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SummaryMost pear (Pyrus L.) cultivars exhibit gametophytic self-incompatibility (GSI), controlled by a single S-locus. Crosscompatible cultivars with different S-genotypes must be inter-planted in an orchard for efficient fruit production.Thus, it is essential to identify S-genotypes in order to choose pollinators for fruit production. The S-genotypes of many Chinese pear cultivars have been identified, but some results remain inconsistent. In this study, 15 Chinese pear cultivars with inconsistent S-genotypes were selected for further identification using 37 leaf samples collected from six different regions in China. The S-genotypes of all 15 pear cultivars were determined by polymerase chain reaction (PCR), cloning, and sequencing of the PCR products. The S-genotypes were as follows: ‘Baozhuli’ (S22S42), ‘Cangxixueli’ (S5S15), ‘Hanxiang’ (S12S31), ‘Hangqing’ (S1S4), ‘Jinqiuli’ (S3S9), ‘Jinfeng’ (S17S19), ‘Longxiang’ (S16S42), ‘Nanguoli’ (S11S17), ‘Pingxiangli’ (S31Sd), ‘Qingkui’ (S1S3), ‘Qingxiang’ (S4S...

Generation of disomic alien chromosome addition lines and alien chromosome substitution lines is important for genetic research and chromosome engineering. After determining the transmission rates ...

SummaryThe nucleus plays an essential role in regulating most biological activities in most living organisms. Pear (Pyrus bretshneideri) pollen is binucleate, possessing a vegetative nucleus and a generative nucleus. To reveal the nuclear proteome and to produce accurate physical maps of DNA sequences using fluorescence in situ hybridisation (FISH) analysis, it is essential to obtain high quality nuclei. Protocols to isolate intact nuclei usually involve liberating them from centrifuged cells, then filtering them using a nylon mesh. Releasing the nuclei is the most critical step to obtain high quality material from small amounts of tissue. Here, we describe an easy and efficient method to isolate and purify both the generative and vegetative nuclei from pear pollen tubes. The nuclei were liberated by grinding pollen tubes, filtered on a cell strainer with a pore size of 40 µm, then passing them through a second cell strainer with a mesh size of 25 µm. After these procedures, and purification through a 28%...

SummaryThe selection of mutants is one of the most important steps in horticultural breeding. Fruit size is an important breeding objective in pear (Pyrus spp.) because of its economic importance. The pear cultivar ‘Da Nanguoli’ (Pyrus ussuriensis Maxim) produces larger fruit than ‘Nanguoli’, from which ‘Da Nanguoli’ was a bud mutation. The molecular basis for this bud mutation remains unknown. In the present study, ploidy analysis showed that ‘Da Nanguoli’ did not exhibit any alteration in the overall ploidy level. Seventy-six transcript-derived fragments (TDFs) of genes that were differentially expressed between the two cultivars were sequenced using cDNA-AFLP technology. BLAST-X analysis showed that 26 (34.2%) of the TDFs had high sequence homology with known proteins in the non-redundant National Center for Biotechnology Information (NCBI) protein sequence database (i.e., E-values < –3.00). In total, 17 TDFs were chosen and their patterns of expression revealed using cDNA-AFLP and confirmed using quan...

SummaryIn an integrated cell system, mitochondria play a pivotal role in energy metabolism and the maintenance of cellular homeostasis. In most cases, protocols for the purification of the mitochondrial fraction are based on blenderhomogenisation of the selected tissue and differential centrifugation of the cell homogenate in a Percoll™ gradient. The method of homogenisation usually represents the critical step which affects the yield and quality of the mitochondria isolated from plant cells. To isolate mitochondria from minute, mg-amounts of plant tissue (e.g., pollen tubes), we have developed a new method for the homogenisation of pollen tubes using a 30-µm pore size cell strainer. The isolated mitochondria were shown to be pure, intact, and functional, based on marker enzyme assays, transmission electron microscopy, and flow cytometry analysis, respectively. Furthermore, the isolated mitochondria were used to extract mitochondrial DNA and to analyse mitochondrial proteins. The new method was easy, reli...

SummaryChinese apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility (GSI) controlled by a single locus containing at least two linked genes [i.e., the S-RNase gene and the pollen-expressed SFB (or SLF) gene] as do other fruit species in the family, Rosaceae. To elucidate the S-genotypes of 14 native Chinese apricot cultivars, PCR was performed using primers designed from Prunus S-RNase and SFB consensus sequences. After cloning and sequencing the PCR products, the S-genotypes of all 14 apricot cultivars were determined, and eight new S-RNase alleles and nine SFB alleles were identified. The S-RNases shared typical structural features with S-RNases from other Prunus spp. exhibiting GSI. The SFB genes showed similar structural characteristics to SFB genes in other Prunus spp. The intron sequences of the SFB genes revealed sequence and length polymorphisms. The deduced level of amino acid sequence identity for the eight new S-RNase alleles was 66.4 – 100% in P. armeniaca, while the similarit...

SummaryChinese pear (Pyrus spp.) exhibits gametophytic self-incompatibility (GSI), as do other fruit species in the family Rosaceae. This work determined S-locus diversity in 20 native Chinese pear cultivars or wild accessions using polymerase chain reaction (PCR) and pollination experiments. After cloning and sequencing the PCR products, the S-genotypes of all 20 pear cultivars or wild accessions were determined. Subsequent sequence analysis showed that P. sinkiangensis Yu ‘Aolian’ (SpS32), P. phaeocarpa Rehd. ‘Diaodan’ (SdSe), P. xerophila Yu ‘Shageda’ (S36Sd) and ‘Xingyeli’ (S22Sc), originating in China, shared some S-RNase genes with P. communis, providing evidence that oriental and occidental Pyrus species may share the same pool of alleles at the S-locus. Two novel S-RNase genes were also discovered in P. ussuriensis ‘Maili’ and ‘Neimenggushanli’, and deposited as S40 and S41, under the accession numbers DQ903313 and DQ988687, respectively. Their deduced amino acid sequences showed high similarity t...

SummaryPrunus mume (Japanese apricot) cultivars are self-incompatible and orchards require pollinator cultivars to guarantee optimal fruit production. Identification of the self-incompatibility (S) genotypes of P. mume cultivars is therefore useful for choosing pollinators and for breeding. To elucidate the S-genotypes of eleven Chinese and one introduced cultivar, PCR was performed with primers amplifying the variable second intron of the S-RNase gene. The S-genotyping results were as follows: S4S12 for ‘Dantaofen’; S3S4 for ‘Duoezhusha’; S12S13 for ‘Fubantiaozhi’; S15S16 for ‘Hongding’; S13S14 for ‘Longyanmei’; S5S11 for ‘Musashino’; S10S14 for ‘Xiaoyezhugan’; and S3S15 for ‘Qingfeng’,‘Qingjia’, ‘Xiyeqing’, ‘Dayeqing’ and ‘Changnong 17’. We identified seven new S-RNase alleles, S10-RNase to S16-RNase, deposited in GenBank under the accession numbers: DQ011150, DQ201191, DQ201192, DQ345781, DQ768219, DQ903312 and EF990750, respectively.

SummaryPolymerase chain reaction (PCR) was conducted with Prunus S-RNase gene-specific primers on 17 Chinese cultivars of Japanese plum (Prunus salicina Lindl.). These primers were designed from the conserved regions of Prunus S-RNase genes. Each cultivar produced two amplicons, apart from two cultivars that had three amplicons. In all, 36 amplicons were cloned and sequenced. Analysis of these sequences revealed 13 novel S-alleles, the amino acid sequences of which showed 62% (S15 vs. S19) to 92% (S22 vs. S24) identity. The sequences also demonstrated several typical structural features of Prunus S-RNase genes: three conserved regions (C1, C2 and C3), one hypervariable region (RHV) with one intron, and another intron located at the junction between the signal peptide and the mature protein. Compared to S2 from apricot, S26 from Japanese plum had only two nucleotide substitutions in the exon region, which resulted in only one amino acid residue difference in the signal peptide. However, there were large nu...