Chloroplast DNA (cpDNA) restriction fragment length polymorphisms were used to study the parentage of lemons [Citrus limon (L.) Burm. f.] and several other putative hybrids. The 30 citrus accessions studied included nine lemons, three pummelos [C. maxima (Burm.) Merrill], three citrons (C. medica L.), three mandarins (C. reticulata Blanco, C. clementina Hort. Ex. Y. Tan., and C. sunki Hort. ex. Tan.), and single representatives of 13 other taxa. Four different fragments of citrus cpDNA were amplified by polymerase chain reaction using four universal chloroplast primers and amplification products were digested with four endonucleases, Rsa I, Dra I, Hae III, and Mbo I. A total of 104 different restriction fragments were scored and used for phylogenetic analysis by parsimony. The three taxa which have been proposed as ancestral Citrus L. sp., C. medica, C. maxima, and C. reticulata, had distinct cpDNA patterns. Pummelo contributed the chloroplast genome to lemons, sweet orange [C. sinensis (L.) Osbeck], and Bergamot orange (C. bergamia Risso and Poit.), and mandarin contributed the chloroplast genome to rough lemons (C. jambhiri Lush.), Rangpur lime (C. limonia Osbeck), and `Mexican' lime [C. aurantifolia (Christm.) Swing.]. Data suggest that the particular accessions of these ancestral species that were studied were not directly involved in the hybridization events that created these hybrids, since they have similar but not identical cpDNA restriction fragments. Using inter-simple sequence repeat markers that amplified from nuclear genomic DNA, a set of samples including pummelos, citrons, mandarins, sour oranges (C. aurantium L.), and `Samuyao' papeda (C. micrantha Wester) were tested as possible parents of lemons. Sour orange and citron together had all nuclear and chloroplast fragments found in lemon and are therefore proposed to be the maternal and paternal parents, respectively, of many commercial lemon cultivars, including `Lisbon', `Eureka', `Villafranca', and `Monachello'.
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