Molecular genetic analysis of nucellar embryony (Apomixis) in Citrus maxima x Poncirus trifoliata

Some citrus varieties express the trait of nucellar embryony (NE). The nucellus tissue surrounding the embryo sac gives rise to embryos genetically identical to the seed-producing tree. The phenotype of the trait is commonly called polyembryony because when NE is expressed, multiple embryos can develop within one seed. The cross Citrus maxima ‘Chandler’ (monoembryonic) x Poncirus trifoliata (polyembryonic) provided a population of 88 siblings segregating for NE. Progeny were classified for polyembryony. Molecular marker data were generated for the parents and progeny using Amplified Fragment Length Polymorphism (AFLP) System II with multiplexed PCR. The extreme monoembryonic and polyembryonic phenotypes of this population were used in a bulked segregant analysis screen of 256 primer combinations that identified AFLP markers linked to a major gene ( N) conferring nucellar embryony in P. trifoliata. No locus in ‘Chandler’ was linked to NE. Three of the markers most closely linked to NE in Poncirus were cloned, sequenced, and used to probe a 9-fold coverage BAC library. Each of thirteen positive BAC clones was confirmed to carry one DNA fragment matching the exact mobility of the original genomic AFLP polymorphism when amplified with the appropriate primer combination.; Scoring 42 AFLP primer combinations in the 88 progeny identified over 600 segregating AFLP markers. Molecular marker linkage maps were constructed for C. maxima and P. trifoliata with JoinMap 3.0® using a pseudo-testcross analysis. The map of C. maxima ‘Chandler’ (Ch) had 257 markers distributed across nine linkage groups. The map of P. trifoliata constructed from P. trifoliata ‘Rubidoux’ and ‘Webber Fawcett’ (RW) had 211 markers distributed across 11 linkage groups.; Quantitative trait values for the percentage of polyembryonic seed produced by each hybrid in the population was used in conjunction with the Ch and RW maps for Quantitative Trait Loci (QTL) mapping. The QTL analysis confirmed the major gene N on RW-10 that confers nucellar embryony. Multiple QTL method mapping provided strong evidence that a second independent gene had a large effect on the percentage of polyembryonic seed that a hybrid produced. That locus, named P1 (polyembryony one), explained ∼63% of the variance in the percentage of polyembryonic seed produced by hybrids with NE.