Transcriptomics reveal the genetic coordination of early defense to Armillaria root rot (ARR) in Prunus spp.

Armillaria root rot (ARR) poses a significant threat to the long-term productivity of stone-fruit and nut crops in the predominant production area of the United States. To mitigate this issue, the development of ARR-resistant and horticulturally-acceptable rootstocks is a crucial step towards the maintenance of production sustainability. To date, genetic resistance to ARR has been found in exotic plum germplasm and a peach/plum hybrid rootstock, 'MP-29'. However, the widely-used peach rootstock Guardian® is susceptible to the pathogen. To understand the molecular defense mechanisms involved in ARR resistance in Prunus rootstocks, transcriptomic analyses of one susceptible and two resistant Prunus spp. were performed using two causal agents of ARR, including Armillaria mellea and Desarmillaria tabescens. The results of in vitro co-culture experiments revealed that the two resistant genotypes showed different temporal response dynamics and fungus-specific responses, as seen in the genetic response. Gene expression analysis over time indicated an enrichment of defense-related ontologies, including glucosyltransferase activity, monooxygenase activity, glutathione transferase activity, and peroxidase activity. Differential gene expression and co-expression network analysis highlighted key hub genes involved in the sensing and enzymatic degradation of chitin, GSTs, oxidoreductases, transcription factors, and biochemical pathways likely involved in Armillaria resistance. These data provide valuable resources for the improvement of ARR resistance in Prunus rootstocks through breeding.

Preventative Root-Collar Excavation Reduces Peach Tree Mortality Caused By Armillaria Root Rot On Replant Sites.

In the southeastern United States, Armillaria root rot (ARR) is caused by Desarmillaria tabescens and has become the primary cause of premature mortality in peach orchards. Most rootstocks used in commercial orchards are susceptible and management options are limited. A postinfection practice known as root-collar excavation (RCE), which involves permanent removal of the soil from the base of the trunk, has been shown to improve yields and prolong the productive life of symptomatic trees. However, symptomatic trees already have an advanced infection at the base of the trunk. This study evaluated the efficacy of preventative RCE on the progression of tree mortality in two orchards that were planted in infested replant sites. To provide convincing data for growers, the study was carried out in a commercial orchard and an experimental orchard for 8 years. Furthermore, representative enterprise budgets and net present value (NPV) analysis were utilized to compare the profitability of the two approaches. Trees were planted shallow on berms (45 by 90 cm) to facilitate RCE with hoes and AirSpade 2 years later. Tree mortality in the RCE treatment of the experimental orchard was first observed in year 6 and increased 8% on average per year thereafter. In contrast, tree mortality in the "Grower Standard" treatment was first observed in year 4 and increased 12.7% on average per year thereafter. At the commercial orchard, tree mortality in the RCE treatment was first observed in year 7 and increased 1.9% on average thereafter, while tree mortality in the Grower Standard treatment first appeared in year 5 and increased 4.3% on average thereafter. The delayed onset of ARR-associated tree mortality and the lower annual tree mortality rate in the RCE treatment led to higher NPVs in both locations. There were no negative effects on yield or fruit quality. However, the new planting system can create horticultural challenges, including the formation of a proper berm, uneven ground around the tree interfering with tree care and harvest, increased erosion due to channeling of rainwater, and increased rootstock suckering. The RCE is a valid option for southeastern growers needing to manage high ARR disease pressure on replant sites or on sites only recently cleared from ARR-infected forest land.

First Report of the Root-Knot Nematode, Meloidogyne floridensis Infecting Guardian® Peach Rootstock in South Carolina, USA.

In 2018 to 2019, soil and root samples from some declining peach orchards were collected in Edgefield County, South Carolina, USA. Excavated roots of Guardian® peach (Prunus persica) rootstock showed strong gall symptoms. Extracted root-knot nematodes (RKN) were identified by both morphological and molecular methods as M. floridensis. This is the first detection of the peach RKN in South Carolina and the third state in the USA after Florida and California.

Genotyping by Sequencing for SNP-Based Linkage Map Construction and QTL Analysis of Chilling Requirement and Bloom Date in Peach [Prunus persica (L.) Batsch].

Low-cost, high throughput genotyping methods are crucial to marker discovery and marker-assisted breeding efforts, but have not been available for many 'specialty crops' such as fruit and nut trees. Here we apply the Genotyping-By-Sequencing (GBS) method developed for cereals to the discovery of single nucleotide polymorphisms (SNPs) in a peach F2 mapping population. Peach is a genetic and genomic model within the Rosaceae and will provide a template for the use of this method with other members of this family. Our F2 mapping population of 57 genotypes segregates for bloom time (BD) and chilling requirement (CR) and we have extensively phenotyped this population. The population derives from a selfed F1 progeny of a cross between 'Hakuho' (high CR) and 'UFGold' (low CR). We were able to successfully employ GBS and the TASSEL GBS pipeline without modification of the original methodology using the ApeKI restriction enzyme and multiplexing at an equivalent of 96 samples per Illumina HiSeq 2000 lane. We obtained hundreds of SNP markers which were then used to construct a genetic linkage map and identify quantitative trait loci (QTL) for BD and CR.

Identification of genes associated with growth cessation and bud dormancy entrance using a dormancy-incapable tree mutant.

BACKGROUND: In many tree species the perception of short days (SD) can trigger growth cessation, dormancy entrance, and the establishment of a chilling requirement for bud break. The molecular mechanisms connecting photoperiod perception, growth cessation and dormancy entrance in perennials are not clearly understood. The peach [Prunus persica (L.) Batsch] evergrowing (evg) mutant fails to cease growth and therefore cannot enter dormancy under SD. We used the evg mutant to filter gene expression associated with growth cessation after exposure to SD. Wild-type and evg plants were grown under controlled conditions of long days (16 h/8 h) followed by transfer to SD (8 h/16 h) for eight weeks. Apical tissues were sampled at zero, one, two, four, and eight weeks of SD and suppression subtractive hybridization was performed between genotypes at the same time points. RESULTS: We identified 23 up-regulated genes in the wild-type with respect to the mutant during SD exposure. We used quantitative real-time PCR to verify the expression of the differentially expressed genes in wild-type tissues following the transition to SD treatment. Three general expression patterns were evident: one group of genes decreased at the time of growth cessation (after 2 weeks in SD), another that increased immediately after the SD exposure and then remained steady, and another that increased throughout SD exposure. CONCLUSIONS: The use of the dormancy-incapable mutant evg has allowed us to reduce the number of genes typically detected by differential display techniques for SD experiments. These genes are candidates for involvement in the signalling pathway leading from photoperiod perception to growth cessation and dormancy entrance and will be the target of future investigations.

Mapping quantitative trait loci associated with chilling requirement, heat requirement and bloom date in peach (Prunus persica).

*Chilling requirement, together with heat requirement, determines the bloom date, which has an impact on the climatic distribution of the genotypes of tree species. The molecular basis of floral bud chilling requirement is poorly understood, despite its importance to the adaptation and production of fruit trees. In addition, the genetic nature of heat requirement and the genetic interrelationships among chilling requirement, heat requirement and bloom date remain unclear. *A peach (Prunus persica) F(2) population of 378 genotypes developed from two genotypes with contrasting chilling requirements was used for linkage map construction and quantitative trait loci (QTL) mapping. The floral bud chilling and heat requirements of each genotype were evaluated over 2 yr and the bloom date was scored over 4 yr. *Twenty QTLs with additive effects were identified for three traits, including one major QTL for chilling requirement and two major QTLs for bloom date. The majority of QTLs colocalized with QTLs for other trait(s). In particular, one genomic region of 2 cM, pleiotropic for the three traits, overlapped with the sequenced peach EVG region. *This first report on the QTL mapping of floral bud chilling requirement will facilitate marker-assisted breeding for low chilling requirement cultivars and the map-based cloning of genes controlling chilling requirement. The extensive colocalization of QTLs suggests that there may be one unified temperature sensing and action system regulating chilling requirement, heat requirement and bloom date together.

Genetic linkage mapping for molecular dissection of chilling requirement and budbreak in apricot (Prunus armeniaca L.).

Commercial production of apricot is severely affected by sensitivity to climatic conditions, an adaptive feature essential for cycling between vegetative or floral growth and dormancy. Yield losses are due to late winter or early spring frosts and inhibited vegetative or floral growth caused by unfulfilled chilling requirement (CR). Two apricot cultivars, Perfection and A.1740, were selected for high and low CR, respectively, to develop a mapping population of F1 individuals using a two-way pseudo-testcross mapping strategy. High-density male and female maps were constructed using, respectively, 655 and 592 markers (SSR and AFLP) spanning 550.6 and 454.9 cM with average marker intervals of 0.84 and 0.77 cM. CR was evaluated in two seasons on potted trees forced to break buds after cold treatments ranging from 100 to 900 h. A total of 12 putative CR quantitative trait loci (QTLs) were detected on six linkage groups using composite interval mapping and a simultaneous multiple regression fit. QTL main effects of additive and additive x additive interactions accounted for 58.5% +/- 6.7% and 66.1% +/- 5.8% of the total phenotypic variance in the Perfection and A.1740 maps, respectively. We report two apricot high-density maps and QTLs corresponding to map positions of differentially expressed transcripts and suggested candidate genes controlling CR.

Dormancy-associated MADS genes from the EVG locus of peach [Prunus persica (L.) Batsch] have distinct seasonal and photoperiodic expression patterns.

Mapping and sequencing of the non-dormant evg mutant in peach [Prunus persica (L.) Batsch] identified six tandem-arrayed DAM (dormancy-associated MADS-box) genes as candidates for regulating growth cessation and terminal bud formation. To narrow the list of candidate genes, an attempt was made to associate bud phenology with the seasonal and environmental patterns of expression of the candidates in wild-type trees. The expression of the six peach DAM genes at the EVG locus of peach was characterized throughout an annual growing cycle in the field, and under controlled conditions in response to a long day-short day photoperiod transition. DAM1, 2, 4, 5, and 6 were responsive to a reduction in photoperiod in controlled conditions and the direction of response correlated with the seasonal timing of expression in field-grown trees. DAM3 did not respond to photoperiod and may be regulated by chilling temperatures. The DAM genes in peach appear to have at least four distinct patterns of expression. DAM1, 2, and 4 are temporally associated with seasonal elongation cessation and bud formation and are the most likely candidates for control of the evg phenotype.

Phylogenetic analysis and molecular evolution of the dormancy associated MADS-box genes from peach.

BACKGROUND: Dormancy associated MADS-box (DAM) genes are candidates for the regulation of growth cessation and terminal bud formation in peach. These genes are not expressed in the peach mutant evergrowing, which fails to cease growth and enter dormancy under dormancy-inducing conditions. We analyzed the phylogenetic relationships among and the rates and patterns of molecular evolution within DAM genes in the phylogenetic context of the MADS-box gene family. RESULTS: The peach DAM genes grouped with the SVP/StMADS11 lineage of type II MIKCC MADS-box genes. Phylogenetic analyses suggest that the peach SVP/StMADS11-like gene family, which contains significantly more members than annual model plants, expanded through serial tandem gene duplication. We found evidence of strong purifying selection acting to constrain functional divergence among the peach DAM genes and only a single codon, located in the C-terminal region, under significant positive selection. CONCLUSION: Because all DAM genes are expressed in peach and are subjected to strong purifying selection we suggest that the duplicated genes have been maintained by subfunctionalization and/or neofunctionalization. In addition, this pattern of selection suggests that the DAM genes are important for peach growth and development.

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/).].

Comparison of peach and Arabidopsis genomic sequences: fragmentary conservation of gene neighborhoods.

We examined the degree of conservation of gene order in two plant species, Prunus persica (peach) and Arabidopsis thaliana (thale cress), whose lineages diverged more than 90 million years ago. In the three peach genomic regions studied, segments with a gene order congruent with A. thaliana were short (two to three genes in length); and for any peach region, corresponding segments were found in diverse locations in the A. thaliana genome. At the gene level and lower, the A. thaliana sequence was enormously useful for identifying likely coding regions in peach sequences and in determining their intron-exon structure. The peach BAC sequence data reported here contained a BLAST-detectable putative coding sequence an average of every 7 kb, and the peach introns identified in this study were, on average, almost twice the length of the corresponding introns in A. thaliana.

High-throughput targeted SSR marker development in peach (Prunus persica).

Simple sequence repeats (SSRs) have proven to be highly polymorphic, easily reproducible, codominant markers. However, developing an SSR map is very time consuming and expensive, and most SSRs are not specifically linked to gene loci of immediate interest. The ideal situation would be to combine a high-throughput, relatively inexpensive mapping technique with rapid identification of SSR loci in mapped regions of interest. For this reason, we coupled the high-throughput technique of AFLP mapping with subsequent direct targeting of SSRs identified in AFLP-marked regions of interest. This approach relied on the availability of peach bacterial artificial chromosome (BAC) library resources. We present examples of using this strategy to rapidly identify SSR loci tightly linked to two important, simply inherited traits in peach (Prunus persica (L.) Batsch): root-knot nematode resistance and control of the evergrowing trait. SSRs developed in this study were also tested for their transportability in other Prunus species and in apricots.