Mapping QTLs for blight resistance and morpho-phenological traits in inter-species hybrid families of chestnut (Castanea spp.).

Chestnut blight (caused by Cryphonectria parasitica), together with Phytophthora root rot (caused by Phytophthora cinnamomi), has nearly extirpated American chestnut (Castanea dentata) from its native range. In contrast to the susceptibility of American chestnut, many Chinese chestnut (C. mollissima) genotypes are resistant to blight. In this research, we performed a series of genome-wide association studies for blight resistance originating from three unrelated Chinese chestnut trees (Mahogany, Nanking and M16) and a Quantitative Trait Locus (QTL) study on a Mahogany-derived inter-species F2 family. We evaluated trees for resistance to blight after artificial inoculation with two fungal strains and scored nine morpho-phenological traits that are the hallmarks of species differentiation between American and Chinese chestnuts. Results support a moderately complex genetic architecture for blight resistance, as 31 QTLs were found on 12 chromosomes across all studies. Additionally, although most morpho-phenological trait QTLs overlap or are adjacent to blight resistance QTLs, they tend to aggregate in a few genomic regions. Finally, comparison between QTL intervals for blight resistance and those previously published for Phytophthora root rot resistance, revealed five common disease resistance regions on chromosomes 1, 5, and 11. Our results suggest that it will be difficult, but still possible to eliminate Chinese chestnut alleles for the morpho-phenological traits while achieving relatively high blight resistance in a backcross hybrid tree. We see potential for a breeding scheme that utilizes marker-assisted selection early for relatively large effect QTLs followed by genome selection in later generations for smaller effect genomic regions.

Cyto-molecular characterization of rDNA and chromatin composition in the NOR-associated satellite in Chestnut (Castanea spp.).

The American chestnut (Castanea dentata, 2n = 2x = 24), once known as the "King of the Appalachian Forest", was decimated by chestnut blight during the first half of the twentieth century by an invasive fungus (Cryphonectria parasitica). The Chinese chestnut (C. mollissima, 2n = 2x = 24), in contrast to American chestnut, is resistant to this blight. Efforts are being made to transfer this resistance to American chestnut through backcross breeding and genetic engineering. Both chestnut genomes have been genetically mapped and recently sequenced to facilitate gene discovery efforts aimed at assisting molecular breeding and genetic engineering. To complement and extend this genomic work, we analyzed the distribution and organization of their ribosomal DNAs (35S and 5S rDNA), and the chromatin composition of the nucleolus organizing region (NOR)-associated satellites. Using fluorescent in situ hybridization (FISH), we have identified two 35S (one major and one minor) and one 5S rDNA sites. The major 35S rDNA sites are terminal and sub-terminal in American and Chinese chestnuts, respectively, originating at the end of the short arm of the chromosome, extending through the secondary constriction and into the satellites. An additional 5S locus was identified in certain Chinese chestnut accessions, and it was linked distally to the major 35S site. The NOR-associated satellite in Chinese chestnut was found to comprise a proximal region packed with 35S rDNA and a distinct distal heterochromatic region. In contrast, the American chestnut satellite was relatively small and devoid of the distal heterochromatic region.

Defining the 'HoneySweet' insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica).

'HoneySweet' plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, 'HoneySweet' DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum 'Improved French'. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the 'HoneySweet' transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.

Dissecting Resistance to Phytophthora cinnamomi in Interspecific Hybrid Chestnut Crosses Using Sequence-Based Genotyping and QTL Mapping.

The soilborne oomycete Phytophthora cinnamomi-which causes root rot, trunk cankers, and stem lesions on an estimated 5,000 plant species worldwide-is a lethal pathogen of American chestnut (Castanea dentata) as well as many other woody plant species. P. cinnamomi is particularly damaging to chestnut and chinquapin trees (Castanea spp.) in the southern portion of its native range in the United States due to relatively mild climatic conditions that are conductive to disease development. Introduction of resistant genotypes is the most practical solution for disease management in forests because treatment with fungicides and eradication of the pathogen are neither practical nor economically feasible in natural ecosystems. Using backcross families derived from crosses of American chestnuts with two resistant Chinese chestnut cultivars Mahogany and Nanking, we constructed linkage maps and identified quantitative trait loci (QTLs) for resistance to P. cinnamomi that had been introgressed from these Chinese chestnut cultivars. In total, 957 plants representing five cohorts of three hybrid crosses were genotyped by sequencing and phenotyped by standardized inoculation and visual examination over a 6-year period from 2011 to 2016. Eight parental linkage maps comprising 7,715 markers were constructed, and 17 QTLs were identified on four linkage groups (LGs): LG_A, LG_C, LG_E, and LG_K. The most consistent QTLs were detected on LG_E in seedlings from crosses with both 'Mahogany' and 'Nanking' and LG_K in seedlings from 'Mahogany' crosses. Two consistent large and medium effect QTLs located ∼10 cM apart were present in the middle and at the lower end of LG_E; other QTLs were considered to have small effects. These results imply that the genetic architecture of resistance to P. cinnamomi in Chinese chestnut × American chestnut hybrid progeny may resemble the P. sojae-soybean pathosystem, with a few dominant QTLs along with quantitatively inherited partial resistance conferred by multiple small-effect QTLs.

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.