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.

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.

Diversity, distribution, and evolution of Solanum bulbocastanum late blight resistance genes.

Knowledge on the evolution and distribution of late blight resistance genes is important for a better understanding of the dynamics of these genes in nature. We analyzed the presence and allelic diversity of the late blight resistance genes Rpi-blb1, Rpi-blb2, and Rpi-blb3, originating from Solanum bulbocastanum, in a set of tuber-bearing Solanum species comprising 196 different taxa. The three genes were only present in some Mexican diploid as well as polyploid species closely related to S. bulbocastanum. Sequence analysis of the fragments obtained from the Rpi-blb1 and Rpi-blb3 genes suggests an evolution through recombinations and point mutations. For Rpi-blb2, only sequences identical to the cloned gene were found in S. bulbocastanum accessions, suggesting that it has emerged recently. The three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their spread is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2, and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2, and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach, some accessions could be identified that contain novel, as yet unknown, late blight resistance factors in addition to the Rpi-blb1, Rpi-blb2, and Rpi-blb3 genes.

Phytophthora infestans isolates lacking class I ipiO variants are virulent on Rpi-blb1 potato.

A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1-mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1-mediated resistance in potato.

Exploiting knowledge of R/Avr genes to rapidly clone a new LZ-NBS-LRR family of late blight resistance genes from potato linkage group IV.

In addition to the resistance to Phytophthora infestans (Rpi) genes Rpi-blb1 and Rpi-blb2, Solanum bulbocastanum appears to harbor Rpi-blb3 located at a major late blight resistance locus on LG IV, which also harbors Rpi-abpt, R2, R2-like, and Rpi-mcd1 in other Solanum spp. Here, we report the cloning and functional analyses of four Rpi genes, using a map-based cloning approach, allele-mining strategy, Gateway technology, and transient complementation assays in Nicotiana benthamiana. Rpi-blb3, Rpi-abpt, R2, and R2-like contain all signature sequences characteristic of leucine zipper nucleotide binding site leucine-rich repeat (LZ-NBS-LRR) proteins, and share amino-acid sequences 34.9% similar to RPP13 from Arabidopsis thaliana. The LRR domains of all four Rpi proteins are highly homologous whereas LZ and NBS domains are more polymorphic, those of R2 being the most divergent. Clear blocks of sequence affiliation between the four functional resistance proteins and those encoded by additional Rpi-blb3 gene homologs suggest exchange of LZ, NBS, and LRR domains, underlining the modular nature of these proteins. All four Rpi genes recognize the recently identified RXLR effector PiAVR2.

Mapping and cloning of late blight resistance genes from Solanum venturii using an interspecific candidate gene approach.

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases of potato. Resistance (R) genes from the wild species Solanum demissum have been used by breeders to generate late-blight-resistant cultivars but resistance was soon overcome by the pathogen. A more recent screening of a large number of wild species has led to the identification of novel sources of resistance, many of which are currently being characterized further. Here, we report on the cloning of dominant Rpi genes from S. venturii. Rpi-vnt1.1 and Rpi-vnt1.3 were mapped to chromosome 9 using nucleotide binding site (NBS) profiling. Subsequently, a Tm-2(2)-based allele mining strategy was used to clone both genes. Rpi-vnt1.1 and Rpi-vnt1.3 belong to the coiled-coil NBS leucine-rich repeat (LRR) class of plant R genes and encode predicted peptides of 891 and 905 amino acids (aa), respectively, which share 75% amino acid identity with the Tomato mosaic virus resistance protein Tm-2(2) from tomato. Compared with Rpi-vnt1.1, Rpi-vnt1.3 harbors a 14-aa insertion in the N-terminal region of the protein and two different amino acids in the LRR domain. Despite these differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum.

Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes.

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.

Iron and zinc deficiencies in China: what is a feasible and cost-effective strategy?

In order to prioritise interventions for micronutrient deficiencies in China, the populations affected by iron and zinc deficiencies were assessed based on data from the 2002 China National Nutrition and Health Survey. The costs and cost-effectiveness of supplementation, food diversification and food fortification were estimated using the standard World Health Organization ingredients approach. Results indicated that 30% of children (60 years), pregnant and lactating women, and 20% of women of reproductive age were anaemic, some 245 million people. Approximately 100 million people were affected by zinc deficiency (zinc intake inadequacy and stunting), the majority living in rural areas. Among interventions on iron and zinc deficiency, biofortification showed the lowest costs per capita, I 0.01 (international dollars), while dietary diversification through health education represented the highest costs at I 1148(international dollars). The cost-effectiveness of supplementation, food fortification and dietary diversification for iron deficiency alone was I 179(international dollars) , I 66 and I 103 (international dollars) per disability-adjusted life-year (DALY), respectively. Data for biofortification were not available. For zinc deficiency, the corresponding figures were I 399(international dollars), I 153(international dollars) and I 103(international dollars) per DALY, respectively. In conclusion, iron and zinc deficiencies are of great public health concern in China. Of the two long-term intervention strategies, i.e. dietary diversification and biofortification with improved varieties, the latter is especially feasible and cost-effective for rural populations. Supplementation and fortification can be used as short-term strategies for specific groups.

Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants.

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.

Agroinfection-based high-throughput screening reveals specific recognition of INF elicitins in Solanum.

SUMMARY We adapted and optimized the use of the Agrobacterium tumefaciens binary PVX expression system (PVX agroinfection) to screen Solanum plants for response to pathogen elicitors and applied the assay to identify a total of 11 clones of Solanum huancabambense and Solanum microdontum, out of 31 species tested, that respond to the elicitins INF1, INF2A and INF2B of Phytophthora infestans. Prior to this study, response to INF elicitins was only known in Nicotiana spp. within the Solanaceae. The identified S. huancabambense and S. microdontum clones also exhibited hypersensitivity-like cell death following infiltration with purified recombinant INF1, INF2A and INF2B, thereby validating the screening protocol. Comparison of INF elicitin activity revealed that Nicotiana plants responded to significantly lower concentrations than Solanum, suggesting variable levels of sensitivity to INF elicitins. We exploited natural variation in response to INF elicitins in the identified Solanum accessions to evaluate the relationship between INF recognition and late blight resistance. Interestingly, several INF-responsive Solanum plants were susceptible to P. infestans. Also, an S. microdontum xSolanum tuberosum (potato) population that segregates for INF response was generated but failed to identify a measurable contribution of INF response to resistance. These results suggest that in Solanum, INF elicitins are recognized as general elicitors and do not have a measurable contribution to disease resistance.

Phytate, calcium, iron, and zinc contents and their molar ratios in foods commonly consumed in China.

A total of 60 food samples commonly consumed in China were analyzed for phytate using the anion-exchange method and for calcium, iron, and zinc using atomic absorption spectrophotometry. The foods analyzed included those based on cereal grains and soybean. Phytate contents expressed on a wet weight basis ranged from 0 for foods made from starches to 1878 mg/100 g for dried stick-shaped soybean milk film. The calcium contents were between 2.08 mg/100 g for ground corn and 760.67 mg/100 g for diced fried soybean curd. The lowest values of iron and zinc were 0.04 mg/100 g for Panjin pearl rice cooked with discarding extra water and 0.08 mg/100 g for potato and bean starches, while the highest values of iron and zinc were observed in dried stick-shaped soybean milk film. Although many foods were relatively rich in calcium, zinc, and iron, many also contained a higher level of phytate. Of the 60 food samples, 34 foods had a phytate/calcium molar ratio >0.24, 53 foods had a phytate/iron molar ratio >1, 31 foods had a phytate/zinc molar ratio >15, and only 7 foods had a phytate x calcium/zinc >200. Phytate in foods impair the bioavailability of calcium, iron, and zinc, which to some extent depends upon food processing and cooking methods.