S. J. 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-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.

Inheritance of Hetero-Diploid Pollen S-Haplotype in Self-Compatible Tetraploid Chinese Cherry (Prunus pseudocerasus Lindl)

The breakdown of self-incompatibility, which could result from the accumulation of non-functional S-haplotypes or competitive interaction between two different functional S-haplotypes, has been studied extensively at the molecular level in tetraploid Rosaceae species. In this study, two tetraploid Chinese cherry (Prunus pseudocerasus) cultivars and one diploid sweet cherry (Prunus avium) cultivar were used to investigate the ploidy of pollen grains and inheritance of pollen-S alleles. Genetic analysis of the S-genotypes of two intercross-pollinated progenies showed that the pollen grains derived from Chinese cherry cultivars were hetero-diploid, and that the two S-haplotypes were made up of every combination of two of the four possible S-haplotypes. Moreover, the distributions of single S-haplotypes expressed in self- and intercross-pollinated progenies were in disequilibrium. The number of individuals of the two different S-haplotypes was unequal in two self-pollinated and two intercross-pollinated progenies. Notably, the number of individuals containing two different S-haplotypes (S1- and S5-, S5- and S8-, S1- and S4-haplotype) was larger than that of other individuals in the two self-pollinated progenies, indicating that some of these hetero-diploid pollen grains may have the capability to inactivate stylar S-RNase inside the pollen tube and grow better into the ovaries.

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.