The complete chloroplast DNA sequence of eleven grape cultivars. simultaneous resequencing methodology

摘要

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. IntroductionGeorgia is home to over 500 grape cultivars (Ketskhoveli et al. 1960). The greater Caucasus region in which Georgia lies is widely believed to be the area where grape domestication began, and the study of genetic diversity in this region is viewed as a key to understanding grape domestication in general (Negrul 1946).The plastid genome is an effective tool for interspecific phylogenetic and intraspecific phylogeographic studies of angiosperms (Aoki et al. 2006; Gutiérrez-Rodríguez et al. 2011). Few papers are devoted to grape chloroplast DNA. Arroyo-Garcia et al. (2006) analyzed chloroplast DNA variation at nine polymorphic microsatellite loci of 1201 V. vinifera genotypes belonging to both sativa and sylvestris subspecies. Genotypic analyses for these microsatellite loci identified eight different chlorotypes (A to H) (Arroyo-Garcia et al. 2006). Among them, only four (A, B, C and D) had global frequencies greater than 5%. The intermediate relationship of chlorotype B to all other chlorotypes suggests that it could be an ancestral V. vinifera chlorotype.Plastid DNA of Caucasian (Georgian) grape varieties was studied by sequencing of some noncoding regions of grape chloroplast DNA (Beridze et al. 2011). During the investigation of 113 samples of a world-wide set of grape cultivars including Georgian cultivars, four plastid DNA haplotypes were evident and were designated by their character-states at each of the three polymorphic positions (Beridze et al. 2011) (Table 1).Table 1. Haplogroup definition for investigated cultivars.View popupExpand inlineCollapse inlineHaplogroupNucleotide positionInvestigated cultivars2058671586721AAA (1 haplotype)AAARkatsiteliATA (2 haplotypes)ATAChardonnay, Gouais Blanc, Meskhuri MtsvaneATT (2 haplotypes)ATTChasselas, SaperaviGTA (1 haplotype)GTAPinot Noir, Maxxa, Chkhaveri, Aladasturi, Krakhuna, Titska, Tsolikuri The AAA plastid haplotype was found only in the cultivars from Georgia. More specifically, twenty-three (57.5%) of the 40 included Georgian cultivars exhibited this haplotype, of which the “Rkatsiteli” cultivars originating from Eastern Georgia prevailed. This contrasts with the nine cultivars (22.5%) of the “Chkhaveri” group (GTA), which are mostly cultivated in Western Georgia near the Black Sea coast. Six other Georgian cultivars exhibited the “Saperavi” (ATT) haplotype. Among these was the well-known Saperavi cultivar, which is now mainly distributed in Eastern Georgia but is believed to have originated in south-west Caucasus. Only two Georgian cultivars exhibited the “Meskhuri” group haplotype (ATA), as this group comprises mainly West European cultivars (Beridze et al. 2011). Both the B and C chlorotypes of Arroyo-Garcia et al. (Arroyo-Garcia et al. 2006) are combined in the haplotype ATA according to these data (Beridze et al. 2011).The genomic DNA of Georgian grape cultivars was also studied by nuclear microsatellite analysis based on 20 nuclear microsatellites and no close relationship between Georgian and Western European cultivars was found (Imazio et al. 2013).In the present investigation,