The genetic maps constructed based on physical positions of SNPs
In this study, SNPs used for constructing the genetic maps of AB and D genomes were aligned based on their physical positions to the sequence assembly of CS [65] and A. tauschii AL8/78 [23], respectively. Moreover, for genetic map construction, the method of ‘nearest neighbor & two-opt’ (nnTwoOpt) was also used for tour construction and its improvement, which is similar to Travelling Salesman Problem (TSP) [66]. For AB genome, chromosomes without any linkage gaps, such as 1A, 1B, 2A, 5A, 5B, 6B, 7A, and 7B, almost had the same genetic lengths of chromosome maps self-organized by nnTwoOpt method to those aligned with CS physical map (Table 3). For the chromosomes 2B, 3A, 3B, 4B and 6A, linkage gaps were found more or less on them, which were mostly caused by the extreme disequilibrium of SNP-polymorphism distribution. The polymorphism SNPs of each chromosome in D genome were much more than that in AB genome, and only one linkage gap was found on genetic map of chromosome 3D self-organized by nnTwoOpt method among the whole genome (Table 5). Considering that only subtle difference was observed in genetic lengths between self-organizing genetic maps and that aligned with CS physical map (Table 3, 5), we used the physical maps of CS and AL8/78 to align SNPs to AB and D genomes, respectively. So, with a physical map for alignment reference, the changes of the genetic length and RF of the diploid and tetraploid genomes after their hexaploidization could be investigated directly and conveniently by comparing them between populations with different polyploidy.
However, linkage gaps were found on some chromosome genetic maps of the tetraploid genome, where the genetic distance between two adjacent SNP loci was larger than 50 cM (Table 3). More double-crossovers often occur between two adjacent SNP loci if their genetic distance is over 50cM. In this situation, the measured RF would be much smaller than the true RF and did not reflect it accurately. Considering this, we only analyzed the genetic distance and RF between two adjacent SNP loci that were also closely linked to each other.
Enhanced genetic recombination of ancestral diploid genome after hexaploidization
On chromosome level, we compared the average RF between adjacent SNP loci in diploid and tetraploid populations with that in SHW population. And significant increase of RF was observed only in the ancestral diploid genome DD after their hexaploidization, rather than that in the ancestral tetraploid genome AABB. These results suggested that the increase extent of RF in ancestral genome depended on polyploidization level (eventual ploidy/initial ploidy), as the increase rate of RF from diploid to hexaploid was much greater than that from tetraploid to hexaploid. Most reported studies focused on the RF change from diploid to tetraploid (polyploidization level = 2). Leflon et al. [59] reported that the total genetic length of A7 linkage group increased from 52 cM in Brassica rapa to 96 cM in B. napus, after their allopolyploidization from diploidy to tetraploidy. The meiotic RF increased from 15.4% in diploid Arabidopsis thaliana to 24.1% in allotetraploid A. suecica [60]. These reported data showed that the RF in diploid genome will raise less than 2 fold (Pecinka et al. [60]: 1.56) when polyploidization level =2. Being limited by their slected plant species, it is difficult to investige the RF change from diploid to hexaploid (polyploidization level = 3), as a trigenomic Brassica (AABBCC) is not known to exist in nature. Specially using wheat as a good model studying polyploidy, we found that the RF in diploid genome increased more than two fold (Table 4: about 2.3-fold on average) when promoting its polyploidization level from diploid to hexaploid. Furthermore, no significant change of RF was observed in tetraploid genome after its hexaploidization, as its polyploidization level was 1.5 (eventual ploidy/initial ploidy=6/4).
However, this situation depending on polyploidization level might be only suitable for euploidy but not for aneuploidy. For example, in B. rapa, A7 linkage group of allotriploid got 4-fold increase of the total genetic length more than both diploid and tetraploid [59]. The possible reasons were as follows: (1) aneuploidy causes greater genome instability than polyploidy for organisms [2,67] and aneuploidy itself can be responsible for the procreation of chromosomal instability [68,69]; (2) chromosomes that remain as univalents in the aneuploidy could lead to a compensatory increase in crossover frequency among unaffected bivalents [59, 70-72].
The RF of the diploid D genome was significantly enhanced after their hexaploidization, and the genetic mechanisms for this has not been clear yet. However, reported QTLs for crossover (CO) have been detected in hexaploid wheat, and most of them were distributed on AB genome. With 13 recombinant inbred mapping populations, Gardiner et al. [73] detected 5 QTLs for CO frequency on AB genome of the common wheat, which were located on 2A, 2B, 4B, 5A and 6A, respectively; Jordan et al. [54] detected 40 QTLs for total CO frequency by nested association mapping, most of which were also mapped to AB genome. These results implied that the genetic factors determining the CO frequency might be present in the ancestral genome AABB, which also lead to the RF increase of diploid genome DD after their hexaploidization. However, the max phenotypic effect of QTLs reported by Gardiner et al. [73] increased CO frequency less than 15%, and all 40 QTLs detected by Jordan et al. [54] acrossing the whole genome effected 7.0% of the overall mean for total COs. The effect size of these QTLs were much lower than the increase extent of >200% for RF in diploid genome caused by hexaploidization in this study. This suggested that the RF increase of the diploid D genome in our study was caused by hexaploidization for the most part, while the contribution of the genetic factors provided by AB genome was very minor.
Polyploidization enhancing variation and adaptive evolution of bread wheat
Allopolyploidy accelerates revolution in wheat often by two ways: (1) it triggers rapid genome changes through the instantaneous generation of a variety of cardinal genetic and epigenetic alterations, which generate heterosis between subgenomes in polypoid plants [1,2], and (2) the allopolyploid condition facilitates sporadic genomic changes that are not attainable at the diploid level, and take the advantages of gene redundancy [5,8,9,61,62]. The hexaploid wheat takes the advantages of heterosis from both the tetraploid wheat and Ae. tauschii. Genome sequence of Ae. tauschii and gene annotation for whole genome reveals that the diploid progenitor of hexaploid wheat D genome serves as a gene repertoire for modern wheat adaptation, which provides possible resistance to disease and pest, tolerance to environmental stresses and grain quality [22,23]. Importantly, these gene could be expressed normally in a hexaploid genetic background, for that lots of related QTLs or genes had been mapped to D genome of synthetic hexaploid wheat [24,30-32,26-29,36-37,41,42,74]. Moreover, the mRNA and small RNA transcriptomes analysis in nascent hexaploid wheat also demonstrate the heterosis generating in the common wheat [4]. Using wheat, our study suggests that the enhanced genetic recombination of the ancestral diploid genome that was caused by allopolyploidization could be regarded as another advantage or a new way to increase evolutionary potential of polyploid.
By allopolyploidization, Ae. tauschii adds its genome into that of tetraploid wheat, and produces hexaploid wheat, a major type of cultivated wheat, which accounting for about 95% of world wheat production, while the tetraploid wheat only accounting for the other 5% [17], suggesting that the added D genome, made bread wheat more adaptive to alterable environments and then spreaded more rapidly than the tetraploid wheat. However, the D genome of the first bread wheats were originated from only a small part of Ae.tauschii population that cannot possess all the superiorities mentioned above in a few lucky individuals. There must be some other reasons underlying more rapid spread of hexaploid common wheat than tetraploid wheat. Interestingly, our study shows that hexaploidization enhanced genetic recombination of the ancestral diploid genome DD in allohexaploid wheat. And the RF throughout the whole D genome in SHW increased more than 2 fold than that in diploidy, which does favor to bread wheat in enhancing variation and adaptive evolution by intercrossing with each other among the first hexaploidy individuals of wheat, as more recombination events has the potential to substantially accelerate the development of new varieties by (1) allowing quick assembly of novel beneficial multi-allelic complexes and (2) breaking the linkage among unfavorable genes and fixing desirable haplotypes in fewer generations [50]. This was more efficient than that in a diploid or tetraploid genetic background, as more recombination events occurred in hexaploid genetic background, with higher possibility to create more phenotypic variations to the selection pools for evolution.