Flow cytometry, SSR and modified AFLP markers for the identification of zygotic plantlets in backcrosses between ‘Femminello’ lemon cybrids (2n and 4n) and a diploid clone of ‘Femminello’ lemon (Citrus limon L. Burm. F.) tolerant to mal secco disease

Abstract

One of the most important goals in the genetic improvement of lemon in the Mediterranean area is certainly the obtainment of new genotypes tolerant or resistant to mal secco disease. Two lemon cybrids (one diploid and one autotetraploid) with an intermediate degree of resistance to mal secco disease, spontaneously obtained by symmetric protoplast fusion between ‘Valencia’ orange and ‘Femminello’ lemon, were used as mother plants in backcrosses with a diploid clone of ‘Femminello’ lemon tolerant to the mal secco disease (LTMS) to improve tolerance and fruit quality. Since ‘Femminello’ lemon normally reproduces apomictically by nucellar embryony, an embryo-rescue technique was applied 105 days after pollination to recover zygotic embryos. Sixty-six plantlets were regenerated as follows: 20 from the 4nX2n cross and 46 from the 2nX2n cross. In order to distinguish zygotic embryos from nucellars, flow cytometry was applied on all the 66 plantlets; and a modified amplified fragment-length polymorphism (AFLP) technique and microsatellite analysis were carried out on a sample of 20 seedlings (six triploids and 14 diploids). Here, we report the usefulness and reliability of the tested techniques and discuss the results obtained. Flow cytometry (FCM) was useful only in inter-ploid crosses, identifying six triploids, four of which were also detected by simple sequence repeat (SSR) analysis and only two by modified AFLP. Among the 14 tested diploids, five zygotic genotypes were identified by SSR and four by modified AFLP. In our hands, the SSR technique appeared to be more suitable than modified AFLP.