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, 117 (4), 565-83

Phylogenetic Origin of Limes and Lemons Revealed by Cytoplasmic and Nuclear Markers

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Phylogenetic Origin of Limes and Lemons Revealed by Cytoplasmic and Nuclear Markers

Franck Curk et al. Ann Bot.

Abstract

Background and aims: The origin of limes and lemons has been a source of conflicting taxonomic opinions. Biochemical studies, numerical taxonomy and recent molecular studies suggested that cultivated Citrus species result from interspecific hybridization between four basic taxa (C. reticulata,C. maxima,C. medica and C. micrantha). However, the origin of most lemons and limes remains controversial or unknown. The aim of this study was to perform extended analyses of the diversity, genetic structure and origin of limes and lemons.

Methods: The study was based on 133 Citrus accessions. It combined maternal phylogeny studies based on mitochondrial and chloroplastic markers, and nuclear structure analysis based on the evaluation of ploidy level and the use of 123 markers, including 73 basic taxa diagnostic single nucleotide polymorphism (SNP) and indel markers.

Key results: The lime and lemon horticultural group appears to be highly polymorphic, with diploid, triploid and tetraploid varieties, and to result from many independent reticulation events which defined the sub-groups. Maternal phylogeny involves four cytoplasmic types out of the six encountered in the Citrus genus. All lime and lemon accessions were highly heterozygous, with interspecific admixture of two, three and even the four ancestral taxa genomes. Molecular polymorphism between varieties of the same sub-group was very low.

Conclusions: Citrus medica contributed to all limes and lemons and was the direct male parent for the main sub-groups in combination with C. micrantha or close papeda species (for C. aurata, C. excelsa, C. macrophylla and C. aurantifolia--'Mexican' lime types of Tanaka's taxa), C. reticulata(for C. limonia, C. karna and C. jambhiri varieties of Tanaka's taxa, including popular citrus rootstocks such as 'Rangpur' lime, 'Volkamer' and 'Rough' lemons), C. aurantium (for C. limetta and C. limon--yellow lemon types--varieties of Tanaka's taxa) or the C. maxima × C. reticulate hybrid (for C. limettioides--'Palestine sweet' lime types--and C. meyeri). Among triploid limes, C. latifolia accessions ('Tahiti' and 'Persian' lime types) result from the fertilization of a haploid ovule of C. limon by a diploid gamete of C. aurantifolia, while C. aurantifolia triploid accessions ('Tanepao' lime types and 'Madagascar' lemon) probably result from an interspecific backcross (a diploid ovule of C. aurantifolia fertilized by C. medica). As limes and lemons were vegetatively propagated (apomixis, horticultural practices) the intra-sub-group phenotypic diversity results from asexual variations.

Keywords: Citrus; SNP; SSR; indel; lime and lemon origin.; phylogenetics.

Figures

F<sc>ig.</sc> 1.
Fig. 1.
Cytoplasmic type of the 133 citrus accessions. Neighbor–Joining tree established from three mitochondrial indels and five chloroplastic simple sequence repeats (SSRs). Blue numbers: bootstrap value given to each edge indicates the frequency of occurrence of this edge in the bootstrapped trees. Blue line: scale of edge lengths.
F<sc>ig</sc>. 2.
Fig. 2.
Distribution of the highest specific GST values. (A) Comparative distribution of the three kinds of markers [simple sequence repeats (SSRs), indels and single nucleotide polymorphisms (SNPs)]. (B) Diagnostic value of the 123 markers for the ancestral taxa. (C) Diagnostic value of the 73 selected markers for STRUCTURE and specific allele homozygosity/heterozygosity (Hom/Het) analysis of the ancestral taxa. N, non-diagnostic markers.
F<sc>ig.</sc> 3.
Fig. 3.
Percentage contribution of the four ancestral taxa to the 90 MLGs. STRUCTURE analyses using 73 indels and SNP markers (average values for ten runs with K = 4) (blue, Citrus maxima; red, C. reticulata; green, C. micrantha; yellow, C. medica). The boxes at the top of each panel show the distribution of lemons and limes in the different nuclear clusters and sub-clusters (A–G) and the different types of cytoplasm (CT1–CT6)
F<sc>ig.</sc> 4.
Fig. 4.
Contribution of the four ancestral taxa to the 90 MLGs. Analyses of the frequency of homozygosity and heterozygosity (%) using the four specific sets of diagnostic markers (total of 73 specific markers). Orange, C. medica homozygosity; yellow, C. medica heterozygosity; red, C. reticulata homozygosity; pink, C. reticulata heterozygosity; deep blue, C. maxima homozygosity; light blue, C. maxima heterozygosity; dark green, C. micrantha homozygosity; light green, C. micrantha heterozygosity).
F<sc>ig</sc>. 5.
Fig. 5.
Pattern of relative allele fluorescence for the 9P25060404 SNP marker and inference of allele dosage for four triploid limes (AGG, ‘Tanepao’ and ‘Coppenrath’ limes; AAG, ‘Tahiti’ and ‘Persian’ limes). Horizontal axis, dose of allele A; vertical axis, dose of allele G; red cluster (Y:Y), GG homozygote accessions; green cluster (Y:X), AG heterozygote accessions; blue cluster (X:X), AA homozygote accessions; black cluster (NTC) AGG, triploid accessions with two doses of allele G and one dose of allele A; black cluster (NTC) AAG, triploid accessions with two doses of allele A and one dose of allele G.
F<sc>ig</sc>. 6.
Fig. 6.
Hierarchical classification of triploid limes using 123 genetic markers. Blue numbers, the bootstrap value given to each edge indicates the frequency of occurrence of this edge in the bootstrapped trees. Blue line, scale of edge lengths.
F<sc>ig</sc>. 7.
Fig. 7.
Contribution of the four ancestral taxa to the ten triploid limes. Analysis of the frequency of homozygosity and heterozygosity doses 2/3, 1/3 for the four specific sets of diagnostic markers (total of 73 markers). C., Citrus; mic, micrantha; med., medica; max., maxima; ret., reticulata.
F<sc>ig.</sc> 8.
Fig. 8.
Origin of the main lime and lemon varietal sub-groups.

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