Sequencing a Juglans regia×J.microcarpa hybrid yields high-quality genome assemblies of parental species

Members of the genus Juglans are monecious wind-pollinated trees in the family Juglandaceae with highly heterozygous genomes,which greatly complicates genome sequence assembly.The genomes of interspecific hybrids are usually comprised of haploid genomes of parental species.We exploited this attribute of interspecific hybrids to avoid heterozygosity and sequenced an interspecific hybrid Juglans microcarpa×J.regia using a novel combination of single-molecule sequencing and optical genome mapping technologies.The resulting assemblies of both genomes were remarkably complete including chromosome termini and centromere regions.Chromosome termini consisted of arrays of telomeric repeats about 8 kb long and heterochromatic subtelomeric regions about 10 kb long.The centromeres consisted of arrays of a centromere-specific Gypsy retrotransposon and most contained genes,many of them transcribed.Juglans genomes evolved by a whole-genome-duplication dating back to the Cretaceous-Paleogene boundary and consist of two subgenomes,which were fractionated by numerous short gene deletions evenly distributed along the length of the chromosomes.Fractionation was shown to be asymmetric with one subgenome exhibiting greater gene loss than the other.The asymmetry of the process is ongoing and mirrors an asymmetry in gene expression between the subgenomes.Given the importance of J.microcarpa×J.regia hybrids as potential walnut rootstocks,we catalogued disease resistance genes in the parental genomes and studied their chromosomal distribution.We also estimated the molecular clock rates for woody perennials and deployed them in estimating divergence times of Juglans genomes and those of other woody perennials.

Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi,and P.pini in Juglans microcarpa×J.regia hybrids

Soil-borne plant pathogens represent a serious threat that undermines commercial walnut(Juglans regia)production worldwide.Crown gall,caused by Agrobacterium tumefaciens,and Phytophthora root and crown rots,caused by various Phytophthora spp.,are among the most devastating walnut soil-borne diseases.A recognized strategy to combat soil-borne diseases is adoption of resistant rootstocks.Here,resistance to A.tumefaciens,P.cinnamomi,and P.pini is mapped in the genome of Juglans microcarpa,a North American wild relative of cultivated walnut.Half-sib J.microcarpa mother trees DJUG 31.01 and DJUG 31.09 were crossed with J.regia cv.Serr,producing 353 and 400 hybrids,respectively.Clonally propagated hybrids were genotyped by sequencing to construct genetic maps for the two populations and challenged with the three pathogens.Resistance to each of the three pathogens was mapped as a major QTL on the long arm of J.microcarpa chromosome 4D and was associated with the same haplotype,designated as haplotype b,raising the possibility that the two mother trees were heterozygous for a single Mendelian gene conferring resistance to all three pathogens.The deployment of this haplotype in rootstock breeding will facilitate breeding of a walnut rootstock resistant to both crown gall and Phytophthora root and crown rots.

PAPP2C Interacts with the Atypical Disease Resistance Protein RPW8.2 and Negatively Regulates Salicylic Acid-Dependent Defense Responses in Arabidopsis

Many fungal and oomycete pathogens differentiate a feeding structure named the haustorium to extract nutrition from the plant epidermal cell. The atypical resistance （R） protein RPW8.2 activates salicylic acid （SA）-dependent, haustorium-targeted defenses against Golovinomyces spp., the causal agents of powdery mildew diseases on multiple plant species. How RPW8.2 activates defense remains uncharacterized. Here, we report that RPWS.2 interacts with the phytochrome-associated protein phosphatase type 2C （PAPP2C） in yeast and in planta as evidenced by co- immunoprecipitation and bimolecular fluorescence complementation assays. Down-regulation of PAPP2C by RNA interfer- ence （RNAi） in Col-0 plants lacking RPWS.2 leads to leaf spontaneous cell death and enhanced disease resistance to powdery mildew via the SA-dependent signaling pathway. Moreover, down-regulation of PAPP2C by RNAi in the RPW8.2 background results in strong HR-like cell death, which correlates with elevated RPWS.2 expression. We further demonstrate that hemagglutinin （HA）-tagged PAPP2C prepared from tobacco leaf cells transiently transformed with HA-PAPP2C possesses phosphatase activity. In addition, silencing a rice gene （Os04g0452000） homologous to PAPP2C also results in spontaneous cell death in rice. Combined, our results suggest that RPW8.2 is functionally connected with PAPP2C and that PAPP2C negatively regulates SA-dependent basal defense against powdery mildew in Arabidopsis.

Sequencing and comparative analyses of Aegilops tauschii chromosome arm 3DS reveal rapid evolution of Triticeae genomes

Bread wheat （Triticum aestivum, AABBDD） is an allohexaploid species derived from two rounds of interspecific hybridizations. A high-quality genome sequence assembly of diploid Aegilops tauschii, the donor of the wheat D genome, will provide a useful platform to study polyploid wheat evolution. A combined approach of BAC pooling and next-generation sequencing technology was employed to sequence the minimum tiling path （MTP） of 3176 BAC clones from the short arm ofAe. tauschii chromosome 3 （At3DS）. The final assembly of 135 super-scaffolds with an N50 of 4.2 Mb was used to build a 247-Mb pseudomolecule with a total of 2222 predicted protein-coding genes. Compared with the orthologous regions of rice, Brachypodium, and sorghum, At3DS contains 38.67% more genes. In comparison to At3DS, the short arm sequence of wheat chromosome 3B （Ta3BS） is 95-Mb large in size, which is primarily due to the expansion of the non-centromeric region, suggesting that transposable element （TE） bursts in Ta3B likely occurred there. Also, the size increase is accompanied by a proportional increase in gene number in Ta3BS. We found that in the sequence of short arm of wheat chromosome 3D （Ta3DS）, there was only less than 0.27% gene loss compared to At3DS. Our study reveals divergent evolution of grass genomes and provides new insights into sequence changes in the polyploid wheat genome.