OENO One (Jun 2014)
The complete chloroplast DNA sequence of eleven grape cultivars. simultaneous resequencing methodology
Abstract
Aims: The chloroplast DNA sequence of eight Georgian grape cultivars (Rkatsiteli, Saperavi, Meskhuri Mtsvane, Chkhaveri, Aladasturi, Krakhuna, Tsitska, Tsolikouri) and three French cultivars (Chardonnay, Gouais Blanc, Chasselas), belonging to four different haplogroups (AAA, ATT, ATA, GTA), was determined by Illumina resequencing of genomic DNA. The chloroplast DNA sequence of the Maxxa cultivar was used as reference. Methods and results: The comparison of sequenced chloroplast DNA gave 100 % identity to Chardonnay and Gouais Blanc, differing from Meskhuri Mtsvane by two insertions/deletions (indels) (all ATA haplogroup). The difference between Chasselas and Saperavi was a single insertion (both ATT haplogroup), while Maxxa, Chkhaveri, Aladasturi, Krakhuna, Tsitska and Tsolikouri were all identical (all members of the GTA haplogroup). Forty-seven identical single nucleotide polymorphisms (SNPs) were detected in the AAA, ATA and ATT haplogroups in comparison to the reference DNA. Additionally, 18 SNPs were detected for the ATT haplogroup, 4 for AAA, 6 for ATA and 11 for both AAA and ATA. The phylogenetic results show that the ATT, AAA and ATA haplogroups are more closely related to each other than to the GTA haplogroup. Conclusion: In the sequencing data of grape genomic DNA at the coverage (read depth) of chromosomal DNA 30-40, the coverage of chloroplast DNA reaches several thousand reads per bp due to the high number of chloroplast DNA copies in genomic DNA, much higher than necessary for resequencing. Based on these data, a new methodology of simultaneous resequencing of large number of chloroplast DNA was developed without preliminary chloroplast isolation or chloroplast enrichment. Significance and impact of the study: This method has great potential for expanding both phylogenetic and population genetic information on the evolution of domesticated crops.
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