Mutant Mechanism Analyses Of Self-Compatible Pear And Creation Of Self-Fruitful Germplasm Resources In Pyrus

Self-incompatibility (SI) is a genetic mechanism employed by flowering plants to prevent inbreeding and promoting out-crossing, which involves a complex set of cell-cell interactions between the pistil and the pollen, thence, a model system for studying of intercellular information transmission, cell-cell recognition and gene spatial-temporal expression. Extensive studies have been carried out in species of Solanaceae, Rosaceae and Scrophulariaceae, the majority of which display an S-RNase-mediated gametophytic self-incompatibility (GSI). Information available indicates that GSI response is under the genetic control of a single multi-allelic locus, the S-locus, which contains at least two separate genes, S-RNase and SFB/SLF, controlling female and male specificity respectively, hence the name "S-haplotype" to describe variants of the S-locus. Recognition between S-RNase and SFB/SLF of the same S-haplotype triggers an SI reaction.Pear (Pyrus) is a commercially important fruit crop worldwide, but exhibits S-RNase-based gametophytic self-incompatibility, as other Rosaceae species do, and most cultivars require pollinators inter-planted to ensure adequate pollination. However, the fructifications often vary with changing environmental conditions. Selection of superior SC pear cultivars has long been one of the priorities to simplify pear growing and to cut down orchard management cost.SI plants might become spontaneously self-compatible (SC) under the action of many natural agents, though very rarely, which makes SC mutants sources of high interest for breeding purposes and the SC nature investigation. One type of SC mutant is now known to be stylar-part mutant (SPM or SM), as a result of stylar S-RNase gene mutation, such as gene deletion, expressional suppression. Another is pollen-part mutant (PPM), resulting from duplication of pollen S-gene and (or) mutation of pollen S-gene per se, say, insertion and (or) deletion mutation.1. Pear cultivar’Sha 01’is a sport from’Kuerlexiangli’. Field pollination data revealed that’Sha 01’displaying self-compatibility (SC), whereas’Kuerlexiangli’showing self-incompatibility (SI) upon self-pollination. Reciprocal pollinations between the two varieties showed that 76% of’Kuerlexiangli’flowers pollinated with’Sha 01’pollen set fruit, but only 7% of’Sha 01’flowers set fruit when pollinated with’Kuerlexiangli’pollen. The pollen performance was monitored with fluorescence microscopy, and it was observed that’Sha 01’accepted self-pollen but rejected’Kuerlexiangli’pollen, whereas ’Kuerlexiangli’rejected self-pollen but accepted’Sha 01’pollen. Taken together,’Sha 01’ showed SC as a pollen-part mutant. Molecular S-genotyping of ’Sha 01’, its’ selfed progeny and wild type’Kuerlexiangli’showed that all contained S22-RNase and S28-RNase alleles, but showed no nucleotide sequence difference or changes in transcription. After flow cytometry and chromosome number analyses,’Sha 01’was found to be a tetraploid (2n=68) and’Kuerlexiangli’a diploid (2n=34). Thus, the S-genotype for’Sha 01’was S22S22S28S28 and for’Kuerlexiangli’was S22S28.Ploidy level of’Sha 01’selfed progeny were also determined from leaves using flow cytometry, all seedlings were tetraploids. The S-genotyping for the progeny identified by S-RNase gene relative optical density for both alleles. Closer inspection of 52 progeny, showed they could be classed into three types S22S22S28S28:S22S22S22S28:S22S28S28S28 with the distribution 28:10:14, no individual homozygous for either allele was found. Therefore, it could be concluded that only heteroallelic diploid pollen S22S28 could achieve’Sha 01’fertilization through a so-called "competitive interaction".2. The pear cultivar’Zaoguan’(S4S34) is the self-compatibility (SC) progeny of’Yali’ x’Qingyun’. Two cultivars’Xinya’and’Yaqing’, also S-genotyped as S4S34 for the S-RNase gene, were used as controls. Field pollination data revealed that’Zaoguan’ displayed SC, whereas ’Xinya’ and ’Yaqing’ showed self-incompatibility (SI) upon self-pollination. Reciprocal pollinations between the varieties showed that most of the ’Zaoguan’ flowers pollinated with ’Xinya’ or ’Yaqing’ pollen set fruits but that few of the ’Xinya’ or ’Yaqing’ flowers set fruit when pollinated with’Zaoguan’pollen. The pollen performance was monitored with fluorescence microscopy, and we observed that’Zaoguan’ accepted self-pollen as well as ’Xinya’ or ’Yaqing’ pollen, whereas ’Xinya’ or’Yaqing’ rejected self-pollen and ’Zaoguan’ pollen. The S34-RNase but not the S4-RNase can be detected in all selfed progeny of ’Zaoguan’. Comparisons of the 2D-PAGE profiles of the stylar extracts from the three cultivars showed that the S4-RNase protein expressed normally, but the S34-RNase of ’Zaoguan’ was not found. Thus, we conclude that the stylar S34 products were defective in ’Zaoguan’ and that the S4-allele functioned normally. The nucleotide sequences of the S4-and S34-RNase of’Zaoguan’showed no differences from those of ’Xinya’ or ’Yaqing’, and they transcribed normally. These results indicate that SC in ’Zaoguan’ was due to the loss of the S34-RNase caused by unknown post-translational factors.3. The genomic DNAs from ’Jinzhuili’ and ’Yali’ were extracted and amplified by PCR with three pairs of primers corresponding to pear SFBB-alpha, SFBB-beta and SFBB-gamma genes, respectively. Blast results showed that ’Jinzhuili’ and ’Yali’ all contained 6 same SFBB gene, such as SFBB21-alpha gene、SFBB21-beta gene、SFBB21-gamma gene、SFBB34-alpha gene、SFBB34-beta gene、SFBB34-gamma gene. Thus, we conclude that the SFBB genes from ’Jinzhuili’ pollen were normal, which was not the reason that caused ’Jinzhui’ self-compatibility. Phylogenetic analysis revealed that 34 rosaceous SFB/SLFs were dived into two subfamily-specific groups, but did not further form species-specific subgroup. The evolutionary pattern of SFB/SLFs concurs with that of rosaceous S-RNases, suggesting that SFB/SLFs occur after divergence of subfamily but before the divergence of species as S-RNases do in Rosaceae.The present study could provide a scientific base for fully clarifying the mechanism of pear GSI at the molecular level.4. A primer pair ’S4smF’and ‘S4sm R’ was designed from the deletion junction sequences in the S4sm-haplotype. Genomic PCR with ’S4smF’ and ’S4smR’ yielded a product of 666 bp from S4sm-haplotype. This indicates that the product of 666 bp can be used as a DNA marker specific for the S4sm-haplotype, except for S4S4sm trees were self-incompatible, and the other trees were selected as self-fruitful trees. From the 26 cross-progeny in’Osa-Nijisseiki’ (S2S4sm)×’Hosui’(S3S5) and 21 cross-progeny in’Osa-Nijisseiki’(S2S/m)× ’Shinseiki’(S3S4), we selected 14 progeny and 4 progeny were as self-fruitful trees, respectively. Therefore, the combination of the S4sm-haplotype specific marker provided an early and reliable selection system for the breeding of self-compatible pear cultivars using the S4sm-haplotype.5. In this paper, bud stage artificial self-pollination and pollen irradiation self-pollination were used to create S-RNase gene homozygote germplasm in Pyrus. Under natural conditions,’Huanghua’ showed the highest fruit set in 43% on the sixth day before flowering by bud stage artificial self-pollination; with the different radiation dose in ’Hosui’ pollen, Co60γ 40Gy dose showed the highest fruit set in 31%. Seven S1-RNase gene homozygous plants and four S2-RNase gene homozygous plants were selected from ’Huanghua’ bud stage selfed progeny. Three S3-RNase gene homozygous plants and two S5-RNase gene homozygous plants were selected from ’Hosui’ pollen irradiation selfed progeny. These S-RNase gene homozygous plants provides valuable experimental material in the future, would be used in pear breeding, pollen and pistil of mutual recognition of cell signal transduction, S gene and its protein product of temporal and spatial expression characteristics of the functional verification and other studies.