Cloning Of Novel Genes Related To Self-Incompatibility And Identification Of Self-Compatibility In Japanese Apricot

Japanese apricot(Prunus mume Sieb. et Zucc.) originates in China and it is a fruit crop with a typical of gametophytic self-incompatibility (GSI), but some self-compatible (SC) varieties appeared during long-term evolution. Previous studies have shown that GSI is controlled by S locus, which consists of a pistil determinant S-ribonuclease gene (S-RNase gene) and a pollen determinant gene (SFB/SLF gene). In order to enrich the information available on S-genotypes and investigate the mechanisms of SC in Japanese apricot, this study cloned new SI genes and identified SC using the materials collected from the national germplasm nursery of Japanese apricot, located at Nanjing Agricultural University. The results of this study could provide a reasonable basis for the appropriate arrangement of pollination trees and the parental set of bybridization in orchard plant of Japanese apricot. In addition, these results provide the foundation for further explore the molecular mechanisms of SC of Japanese apricot. The main results of this study are as follows:1. First, AS-PCR was conducted on the genomic DNA of each of the18Japanese apricot cultivars using the Prunus S-RNase consensus primer pair, Pru-C2and PCE-R, which was designed from the second and third conserved regions of Prunus S-RNases, respectively. The PCR products were analysed by1.5%agarose gel electrophoresis, two distinct bands were generated in each of eight cultivars and one amplification band was observed in other five cultivars. Then, the PCR products which had one or none band were analysed by6.0%polyacrylamide gel electrophoresis (PAGE) possessing a better separation and resolution. Finally, two bands were gained by PCR of genomic DNA of each of the14Japanese apricot cultivars, only one band was observed in each of the other four cultivars. By cloning and sequencing,13S-RNase alleles (S1, S2, S7, Sp, S12, S14, S15, S18, S20, S22, S24, S23and S26) and seven new S-RNase alleles (S30, S31, S32, S33, S34, S35and S36) with GenBank Accession Numbers JN232975, JN232976, JN232977, JN232978, JX065222, JX065223, JX065224were identified.2. For the identification of SFB genes, PCR was carried out on the genomic DNA of each of the12Japanese apricot cultivars using the Prunus SFB primer pair. SFB-ClF and Pm-Vb, which was designed from the conserved regions of Prunus SFB: the F-box motif and region downstream of variable HVb, respectively. Only one PCR amplification fragment of approximately1000bp was obtained from each cultivar by agarose gel electrophoresis. Cloning and sequencing results revealed that each of the12cultivars possessed two different SFB alleles and12new SFB alleles were identified. The new SFB alleles with GenBank Accession Numbers were as follows:PmSFB2(JQ356589), PmSFB12(JQ356586), PmSFB14(JQ356596), PmSFB18(JX065217), PmSFB22(JQ356584), PmSFB24(JQ356595), PmSFB31(JQ356588), PmSFB34(JQ356597), PmSFB40(JQ356585), PmSFB41(JQ356593), PmSFB42(JQ356581) and PmSFB43(JQ356578).For PmS-RNase and PmSFB genes identified in this study, sequence analysis and expression analysis of RT-PCR showed that they shared typical structural features with S-RNase and SFB genes from other Prunus species, respectively. PmS-RNase genes were expressed specifically in the style, and PmSFB alleles were specifically expressed in pollen.3. Two Japanese apricot cultivars’SichuanBaimei’and’Changnong17’native to China were confirmed to be SC through field self-pollination tests. Further analysis of AS-PCR showed that there were insertion mutation in pollen SFB genes of the two cultivars, which possibly resulted in self-compatibility of these two cultivars.4. In this study, we confirmed SC of ’Zaohong’ through replicated self-pollination tests. Cross-pollination tests showed that SC of’Zaohong’was caused by a loss of pollen function. Sequence analysis and expression analysis based on RT-PCR of the S-haplotypes of’Zaohong’ showed there were no mutations which were likely to alter gene function and no differences at the expressive level when compared with ’Xiyeqing’, a self-incompatible (SI) cultivar with the same S haplotypes. In addition, a new type of F-box gene encoding a previously uncharacterised protein was identified. These results suggest that SC in’Zaohong’occurs in pollen, and that other factors outside the S-locus, including PmF-box genes, might be associated with the loss of function of pollen S genes.