| 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.In pear, to my knowledge, only one stylar-part SC mutant of Japanese pear Osa-Nijisseiki has been extensively studied, however, there were some discrepancies regarding the exact cause of SC mutation. Many china-native pear cultivars could set economic amount of fruits on self-pollination, but the reason for it is not yet known. In the present study, genetic and molecular approaches were used to investigate the mutational mechanism of three naturally occurring SC mutant.Previous investigations have shown that the S4-allele in Osa-Nijisseiki (S2S4SM; SM= stylar-part mutant), a sport from Nijisseiki, exhibits style-specific inactivation, but behaves as a functional S4-allele in pollen. The S2 allele of Osa-Nijisseiki is functional in both the style and pollen. But how S4 mutated to S4SM, e g, whether S4-RNase gene deleted in the genome of Osa-Nijisseiki, expressed and transmitted to its progeny were not dear. In this paper, field pollination and microscopic observations of pollen tube growth were carried out, and results confirmed that Osa-Nijisseiki and its SC progeny, Akibae (S4SMS5) and 54S-135 (S4SMS4SM), displayed SC character, only on the stylar part, the pollen function normally in SI response. Molecular S-RNase genotyping detected both S2- and S4-RNase gene fragments in both Osa-Nijisseiki and Nijisseiki genome, with S4-RNase in a highly reduced level in Osa-Nijisseiki compared with that in Nijisseiki, and without perceivable difference in S2-RNase gene signal. However, in Akibae, one fragment was found which turned out to be S5-RNase, as the fragment could not be digested by S4-RNase gene specific restriction endonucleases (NdeI). As for 54S-135 and seedlings derived from its selfed progeny, no PCR products was detected, which indicated that S4-RNase did not exist in their genomes, as well as in that of Akibae. S-allele-specific detection of S-RNase transcripts also confirmed the deletion of S4-RNase gene in 54S-135 and Akibae, of course, not in Nijisseiki, the presence of S2-RNase gene in Osa-Nijisseiki and Nijisseiki, and S5-RNase gene in Akibae. Nevertheless, a faint band of transcripts was found in Osa-Nijisseiki style mRNA, which showed the normal S4-RNase transcription in Osa-Nijisseiki style. IEF-PAGE analyses of stylar proteins further evidenced the S4-RNase protein in Osa-Nijisseiki style (especially in stigma), though less than that in Nijisseiki. When selfed Osa-Nijisseiki progeny were S-genotyped, they segregated into two class: S2S4SM and S4SMS4SM, with only S2-RNase gene fragments detected in S2S4SM class, and no signal found in S4SMS4SM one. All these results pointed to the conclusion that S4-RNase exist chimerically in the L1 layers of apical meristem of Osa-Nijisseiki, and functioned normally at transcriptional and translational level, but lacked in L2 layers, in that no S4-RNase was detected in its progeny.Yali(Y), a China-native leading pear cultivar, displaying typical S-RNase-mediated GSI. However, Jinzhuili (Z), a sport from Yali, is self-compatible (SC). How Z mutated to SC is unclear to date. In this study, field pollination test were carried out under four crosses: A (Z selfed), B (Y selfed), C (Z×Y), and D (Y×Z), and fruit-set percentage determined and pollen tube growth inspected under fluorescent microscope. It were observed that more than 75% flowers set fruit and many pollen tubes grew down to the base of style in crosses of A and D, whereas fruit setting did not effected, and most pollen tubes were arrested at the upper part of style in the cross B and C. These results indicated that both Y and Z functioned normally in SI response on the stylar part and could reject competent Y pollen, but accept Z pollen。Further molecular analyses revealed that both Y and Z encompass S21- and S34-RNase, behaved normally at the transcriptional level, and were identical to one another in mRNA sequences of two S-RNase genes. All these evidence prompted us conclude that Z lost pollen SI function, and its style behave normally in SI response as that of its wild type Y does. After molecular S-genotyping of progeny derived from selfing Z, they grouped into two types: one genotyped as heterozygous S21S34, another as homozygous S34S34, with an approximate ratio of 1:1, whereas no homozygous S21S21 was found. The results suggested that S21-allele pollens functioned in SI, and S34-allele pollens failed, thus Z is S-genotyped as S21S34PPM. How S34-allele pollen of Z mutated await further investigations.Daguohuanghua (D) is a naturally occurring larger-fruit-sized bud mutant from Huanghua (H). After field pollination, fruit set percentage were determined and pollen tube growth inspected under fluorescent microscope. It were observed that 60% flowers of D set fruits and many pollen tubes grew down to the base of style, whereas 1.5% flowers of H did and most pollen tubes were arrested at the upper part of style, after self pollination. The results indicated that H were SI, and D SC. Interestingly, styles of H accepted pollens from D, but pollens from H were rejected by styles of D, which indicated that D lost pollen SI function, its style function normally. Molecular S-genotyping revealed that both H and D harbour S1- and S2-RNase, and behaved normally at the transcriptional level. These results confirmed that D performed normally as H did in SI response on the stylar part. After chromosome preparation were made of both cultivars and chromosome numbers of both counted, it were observed that D contained 68 chromosomes, and H 34, which indicated that D were tetraploid mutant (4n=68) from H (2n=34), thus D were genotyped as S1S1S2S2. Comparison on ovary and anther size of both shown that D were significantly larger than H in ovary and anther size, further confirming the tetraploidy of D. After investigation of S-genotypes of progeny obtained by selfing D, all the progeny segregated into three classes of heterozygous genotypes: S1S1S1S2, S1S1S2S2 and S1S2S2S2, but no individual of homozygous genotype (S1S1S1S1 and S2S2S2S2) were found, which indicated that only heteroallelic pollen (S1S2) can overcome self-incompatibility. All these evidences prompted me to concluded that tetraploidization of D leaded to the breakdown of self-incompatibility through a so-called "competitive interaction".In this paper, stylar part SC mutant of Osa-Nijisseiki were systematically investigated, and provided with evidence that S4-RNase did exist in Osa-Nijisseiki, though less than that in Nijisseiki, and expressed normally at both transcriptional and translational level. However the little S4-RNase could not transmitted to its progeny. Besides, two pollen part mutants were studied for the first time, and results showed that the two PPMs employed different strategy to overcome self-incompatibility. One likely resulted from pollen-S mutation, and the other chromosome duplication. They were the first two cases of PPMs investigated in Pyrus to date.
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