Thomas M. Gradziel

Structural and Transcriptional Analysis of the Self-Incompatibility Locus of Almond: Identification of a Pollen-Expressed F-Box Gene with Haplotype-Specific Polymorphism

Gametophytic self-incompatibility in Rosaceae, Solanaceae, and Scrophulariaceae is controlled by the S locus, which consists of an S-RNase gene and an unidentified “pollen S” gene. An ∼70-kb segment of the S locus of the rosaceous species almond, the S haplotype–specific region containing the S-RNase gene, was sequenced completely. This region was found to contain two pollen-expressed F-box genes that are likely candidates for pollen S genes. One of them, named SFB (S haplotype–specific F-box protein), was expressed specifically in pollen and showed a high level of S haplotype–specific sequence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the S specificity of pollen because it showed little allelic sequence polymorphism and was expressed also in pistil. Three other S haplotypes were cloned, and the pollen-expressed genes were physically mapped. In all four cases, SFBs were linked physically to the S-RNase genes and were located at the S haplotype–specific region, where recombination is believed to be suppressed, suggesting that the two genes are inherited as a unit. These features are consistent with the hypothesis that SFB is the pollen S gene. This hypothesis predicts the involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based gametophytic self-incompatibility system.

Cloning and characterization of cDNAs encoding S-RNases from almond (Prunus dulcis) : primary structural features and sequence diversity of the S-RNases in Rosaceae

Abstract cDNAs encoding three S-RNases of almond (Prunus dulcis), which belongs to the family Rosaceae, were cloned and sequenced. The comparison of amino acid sequences between the S-RNases of almond and those of other rosaceous species showed that the amino acid sequences of the rosaceous S-RNases are highly divergent, and intra-subfamilial similarities are higher than inter-subfamilial similarities. Twelve amino acid sequences of the rosaceous S-RNases were aligned to characterize their primary structural features. In spite of␣their high level of diversification, the rosaceous S-RNases were found to have five conserved regions, C1, C2, C3, C5, and RC4 which is Rosaceae-specific conserved region. Many variable sites fall into one region, named RHV. RHV is located at a similar position to that of the hypervariable region a (HVa) of the solanaceous S-RNases, and is assumed to be involved in recognizing S-specificity of pollen. On the other hand, the region corresponding to another solanaceous hypervariable region (HVb) was not variable in the rosaceous S-RNases. In the phylogenetic tree of the T2/S type RNase, the rosaceous S-RNase fall into two subfamily-specific groups (Amygdaloideae and Maloideae). The results of sequence comparisons and phylogenetic analysis imply that the present S-RNases of Rosaceae have diverged again relatively recently, after the divergence of subfamilies.

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

Abstract In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (Sa, Sb, Sc, Sd). We previously reported the cDNA cloning of three of these alleles, namely Sb, Sc and Sd. In this paper we report the cloning and DNA sequence analysis of the Sa allele. The Sa-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (Sb, Sc, and Sd) and this similarity was lower than that observed among the Sb, Sc and Sd-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; Sa (602 bp), Sb (1083 bp), Sc (221 bp) and Sd (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the Sa- and Sb-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the Sa-allele and 556 bp in the Sb-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage.

Endopolygalacturonase: a Candidate Gene for Freestone and Melting Fleshin Peach

Peach fruit are handled, processed, and marketed according to their stone adhesion and fruit softening type. Uncertainty exists over whether these simply inherited traits are controlled by two linked loci, Freestone (F) and Melting flesh (M) or one multi-allelic locus, and whether M is controlled by the cell wall degrading enzyme, endopolygalacturonase. From morphological and molecular analysis of two related segregating populations of peach, we conclude that a single locus containing at least one gene for endopolygalacturonase, controls both F and M with at least three effective alleles. A simple diagnostic PCR test is now available for the three major phenotypes of freestone melting flesh (FMF), clingstone melting flesh (CMF), and clingstone non-melting flesh (CNMF).

Amplified fragment length polymorphism as a tool for molecular characterization of almond germplasm: genetic diversity among cultivated genotypes and related wild species of almond, and its relationships with agronomic traits

Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient method for producing DNA fingerprints and molecular characterization. Our objectives were to: estimate genetic similarities (GS), marker indices, and polymorphic information contents (PICs) for AFLP markers in almond cultivars; assess the genetic diversity of almond cultivars and wild species, using GS estimated from AFLP fingerprints and molecular characterization; and facilitate the use of markers in inter-specific introgression and cultivar improvement. The genetic diversity of 45 almond cultivars from Iran, Europe, and America, were studied assaying 19 primer combinations. In addition, several agronomic traits were evaluated, including flowering and maturity times, self-incompatibility, and kernel and fruit properties. Out of the 813 polymerase chain reaction fragments that were scored, 781 (96.23%) were polymorphic. GS ranged from 0.5 to 0.96, marker indices ranged from 51.37 to 78.79, and PICs ranged from 0.56 to 0.86. Results allowed the unique molecular identification of all assayed genotypes. However, the correlation between genetic similarity clustering as based on AFLP and clustering for agronomic traits was low. Cluster analysis based on AFLP data clearly differentiated the genotypes and wild species according to their origin and pedigree, whereas, cluster analysis based on agronomic data differentiated according the pomological characterization. Our results showed the great genetic diversity of the almond cultivars and their interest for almond breeding.

Development of ChillPeach genomic tools and identification of cold-responsive genes in peach fruit

The ChillPeach database was developed to facilitate identification of genes controlling chilling injury (CI), a global-scale post-harvest physiological disorder in peach. It contained 7,862 high-quality ESTs (comprising 4,468 unigenes) obtained from mesocarp tissues of two full-sib progeny contrasting for CI, about 48 and 13% of which are unique to Prunus and Arabidopsis, respectively. All ESTs are in the Gateway® vector to facilitate functional assessment of the genes. The data set contained several putative SNPs and 184 unigenes with high quality SSRs, of which 42% were novel to Prunus. Microarray slides containing 4,261 ChillPeach unigenes were printed and used in a pilot experiment to identify differentially expressed genes in cold-treated compared to control mesocarp tissues, and in vegetative compared to mesocarp tissues. Quantitative RT-PCR (qRT-PCR) confirmed microarray results for all 13 genes tested. The microarray and qRT-PCR analyses indicated that ChillPeach is rich in putative fruit-specific and novel cold-induced genes. A website (http://bioinfo.ibmcp.upv.es/genomics/ChillPeachDB) was created holding detailed information on the ChillPeach database.

AN EXTENDED INTERSPECIFIC GENE POOL AVAILABLE TO PEACH AND ALMOND BREEDING AS CHARACTERIZED USING SIMPLE SEQUENCE REPEAT (SSR) MARKERS

Genetic diversity, as revealed by eighteenSimple Sequence Repeat (SSR) markers inthirty almond [P. dulcis (Mill.) D.A.Webb], twenty fresh-market peach [Prunus persica (L.), Basch], fifteenprocessing clingstone peach cultivars, andten rootstocks, established the geneticrelatedness among cultivars andcharacterized the variation within andbetween species. One accession each of thewild Prunus species, P.davidiana [(Carriere) Franch] and P.webbii [(Spach.) Vieh.], was included inthe analysis. The number of presumedalleles revealed by the SSR analysis rangedfrom one to six in peach whereas almondcultivars showed a range of three to nine.Peach cultivars clustered into ten groups,which are in general agreement withdocumented origin. Most processingclingstone peach cultivars clusteredseparately from fresh-market freestonecultivars supporting a distinct origin. Twomajor clusters were observed in almond withone containing California cultivars and theother containing European cultivars and theimportant California cultivar Mission.Results establish the value of SSR markersfor distinguishing different geneticlineages and characterize an extensive andlargely unexploited inter-species gene poolavailable to peach and almond breedingprograms.

Phenotypic diversity within native Iranian almond ( Prunus spp.) species and their breeding potential

A total of 137 accessions from 18 wild almond species were collected from Iran and leaf and fruit traits were characterized. Also evaluated were flowering and ripening date, self-incompatibility and kernel bitterness. An extensive phenotypic diversity was found both among and within species. Differences in average leaf dimensions among and within species were associated with average rainfall but not altitude of collection site. Adjacent accessions located in drier areas had smaller leaf dimensions than those located in semi-humid or humid regions. No relation was found between average fruit dimensions and collection site conditions. Principal component analysis revealed that the nut weight and width, and the kernel weight had highest loading in the first component accounting for 45.8% of total variation. In contrast, leaf traits in the second component accounted for 22.3% of total variation. No significant correlations were detected between leaf dimensions and fruit traits in all species evaluated. Results document a rich source of new germplasm for almond improvement programs. Small fruit size, pollen-pistil self-incompatibility, and bitter kernel flavour are the most common obstacles to the utilization of this wild germplasm in breeding.

Leucoanthocyanidin dioxygenase gene (PpLDOX): a potential functional marker for cold storage browning in peach

Enzymatic browning of the peach fruit mesocarp is a major component of the postharvest physiological disorder commonly called chilling injury or internal breakdown (IB). Previously, we detected a major quantitative trait locus (QTL; qP-Brn5.1m) affecting browning in peach using two related progeny populations (Pop-DG and Pop-G). In this report, a gene encoding the leucoanthocanidin dioxygenase (PpLDOX) enzyme was identified as the gene potentially responsible for this QTL. PpLDOX has a high similarity with the LDOX gene of the anthocyanin biosynthesis pathway of Arabidopsis thaliana. It was co-located with qP-Brn5.1m via the bin mapping technique with the Prunus reference T×E map. A silent SNP within the PpLDOX coding sequence was used to locate the gene more precisely on the Pop-DG map and confirm its bin assignment. These results demonstrate both the utility of comparative mapping within Prunus using the T×E reference map and the power of the bin mapping approach for easily mapping genes in the Prunus genome. An SSR polymorphism was observed in the intron of PpLDOX gene sequence. The SSR co-segregated with the SNP and was used to assess association of PpLDOX with browning in 27 peach and nectarine cultivars. Cumulative evidence obtained indicates that PpLDOX partially explains genetic variation for cold storage browning susceptibility in peach and nectarine. This functional gene has potential use in marker-assisted breeding of new cultivars with lower IB susceptibility and for genotyping current cultivars for possible differential handling during storage to reduce symptom incidence.

A practical method for almond cultivar identification and parental analysis using simple sequence repeat markers

Early and accurate identification of almond [Prunus dulcis (Mill.) D.A. Webb] cultivars is critical to commercial growers and nurseries. Previously published simple sequence repeat loci were examined for their ability to distinguish commonly grown almond cultivars. Twelve highly polymorphic loci were selected for their ability to uniquely identify a set of 18 almond cultivars commonly grown in California, many of which are closely related. These markers also allow an accurate assessment of parent/progeny relationships among cultivars. This system can reliably identify at an early stage of development all major California almond cultivars in current production.