Genomic Biosurveillance Detects A Sexual Hybrid in the Sudden Oak Death Pathogen

Invasive exotic pathogens pose a threat to trees and forest ecosystems worldwide1, hampering the provision of essential ecosystem services such as carbon sequestration and water purification2. Hybridization is a major evolutionary force that can drive the emergence of pathogens3. Phytophthora ramorum, an emergent pathogen that causes the sudden oak and larch death, spreads as reproductively isolated divergent clonal lineages. Sexual recombination has never been reported in this pathogen under natural conditions and laboratory crosses have yielded unfit progenies, suggesting postzygotic barriers to hybridization. Here we report the discovery in a plant nursery of novel variants of P. ramorum that are the result of homoploid hybridization via sexual recombination between North American and European lineages of the pathogen. We show that these hybrids are viable, can infect plants and produce spores for long-term survival and propagation. Genome sequencing revealed novel genotypic combinations, not present in the parental lineages, at 54,515 single nucleotide polymorphism loci. More than 6000 of the novel genotypes at these loci are predicted to have a functional impact in genes associated with host infection, including effectors, carbohydrate-active enzymes and proteases. We also observed post-meiotic mitotic recombination that could generate additional genotypic and phenotypic variation and contribute to homoploid hybrid speciation. Our study highlights the importance of plant pathogen biosurveillance to detect novel variants and inform management and control.

A sexual hybrid and autopolyploids detected in seed from crosses between Neslia paniculata and Camelina sativa (Brassicaceae)

It is important to understand the probability of hybridization and potential for introgression of transgenic crop alleles into wild populations as part of pre-release risk assessment. Here we completed bidirectional crosses between the emerging crop, camelina [Camelina sativa (L.) Crantz] and its weedy relative, ball mustard [Neslia paniculata (L.) Desv.]. Ball mustard is a self-compatible annual that produces hard ball-like seeds similar to canola or mustard seed in size and shape. A total of 1593 crosses were completed and collected with camelina as the maternal parent, while 3253 crosses were successfully collected in the reverse direction. Putatively hybrid seedlings were screened with flow cytometry and species-specific nuclear ribosomal internal transcribed spacer (ITS) markers. Three plants had DNA contents close to expectations for hybrids, but only one of these, formed on camelina, had the expected ITS markers. This hybrid exhibited low fertility, and neither self-pollination nor backcrossing produced viable progeny. The other two plants, formed on ball mustard, had high pollen and seed fertility and were identified as ball mustard neoautotetraploids. Therefore, the hybridization rate between camelina and ball mustard is relatively low at one in 20 000 ovules pollinated when camelina is the maternal parent. However, autotetraploids may form frequently in ball mustard, and tetraploid populations may exist in nature.

Meiotic behavior of interspecific hybrids between artificially tetraploidized sexual Brachiaria ruziziensis and tetraploid apomictic B. brizantha (Poaceae)

The meiotic behavior of four interspecific promising hybrids was evaluated by conventional cytological methods. The female genitors were two artificially tetraploidized sexual accessions of B. ruziziensis (R41 and R44, 2n = 4χ = 36), which were crossed to an agronomically superior natural tetraploid apomictic genotype of B. brizantha (B140 - BRA003395). Three of them (HBGC313, HBGC 315, and HBGC324) were sexual and one (HBGC325) apomictic. Analyses of some cells in diakinesis revealed multivalent chromosome configurations, suggesting that genetic recombination and introgression of some genes could be present. The four hybrids had different types of meiotic abnormalities at various frequencies. Abnormalities related to irregular chromosome segregation due to polyploidy were common among these hybrids, and characterized by precocious chromosome migration to the poles, laggard chromosomes, both generating micronuclei in telophases and tetrads and, as a consequence, unbalanced gametes. One abnormality genotype-specific, related to spindle orientation (a putative divergent spindle mutation), was recorded for the first time in two of the hybrids, HBGC313 and HBGC325. The sexual hybrid HBGC324 had the lower rate of abnormalities, and it could be used as a female genitor in future crosses in the breeding program. The abnormalities present in these hybrids may impact fertility and affect seed production. Based on the results, HBGC324 is the single hybrid recommended to the breeding program. Hybrids must produce a good amount of viable seeds, besides good overall dry matter production and nutritive value, in order to be widely utilized and adopted in production systems. Due to pseudogamy, the desirable superior apomictic hybrids need viable pollen grains to fertilize the secondary nucleus of the embryo sac and thus ensure normal and vigorous endosperm development and plenty of seed set.

Production of Sexual Hybrid Progenies for Clarifying the Phylogenic Relationship between Citrus and Citropsis species

The reciprocal crosses between two citrus cultivars and Citropsis schweinfurthii (Engl.) Swing. & M. Kell. were conducted. The cross between `Nanpu' tangor {`Kiyomi' tangor (Citrus unshiu Marc. × C. sinensis Osbeck) × `Fairchild' tangerine-tangelo [clementine (C. clementina hort. ex Tanaka) × `Orlando' tangelo (C. paradisi Macf. × C. reticulata Blanco)]} and C. schweinfurthii produced some developed seeds with an average weight approximately 1/10 of that of the seeds obtained from open pollination in `Nanpu' tangor. These seeds germinated on Murashige and Tucker medium, and three and 28 seedlings were obtained from crosses using C. schweinfurthii as the female and the male parent, respectively. The absolute nuclear genome size of these seedlings [∼0.84 pg of DNA content per somatic nucleus (2C)] was intermediate of that of the `Nanpu' tangor (0.78 pg/2C) and C. schweinfurthii (0.90 pg/2C) seedlings. The chromosome counts of the young leaves revealed that they were diploids (2n = 2X = 18). Furthermore, the hybridity of the seedlings obtained from the reciprocal crosses between `Nanpu' tangor and C. schweinfurthii was confirmed by randomly amplified polymorphic DNA (RAPD) analysis and cleaved amplified polymorphic sequence (CAPS) analysis. These hybrids will be utilized as important materials for investigating the phylogenic relationships between these genera in the subfamily Aurantioideae.

Production of a self-inhibitor of urediospore germination in Melampsora lini (flax rust) segregates as a recessive, single gene trait

Two strains of Melampsora lini (flax rust) were identified that differed in their ability to produce a self-inhibitor of urediospore germination. Analysis of germination self-inhibitor production amongst 63 F2 progeny derived from a sexual hybrid of these two strains revealed segregation of this trait. Segregation of urediospore germination self-inhibitor production was consistent with single-locus control with inhibitor production being recessively inherited. Key words: rust, germination, spore, inhibitor.

Somatic Hybridization of Complementary Citrus Rootstock:Five New Hybrids

Protoplasm culture following polyethylene glycol-induced fusion resulted in the regeneration of tetraploid somatic hybrid plants from the following attempted parental combinations: Cleopatra mandarin (Citrus reticulata Blanco) + Argentine trifoliate orange [Poncirus trifoliata (L.) Raf.]; `Succari' sweet orange [C. sinensis (L.) Osb.] + Argentine trifoliate orange; sour orange (C. aurantium L.) + Flying Dragon trifoliate orange (P. trifolita); sour orange + Rangpur (C. limonia Osb.); and Milam lemon (purported sexual hybrid of C. jambhiri Lush × C. sinensis) + Sun Chu Sha mandarin (C. reticulate Blanco). Protoplasm isolation, fusion, and culture were conducted according to previously published methods. Regenerated plants were classified according to leaf morphology, chromosome number, and peroxidase, phosphoglucomutase, and phosphoglucose isomerase leaf isozyme profiles. All of the somatic hybrid plants were tetraploid, as expected (2n = 4x = 36), and all five selections have been propagated and entered into commercial citrus rootstock trials.

Reexamination of Corvospongilla novaeterrae (Porifera, Spongillidae), an environmentally restricted freshwater sponge from eastern Canada

Corvospongilla novaeterrae (Potts, 1886) (Demospongiae, Spongillidae) is known only from a few acidic lakes on the eastern coast of Canada. It is considered by some to be a sexual hybrid and thus an invalid species. This assumption is based upon an erroneous interpretation of C. novaeterrae's highly variable gemmoscleres and the abundant of foreign spicules (predominantly those of Duosclera mackayi) in the type specimens. We have examined a new specimen from Nova Scotia that has no foreign spicules and fits the original species description. We evaluate three hypotheses concerning this species' status: (1) it is a hybrid of Corvomeyenia everetti and another species; (2) it is an unusual ecomorphic form of C. everetti resulting from atmospheric inputs of sea salts; (3) it is a valid Corvospongilla species, closely related to C. seckti, C. volkmeri, and C. boehmi. Corvospongilla novaeterrae's morphologically variable gemmoscleres are quite similar to those of C. seckti, C. volkmeri, and C. boehmi, and appear to be evolutionary transients between birotulates and amphioxea (as in Radiospongilla and Pectispongilla). Other characters linking C. novaeterrae with other Corvospongilla species include (i) large gemmules (diameter > 1000 μm) with simple multiple foramina, (ii) a weakly developed or absent pneumatic layer, (iii) the size and shape of the birotulate microscleres, and (iv) the predominantly rod-shaped, tangentially arranged gemmoscleres. Evidence supports the acceptance of C. novaeterrae as a valid, environmentally restricted species.

Production of Somatic Hybrid and Autotetraploid Breeding Parents for Seedless Citrus Development

Allotetraploid somatic hybrid plants of `Nova' tangelo [a sexual hybrid of `Clementine mandarin (C. reticulata Blanco) × `Orlando' tangelo (C. reticulata × C. paradisi Macf.)] + `Succari' sweet orange (C. sinensis L. Osbeck), and `Hamlin' sweet orange (C. sinensis L. Osbeck) + `Dancy' tangerine (C. reticulata) were regenerated following protoplast fusion. `Nova' and `Hamlin' protoplasts were isolated from ovule-derived embryogenic callus and suspension cultures, respectively, and fused using a polyethylene glycol method with seedling leaf-derived protoplasts of `Succari' and `Dancy', respectively. Plants were regenerated via somatic embryogenesis, and somatic hybrids were identified on the basis of leaf morphology, root-tip cell chromosome number, and electrophoretic analysis of peroxidase and phosphoglucose mutase isozyme banding patterns. Diploid plants were regenerated from unfused protoplasts of `Hamlin', `Nova', and `Succari'. Tetraploid plants of `Hamlin' and `Succari' were also recovered, apparently resulting from homokaryotic fusions. No `Dancy' plants were recovered. The somatic hybrid and autotetraploid plants can be used for interploid hybridization with selected monoembryonic scions to generate improved seedless triploid tangor/tangelo cultivars. The lack of suitable tetraploid breeding parents has previously inhibited the development of quality seedless cultivars by this method.