Dramatically regulated by HT stress, GrKMT1A;1a, GrKMT1A;2, GrKMT3;3, GrKMT6B;1, and GrKMT6B;2 highly expressed in anther and ovary (Figs 5 and 6), suggesting that if the roles of GrKMTs and GrRBCMTs were further investigated in reproductive tissues or organs, it would be able to mine novel resistant genes and provide new understanding for plant HT stress response. Evolution of GrKMTs and GrRBCMTs impacts differentially on their functions. It has been our main interest how the evolution of XAV-939MedChemExpress XAV-939 duplicated genes affects their biological functions, since gene duplication has played a vital role in the evolution of new gene functions and is one of the primary driving forces in the evolution of genomes and genetic systems52. Gene families may evolve primarily through tandem duplication and polyploidy or large-scale segmental duplications52. Arabidopsis genome has undergone about two rounds of duplications before Arabidopsis/Brassica rapa split and after the monocot/dicot divergence53. The outcomes of duplicated genes include nonfunctionalization, neofunctionalization and subfunctionalization54. The nonfunctionalization of one copy is the most likely fate due to deleterious mutation, functionally redundant and dosage constraints54. G. ramondii undergone independent whole-genome duplication event approximately 13.3 to 20.0 million years ago, and shared one paleohexaploidization event with eudicots, but has a higher gene number and lower mean gene density compared with Arabidopsis36, meaning many genes were lost after duplication. We identified 46 KMTs and RBCMTs in Arabidopsis (2n = 10) and only 52 members in G. ramondii (2n = 26). Based on the canonical criteria21,22, seven pairs of GrKMT or GrRBCMT genes were created by the duplication of homologous genes. GrKMT1B;2a/2b, GrKMT1B;3a/3d, GrKMT2;3b/3c, GrKMT6A;1a/1b, GrRBCMT;9a/9b, GrKMT1A;4b/4c/4d might be due to ancient large-scale duplication event, while GrKMT1B;3b/3c may formed by tandem duplication (Supplementary Table S4). Even though GrKMT1B;3a was also shown to meet the parameters of duplicated genes for GrKMT1B;3b/3c/3d in NCBI, they were not considered as duplicated genes since GrKMT1B;3d is much shorter than GrKMT1B;3b/3c (Fig. 4; Supplementary Table S4). GrRBCMT;9a/9b as duplicated genes also could not be confirmed, because GrRBCMT;9b (Gorai. N022300) still not be SB 202190 site mapped on any chromosome (Fig. 1). Duplicated genes can generally be grouped into one clade of phylogenetic tree (Fig. 2); most of these genes exist in sister pairs or triplets and have similar gene structure with possible similar functions, whereas others are divergent in the distribution of introns/exons, suggesting the possibility of functional diversification22. We foundScientific RepoRts | 6:32729 | DOI: 10.1038/srepwww.nature.com/scientificreports/that the gene structure was conserved in most of GrKMT genes, except GrKMT6A;1a/1b and GrRBCMT;9a/9b with one exon difference; domain organization of GrKMT1A;4b/4c/4d and GrKMT2;3b/3c were conserved, but GrKMT1B;2a/2b, GrKMT6A;1a/1b and GrRBCMT;9a/9b are divergent (Figs 3 and 4, Supplementary Table S3); only sisters genes of GrKMT6A;1a/1b and GrRBCMT;9a/9b showed similar expression patterns in different tissues and organs. For example, GrKMT1;3b/3c have same gene structure, domain organization, but GrKMT1;3b only highly expresses in anther, and is not involved in HT stress, and GrKMT1; 3c strongly expresses in root, stem and leaf and is sensitive to HT stress (Figs 3?; Supplem.Dramatically regulated by HT stress, GrKMT1A;1a, GrKMT1A;2, GrKMT3;3, GrKMT6B;1, and GrKMT6B;2 highly expressed in anther and ovary (Figs 5 and 6), suggesting that if the roles of GrKMTs and GrRBCMTs were further investigated in reproductive tissues or organs, it would be able to mine novel resistant genes and provide new understanding for plant HT stress response. Evolution of GrKMTs and GrRBCMTs impacts differentially on their functions. It has been our main interest how the evolution of duplicated genes affects their biological functions, since gene duplication has played a vital role in the evolution of new gene functions and is one of the primary driving forces in the evolution of genomes and genetic systems52. Gene families may evolve primarily through tandem duplication and polyploidy or large-scale segmental duplications52. Arabidopsis genome has undergone about two rounds of duplications before Arabidopsis/Brassica rapa split and after the monocot/dicot divergence53. The outcomes of duplicated genes include nonfunctionalization, neofunctionalization and subfunctionalization54. The nonfunctionalization of one copy is the most likely fate due to deleterious mutation, functionally redundant and dosage constraints54. G. ramondii undergone independent whole-genome duplication event approximately 13.3 to 20.0 million years ago, and shared one paleohexaploidization event with eudicots, but has a higher gene number and lower mean gene density compared with Arabidopsis36, meaning many genes were lost after duplication. We identified 46 KMTs and RBCMTs in Arabidopsis (2n = 10) and only 52 members in G. ramondii (2n = 26). Based on the canonical criteria21,22, seven pairs of GrKMT or GrRBCMT genes were created by the duplication of homologous genes. GrKMT1B;2a/2b, GrKMT1B;3a/3d, GrKMT2;3b/3c, GrKMT6A;1a/1b, GrRBCMT;9a/9b, GrKMT1A;4b/4c/4d might be due to ancient large-scale duplication event, while GrKMT1B;3b/3c may formed by tandem duplication (Supplementary Table S4). Even though GrKMT1B;3a was also shown to meet the parameters of duplicated genes for GrKMT1B;3b/3c/3d in NCBI, they were not considered as duplicated genes since GrKMT1B;3d is much shorter than GrKMT1B;3b/3c (Fig. 4; Supplementary Table S4). GrRBCMT;9a/9b as duplicated genes also could not be confirmed, because GrRBCMT;9b (Gorai. N022300) still not be mapped on any chromosome (Fig. 1). Duplicated genes can generally be grouped into one clade of phylogenetic tree (Fig. 2); most of these genes exist in sister pairs or triplets and have similar gene structure with possible similar functions, whereas others are divergent in the distribution of introns/exons, suggesting the possibility of functional diversification22. We foundScientific RepoRts | 6:32729 | DOI: 10.1038/srepwww.nature.com/scientificreports/that the gene structure was conserved in most of GrKMT genes, except GrKMT6A;1a/1b and GrRBCMT;9a/9b with one exon difference; domain organization of GrKMT1A;4b/4c/4d and GrKMT2;3b/3c were conserved, but GrKMT1B;2a/2b, GrKMT6A;1a/1b and GrRBCMT;9a/9b are divergent (Figs 3 and 4, Supplementary Table S3); only sisters genes of GrKMT6A;1a/1b and GrRBCMT;9a/9b showed similar expression patterns in different tissues and organs. For example, GrKMT1;3b/3c have same gene structure, domain organization, but GrKMT1;3b only highly expresses in anther, and is not involved in HT stress, and GrKMT1; 3c strongly expresses in root, stem and leaf and is sensitive to HT stress (Figs 3?; Supplem.