In crop plant genetics, linkage maps provide the basis for the mapping of loci that affect important traits and for the selection of markers to be applied in crop improvement. In outcrossing species such as almond [Prunus dulcis (Mill.) D.A. Webb], application of a double pseudo-testcross mapping approach to the F₁ progeny of a bi-parental cross leads to the construction of a linkage map for each parent. Here, we report on the application of genotyping-by-sequencing to discover and map single-nucleotide polymorphisms in the almond cultivars 9Nonpareil9 and 9Lauranne9. Allele-specific marker assays were developed for 309 tag pairs. Application of these assays to 231 9Nonpareil9 × 9Lauranne9 F₁ progeny provided robust linkage maps for each parent. Analysis of phenotypic data for shell hardness demonstrated the utility of these maps for quantitative trait locus mapping. Comparison of these maps to the peach genome assembly confirmed high synteny and collinearity between the peach and almond genomes. The marker assays were applied to progeny from several other 9Nonpareil9 crosses, providing the basis for a composite linkage map of 9Nonpareil9. Applications of the assays to a panel of almond clones and a panel of rootstocks used for almond production demonstrated the broad applicability of the markers and providing subsets of markers that could be used to discriminate among accessions. The sequence-based linkage maps and single nucleotide polymorphism assays presented here could be useful resources for the genetic analysis and genetic improvement of almond.

Almond shell hardness is an economically important quality trait that protects the integrity of the kernel. It is directly related with shelling or crack-out percent. In the U.S. and Australian markets, soft to semi-hard shells varieties have been used in the industry, while in the European market, hard shells are dominantly sold and distributed. The origin of a hard shell is from wild almond species. Soft shells in the USA and Australia have been selected for a higher crack-out percent or larger kernel and ease of processing. Here, we examined breeding populations totaling 264 trees that were genotyped with genotyping-by-sequencing (GBS) where shell hardness was measured with a texture analyzer, and shell weight, in-shell weights and shell thickness were measured and crack-out percent was calculated. In a genome-wide association, seven genetic loci were found to be associated with shell hardness, crack-out percent, shell thickness and shell weight. Of these seven, one was shared between multiple traits. Kompetitive allele-specific PCR (KASP) assays were developed and tested in unrelated breeding lines from Australia, Spain, France and Italy. One KASP assay was found to be highly predictive of shell hardness in this diverse panel and can be applied for use in marker-assisted breeding.

Preservation of lutein concentrations in wheat-based end-products during processing is important both for product quality and nutritional value. A key constituent involved in lutein degradation is endogenous lipoxygenase. Lutein and lutein ester concentrations were compared at intervals during storage of noodle sheets prepared from flour of wheat varieties representing a range in lipoxygenase activity, as well as in different mill streams and in different grain tissues. Higher lipoxygenase concentration was associated with an increased loss of free lutein and lutein mono-esters whereas lutein diesters appeared to be more resistant to degradation. Lutein degradation was reduced in the presence of a lipoxygenase inhibitor, when noodle sheets were heated to destroy enzyme activity or when pH was increased. In addition, three populations were used to investigate the genetic control of lipoxygenase. A previously reported mutation of Lpx-B1.1 was associated with a reduction in activity from high to intermediate whilst a new locus on chromosome 4D was associated with variation between intermediate and near-zero. The gene underlying the 4D locus is a putative lipoxygenase. Stability of lutein could be improved by deployment of the mutations at the 4B and 4D loci and/or by post-harvest storage of grain under conditions that promote esterification.

In many plant species, self-incompatibility systems limit self-pollination and mating among relatives. This helps maintain genetic diversity in natural populations but imposes constraints in agriculture and plant breeding. In almond [Prunus dulcis (Mill.) D.A. Webb], the specificity of self-incompatibility is mainly determined by stylar ribonuclease (S-RNase) and S-haplotype-specific F-box (SFB) proteins, both encoded within a complex locus, S. Prior to this research, a nearly complete sequence was available for one S-locus haplotype. Here, we report complete sequences for four haplotypes and partial sequences for 11 haplotypes. Haplotypes vary in sequences of genes (particularly S-RNase and SFB), distances between genes and numbers and positions of long terminal repeat transposons. Haplotype variation outside of the S-RNase and SFB genes may help maintain functionally important associations between S-RNase and SFB alleles. Fluorescence-based assays were developed to distinguish among some S-RNase alleles. With three-dimensional modelling of five S-RNase proteins, conserved active sites were identified and variation was observed in electrostatic potential and in the numbers, characteristics and positions of secondary structural elements, loop anchoring points and glycosylation sites. A hypervariable region on the protein surface and differences in the number, location and types of glycosylation sites may contribute to determining S-RNase specificity.

Para rubber trees (Hevea brasiliensis) are the largest major source of natural rubber in the world. Its major pathogens are Phytophthora spp., Corynespora cassiicola, and Colletotrichum spp. A rubber diversity panel of 116 clones using over 12,000 single nucleotide polymorphisms (SNPs) from DArTSeq genotyping revealed clear phylogenetic differences in clones that originated from different geographical regions of the world. An integrated linkage map constructed with an F₁ progeny of 86 from an interspecific cross between H. brasiliensis and H. benthamiana using 23,978 markers [10,323 SNPs and 13,655 SilicoDArTs] spanned 3947.83 cM with 0.83 cM average marker-interval. The genome scaffolds that were anchored to the linkage map, covering 1.44 Gb of H. brasiliensis reference genome, revealed a high level of collinearity between the genetic map and reference genome. Association analysis identified 12 SNPs significantly associated with the resistance against Phytophthora, Corynespora, and Colletotrichum in six linkage groups: 2, 6, 12, 14, 17, and 18. Kompetitive Allele-Specific PCR marker assays were developed for those 12 SNPs, screened with 178 individuals, and detected clear separation between two genotypes. Within the proximity to those SNPs, 41 potentially key genes that have previously been reported to associate with plant disease resistance were predicted with high confidence.