The almond, ['Prunus dulcis' (Mill) D.A. Webb syn. 'P. amygdalus' Batsch] is an important nut crop belonging to the family Rosaceae. Almond consumption has shown steady growth during the last decades due to the increased awareness of its excellent flavour, nutritional value and health benefits. The Australian climate provides great potential for growing almonds and the Australian almond breeding program was initiated in 1997 to develop cultivars better suited to growing conditions and to meet market demands. The construction of a genetic linkage map for one of the breeding populations (Nonpareil x Lauranne) was a part of this breeding program. This population comprised 181 individuals, of which 93 were used for map construction and quantitative trait loci (QTL) analysis in this study. Important traits that were investigated included flowering time, flower structures in relation to fruit set and autogamy and shell and kernel traits.

Developed recently, high resolution melting (HRM) analysis is an efficient, accurate and inexpensive method for distinguishing DNA polymorphisms. HRM has been used to identify mutations in human genes, and to detect SNPs, INDELs and microsatellites in plants. However, its capacity to discriminate DNA variants in the context of complex haplotypes involving INDEL as well as SNP variants has not been examined until now. In this study, we genotyped an almond (Prunus dulcis (Mill.) D. A. Webb, syn. Prunus amygdalus Batsch) pseudotestcross mapping population that showed segregation of complex haplotypes associated with CYP79D16 promoter sequence. The 175 bp region in question included a 7 bp INDEL and 3 SNPs, and manifested as three different haplotypes in the parents. Thus, with one homozygous and one heterozygous parent, two relevant genotypes were identified in the mapping population. Although the population displayed monomorphism with respect to the INDEL and one of the SNPs, HRM was sufficiently sensitive to distinguish genotypes on the basis of the two informative SNPs, and the resulting data were used to map CYP79D16 to linkage group 6 of the almond genome. Thus the capacity of HRM to resolve genotypes arising from complex haplotypes has been demonstrated, and this has important implications for the design of efficient HRM markers for various genetic applications including mapping, population studies and biodiversity analyses.

Almond ('Prunus dulcis') is the only nut crop of the Rosaceae. As one of the earliest species to bloom in spring, almond trees can experience severe crop loss due to late frosts and unfavourable climatic conditions. The Australian almond breeding program is one of the world's major almond breeding programs and has developed a genetic linkage map. One of the objectives of the project is to investigate the inheritance of flowering time. The flowering time of a 'Nonpareil' × 'Lauranne' population was evaluated for two years. The proportion of buds and flowers at seven stages of bud and flower development were recorded for each tree using the same procedures in each of two years. A proportional odds logistic model was used to analyse the data, considering the timing of development of buds and flowers as a latent variable that was estimated for each tree on each occasion recorded. These estimates of flower timing were then used in QTL analysis of flowering time. The goal of this project is to identify genetic markers useful in breeding for altered flowering time in almond.

Almond (Prunus dulcis) is the only nut crop of the Rosaceae. As one of the earliest species to bloom in spring, almond trees can experience severe crop loss due to late frosts and unfavourable climatic conditions. The Australian almond breeding program is one of the world's major almond breeding programs and has developed a genetic linkage map. One of the objectives of the project is to investigate the inheritance of flowering time. The flowering time of a 'Nonpareil' x 'Lauranne' population was evaluated for two years. The proportion of buds and flowers at seven stages of bud and flower development were recorded for each tree using the same procedures in each of two years. A proportional odds logistic model was used to analyse the data, considering the timing of development of buds and flowers as a latent variable that was estimated for each tree on each occasion recorded. These estimates of flower timing were then used in QTL analysis of flowering time. The goal of this project is to identify genetic markers useful in breeding for altered flowering time in almond.

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.

Peach and almond have been considered as model species for the family Rosaceae and other woody plants. Consequently, mapping and characterisation of genes in these species has important implications. High-resolution melting (HRM) analysis is a recent development in the detection of SNPs and other markers, and proved to be an efficient and cost-effective approach. In this study, we aimed to map genes corresponding to known proteins in other species using the HRM approach. Prunus unigenes were searched and compared with known proteins in the public databases. We developed single-nucleotide polymorphism (SNP) markers, polymorphic in a mapping population produced from a cross between the cloned cultivars Nonpareil and Lauranne. A total of 12 SNP-anchored putative genes were genotyped in the population using HRM, and mapped to an existing linkage map. These genes were mapped on six linkage groups, and the predicted proteins were compared to putative orthologs in other species. Amongst those genes, four were abiotic stress-responsive genes, which can provide a starting point for construction of an abiotic resistance map. Two allergy and detoxification related genes, respectively, were also mapped and analysed. Most of the investigated genes had high similarities to sequences from closely related species such as apricot, apple and other eudicots, and these are putatively orthologous. In addition, it was shown that HRM can be an effective means of genotyping populations for the purpose of constructing a linkage map. Our work provides basic genomic information for the 12 genes, which can be used for further genetic and functional studies.