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Aegilops species represent the most important gene pool for breeding bread wheat (Triticum aestivum). Thus, understanding the genome evolution, including chromosomal structural rearrangements and syntenic relationships among Aegilops species or between Aegilops and wheat, is important for both basic genome research and practical breeding applications. In the present study, we attempted to develop subgenome D-specific fluorescence in situ hybridization (FISH) probes by selecting D-specific oligonucleotides based on the reference genome of Chinese Spring. The oligo-based chromosome painting probes consisted of approximately 26 000 oligos per chromosome and their specificity was confirmed in both diploid and polyploid species containing the D subgenome. Two previously reported translocations involving two D chromosomes have been confirmed in wheat varieties and their derived lines. We demonstrate that the oligo painting probes can be used not only to identify the translocations involving D subgenome chromosomes, but also to determine the precise positions of chromosomal breakpoints. Chromosome painting of 56 accessions of Ae. tauschii from different origins led us to identify two novel translocations: a reciprocal 3D-7D translocation in two accessions and a complex 4D-5D-7D translocation in one accession. Painting probes were also used to analyze chromosomes from more diverse Aegilops species. These probes produced FISH signals in four different genomes. Chromosome rearrangements were identified in Aegilops umbellulata, Aegilops markgrafii, and Aegilops uniaristata, thus providing syntenic information that will be valuable for the application of these wild species in wheat breeding., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cold stress at the reproductive stage severely affects the production and geographic distribution of rice. The Geng/japonica subpopulation gradually developed stronger cold adaptation than the Xian/indica subpopulation during the long-term domestication of cultivated rice. However, the evolutionary path and natural alleles underlying the cold adaptability of intra-Geng subspecies remain largely unknown. Here, we identified MITOGEN-ACTIVATED PROTEIN KINASE 3 (OsMAPK3) and LATE EMBRYOGENESIS ABUNDANT PROTEIN 9 (OsLEA9) as two important regulators for the cold adaptation of Geng subspecies from a combination of transcriptome analysis and genome-wide association study. Transgenic validation showed that OsMAPK3 and OsLEA9 confer cold tolerance at the reproductive stage. Selection and evolution analysis suggested that the Geng version of OsMAPK3 (OsMAPK3 Geng ) directly evolved from Chinese Oryza rufipogon III and was largely retained in high-latitude and high-altitude regions with low temperatures during domestication. Later, the functional nucleotide polymorphism (FNP-776) in the Kunmingxiaobaigu and Lijiangxiaoheigu version of the OsLEA9 (OsLEA9 KL ) promoter originated from novel variation of intra-Geng was selected and predominantly retained in temperate Geng to improve the adaptation of Geng together with OsMAPK3 Geng to colder climatic conditions in high-latitude areas. Breeding potential analysis suggested that pyramiding of OsMAPK3 Geng and OsLEA9 KL enhanced the cold tolerance of Geng and promotes the expansion of cultivated rice to colder regions. This study not only highlights the evolutionary path taken by the cold-adaptive differentiation of intra-Geng, but also provides new genetic resources for rice molecular breeding in low-temperature areas., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Distant species producing the same secondary metabolites is an interesting and common phenomenon in nature. A classic example of this is scutellarein whose derivatives have been used clinically for more than 30 years. Scutellarein occurs in significant amounts in species of two different orders, Scutellaria baicalensis and Erigeron breviscapus, which diverged more than 100 million years ago. Here, according to the genome-wide selection and functional identification of 39 CYP450 genes from various angiosperms, we confirmed that only seven Scutellaria-specific CYP82D genes and one Erigeron CYP706X gene could perform the catalytic activity of flavone 6-hydroxylase (F6H), suggesting that the convergent evolution of scutellarein production in these two distant species was caused by two independently evolved CYP450 families. We also identified seven Scutellaria-specific CYP82D genes encoding flavone 8-hydroxylase (F8H). The evolutionary patterns of CYP82 and CYP706 families via kingdom-wide comparative genomics highlighted the evolutionary diversity of CYP82D and the specificity of CYP706X in angiosperms. Multi-collinearity and phylogenetic analysis of CYP82D in Scutellaria confirmed that the function of F6H evolved from F8H. Furthermore, the SbaiCYP82D1 A319D , EbreCYP706X R130A , EbreCYP706X F312D and EbreCYP706X A318D mutants can significantly decrease the catalytic activity of F6H, revealing the contribution of crucial F6H amino acids to the scutellarein biosynthesis of distant species. This study provides important insights into the multi-origin evolution of the same secondary metabolite biosynthesis in the plant kingdom., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Karyotype dynamics driven by complex chromosome rearrangements constitute a fundamental issue in evolutionary genetics. The evolutionary events underlying karyotype diversity within plant genera, however, have rarely been reconstructed from a computed ancestral progenitor. Here, we developed a method to rapidly and accurately represent extant karyotypes with the genus, Cucumis, using highly customizable comparative oligo-painting (COP) allowing visualization of fine-scale genome structures of eight Cucumis species from both African-origin and Asian-origin clades. Based on COP data, an evolutionary framework containing a genus-level ancestral karyotype was reconstructed, allowing elucidation of the evolutionary events that account for the origin of these diverse genomes within Cucumis. Our results characterize the cryptic rearrangement hotspots on ancestral chromosomes, and demonstrate that the ancestral Cucumis karyotype (n = 12) evolved to extant Cucumis genomes by hybridizations and frequent lineage- and species-specific genome reshuffling. Relative to the African species, the Asian species, including melon (Cucumis melo, n = 12), Cucumis hystrix (n = 12) and cucumber (Cucumis sativus, n = 7), had highly shuffled genomes caused by large-scale inversions, centromere repositioning and chromothripsis-like rearrangement. The deduced reconstructed ancestral karyotype for the genus allowed us to propose evolutionary trajectories and specific events underlying the origin of these Cucumis species. Our findings highlight that the partitioned evolutionary plasticity of Cucumis karyotype is primarily located in the centromere-proximal regions marked by rearrangement hotspots, which can potentially serve as a reservoir for chromosome evolution due to their fragility., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double-stranded labelled oligos, which produced much stronger signals than single-stranded labelled oligos, by amplification using fluorophore-conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross-species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo-painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

The important role of polyploidy in plant evolution is widely recognized. However, many questions remain to be explored to address how polyploidy affects the phenotype of the plant. To shed light on the phenotypic and molecular impacts of allopolyploidy, we investigated the leaf development of a synthesized allotetraploid (Cucumis × hytivus), with an emphasis on chlorophyll development. Delayed leaf maturation was identified in C. × hytivus, based on delayed leaf expansion, initial chlorophyll deficiency in the leaves and disordered sink-source transition. Anatomical observations also revealed disturbed chloroplast development in C. ×hytivus. The determination of chlorophyll biosynthesis intermediates suggested that the chlorophyll biosynthesis pathway of C. × hytivus is blocked at the site at which uroporphyrinogen III is catalysed to coproporphyrinogen III. Three chlorophyll biosynthesis-related genes, HEMA1, HEME2 and POR, were significantly repressed in C. × hytivus. Sequence alignment showed both synonymous and non-synonymous substitutions in the HEMA1, HEME2 and POR genes of the parents. Cloning of the chlorophyll biosynthetic genes suggested the retention of homoeologs. In addition, a chimeric clone of the HEMA1 gene that consisted of homologous genes from the parents was identified in C. × hytivus. Overall, our results showed that allopolyploidization in Cucumis has resulted in disturbed chloroplast development and reduced chlorophyll biosynthesis caused by the repressed expression of duplicated homologous genes, which further led to delayed leaf maturation in the allotetraploid, C. × hytivus. The preferential retention/loss of certain types of genes and non-reciprocal homoeologous recombination were also supported in the present study, which provides new insights into the impact of allopolyploidy., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single-copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome-specific gene probes. This single-copy gene-based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis-aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross-species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Copy number variation (CNV) has been revealed as a significant contributor to the genetic variation in humans. Although CNV has been reported in several model animal and plant species, the presence of CNV and its biological impact in polyploid species has not yet been documented. We conducted a fluorescence in situ hybridization (FISH)-based CNV survey in potato, a vegetatively propagated autotetraploid species (2n = 4x = 48). We conducted FISH analysis using 18 randomly selected potato bacterial artificial chromosome (BAC) clones in a set of 16 potato cultivars with diverse breeding backgrounds. Six BACs (33%) with insert sizes of 137-145 kb were found to be associated with large CNV events detectable at the cytological level. We demonstrate that the large CNVs associated with two specific BACs (RH102I10 and RH83C08) were widespread among potato cultivars developed in North America and Europe. We measured the transcript abundance of four genes associated with the CNV spanned by BAC RH102I10. All four genes displayed a dosage effect in transcription. Although potato is vegetatively propagated, we observed that female gametes lacking the RH102I10-associated CNV were inferior to those with at least one copy of this CNV, indicating that the RH102I10-associated CNV can impact on the growth and development of the potato plants. Our results show that CNV is highly abundant in the potato genome and may play a significant role in genetic variation of this important food crop., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)