Observation And Related-genes Isolation Of Self-incompatibility In Guanxi Pomelo And Duwei Pomelo (Citrus Grandis Cv.)

Self-incompatibility (SI) is a endemic ability of carpophyte which inhibits self-fertilization. SI systems are controlled by a single genetic locus called S-locus in heredity. Molecular information shows that there are three types of SI at present, the single -locus sporophytic SI and two distinctforms of single-locus gametophytic SI.)Seed abortions in Guanxi pomelo and Duwei pomelo (Citrus grandis cv.) are closely correlated with pollination and fertilization failure caused by self-incompatibility. Therefore, in this paper, we have studied and discussed on self-incompatibility in these two pomelo cultivars. The results are as follows:1. With the technology of fluorescent microscopy, the compatibility of self- and cross-pollination of two pomelo cultivars, Guanxi pomelo and Duwei pomelo, was studied in the experiment. Results showed that both of self- and cross- pollens could germinate normally after pollination on the stigma. In the case of self-pollination, most of the pollen tubes had ruptured when elongating with appearance of exudant after the pollen tubes growed into the stigma tissue. Some callose was found along the inner fringe of stylar canals, some pollen tubes could get into the ovaries , then into ovules through suspensor. The callose spots accumulated in nucellar tissue of both varieties. In most embryo sacs of Guanxi pomelo and some embryo sacs of Duwei pomelo there were high-intensity fluorescent lightspots in self-pollination. However all these phenomena could not be observed in cross-pollination. These results indicated that there were a series of self-incompatible reactions occurred in the whole process of elongating from stigma to ovaries and ovules in these two varieties.2. The pollen germination and growth of Guanxi pomelo and Duwei pomelo was studied in extractions from different Pistil parts. Results showed that there could be a series of self-incompatible reactions mainly occurred in the ovaries and only low-grade reactions in the stigma, which was defferent from the results showed that these reactions of self-incompatiblity in other pomelo cultivars mainly ocurred in the styles.3. To observe the effect of different pistil parts, we cut pistil into stigma, stylar and ovaries respectively which were replanted on culture medium, and pollinated on them. Our study showed that both of self and cross pollens could germinate and elongate normally after pollination on different pistil parts. There were some intensive self-incompatible reactions on the ovaries of Guanxi pomelo and Duwei pomelo in self-pollination, and also high-intensity fluorescent lightspots in most embryo sacs of Guanxi pomelo. These demonstrated that the ovaries was a main area of self-incompatible reactions of Guanxi pomelo and Duwei pomelo, and embryo sac was the main inhibition area of Guanxi pomelo that lead to abnormal fertilization.4. The development of ovule was studied after self- and cross-pollination in Guanxi pomelo and Duwei pomelo by paraffin section. Guanxi pomelo after self-pollination had two ovular developmental patterns (Type I and Type II). In Type I, nucellar cells were deformed 5 days after pollination (ADP), followed by the atrophy of embryo sac, but outer integument continued to develop for 50 ADP. in Type II, endosperm developed abnormally and eventually aborted after pollination. There were no endosperm in Duwei pomelo after self- pollination. Nucellar cells and embryo sac simultaneously atrophied 35 ADP. The development of ovule in crosses between Guanxi pomelo and Duwei pomelo was normal and result in normal seed formation. These results demonstrate that Guanxi pomelo and Duwei pomelo are self-incompatible, which is the main reason for seedless in fruit. The recognition response and inhibition process occurred in different development stage, before fertilization in Type I and after fertilization in Type II, respectively. In contrast, self-cross pollen tubes in Duwei pomelo are unable to reach embryo sac and self-incompatible reaction occurred only before fertilization. It appears that aborted seed sizes are related to the time of self-incompatible reaction.5. To identify the genes controlling self-incompatibility in Guanxi pomelo, sup-pression subtractive hybridization (SSH) was performed using cDNAs prepared from Guanxi pomelo Pistil treated with self-pollination as testers and cDNAs from pollen as drivers. A forward subtractive cDNA library was constructed, from which 80 recombin- ant colonies were picked and amplified. After sequencing, 42 unique expressed sequence tags (ESTs) were obtained. By clustering and blast analysis in genebank, there are 23 transcripts with significant protein homology were sorted into 16 functional categories, which included 4 transcripts that had previously been reported as responsive to self-incompatibility: myo-inositol 1-phosphate synthase(CGF2030,CGF2011),calcium-dependent protein kinase (CDPK, CGDH2035),phosphatase (CGDF107),GTPase binding (CGS10601). With the exclusion of them, there are also some other functionally unknown transcripts were obtained from the subtractive cDNA library.It is important to analyze the functions of their corresponding full-length genes not only for further understanding of the molecular mechanisms of self-incompatibility in Guanxi pomelo, but also for modulating self-incompatibility of high plants by genetic manipulation.