Construction of a collection of introgression lines of "Texas" almond DNA fragments in the "Earlygold" peach genetic background.

Peach [Prunus persica L. Batsch] is one of the major temperate fruit tree species, the commercial materials of which have a low level of genetic variability. Almond [P. dulcis (Mill) DA Webb], a close relative of peach cultivated for its kernels, has a much higher level of diversity. The species are inter-compatible and often produce fertile hybrids, almond being a possible source of new genes for peach that could provide biotic and abiotic stress tolerance traits. In this paper we describe the development of a collection of peach-almond introgression lines (ILs) having a single fragment of almond (cv. Texas) in the peach background (cv. Earlygold). Lines with few introgressions were selected with markers from successive generations from a "Texas" × "Earlygold" F1 hybrid, initially using a set of SSRs and later with the 18 k peach SNP chip, allowing for the final extraction of 67 lines, 39 with almond heterozygous introgressions covering 99% of the genome, and 28 with homozygous introgressions covering 83% of the genome. As a proof of concept, four major genes and four quantitative characters were examined in the selected ILs giving results generally consistent with previous information on the genetics of these characters. This collection is the first of its kind produced in a woody perennial species and promises to be a valuable tool for genetic analyses, including dissection of quantitative traits, positional cloning, epistasis and as prebreeding material to introgress almond genes of interest into the peach commercial gene pool.

Phenotypic diversity and distinctiveness of the Belltall garlic landrace.

Among the Mediterranean horticultural landraces, garlic is one of the crops most threatened by genetic erosion. Due to its sexual sterility and to the incidence of seed-borne diseases, historical varieties have been widely replaced by commercial cultivars. In Catalonia, despite the historical relevance of the crop, solely the Belltall garlic landrace is cultivated for commercial purposes. To assess the genotypic and phenotypic diversity within the Belltall garlic, we evaluated sixteen local accessions and five recognized traditional and modern varieties as controls. Genetic analysis with SSR and InDel markers showed low genetic diversity within the Belltall population, grouping modern and traditional varieties separately. Farmers and consumers were involved in the definition of the landrace ideotype and classified the materials by means of projective mapping. Scant phenotypic diversity was found within the Belltall landrace, which is characterized by its color profile and the small size of bulb and cloves. The Belltall landrace grown outside its area of origin lost the distinctive quality signals that differentiate the landrace from the commercial cultivars (clove appearance), indicating that the high quality of the landrace is under genotype-by-environment effects (i.e. local adaptation). Moreover, the size of the Belltall sowing clove had a strong effect on the harvested bulb size. Our research represents a case study for the description of the variability within garlic landraces and an approach to quantify the phenomenon of local adaptation that currently drives their conservation.

Characterization of Japanese Plum (Prunus salicina) PsMYB10 Alleles Reveals Structural Variation and Polymorphisms Correlating With Fruit Skin Color.

The red to blue hue of plant organs is caused due to anthocyanins, which are water-soluble flavonoid pigments. The accumulation of these pigments is regulated by a complex of R2R3-MYB transcription factors (TFs), basic-helix-loop-helix (bHLH), and WD-repeat (WDR) proteins (MBW complex). In Rosaceae species, R2R3-MYBs, particularly MYB10 genes, are responsible for part of the natural variation in anthocyanin colors. Japanese plum cultivars, which are hybrids of Prunus salicina, have high variability in the color hue and pattern, going from yellow-green to red and purple-blue, probably as a result of the interspecific hybridization origin of the crop. Because of such variability, Japanese plum can be considered as an excellent model to study the color determination in Rosaceae fruit tree species. Here, we cloned and characterized the alleles of the PsMYB10 genes in the linkage group LG3 region where quantitative trait loci (QTLs) for the organ color have been mapped to other Prunus species. Allele segregation in biparental populations as well as in a panel of varieties, combined with the whole-genome sequence of two varieties with contrasting fruit color, allowed the organization of the MYB10 alleles into haplotypes. With the help of this strategy, alleles were assigned to genes and at least three copies of PsMYB10.1 were identified in some varieties. In total, we observed six haplotypes, which were able to characterize 91.36% of the cultivars. In addition, two alleles of PsMYB10.1 were found to be highly associated with anthocyanin and anthocyanin-less skin. Their expression during the fruit development confirms their role in the fruit skin coloration. Here, we provide a highly efficient molecular marker for the early selection of colored or non-colored fruits in Japanese plum breeding programs.

Pedigree analysis of 220 almond genotypes reveals two world mainstream breeding lines based on only three different cultivars.

Loss of genetic variability is an increasing challenge in tree breeding programs due to the repeated use of a reduced number of founder genotypes. However, in almond, little is known about the genetic variability in current breeding stocks, although several cases of inbreeding depression have been reported. To gain insights into the genetic structure in modern breeding programs worldwide, marker-verified pedigree data of 220 almond cultivars and breeding selections were analyzed. Inbreeding coefficients, pairwise relatedness, and genetic contribution were calculated for these genotypes. The results reveal two mainstream breeding lines based on three cultivars: "Tuono", "Cristomorto", and "Nonpareil". Descendants from "Tuono" or "Cristomorto" number 76 (sharing 34 descendants), while "Nonpareil" has 71 descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.041, with 14 genotypes presenting a high inbreeding coefficient, over 0.250. Breeding programs from France, the USA, and Spain showed inbreeding coefficients of 0.075, 0.070, and 0.037, respectively. According to their genetic contribution, modern cultivars from Israel, France, the USA, Spain, and Australia trace back to a maximum of six main founding genotypes. Among the group of 65 genotypes carrying the Sf allele for self-compatibility, the mean relatedness coefficient was 0.125, with "Tuono" as the main founding genotype (24.7% of total genetic contribution). The results broaden our understanding about the tendencies followed in almond breeding over the last 50 years and will have a large impact into breeding decision-making process worldwide. Increasing current genetic variability is required in almond breeding programs to assure genetic gain and continuing breeding progress.

Transposons played a major role in the diversification between the closely related almond and peach genomes: results from the almond genome sequence.

We sequenced the genome of the highly heterozygous almond Prunus dulcis cv. Texas combining short- and long-read sequencing. We obtained a genome assembly totaling 227.6 Mb of the estimated almond genome size of 238 Mb, of which 91% is anchored to eight pseudomolecules corresponding to its haploid chromosome complement, and annotated 27 969 protein-coding genes and 6747 non-coding transcripts. By phylogenomic comparison with the genomes of 16 additional close and distant species we estimated that almond and peach (Prunus persica) diverged around 5.88 million years ago. These two genomes are highly syntenic and show a high degree of sequence conservation (20 nucleotide substitutions per kb). However, they also exhibit a high number of presence/absence variants, many attributable to the movement of transposable elements (TEs). Transposable elements have generated an important number of presence/absence variants between almond and peach, and we show that the recent history of TE movement seems markedly different between them. Transposable elements may also be at the origin of important phenotypic differences between both species, and in particular for the sweet kernel phenotype, a key agronomic and domestication character for almond. Here we show that in sweet almond cultivars, highly methylated TE insertions surround a gene involved in the biosynthesis of amygdalin, whose reduced expression has been correlated with the sweet almond phenotype. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond and its close relative peach.

High-density mapping suggests cytoplasmic male sterility with two restorer genes in almond × peach progenies.

Peach (Prunus persica) and almond (Prunus dulcis) are two sexually compatible species that produce fertile offspring. Almond, a highly polymorphic species, is a potential source of new genes for peach that has a strongly eroded gene pool. Here we describe the genetics of a male sterile phenotype that segregated in two almond ('Texas') × peach ('Earlygold') progenies: an F2 (T×E) and a backcross one (T1E) to the 'Earlygold' parent. High-density maps were developed using a 9k peach SNP chip and 135 simple-sequence repeats. Three highly syntenic and collinear maps were obtained: one for the F2 (T×E) and two for the backcross, T1E (for the hybrid) and E (for 'Earlygold'). A major reduction of recombination was observed in the interspecific maps (T×E and T1E) compared to the intraspecific parent (E). The E map also had extensive monomorphic genomic regions suggesting the presence of large DNA fragments identical by descent. Our data for the male sterility character were consistent with the existence of cytoplasmic male sterility, where individuals having the almond cytoplasm required the almond allele in at least one of two independent restorer genes, Rf1 and Rf2, to be fertile. The restorer genes were located in a 3.4 Mbp fragment of linkage group 2 (Rf1) and 1.4 Mbp of linkage group 6 (Rf2). Both fragments contained several genes coding for pentatricopeptide proteins, demonstrated to be responsible for restoring fertility in other species. The implications of these results for using almond as a source of novel variability in peach are discussed.

Cell wall polysaccharide chemistry of peach genotypes with contrasted textures and other fruit traits.

Cell wall composition, pectin, and hemicellulose fine structure variation were assessed in peach and related genotypes with contrasted texture and fruit shape. Cell walls were prepared from four commercial peaches, eight genotypes from the Jalousia × Fantasia peach cross, and six genotypes from the Earlygold peach × Texas almond cross. Sugar composition was determined chemically while fine structure of homogalacturonan pectin and xyloglucan hemicellulose were assessed by coupling pectin lyase and glucanase degradation, respectively, with MALDI-TOF MS analysis of the degradation products. The results indicate clear compositional and structural differences between the parents and their related genotypes on the basis of pectin versus cellulose/hemicellulose content and on the fine structure of homogalacturonan and xyloglucan. A relation between methyl- and acetyl-esterification of pectin with fruit shape is revealed in the Fantasia × Jalousia peach genotypes.

Genetic variation, population structure and linkage disequilibrium in peach commercial varieties.

BACKGROUND: Peach [Prunus persica (L.) Batsch] is one of the most economically important fruit crops that, due to its genetic and biological characteristics (small genome size, taxonomic proximity to other important species and short juvenile period), has become a model plant in genomic studies of fruit trees. Our aim was an in-depth study of the extent, distribution and structure of peach genetic variation in North American and European commercial varieties as well as old Spanish varieties and several founders used in the early USA peach breeding programmes. For this we genotyped 224 peach cultivars using 50 SSRs evenly distributed along the 8 linkage groups of the Prunus reference map. RESULTS: Genetic distance analysis based on SSRs divided the peach cultivars in three main groups based mainly on their fruit characteristics: melting flesh peaches, melting flesh nectarines and non-melting varieties. Whereas non-melting flesh peaches had a higher number of alleles than melting peaches and nectarines, they were more homozygous. With some exceptions ('Admiral Dewey', 'Early Crawford' and 'Chinese Cling'), the founder US cultivars clustered together with the commercial melting peaches, indicating that their germplasm is well represented in modern cultivars. Population structure analysis showed a similar subdivision of the sample into subpopulations. Linkage disequilibrium (LD) analysis in three unstructured, or barely structured, subpopulations revealed a high level of LD conservation in peach extending up to 13-15 cM. CONCLUSIONS: Using a much larger set of SSRs, our results confirm previous observations on peach variability and population structure and provide additional tools for breeding and breeders' rights enforcement. SSR data are also used for the estimation of marker mutation rates and allow pedigree inferences, particularly with founder genotypes of the currently grown cultivars, which are useful to understand the evolution of peach as a crop. Results on LD conservation can be explained by the self-pollinating nature of peach cultivated germplasm and by a bottleneck that occurred at the beginning of modern breeding practices. High LD suggests that the development of whole-genome scanning approaches is suitable for genetic studies of agronomically important traits in peach.

Development and bin mapping of a Rosaceae Conserved Ortholog Set (COS) of markers.

BACKGROUND: Detailed comparative genome analyses within the economically important Rosaceae family have not been conducted. This is largely due to the lack of conserved gene-based molecular markers that are transferable among the important crop genera within the family [e.g. Malus (apple), Fragaria (strawberry), and Prunus (peach, cherry, apricot and almond)]. The lack of molecular markers and comparative whole genome sequence analysis for this family severely hampers crop improvement efforts as well as QTL confirmation and validation studies. RESULTS: We identified a set of 3,818 rosaceaous unigenes comprised of two or more ESTs that correspond to single copy Arabidopsis genes. From this Rosaceae Conserved Orthologous Set (RosCOS), 1039 were selected from which 857 were used for the development of intron-flanking primers and allele amplification. This led to successful amplification and subsequent mapping of 613 RosCOS onto the Prunus TxE reference map resulting in a genome-wide coverage of 0.67 to 1.06 gene-based markers per cM per linkage group. Furthermore, the RosCOS primers showed amplification success rates from 23 to 100% across the family indicating that a substantial part of the RosCOS primers can be directly employed in other less studied rosaceaous crops. Comparisons of the genetic map positions of the RosCOS with the physical locations of the orthologs in the Populus trichocarpa genome identified regions of colinearity between the genomes of Prunus-Rosaceae and Populus-Salicaceae. CONCLUSION: Conserved orthologous genes are extremely useful for the analysis of genome evolution among closely and distantly related species. The results presented in this study demonstrate the considerable potential of the mapped Prunus RosCOS for genome-wide marker employment and comparative whole genome studies within the Rosaceae family. Moreover, these markers will also function as useful anchor points for the genome sequencing efforts currently ongoing in this family as well as for comparative QTL analyses.

Genomic characterization of putative allergen genes in peach/almond and their synteny with apple.

BACKGROUND: Fruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis)Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica). RESULTS: Eight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01-3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2. CONCLUSION: A total of 18 putative peach/almond allergen genes have been mapped on five linkage groups. Their positions confirm the high macro-synteny between peach/almond and apple. The insight gained will help to identify key genes causing differences in allergenicity among different cultivars of peach and other Prunus species.

Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map.

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond x peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.

Candidate gene database and transcript map for peach, a model species for fruit trees.

Peach (Prunus persica) is a model species for the Rosaceae, which includes a number of economically important fruit tree species. To develop an extensive Prunus expressed sequence tag (EST) database for identifying and cloning the genes important to fruit and tree development, we generated 9,984 high-quality ESTs from a peach cDNA library of developing fruit mesocarp. After assembly and annotation, a putative peach unigene set consisting of 3,842 ESTs was defined. Gene ontology (GO) classification was assigned based on the annotation of the single "best hit" match against the Swiss-Prot database. No significant homology could be found in the GenBank nr databases for 24.3% of the sequences. Using core markers from the general Prunus genetic map, we anchored bacterial artificial chromosome (BAC) clones on the genetic map, thereby providing a framework for the construction of a physical and transcript map. A transcript map was developed by hybridizing 1,236 ESTs from the putative peach unigene set and an additional 68 peach cDNA clones against the peach BAC library. Hybridizing ESTs to genetically anchored BACs immediately localized 11.2% of the ESTs on the genetic map. ESTs showed a clustering of expressed genes in defined regions of the linkage groups. [The data were built into a regularly updated Genome Database for Rosaceae (GDR), available at (http://www.genome.clemson.edu/gdr/).].

Comparative mapping and marker-assisted selection in Rosaceae fruit crops.

The development of saturated linkage maps using transferable markers, restriction fragment length polymorphisms, and micro-satellites has provided a foundation for fruit tree genetics and breeding. A Prunus reference map with 562 such markers is available, and a further set of 13 maps constructed with a subset of these markers has allowed genome comparison among seven Prunus diploid (x = 8) species (almond, peach, apricot, cherry, Prunus ferganensis, Prunus davidiana, and Prunus cerasifera); marker colinearity was the rule with all of them. Preliminary results of the comparison between apple and Prunus maps suggest a high level of synteny between these two genera. Conserved genomic regions have also been detected between Prunus and Arabidopsis. By using the data from different linkage maps anchored with the reference Prunus map, it has been possible to establish, in a general map, the position of 28 major genes affecting agronomic characters found in different species. Markers tightly linked to the major genes responsible for the expression of important traits (disease/pest resistances, fruit/nut quality, self-incompatibility, etc.) have been developed in apple and Prunus and are currently in use for marker-assisted selection in breeding programs. Quantitative character dissection using linkage maps and candidate gene approaches has already started. Genomic tools such as the Prunus physical map, large EST collections in both Prunus and Malus, and the establishment of the map position of high numbers of ESTs are required for a better understanding of the Rosaceae genome and to foster additional research and applications on fruit tree genetics.