S. L. Zhang

Competitive interaction between two functional S-haplotypes confer self-compatibility on tetraploid Chinese cherry (Prunus pseudocerasus Lindl. CV. Nanjing Chuisi)

Self-incompatibility (SI) has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility (GSI). In the present study, four PpsS-haplotypes (Prunus pseudocerasus S-haplotypes) comprising at least two genes, i.e., PpsS-RNase (P. pseudocerasus S-RNase) and PpsSFB (P. pseudocerasus S-haplotype-specific F-box) have been successfully isolated in tetraploid P. pseudocerasus Lindl. CV. Nanjing Chuisi (“NC”) which exhibited self-compatibility (SC), and its S-genotype was determined as S-1/S-3′/S-5/S-7. These PpsS-RNases, which were expressed exclusively in style, shared the typical structural features with S-RNases from other Prunus species exhibiting GSI. All PpsSFBs showed similar structure characteristics of SFBs from other Prunus species, and matched with the necessary conditions for pollen S-determinant. No mutations leading to dysfunction of S-haplotype were found in their full-length c-DNA sequences, except for PpsS-3′-haplotype which was not amplified by PCR. These four S-haplotypes complied with tetrasomic inheritance. Diploid pollen grains with S-genotypes S-7/S-1, S-7/S-5 and S-1/S-5 can grow the full length of the style after self-pollination, while pollen grains with S-3′/S-7, S-3′/S-1 and S-3′/S-5 cannot. These results suggest that PpsS-haplotypes-1, -5 and -7 are functional, and that competitive interaction between two of them confer self-compatibility on cultivar “NC”. Furthermore, in terms of recognition specificity, diploid pollen grains carrying PpsS-3′-haplotype are equal to monoploid pollen grains carrying the other functional S-haplotype.

Identification of S-haplotype-specific S-RNase and SFB alleles in native Chinese apricot (Prunus armeniaca L.).

Summary Chinese 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 similarity of the SFB alleles was 73.7 – 98.6%. The physical distances between the SFB and S-RNase genes was determined exactly in the S9,S11,S17, and S26-haplotypes, confirming that the S-RNase and SFB genes were linked. The range of distances between the two genes was 299 – 1,061 bp. This study increases our knowledge on the S-genotypes of apricot native to China, and enriches our genomic information on GSI in the Prunus genus.

Identification of S-genotypes in 17 Chinese cultivars of Japanese plum (Prunus salicina Lindl.) and molecular characterisation of 13 novel S-alleles

Summary Polymerase 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 numbers of nucleotide differences in the intron regions. Phylogenetic analysis of the 13 novel S-alleles, and those of other species in the family Rosaceae, resulted in two distinct groups which correlated with their sub-family classification (i.e., the Maloideae and the Prunoideae). These data should be useful in breeding programmes, for choosing suitable pollinators, and may also contribute to studies on S-allele function, the evolution of new allele specificities, and the taxonomy and speciation of Prunus.

Identification of S-genotypes and novel S-RNase alleles in Prunus mume

Summary Prunus 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.

Identification of S-genotypes and novel S-RNases in native Chinese pear

Summary Chinese 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 Yü ‘Aolian’ (SpS32), P. phaeocarpa Rehd. ‘Diaodan’ (SdSe), P. xerophila Yü ‘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 to S11-RNase (100% ) and S6-RNase (94.4%) between the C1 and C3 exons in Malus. The high similarity scores between S-RNases in Pyrus and Malus indicate that the existence of S-RNases predated speciation between Pyrus and Malus.

Identification of differentially expressed genes in a spontaneous mutant of ‘Nanguoli’ pear (Pyrus ussuriensis Maxim) with large fruit

Summary The 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 quantitative real-time polymerase chain reaction (qRT-PCR). In addition, 98 TDFs, representing candidate genes, were studied in more detail to determine their functions in order to dissect the complex molecular mechanisms involved in the fruit-size mutation.

Two Different Prunus SFB Alleles Have the Same Function in the Self-incompatibility Reaction

Many species in the families of Rosaceae, Solanaceae, and Scrophulariaceae exhibit gametophytic self-incompatibility, a phenomenon controlled by two polymorphic genes at the S-locus, style-S (S-RNase) and pollen-S (SFB). Sequences of both genes show high levels of diversity, characteristic of genes involved in recognition of self-incompatibility systems in plants. In this study, S24-RNase and SFB24 alleles were cloned from Prunus armeniaca cv. Chuanzhihong (Chinese apricot). Sequence comparisons of deduced amino acid sequences revealed that the P. armeniaca S24-haplotype has different SFB alleles, but shares a single S-RNase allele with P. armeniaca S4-haplotype. Moreover, P. armeniaca S24-RNase haplotype has a single and three different alleles with S1-RNase of P. tenella (dwarf almond) and S1-RNase of P. mira (smooth pit peach), respectively. The functionalities of SFB24 and SFB4 have been evaluated by pollen tube growth and controlled field tests of P. tenella and P. mira. Genetic analysis of the two intercrosses showed that progenies segregated 1:1 into two S-genotype classes, which is consistent with the expected ratio for semi-compatibility. These findings imply that the allelic function of the S24-haplotype is identical to that of the S4-haplotype in a self-incompatibility reaction. Thus, these two Prunus S-haplotypes are in fact two neutral variants of the same S-haplotype. The evolution of the S-allele is also discussed in terms of both functions and differences between S24- and S4-haplotypes in Prunus.

A cDNA Clone of BcHSP81-4 from the Sterility Line (Pol CMS) of Non-heading Chinese Cabbage (Brassica campestris ssp. chinensis)

BcHSP81-4 gene, a member of heat shock proteins, was identified from a suppression subtractive hybridization cDNA library in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). The deduced amino acid sequence of the BcHSP81-4 cDNA revealed that it has high homology to other plant organelle isoforms and similar homology to both cytoplasmic and prokaryotic HSP90s. To study the regulation of gene expression, BcHSP81-4 genes in maintainer and sterility lines were monitored at different development stages and at different stress treatments. Real-time PCR was used for quantification of BcHSP81-4 mRNA. These results indicate that BcHSP81-4 is not responsive to heat shock at least at 35°C, while it is very responsive to salt and cold stress. And high expression of BcHSP81-4 in the bud of sterile line suggests that it may play prominent roles in sterility of pol CMS in non-heading Chinese cabbage.

Instability in mitochondrial membranes in Polima cytoplasmic male sterility of Brassica rapa ssp. chinensis

Cytoplasmic male sterility (CMS) is an important factor to observe heterosis in Brassica rapa. Although several studies have documented the rearrangements of mitochondrial DNA and dysfunction in the mitochondria have been observed in most types of CMS, the basis of the molecular mechanisms involved in these processes and other effects on CMS remain unclear. In this study, suppression subtractive hybridization was performed in the flowers of an alloplasmic Polima CMS system from B. rapa ssp. chinensis to identify genes that are differentially expressed between fertile and sterile plants. A total of 443 clones were isolated (156 were upregulated in fertile buds, and 287 were upregulated in sterile ones). Real-time RT-PCR further demonstrated the credibility of SSH. Among these genes, many membrane protein genes (LTP12, PIP2A, and GRP14) were inhibited in the sterile male line. Mitochondrial membrane potential (MMP) assay was then performed. Results showed that the sterile MMP was unstable and failed to create a potential difference; thus, mitochondrial dysfunction occurred. Moreover, abnormal microtubules and photosynthetic pathways were found in sterile male cells. Unstable MMP, nutritional deficiency, and abnormal microtubules were the causes of Polima CMS in Brassica campestris. H2O2, MDA, and O2–, accumulated as byproducts of energy metabolism disorder in sterile male cells.

A method to isolate male gametic nuclei from pear pollen tubes

Summary The 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% (v/v) PercollTM gradient by centrifugation, the isolated nuclei exhibited a high level of purity and integrity, with little debris, as confirmed using enzyme markers and 4’,6-diamidino-2-phenylindole (DAPI) staining. The purified nuclei were used to prepare nuclear proteins and DNA fibres. The present protocol proved suitable for the isolation of male gametic nuclei from in vitro-cultivated pear pollen tubes, for nuclear proteome analysis, and for the preparation of DNA fibres for FISH analysis.