The challenge now is to develop crops and agricultural systems that will continue to provide good yields in the face of continuous evolution and global dissemination of pests, pathogens, and weeds as well as changing and more stressful growing environments. Sustaining the world food supply requires excellence in both foundational and translational research in parallel with agriculture becoming more data-driven. The necessary technologies and expertise are available such that, with sufficient investment, the future is bright for improving plant health as part of integrated and sustainable agricultural systems. Online feedback provided by the international community at large within the first four weeks of the paper’s online publication will be collated and included as an addendum.Cytological, morphological , and molecular data suggest that tetraploid Sorghum halepense arose as a naturally occurring hybrid between S. bicolor , an annual, polytypic African species which includes cultivated sorghum; and S. propinquum , a perennial southeast Asian native of moist habitats. While a firm estimate of its antiquity is lacking, S. propinquum is thought to have shared ancestry with S. bicolor ∼1–2 million years ago , roughly circumscribing the maximum age of S. halepense. Occasionally used as forage and even food , S. halepense has spread in postColumbian times from its hypothesized west Asian center of origin across much of Asia, Africa, Europe, North and South America, and Australia. Its establishment in the U.S. is probably typical of its spread to other continents, hydroponpic rack system being introduced intentionally as a prospective forage and unintentionally as a contaminant of seedlots .

However, while sorghum largely remained confined to cultivation, S. halepense readily naturalized and has spread across much of North America, both to agricultural and non-agricultural habitats – suggesting capabilities for adaptation well beyond those of sorghum. Its common name thought to be a misnomer [the eponymous Col. Johnson may have obtained propagules from his wife’s family, who accidentally introduced it to South Carolina shortly after the Revolutionary War ], ‘Johnsongrass’ has the rare distinction of being both a noxious weed in 20 U.S. states and an invasive species in 16 . With at least 24 herbicide-resistant biotypes now known , Johnsongrass appears likely to become even more problematic in the future. For example, a glyphosate resistant biotype discovered in Argentina in 2002 covered 10,000 ha by 2009 . Its ability to cross with sorghum despite a ploidy barrier makes Johnsongrass a paradigm for the dangers of crop ‘gene escape,’ and restricts deployment of many transgenes that could reduce the cost and increase the stability of sorghum production. Here, we integrate several diverse data types to elucidate the evolution of S. halepense, its invasiveness as exemplified by rapid spread across the United States in post-Columbian times, and the roles of polyploidy and interspecific hybridity in distinctive features of its growth and development. As the first surviving polyploid in its lineage in ∼96 million years , S. halepense may also open new doors to sorghum improvement, with synergy between gene duplication and interspecific hybridity nurturing the evolution of genes with new or modified functions .Sorghum halepense, S. propinquum, S. timorense and representatives of each of the wild S. bicolor races were sequenced using standard methods implemented at the US Department of Energy Joint Genome Institute, as part of a larger project including 27 genomes and 39 transcriptomes in total.

From each accession, 76-bp paired-end reads were aligned to the Sorghum bicolor reference genome using BWA version 0.5.9 . Multiple-sample SNP calling was performed using the mpileup program in the samtools package and bcftools . Reads with mapping quality score > = 25 and base quality > = 20 are used for SNP calling. Raw SNPs are further filtered according to read depth distribution to avoid paralog contamination and low coverage regions. Each accession’s genotype is calculated using maximum likelihood estimation using reads with coverage between 4 and 30X. The genotype with the largest likelihood is assigned to each individual. SNPs with allele frequency > = 0.01 are used for downstream analysis. As tandem genes are often recently derived and share high sequence similarity, they can complicate short read alignment and introduce ‘false SNPs’ from paralogs. To address this, the coverage of genomic reads was examined for every tandem gene in the sorghum genome. The average coverage of the whole genome across the 27 genomes studied is about 553X. There were 31 tandem genes with more than twice the genome coverage , of which 7 have coverage more than 2500X . A total of 14 of the 31 high coverage tandem genes have SNPs called, and were removed from further analysis.To identify S. halepense SNPs, reads from S. halepense were aligned to the reference S. bicolor genome by BWA and SNPs determined with nucleotide groups for each reference S. bicolor genomic position by an in-house script. False positive S. halepense SNPs for each position of the reference S. bicolor genome were inferred and removed, based on three criteria: if the top two nucleotide groups are the same as reference S. bicolor and S. propinquum, respectively, there are no false positive SNPs; if read depth of an SNP is 1 , a false positive was inferred; if p-value calculated by the Fisher exact test for the actual and theoretical read depths , is less than 0.1, a false positive was inferred. The full SNP table with the reference S. bicolor, S. propinquum, and S. halepense SNPs as well as wild S. bicolor and S. timorense SNPs determined with total RNA and genomic DNA, respectively, against the reference S. bicolor genome, is provided .

Classifications of duplicated genes into paralogs versus homologs followed the S. bicolor reference genome .Arabidopsis GO-slim gene annotation was used for function enrichment analysis. GO-slim terms are assigned to sorghum genes based on sequence similarity inferred from best blastp hit. Binomial distribution based on the proportion of a GO-slim term among all annotated genes in the sorghum genome is used asthe null distribution. Test significance threshold is defined as p < 0.05, unless specified otherwise.Despite a presumed genetic bottleneck during polyploid formation, S. halepense is richly polymorphic. A survey of 182 genetically-mapped restriction fragment length polymorphism loci found 18 S. halepense or ‘Sorghum x almum’ genotypes to average 6.13 alleles per locus, versus 3.39 for a worldwide sample of 55 landrace and wild sorghum accessions and 1.9 for 16 F1 hybrid sorghums from eight commercial breeding programs . While some apparently novel alleles in the draft genome may reflect intraspecific polymorphism, a remarkable 67.1% of CDS polymorphisms differentiate S. halepense from representatives of both putative progenitor species and the outgroup S. timorense . The functional impact of these non-synonymous single-nucleotide polymorphisms was assessed by comparison to an evolutionary conservation profile of amino acids from orthologous genes in a panel of diverse plant species, calculating a ‘functional impact score’ using a modified entropy function – 8738 SNPs with high inferred functional impact score’ suggest important consequences for protein function in 5957 S. halepense genes . SNPs causing premature protein translation termination are most abundant, followed by loss of stop codons and loss of translation initiation site . These functionally important mutations are significantly enriched in plasma membrane genes with kinase activity, suggesting changes in environmental sensing and associated intracellular processes such as cell differentiation and metabolism .Rhizomes, subterranean stems that can comprise 70% of its dry weight , are a key link between morphology and ecology of S. halepense. Rhizome growth of polyploid S. halepense transgresses that of its rhizomatous diploid progenitor, S. propinquum. We conducted a field trial in Bogart, GA during 2012-3 of widely spaced tetraploid F2 progeny from a cross between S. bicolor BTx623 and S. halepense Gypsum 9E ; side by side with plots of 161 diploid recombinant inbred lines from a cross between BTx623 and S. propinquum . SbxSh progeny had a higher frequency of rhizome-derived shoots emerging from the soil , larger average number of rhizomes producing above-ground shoots , and greater distance of rhizome-derived shoots from the crown than SbxSp . Rhizome number showed heritabilities of 0.077 and 0.34 in SbxSh and 0.44 in SbxSp . Rhizomatousness is closely related to the ability of S. halepense to overwinter in the temperate United States. In the Bogart,GA field trial, 139 SbxSh progeny showed regrowth after overwintering, air racking while there was no survival of SbxSp in 2012-3 or in two additional years. Moreover, in SbxSh BC1F1- derived BC1F2 families grown in 3 m plots with two replications following conventional sorghum recommendations, those with rhizomes had significantly higher frequencies of survival than those lacking rhizomes . Survival in Salina, KS among replica plots of the same BC1F2 families was too low to evaluate statistically. More extensive rhizome growth than its rhizomatous diploid progenitor is also related to the ability of S. halepense to survive tropical dry seasons. From a total of 96 BC1F2 families selected for rhizome growth in Bogart GA, single 3 m rows were tested for 15 months at the ICRISAT research station in Samanko, Mali . A total of 45 families contained one or more plants that survived the dry season of 8 month duration with zero rainfall. A logistic regression model showed that for each 1 cm increase in rhizome spread from the crown based on Bogart GA trials, the probability of surviving the Malian dry season increased ∼3%. Factors other than rhizomes are also important to perenniality – lines surviving the tropical dry season were only randomly associated with those surviving the mild 2014-15 temperate winter in Bogart, GA , survivors of the harsh 2013-14 winter being more closely associated with dry season survival but too few in overall number to be conclusive .Curiously, rhizome growth is correlated negatively with that of other vegetative organs but positively with reproductive growth. Across four environments , early flowering is correlated with reduced above ground vegetative biomass , but increased rhizome growth in tetraploid SbxSh progeny. Because rhizomes are a vegetative organ, our a priori expectation was that increased vegetative biomass above ground would be correlated with increased rhizome growth. However, we measured rhizome growth primarily based on counting above ground shoots derived from rhizomes. In another rhizomatous grass , rhizome axillary buds experience apical dominance until anthesis, being suppressed by auxins . By excising S. halepense rhizomes from the plant, we found that axillary buds consistently develop as vertical shoots and not as rhizomes . So, once flowering of the primary stalk is initiated, a rhizomatous plant permits the development of additional ramets – which in principle should be able to exert apical dominance themselves. Moreover, our observation that these new buds invariably become ramets and not rhizomes raises questions about their additional dependence on a mobile ‘florigen’ such as that translocated to the plant apex . There may be much to be learned about nature of signaling among ramets at different developmental stages that are interconnected by rhizomes.While ∼80% of annotated sorghum genes are expressed in S. halepense rhizomes, many alleles with striking enrichment of expression more closely resemble the sequences of the non-rhizomatous S. bicolor progenitor than rhizomatous Sp. By laser capture microdissection, we collected meristems and compared transcripts from buds induced to develop as rhizomes or leafy shoots , respectively obtaining 163,264,254 and 152,162,240 Illumina Hiseq reads, of which 67.7% and 67.2% could be anchored to 27,566 and 27,183 sorghum gene models. About 1% of genes showed differential expression between rhizome buds and shoot buds . Appreciable recruitment of alleles from non-rhizomatous S. bicolor to rhizome-enriched expression is indicated by 44 S. bicolor versus only 23 S. propinquum derived transcripts with at least two SNPs supporting these origins and no contradictory SNPs . Consistent with rhizomes being ∼70% of the mass of a Johnsongrass plant , genes highly expressed in rhizome buds were enriched for diverse functions associated with rapid cell division and maturation. Cellulose synthase, Sb06g016760, was the most rhizome enriched gene, also implicated in rapid cell growth. Shoot-bud enriched genes were over-represented in three gene ontology categories associated with cell recognition , perhaps in preparation for new biotic interactions after emergence from the soil. The most shoot-enriched genes were glutathione S-transferase , catalyzing conjugation of the reduced form of glutathione to xenobiotic substrates for detoxification; a glycoside hydrolase , suggesting cell wall loosening during the rhizome-to-shoot transition; and a member of the major facilitator superfamily of transmembrane single-polypeptide secondary carriers implicated in control of sorghum seed size , a trait that shows strong negative correlation with both rhizome development and winter survival .