cultivated mints. Using sequence-capture via DNA hybridization to target single nucleotide polymorphisms (SNPs), we successfully genotyped ∼5000 SNPs within the genome of >400 individuals derived from a self cross. After stringent quality control, and identification of nonredundant SNPs, 1919 informative SNPs were retained for linkage map construction. The resulting linkage map defined a total genetic space of 942.17 cM divided among 12 linkage groups, ranging from 56.32 to 122.61 cM in length. The linkage map is in good agreement with pseudomolecules from our preliminary genome assembly, proving this resource effective for the correction and validation of the reference genome. We discuss the advantages of this method for the rapid creation of linkage maps.

Combining Genome and Gene Co-expression Network Analyses for the Identification of Genes Potentially Regulating Salt Tolerance in Rice.

Salinity stress tolerance is a complex polygenic trait involving multi-molecular pathways. This study aims to demonstrate an effective transcriptomic approach for identifying genes regulating salt tolerance in rice. The chromosome segment substitution lines (CSSLs) of "Khao Dawk Mali 105 (KDML105)" rice containing various regions of DH212 between markers RM1003 and RM3362 displayed differential salt tolerance at the booting stage. CSSL16 and its nearly isogenic parent, KDML105, were used for transcriptome analysis. Differentially expressed genes in the leaves of seedlings, flag leaves, and second leaves of CSSL16 and KDML105 under normal and salt stress conditions were subjected to analyses based on gene co-expression network (GCN), on two-state co-expression with clustering coefficient (CC), and on weighted gene co-expression network (WGCN). GCN identified 57 genes, while 30 and 59 genes were identified using CC and WGCN, respectively. With the three methods, some of the identified genes overlapped, bringing the maximum number of predicted salt tolerance genes to 92. Among the 92 genes, nine genes, OsNodulin, OsBTBZ1, OsPSB28, OsERD, OsSub34, peroxidase precursor genes, and three expressed protein genes, displayed SNPs between CSSL16 and KDML105. The nine genes were differentially expressed in CSSL16 and KDML105 under normal and salt stress conditions. OsBTBZ1 and OsERD were identified by the three methods. These results suggest that the transcriptomic approach described here effectively identified the genes regulating salt tolerance in rice and support the identification of appropriate QTL for salt tolerance improvement.

Chromoanagenesis from radiation-induced genome damage in Populus.

Chromoanagenesis is a genomic catastrophe that results in chromosomal shattering and reassembly. These extreme single chromosome events were first identified in cancer, and have since been observed in other systems, but have so far only been formally documented in plants in the context of haploid induction crosses. The frequency, origins, consequences, and evolutionary impact of such major chromosomal remodeling in other situations remain obscure. Here, we demonstrate the occurrence of chromoanagenesis in poplar (Populus sp.) trees produced from gamma-irradiated pollen. Specifically, in this population of siblings carrying indel mutations, two individuals exhibited highly frequent copy number variation (CNV) clustered on a single chromosome, one of the hallmarks of chromoanagenesis. Using short-read sequencing, we confirmed the presence of clustered segmental rearrangement. Independently, we identified and validated novel DNA junctions and confirmed that they were clustered and corresponded to these rearrangements. Our reconstruction of the novel sequences suggests that the chromosomal segments have reorganized randomly to produce a novel rearranged chromosome but that two different mechanisms might be at play. Our results indicate that gamma irradiation can trigger chromoanagenesis, suggesting that this may also occur when natural or induced mutagens cause DNA breaks. We further demonstrate that such events can be tolerated in poplar, and even replicated clonally, providing an attractive system for more in-depth investigations of their consequences.