The role of Bh4 in parallel evolution of hull colour in domesticated and weedy rice

The two independent domestication events in the genus Oryza that led to African and Asian rice offer an extremely useful system for studying the genetic basis of parallel evolution. This system is also characterized by parallel de‐domestication events, with two genetically distinct weedy rice biotypes in the US derived from the Asian domesticate. One important trait that has been altered by rice domestication and de‐domestication is hull colour. The wild progenitors of the two cultivated rice species have predominantly black‐coloured hulls, as does one of the two U.S. weed biotypes; both cultivated species and one of the US weedy biotypes are characterized by straw‐coloured hulls. Using Black hull 4 (Bh4) as a hull colour candidate gene, we examined DNA sequence variation at this locus to study the parallel evolution of hull colour variation in the domesticated and weedy rice system. We find that independent Bh4‐coding mutations have arisen in African and Asian rice that are correlated with the straw hull phenotype, suggesting that the same gene is responsible for parallel trait evolution. For the U.S. weeds, Bh4 haplotype sequences support current hypotheses on the phylogenetic relationship between the two biotypes and domesticated Asian rice; straw hull weeds are most similar to indica crops, and black hull weeds are most similar to aus crops. Tests for selection indicate that Asian crops and straw hull weeds deviate from neutrality at this gene, suggesting possible selection on Bh4 during both rice domestication and de‐domestication.     

Evolution and selection of Rhg1, a copy‐number variant nematode‐resistance locus

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2‐kb unit contains four genes. One allele of Rhg1, Rhg1‐b, is responsible for protecting most US soybean production from SCN. Whole‐genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2‐kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high‐density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non‐neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Improving panicle exsertion of rice cytoplasmic male sterile line by combination of artificial microRNA and artificial target mimic

The adoption of hybrid rice caused the second leap in rice yield after the ‘green revolution’ and contributes substantially to food security of China and the world. However, almost all cytoplasmic male sterile lines (A lines) as females of hybrid rice have a natural deficiency of ‘panicle enclosure’, which blocks pollination between the A line and the fertility restorer line as the male (R line) of hybrid rice and decreases seed yield. In hybrid rice seed production, exogenous ‘920’ (the active ingredient is gibberellin A₃) must be applied to eliminate or alleviate panicle enclosure of the A line; however, this not only increases production cost and pollutes the environment, it also decreases seed quality. In this study, we designed a transgenic approach to improve plant height and panicle exsertion of the A line to facilitate hybrid rice production and maintain the semi‐dwarf plant type of the hybrid. This approach comprising two components—artificial microRNA (amiRNA) and artificial target mimicry—can manipulate the differential expression of the endogenous Eui1 gene that is associated with rice internode elongation in the A line and the hybrid. amiRNA is a recently developed gene silencing method with high specificity, while target mimicry is a natural mechanism inhibiting the miRNA function that was also recently characterized. This approach provides a paradigm to tune the expression of endogenous genes to achieve the desired phenotype by combining amiRNA and artificial target mimicry technologies.     

Arms race co‐evolution of Magnaporthe oryzae AVR‐Pik and rice Pik genes driven by their physical interactions

Attack and counter‐attack impose strong reciprocal selection on pathogens and hosts, leading to development of arms race evolutionary dynamics. Here we show that Magnaporthe oryzae avirulence gene AVR‐Pik and the cognate rice resistance (R) gene Pik are highly variable, with multiple alleles in which DNA replacements cause amino acid changes. There is tight recognition specificity of the AVR‐Pik alleles by the various Pik alleles. We found that AVR‐Pik physically binds the N‐terminal coiled‐coil domain of Pik in a yeast two‐hybrid assay as well as in an in planta co‐immunoprecipitation assay. This binding specificity correlates with the recognition specificity between AVR and R genes. We propose that AVR‐Pik and Pik are locked into arms race co‐evolution driven by their direct physical interactions.     

Rapid evolutionary dynamics in a 2.8‐Mb chromosomal region containing multiple prolamin and resistance gene families in Aegilops tauschii

Prolamin and resistance gene families are important in wheat food use and in defense against pathogen attacks, respectively. To better understand the evolution of these multi‐gene families, the DNA sequence of a 2.8‐Mb genomic region, representing an 8.8 cM genetic interval and harboring multiple prolamin and resistance‐like gene families, was analyzed in the diploid grass Aegilops tauschii, the D‐genome donor of bread wheat. Comparison with orthologous regions from rice, Brachypodium, and sorghum showed that the Ae. tauschii region has undergone dramatic changes; it has acquired more than 80 non‐syntenic genes and only 13 ancestral genes are shared among these grass species. These non‐syntenic genes, including prolamin and resistance‐like genes, originated from various genomic regions and likely moved to their present locations via sequence evolution processes involving gene duplication and translocation. Local duplication of non‐syntenic genes contributed significantly to the expansion of gene families. Our analysis indicates that the insertion of prolamin‐related genes occurred prior to the separation of the Brachypodieae and Triticeae lineages. Unlike in Brachypodium, inserted prolamin genes have rapidly evolved and expanded to encode different classes of major seed storage proteins in Triticeae species. Phylogenetic analyses also showed that the multiple insertions of resistance‐like genes and subsequent differential expansion of each R gene family. The high frequency of non‐syntenic genes and rapid local gene evolution correlate with the high recombination rate in the 2.8‐Mb region with nine‐fold higher than the genome‐wide average. Our results demonstrate complex evolutionary dynamics in this agronomically important region of Triticeae species.     

Genetic variation at the MHC DRB1 locus is similar across Gunnison's prairie dog (Cynomys gunnisoni) colonies regardless of plague history

Yersinia pestis was introduced to North America around 1900 and leads to nearly 100% mortality in prairie dog (Cynomys spp.) colonies during epizootic events, which suggests this pathogen may exert a strong selective force. We characterized genetic diversity at an MHC class II locus (DRB1) in Gunnison's prairie dog (C. gunnisoni) and quantified population genetic structure at the DRB1 versus 12 microsatellite loci in three large Arizona colonies. Two colonies, Seligman (SE) and Espee Ranch (ES), have experienced multiple plague‐related die‐offs in recent years, whereas plague has never been documented at Aubrey Valley (AV). We found fairly low allelic diversity at the DRB1 locus, with one allele (DRB1*01) at high frequency (0.67–0.87) in all colonies. Two other DRB1 alleles appear to be trans‐species polymorphisms shared with the black‐tailed prairie dog (C. ludovicianus), indicating that these alleles have been maintained across evolutionary time frames. Estimates of genetic differentiation were generally lower at the MHC locus (F_ST = 0.033) than at microsatellite markers (F_ST = 0.098). The reduced differentiation at DRB1 may indicate that selection has been important for shaping variation at MHC loci, regardless of the presence or absence of plague in recent decades. However, genetic drift has probably also influenced the DRB1 locus because its level of differentiation was not different from that of microsatellites in an F_ST outlier analysis. We then compared specific MHC alleles to plague survivorship in 60 C. gunnisoni that had been experimentally infected with Y. pestis. We found that survival was greater in individuals that carried at least one copy of the most common allele (DRB1*01) compared to those that did not (60% vs. 20%). Although the sample sizes of these two groups were unbalanced, this result suggests the possibility that this MHC class II locus, or a nearby linked gene, could play a role in plague survival.     

Segregation of male‐sterility alleles across a species boundary

Hybrid zones may serve as bridges permitting gene flow between species, including alleles influencing the evolution of breeding systems. Using greenhouse crosses, we assessed the likelihood that a hybrid zone could serve as a conduit for transfer of nuclear male‐sterility alleles between a gynodioecious species and a hermaphroditic species with very rare females in some populations. Segregation patterns in progeny of crosses between rare females of hermaphroditic Schiedea menziesii and hermaphroditic plants of gynodioecious Schiedea salicaria heterozygous at the male‐sterility locus, and between female S. salicaria and hermaphroditic plants from the hybrid zone, were used to determine whether male‐sterility was controlled at the same locus in the parental species and the hybrid zone. Segregations of females and hermaphrodites in approximately equal ratios from many of the crosses indicate that the same nuclear male‐sterility allele occurs in the parent species and the hybrid zone. These rare male‐sterility alleles in S. menziesii may result from gene flow from S. salicaria through the hybrid zone, presumably facilitated by wind pollination in S. salicaria. Alternatively, rare male‐sterility alleles might result from a reversal from gynodioecy to hermaphroditism in S. menziesii, or possibly de novo evolution of male sterility. Phylogenetic analysis indicates that some species of Schiedea have probably evolved separate sexes independently, but not in the lineage containing S. salicaria and S. menziesii. High levels of selfing and expression of strong inbreeding depression in S. menziesii, which together should favour females in populations, argue against a reversal from gynodioecy to hermaphroditism in S. menziesii.     

Plastid phylogenetics of Oceania yams (Dioscorea spp., Dioscoreaceae) reveals natural interspecific hybridization of the greater yam (D. alata)

Phylogenetic relationships of Oceanian staple yams (species of Dioscorea section Enantiophyllum) were investigated using plastid trnL‐F and rpl32‐trnL^(UAG) sequences and nine nuclear co‐dominant microsatellites. Analysis of herbarium specimens, used as taxonomic references, allowed the comparison with samples collected in the field. It appears that D. alata, D. transversa and D. hastifolia are closely related species. This study does not support a direct ancestry from D. nummularia to D. alata as previously hypothesized. The dichotomy in D. nummularia previously described by farmers in semi‐perennial and annual types was reflected by molecular markers, but the genetic structure of D. nummularia appears more complex. Dioscorea nummularia displayed two haplotypes, each corresponding to a different genetic group. One, including a D. nummularia voucher from New Guinea, is closer to D. tranversa, D. alata and D. hastifolia and encompasses only semi‐perennial types. The second group is composed of semi‐perennial and annual yams. However, some of these annual yams also displayed D. alata haplotypes. Nuclear markers revealed that some annual yams shared alleles with D. alata and semi‐perennial D. nummularia, suggesting a hybrid origin, which may explain their intermediate morphotypes and the difficulty met in classifying them.     

The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ring‐spot virus resistance locus

Bottle gourd (Lagenaria siceraria) is an important vegetable crop as well as a rootstock for other cucurbit crops. In this study, we report a high‐quality 313.4‐Mb genome sequence of a bottle gourd inbred line, USVL1VR‐Ls, with a scaffold N50 of 8.7 Mb and the longest of 19.0 Mb. About 98.3% of the assembled scaffolds are anchored to the 11 pseudomolecules. Our comparative genomic analysis identifies chromosome‐level syntenic relationships between bottle gourd and other cucurbits, as well as lineage‐specific gene family expansions in bottle gourd. We reconstructed the genome of the most recent common ancestor of Cucurbitaceae, which revealed that the ancestral Cucurbitaceae karyotypes consisted of 12 protochromosomes with 18 534 protogenes. The 12 protochromosomes are largely retained in the modern melon genome, while have undergone different degrees of shuffling events in other investigated cucurbit genomes. The 11 bottle gourd chromosomes derive from the ancestral Cucurbitaceae karyotypes followed by 19 chromosomal fissions and 20 fusions. The bottle gourd genome sequence has facilitated the mapping of a dominant monogenic locus, Prs, conferring Papaya ring‐spot virus (PRSV) resistance in bottle gourd, to a 317.8‐kb region on chromosome 1. We have developed a cleaved amplified polymorphic sequence (CAPS) marker tightly linked to the Prs locus and demonstrated its potential application in marker‐assisted selection of PRSV resistance in bottle gourd. This study provides insights into the paleohistory of Cucurbitaceae genome evolution, and the high‐quality genome sequence of bottle gourd provides a useful resource for plant comparative genomics studies and cucurbit improvement.     

Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae

Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map‐based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST–OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower‐related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild‐type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast‐induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.     

Knockout of two BnaMAX1 homologs by CRISPR/Cas9‐targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.)

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T₀ plants were edited very efficiently (56.30%–67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T₁ and T₂ progeny. We also obtained transgene‐free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.