| Solanum tuberosum L.belongs to Solanum L. section Petota Dumortier, of which 36% in sect. Petota were polyploid. Many different kind of enzymes or markers were used for the phylogeny of wild diploid and polyploid potatoes, e.g., plastid DNA restriction site, Granule-bound starch synthase (GBSSI), Nitrate Reductase (NIA), Conserved Ortholog setâ…¡(COSII). But not so much wild polyploid potatoes were used in these researches. We here conduct a DNA sequencing study, using six nuclear orthologs, of 54 accessions of 11 polyploid species, and 37 diploid species representing possible progenitors to address questions species and genome origins of the polyploids, possible multiple origins, and concordance of different molecular markers from different regions of the genome. The main results were as follows:The protocols of asymmetric PCR-SSCP were optimized by different kind of tests, later the optimized protocols for each individual COSII were set up. Compared to the traditional cloning, the optimal asymmetric PCR-SSCP protocols saved time and labors, decreased PCR recombination and heteroduplex molecules during PCR processing and had the same base types and base numbers in polymorphic sites with clone sequencing results. In some extent, it could replace cloning method. Also the cost could be reduced by 18.9%-33.3% for polyploid species.The optimal protocols and six COSII were applied in all the materials in this study to construct phylogeny of the wild polyploid potatoes. The aligned length of the individual six COSII ranged from 461 characters for C2_Atlg32130 to 1473 for C2_At1g20050. The concatenated length of all six COSII was 4719 characters and for five COSII 4031 (without C2_At5g47390), of which parsimony informative characters were 673 and 551 respectively. The consistency indices of the individual COSII datasets ranged from 0.73-0.86 and the retention indices from 0.94-0.97. The consistency/retention indices were lower for both concatenated dataset,0.63/0.92. Of the 222 combinations (37 diploid accessions x 6 COSII),12.6% had more than one minor variant allele. After elimination alleles with minor allelic variants, only 5.9% of the diploids had allelic variants. Only 1.8% of the alleles from the tetraploids had less than two retained (divergent) alleles and only 1.8% the hexaploids had less than three divergent alleles.The phylogenetic analysis were performed by using different kind of datasets (six individual COS datasets of diploid+polyploid, six-COSII concatenated dataset of diploid, six-GOSII concatenated dataset of diploid+polyploid, five-COSII concatenated dataset of diploid and five-COSII concatenated dataset of diploid+polyploid). The results of six individual COS datasets of all diploid and polyploid indicated Though various degrees of incongruence were found in all six individual COSII phylogenetic trees, the diploid phylogeny was well solved. The 6COS concatenated dataset of diploid+polyploid result differs from prior phylogenies, in separating species from clade 3 into two clades. However, removing data from COSII C2At5g47390 from the dataset (retaining data from the remaining five COSII) recovered a three clade topology as did all three prior results.The five-COSII concatenated dataset of diploid+polyploid results concurred with the cladistic relationships of potato allopolyploids using GBSSI and NIA in 29 of the 54 cases examined:all polyploids were allopoplyploid origins; polyploids and more related diploids were in the same clades The remaining 25 of 54 accessions show three classes of discordance not seen in prior results:1) polyploid alleles in clades of unknown origins not recovered before (3p,4ap1,4p2), without diploid alleles in these new clades; 2) new polyploid alleles in the prexisting three main clades, relative to the studies of GBSSI and NIA; 3) 23 accession/COS combinations, alleles were apparently lost and phylogenetic associations stand out, and 4) multiple origins were found in wild polyploid potatoes. |
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