Douglas G. Bielenberg

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation

Buds are specialized structures that protect fragile meristematic regions during dormancy and are part of the mechanism that plants use to survive unfavorable environmental conditions such as low temperature or dessication stress. The evergrowing (evg) mutant of peach [Prunus persica (L.) Batsch] does not form terminal vegetative buds in response to dormancy-inducing conditions such as short days and low temperatures, and the terminal meristems maintain constant growth (leaf addition and internode elongation). We genetically mapped the evg trait and identified the corresponding genomic region in a wild-type genome. We sequenced and annotated the 132-kb region. Nineteen genes were predicted to be in the sequenced region. Ten of the predicted genes were demonstrated to be expressed in the wild-type germplasm but six of these were not expressed in mutant tissues. These six genes are a cluster of MIKC-type MADS-box transcription factors similar to genes from Ipomoea batatas and Solanum tuberosum MADS-box, which also regulate meristem growth in vegetative tissues. A 41,746-bp deletion is present in this region of the mutant genome which results in the loss of all or part of four of the six MADS-box genes. The six MADS-box genes that are not expressed in the mutant are candidates for the regulation of growth cessation and terminal bud formation in peach in response to dormancy-inducing conditions and have been named dormancy-associated MADS-box (DAM) genes.

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.

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.

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

BackgroundDormancy 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.ResultsThe 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.ConclusionBecause 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.

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

BackgroundIn 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.ResultsWe 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.ConclusionsThe 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.

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.

Acclimation of Palmer Amaranth (Amaranthus palmeri) to Shading

Abstract Experiments were conducted to investigate the acclimation of Palmer amaranth to shading. Plants were grown in the field beneath black shade cloths providing 47 and 87% shade and in full sunlight (no shading). All photosynthetic measurements were taken 4 wk after initiating the shade treatments. Photosynthetic rates of Palmer amaranth grown under 47% shade increased with increasing photosynthetic active radiation (PAR) similar to 0% shade-grown plants. Light-saturated photosynthetic rates were predicted beyond the highest measured PAR of 1,200 µmol m−2 s−1 for plants grown under 0 and 47% shade. Plants acclimated to increased shading by decreasing light-saturated photosynthetic rates from 60.5 µmol m−2 s−1 under full sun conditions to 26.4 µmol m−2 s−1 under 87% shade. Plants grown under 87% shade lowered their light compensation point. Rate of increase in plant height was similar among shade treatments. Plants responded to increased shading by a 13 to 44% reduction in leaf appearance rate (leaf number growing degree days [GDD]−1) and a 22 to 63% reduction in main-stem branch appearance rate (main-stem branch number GDD−1) compared with full sunlight. Palmer amaranth specific leaf area increased from 68 to 97 cm2 g−1 as shading increased to 87%. Plants acclimated to 47% shade by increasing total leaf chlorophyll from 22.8 µg cm−2 in full sunlight to 31.7 µg cm−2 when shaded; however, the increase was not significant at 87% shading. Thus, it is concluded that Palmer amaranth shows photosynthetic and morphological acclimation to 87% or less shading. Nomenclature: Palmer amaranth, Amaranthus palmeri S. Wats. AMAPA.

A genome-wide analysis of MADS-box genes in peach [Prunus persica (L.) Batsch]

BackgroundMADS-box genes encode a family of eukaryotic transcription factors distinguished by the presence of a highly-conserved ~58 amino acid DNA-binding and dimerization domain (the MADS-box). The central role played by MADS-box genes in peach endodormancy regulation led us to examine this large gene family in more detail. We identified the locations and sequences of 79 MADS-box genes in peach, separated them into established subfamilies, and broadly surveyed their tissue-specific and dormancy-induced expression patterns using next-generation sequencing. We then focused on the dormancy-related SVP/AGL24 and FLC subfamilies, comparing their numbers and phylogenetic relationships with those of other sequenced woody perennial genomes.ResultsWe identified 79 MADS-box genes distributed across all eight peach chromosomes and frequently located in clusters of two or more genes. They encode proteins with a mean length of 248 ± 72 amino acids and include representatives from most of the thirteen Type II (MIKC) subfamilies, as well as members of the Type I Mα, Mβ, and Mγ subfamilies. Most Type I genes were present in species-specific monophyletic lineages, and their expression in the peach sporophyte was low or absent. Most Type II genes had Arabidopsis orthologs and were expressed at much higher levels throughout vegetative and fruit tissues. During short-day-induced growth cessation, seven Type II genes from the SVP/AGL24, AGL17, and SEP subfamilies showed significant changes in expression. Phylogenetic analyses indicated that multiple, independent expansions have taken place within the SVP/AGL24 and FLC lineages in woody perennial species.ConclusionsMost Type I genes appear to have arisen through tandem duplications after the divergence of the Arabidopsis and peach lineages, whereas Type II genes appear to have increased following whole genome duplication events. An exception to the latter rule occurs in the FLC and SVP/AGL24 Type II subfamilies, in which species-specific tandem duplicates have been retained in a number of perennial species. These subfamilies comprise part of a genetic toolkit that regulates endodormancy transitions, but phylogenetic and expression data suggest that individual orthologs may not function identically across all species.

Dissection of chilling requirement and bloom date QTLs in peach using a whole genome sequencing of sibling trees from an F2 mapping population

Chilling requirement (CR) for floral bud dormancy release is one of the major limiting factors for geographical adaptation of fruiting trees. Using a whole genome sequencing approach (Illumina platform), we explored polymorphism underlying phenotypic differences among individuals in a peach F2 cross segregating for chilling requirement and bloom date. Allelic configuration of individuals, which represented phenotypic extremes in the cross (300 vs. 1,100 chill hours) allowed reconstruction of low- and high-chill haplotypes within three most significant quantitative trait locus (QTL) intervals on the Prunus G1, G4, and G7. We detected single nucleotide polymorphic sites (SNPs), small deletions and insertions (DIPs), and large structural variants (SVs) associated with low-chill haplotypes and created a prioritized list of candidate genes based on functionally characterized homologs from Arabidopsis thaliana. Two dormancy associated genes PpeDAM5 and PpeDAM6 are the strongest candidate genes for the major QTL signal at the lower end of G1. Also, key functional genes involved in the Polycomb repressive mechanism, cell cycle progression, and hormone regulation were evident as strong candidate genes underlying QTL intervals in this peach cross.