As to intergenomic gene collinearity, 1,631 blocks containing 18,626 collinear gene pairs were revealed between kiwifruit and grape and 1,869 blocks containing 18,869 collinear gene pairs were revealed between kiwifruit and coffee, showing similar collinearity between kiwifruit and the 2 referenced plants ( Tables S1 and S2 ).

Kiwifruit has a much more highly preserved intragenomic homology than either grape or coffee genomes ( Tables S1 and S2 ). We revealed 964 homologous blocks with ≥4 collinear genes, containing 9,998 collinear gene pairs in total. At the same parameter setting, we found 214 and 279 homologous blocks in grape and coffee genomes, containing 2,099 and 2,502 collinear gene pairs, respectively. The number of homologous genes in kiwifruit was about 4–5 times as many as those in grape and coffee, supporting the fact that the kiwifruit genome has undergone additional polyploidy events.

Gene collinearity, showing the preservation of ancestral genome structure in the modern genome, is an important means of unveiling the cryptic nature of genomic evolution. We inferred collinear genes within the kiwifruit, coffee, and grape genomes, and between them, via ColinearScan (), which is an effective way to evaluate genomic blocks of collinear genes. The grape and coffee genomes were used as outgroup references because these genomes are relatively simple and have been affected only by ECH. Thus, we also inferred collinear genes within each of these genomes and all 3 genomes ( Tables S1 and S2 ).

Evidence for Two Paleo-Tetraploidization Events

Figure 1 Original and Corrected Synonymous Nucleotide Substitutions between Collinear Genes (Ks) Show full caption ART, Actinidiaceae recent tetraploidization; AAT, Actinidiaceae ancient tetraploidization; ECH, core-eudicot-common hexaploidization; Mya, million years ago. Continuous curves are used to show Ks distribution in a genome, and broken ones are between genomes. (A) Density fitted by using original Ks values; (B) Inferred means; (C) Density fitted by using corrected Ks values; (D) Inferred evolutionary dates. Our inference of ancient polyploidization features integration of sequence divergence and homologous gene dotploting. We first characterized the synonymous nucleotide substitutions on synonymous substitution sites (Ks) between the collinear genes inferred above. If all the Ks of collinear genes are pooled together, clustering analysis may result in absorbing effects. That is, a smaller cluster produced by one event (often an ancient one) would be covered by a bigger cluster produced by another event (often a recent one). Therefore, we calculated the median (relatively more stable than the mean) value of each block and used them to perform clustering analysis. The kiwifruit collinear blocks clearly produced 3 peaks ( Figure 1 A), and a curve-fitting process located them at 0.164 (+/−0.093), 0.462 (+/−0.156), and 1.55 (+/−0.297), showing 3 events. Then by mapping gene sequence onto whole-genome-based similarity inferred by BLASTN, we constructed the homologous gene dotplot within kiwifruit genome ( Figure S1 ). We also mapped block median Ks values onto the dotplot. As to Ks and checking the dotplot, we could divide most of the blocks produced by 3 events, especially the longer blocks. In fact, if allowing the longer blocks to absorb their nearby smaller blocks, which could have been produced by genomic fractionation, we would distinguish all blocks by 3 events. Blocks produced by the most recent event covered 33.34% of the genomic DNA and 17.36% of total genes, and a covered region has a 1:1 correspondence, showing that it is tetraploidization. We called it as an Actinidiaceae recent tetraploidization (ART). Blocks produced by the mid-aged event covered 47.92% of the genomic DNA and 10.34% of total genes, and a covered region has a 1:1 correspondence, showing that it also is a tetraploidization event. We called it as an Actinidiaceae ancient tetraploidization (AAT). In contrast, we found that the blocks surely produced by the ECH covered 40.76% of the genomic DNA and only 6.96% of total genes.

Jiao et al., 2012 Jiao Y.

Leebens-Mack J.

Ayyampalayam S.

Bowers J.E.

McKain M.R.

McNeal J.

Rolf M.

Ruzicka D.R.

Wafula E.

Wickett N.J.

et al. A genome triplication associated with early diversification of the core eudicots. Figure 2 Examples of Homologous Gene Dotplots between Kiwifruit, Coffee, and Grape Show full caption Chromosome numbers and regions (in Mbp) were shown. Best-hit genes make red dots, secondary hits make blue dots, and the others are shown in gray. Highlights show the best matched chromosomal regions. Arrows show complement correspondence produced by chromosome breakages during evolution. (A) Homologous dotplot between selected grape and kiwifruit chromosomes. (B) Homologous dotplot between selected coffee and kiwifruit chromosomes. (C) Homologous dotplot within selected kiwifruit chromosomes. To verify our inference of the 3 events ART, AAT, and ECH, we used grape as a reference to decipher kiwifruit genome, a rosid eudicot preserving the ECH-triplicated genome structure of a core-eudicot common ancestor. Besides, we used an asteroid plant, coffee, as another reference, which is more closely related to kiwifruit than grape and has not been affected by other polyploidizations after the ECH. Homologous gene dotplots generated from the BLAST results between genomes allowed us to locate the corresponding homologous regions and were used to distinguish orthologous regions, established due to the grape-kiwifruit split, and outparalogous regions, established due to different duplications ( Figures 2 A, S2 , and S3 ). We mapped the inferred median Ks values of each collinear block onto the dotplots ( Figure S1 ). Integrating the aforementioned 2 lines of information helps distinguish orthologous and outparalogous blocks. The grape chromosomes were denoted with blocks in 7 colors, corresponding to the 7 ancestral eudicot chromosomes before the ECH, therefore a grape genomic region mostly has 2 paralogous regions in the same color ().

Maere et al., 2005 Maere S.

De Bodt S.

Raes J.

Casneuf T.

Van Montagu M.

Kuiper M.

Van de Peer Y. Modeling gene and genome duplications in eukaryotes. Figure 3 Species and Gene Phylogenetic Trees Show full caption (A) Phylogenetic tree of kiwifruit (K), coffee (C), and grape (V). The core-eudicot common hexaploidy (ECH) is denoted by a blue flash, and the two kiwifruit paleo-tetraploidizations are denoted by red flashes. (B) Gene phylogeny: three paralogous genes in the grape and coffee genomes are denoted by V1, V2, and V3, and C1, C2, and C3, respectively, produced by the ECH, and each has 4 orthologs and 8 outparalogs in the kiwifruit genome (e.g., V1 has 4 orthologs, K111, K112, K121, and K122 and 8 outparalogs, K211, K212, K221, K222, K311, K312, K321, and K322 in kiwifruit). The species tree is produced based on our present analysis of homologous genes. As to the Ks of grape-kiwifruit collinear gene pairs, there is a bimodal distribution, and therefore can be decomposed into 2 distinct distributions that have means (0.764 ± 0.0802) and (1.034 ± 0.405) ( Figure 1 A), corresponding to the orthologous genes (originated through their split) and outparalogous genes (through the common ECH), respectively. According to the median Ks value of each block, it was not difficult to distinguish the orthologous and the outparalogous blocks, especially for the long ones with ≥10 collinear genes. Allowing the long blocks to absorb the nearby short ones, whose Ks often change much, we inferred orthology and outparalogy for all the predicted collinear blocks ( Figures S2 and S3 ). With the orthologous blocks, by relating them to the AAT and ART blocks in kiwifruit dotplot, as to their locations on kiwifruit chromosomes, we separated the orthologous regions produced by different tetraploidization. For certain outparalogous regions lacking collinear genes, due to widespread and complementary gene losses (), we transitively used the paralogy between grape chromosomes and orthology between grape and kiwifruit chromosomes to locate the regions where outparalogy should exist. The coffee-kiwifruit gene dotplot and Ks analysis revealed orthology and outparalogy between the 2 genomes ( Figures 1 2 B, and S3 ). As expected, each grape or coffee genomic region often has 4 orthologous regions, 2 corresponding to the AAT and the other 2 to ART ( Figure 3 ). These intergenomic analyses agree with the above inference of 2 sequential tetraploidizations in kiwifruit lineage.