Polyploid Isolation Of Ponkan (Citrus Reticulata) And Genetic Diversity Analysis Of Navel Orange (C.sinensis) Cultivars

Somatic mutation, a main biological phenomenon of citrus and other fruits, is often used for breeding as a major variation source. Spontaneous mutations often occur in citrus. In fact, many new cultivars were derived from selection of bud mutation. However, it is difficult to distinguish the mutants by morphology. In present research, 24 cultivars of navel oranges (Citrus sinensis) were analyzed by MSAP (methylation sensitive amplified polymorphism) and other molecular markers to study their genetic diversity. At the same time, based on the properties of ploidy variations in the somatic cells and the polyembryony in Ponkan (C. reticulata), tetraploid plants were screened out from the seedlings in vitro of this variety, which provides useful materials for seedless triploid breeding. The main results were as follows:1,By tissue culture, 449 Ponkan seedlings from 379 seeds were obtained; out of them 7 natural tetraploid plants were screened out by flow cytometry and chromosome counting. SSR (simple sequence repeat) analyses indicated that the 7 tetraploid seedlings were autotetraploid from nucellar cells. The leaves of these autotetraploid seedlings appeared larger than diploid's, while the content levels of chlorophyll a and carotenoids were lower. RAPD, SSR and AFLP techniques were ultilized to analyze variations during somatic doubling. No diversities were found by RAPD and SSR analyses, indicating infrequent recombination. One unique band was observed in one tetraploid by AFLP analysis, which was supposed to be related with the changing of nucleotide sequences or the dispersing of some autotetraploid allelic genes.2,RAPD, SSR, AFLP, SRAP and IRAP analyses were used to detect the variabilites among 24 navel orange cultivars. No polymorphism was found by RAPD, SSR and IRAP. AFLP marker was ineffective, out of 130 pairs of primers, only 5 revealed polymorphisms, distinguishing 4 cultivars. Only one out of the 36 pairs of SRAP primers showed one missing band in 'Skaggs Bonnaza' navel orange.3,Eight Mse I/Pst I (M/P ) primer combinations and eight EcoR I/Hpa II+EcoR I/Msp I(E/H+M) primer combinations generated 36 and 43 polymorphic bands respectivelyamong 24 navel oranges. The average polymorphic ratio (Pi) of M/P and E/H+M were7.56% and 15.19%, respectively; the average polymorphism information content (PIC)were 0.086 and 0.249, marker index (MI) were 0.387 and 1.070, respectively. With...