Genome architecture and tetrasomic inheritance of autotetraploid potato.

Potato (Solanum tuberosum) is the most consumed non-cereal food crop. Most commercial potato cultivars are autotetraploids with highly heterozygous genomes, severely hampering genetic analyses and improvement. By leveraging the state-of-the-art sequencing technologies and polyploid graph binning, we achieved a chromosome-scale, haplotype-resolved genome assembly of a cultivated potato, Cooperation-88 (C88). Intra-haplotype comparative analyses revealed extensive sequence and expression differences in this tetraploid genome. We identified haplotype-specific pericentromeres on chromosomes, suggesting a distinct evolutionary trajectory of potato homologous centromeres. Furthermore, we detected double reduction events that are unevenly distributed on haplotypes in 1021 of 1034 selfing progeny, a feature of autopolyploid inheritance. By distinguishing maternal and paternal haplotype sets in C88, we simulated the origin of heterosis in cultivated tetraploid with a survey of 3110 tetra-allelic loci with deleterious mutations, which were masked in the heterozygous condition by two parents. This study provides insights into the genomic architecture of autopolyploids and will guide their breeding.

Transcriptome analysis reveals that exogenous ethylene activates immune and defense responses in a high late blight resistant potato genotype.

Ethylene (ET) is one of the many important signaling hormones that functions in regulating defense responses in plants. Gene expression profiling was conducted under exogenous ET application in the high late blight resistant potato genotype SD20 and the specific transcriptional responses to exogenous ET in SD20 were revealed. Analysis of differentially expressed genes (DEGs) generated a total of 1226 ET-specific DEGs, among which transcription factors, kinases, defense enzymes and disease resistance-related genes were significantly differentially expressed. GO enrichment and KEGG metabolic pathway analysis also revealed that numerous defense regulation-related genes and defense pathways were significantly enriched. These results were consistent with the interaction of SD20 and Phytophthora infestans in our previous study, indicating that exogenous ET stimulated the defense response and initiated a similar defense pathway compared to pathogen infection in SD20. Moreover, multiple signaling pathways including ET, salicylic acid, jasmonic acid, abscisic acid, auxin, cytokinin and gibberellin were involved in the response to exogenous ET, which indicates that many plant hormones work together to form a complex network to resist external stimuli in SD20. ET-induced gene expression profiling provides insights into the ET signaling transduction pathway and its potential mechanisms in disease defense systems in potato.

Aldehyde dehydrogenase plays crucial roles in response to lower temperature stress in Solanum tuberosum and Nicotiana benthamiana.

The aim of this study is to elucidate the role of ALDH2B7a during the response to lower temperature in Solanum tuberosum. This gene was found to have altered intragenic DNA methylation status in our previous reports. A total of 18 orthologs of StALDH2B7a were identified in the S. tuberosum genome, which were then divided into 8 aldehyde dehydrogenase (ALDH) subfamilies. The methylation statuses of four intragenic cytosine sites in intron 5 and exon 6 of genomic StALDH2B7a were altered by lower temperature stress, resulting in changes in the expression of StALDH2B7a. Silencing of NbALDH2C4, a homolog of StALDH2B7a in Nicotiana benthamiana, resulted in plants which were sensitive to lower temperature and accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA). These data suggested that the expression of StALDH2B7a was upregulated by alteration of its intragenic cytosine methylation status during lower temperature stress, and additional StALDH2B7a enzymes scavenged excess aldehydes resulting from ROS in a response to cold stress in potato. Our study expands the understanding of the mechanisms involved in plant responses to lower temperature, and provides a new gene source to improve potato tolerance to cold stress in northern China, where lower temperature is one of the key limiting factors for crop production.

Cysteine Protease Inhibitors Reduce Enzymatic Browning of Potato by Lowering the Accumulation of Free Amino Acids.

Enzymatic browning is a major issue affecting the quality of processed potato (Solanum tuberosum L.). To understand the molecular mechanism of browning, transcriptional analyses were performed by employing potatoes that differed in browning. Coexpression analysis indicated that 9 out of 15 upregulated genes in browning-less groups encoded for potato protease inhibitors (StPIs). In addition, gene otology analysis showed that the enriched terms were mainly involved in protease inhibitors. Overexpression of cysteine StPI 143 and StPI 146 individually reduced browning and lowered protease activities and tyrosine and total free amino acid (FAA) contents, but they could not decrease polyphenol oxidase activity. Moreover, supplementing exogenous tyrosine or total FAAs into transgenic potato mash to wild-type amounts promoted mash browning, browning with total FAAs, more than with tyrosine, resembling wild-type levels. These results implied that cysteine StPIs reduced browning via lowering the accumulation of FAAs in addition to tyrosine. Our findings have enriched the knowledge about the roles and mechanisms of protease inhibitors in regulating enzymatic browning of potato, which provide new ways for controlling potato browning.

A high-throughput BAC end analysis protocol (BAC-anchor) for profiling genome assembly and physical mapping.

Traditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto- or allopolyploid species. Here, we developed a highly efficient and low-cost BAC end analysis protocol, named BAC-anchor, to identify paired-end reads containing large internal gaps. Our approach mainly focused on the identification of high-throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60-100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I- and 165 Mlu I-derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high-coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies.

Cloning and characterization of r3b; members of the r3 superfamily of late blight resistance genes show sequence and functional divergence.

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.