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We developed 708 gene-based markers for citrus genome analysis. Sequence-tagged site (STS) primers were designed that were located in conserved exon regions and whose PCR products spanned genomic introns. Of these, 79.7 % comprised cleaved amplified polymorphic sequence markers. The gene-based markers and their annotation and position on Clementine scaffolds ver. 1.0 permitted comparison of the genetic map and the Clementine genome sequence. The 708 gene-based markers were used to construct a genetic map using the 87 progenies (AG population) from the cross between ‘Okitsu 46 gou’ (‘Sweet Spring’ (‘Ueda unshiu’ (Citrus unshiu) × Hassaku (Citrus hassaku Hort. ex Tanaka)) × ‘Trovita’ orange) × ‘Kankitsu Chukanbohon Nou 5 gou’ (‘Lee’ (Citrus clementina × tangelo) × Citrus kinokuni). The markers were integrated using common STSs on different phase maps in cross-pollination mode. The integrated map (AGI map) comprised 706 loci, including two morphological traits, and spanned 990.9 centimorgans (cM) with an average marker distance of 1.40 cM. These markers formed nine linkage groups (LGs) (corresponding to citrus physical chromosomes): LG-01 to LG-09 corresponded to Scaffold_01, Scaffold_07, Scaffold_09, Scaffold_06, Scaffold_03, Scaffold_02, Scaffold_04, Scaffold_08, and Scaffold_05, respectively. LG-08 and LG-09 contained morphological traits controlling embryo color and seedlessness. Eighty-eight loci comprised three or more alleles on the AGI map; 36.4 % of them were related to transcription factors and DNA-binding proteins. The 708 gene-based markers and the AGI map are valuable for integrating various citrus genetic maps, alignment of genomic sequences, chromosome assignment, and understanding the diversity of citrus germplasms.

Citrus tristeza virus (CTV) is an acute pathogen that causes serious damage to the citrus industry. Poncirus trifoliata (L.) Raf., a sexually compatible species with Citrus, has resistance against a broad range of CTV strains. Breeding programs have been conducted to introduce the CTV resistance gene from P.trifoliata to Citrus, but no commercial cultivar has yet been developed. In this study, we developed four selection markers linked to CTV resistance to enable marker assisted selection to efficiently introduce CTV resistance into Citrus. The four CTV resistance-linked markers were composed of a co-dominant single nucleotide polymorphism (SNP) marker and three dominant sequence tagged site (STS) markers. All four markers were fitted to the progeny and were linked to CTV resistance, with only 2.8% exceptions. We also developed 46 P.trifoliata allele identification markers from alleles of 35 Citrus species. Among the 46 markers, nine were located in linkage group 2, on which the CTV resistance locus is located. We located the other 31 markers on the rest of the linkage groups so that these markers could be used to distinguish P.trifoliata genome regions remaining in the hybrid progenies. The set of PCR primers developed in this study will be useful for marker assisted backcrossing to introduce the P.trifoliata CTV resistance gene into Citrus.

Most citrus tristeza virus (CTV) strains infect almost all Citrus species and have caused serious damage on citrus production in many countries. Poncirus trifoliata (trifoliate orange) has a single dominant resistance gene effective against a broad range of CTV strains. P. trifoliata is sexually compatible with Citrus species, and introgression breeding programs have been initiated to introduce the CTV resistance into Citrus. “Kankitsu Chukanbohon Nou 8 Gou” (Nou-8) was developed by crossing Citrus sp. “Kiyomi” with an F1 hybrid derived from Citrus hassaku × P. trifoliata, but it still had undesirable eating traits. In aim to progress marker-assisted introgression breeding for CTV resistance efficiently, we selected 189 DNA markers which could be applied to genotyping for Citrus and P. trifoliata and constructed a genetic linkage map of Nou-8. The map included 189 DNA markers on 10 linkage groups and extended about 722.8 cM in total length. CTV resistance locus was located at 2.4 cM position in linkage group 02_(1), with the same location found when progeny were scored for resistance using an ELISA assay and for DNA marker for CTV resistant (CTV103). Graphical genotypes of Nou-8 and its progeny suggested that (1) 36 % of the chromosome in the BC’2 population were inherited from Nou-8 without any recombination sites, (2) Nou-8 had been already completely substituted by Citrus genotype in two linkage groups, and (3) four of 93 progeny of Nou-8 had recombination sites in the flanking region of the CTV resistance gene locus.