There are two cultivated species of rice: African rice , which was domesticated from the wild progenitor O. barthii in Africa, and Asian rice , which was domesticated from the wild progenitors O. rufipogon and O. nivara in Asia. Asian rice is classified under two major subgroups, japonica and indica. The japonica subgroup includes tropical japonica, temperate japonica, and aromatic rice, while the indica subgroup includes aus and indica rice. Rice cultivation in the US includes primarily tropical japonica cultivars in the Southern rice belt and temperate japonica in northern California. Recently, rice production in California has included ‘specialty’ varieties of temperate japonica rice . Although gourmet rice varieties are brought in, control of imported and specialty seed stocks in California has been tightly regulated to prevent the accidental introduction and dissemination of wild or weedy rice . Weedy rice interacts with rice in the US, mainly across the Southern rice belt in Arkansas, Louisiana, Mississippi, Missouri, and Texas. This weed most likely originated from early domesticated Asian rice that reverted to wild/weedy traits and was later introduced into rice cultivation in the US . In the southern US, there are two major weedy rice ecotypesthat have been consistently well-defined,strawhull awnless and blackhull awned . SH and BHA weedy rice are most similar genetically to indica and aus rice varieties,respectively. Hybridization between weedy rice ecotypes and between weedy rice and cultivated rice has been shown to increase genetic diversity in these groups. Neither indica nor aus varieties were grown in the US at or before the time weedy rice was reported in southern US rice, vertical grow system indicating that both ecotypes arose in Asia and were brought in as contaminants of seed stocks during early rice production. Rice cultivated in California is largely of the straw hull variety, while the weedy rice infesting this region is straw hull awned .

Morphologically, California SHA weedy rice is distinct from both SH and BHA weedy rice in the southern US , as it has a straw-colored hull with long awns . Moreover, California SHA weedy rice is morphologically distinct from cultivated rice in California as it has colored pericarp and fully developed awns. SHA weedy rice was widespread in California rice fields from the 1920s into the 1940s . Bellue proposed that California weedy rice in the early 1900s originated from contamination from other parts of the US . These Asian tropical japonica varieties are not present in the US, and no evidence has supported southern US weedy rice de-domestication from the co-occurring cultivated rice they infest. Swift management efforts through a direct,water-seeding system, herbicides, and certified seed mitigated infestations in the Sacramento Valley until complete elimination of weedy rice in California in the 1970. California SHA weedy rice was eradicated until 2003 when a single field was infested. Since 2003, this weed has spread to several other fields in Colusa and Glenn counties. Possibilities for the origin of California weedy rice include hybridization between cultivated rice and other relatives, reversion of cultivated rice to weediness, or introduction of an already established weedy rice lineage by contamination of seed stock entering California. Because the southern US grows tropical japonica cultivars and California grows temperate japonica cultivars, contamination of seed stocks would most likely occur outside of the US. Weedy rice is commonly referred to as red rice when characterized by a red-pigmented pericarp. Contamination of commercial rice with pigmented weedy red rice seed significantly lowers its commercial value. Weedy rice is very similar to the cultivated crops with which it grows, both genetically and phenotypically. The low genetic distance between cultivated, weedy, and wild forms maintains intermediates and in turn perpetuates hybridization between crop and weedy/wild rice.

These similarities can result from the loss of crop-specific alleles in crops, resulting in weediness, hybridization between crops and wild relatives , or by selection for phenotypic mimicry of the cultivated plant growing in rice fields. When domesticated plants and weedy plants are genetically compatible, hybridization can potentially transfer alleles for weedy characteristics to the cultivated populations and cultivar-specific alleles into weedy populations. One important example of this is when herbicide resistance alleles move into weedy species that hybridize with resistant crops. Indeed, interactions among crops and weeds can impact the adaptive potential of a weed to a new environment by simultaneously increasing genetic diversity in the weed and imparting alleles from the crop that are already suited to survival in an agro-ecosystem. Although rice is a ‘model system’ for domestication studies and the evolutionary history of many global weedy rice ecotypesis well-established, the origin of this recently emergent weedy Oryza population in areas without endemic species is poorly understood.In this study, we elucidate the origins of California weedy rice and attempt to identify morphologies that confer weedinessin the de-domestication process. We used a genome-wide panel of48 sequence tagged sites , which are 400–500 bp portions of expressed genes that have already been sequenced in a thorough sampling of AA genome Oryza species. The STS loci we use in this study are an established and effective tool in the rice community for recapturing the population structure of weedy rice, and represent an unbiased sample of genomic SNP diversity across diverse Oryza, including similar varieties. Indeed, the data from these 48 STS markers enables quantification of nucleotide variation in weedy, cultivated, and wild rice, and enable the robust quantification of US weedy rice nucleotide variation and population structure as well as the determination of which Oryza have contributed to US weedy rice genomes and the role of de-domestication in weedy rice evolution.

The Olsen et al. publication provides thorough information as to how loci are distributed among the 12 rice chromosomes and the suitability of estimation of FST and all other genetic diversity parameters. The diverse panel of Oryza used in this study included wild speciesfrom Asia , Africa , Central America , and Australia , along with cultivated Asian rice and cultivated African rice . To these sequences,we added sequence information at the same loci for weedy and cultivated rice collected from California in order to identify the origin of this newly established weedy population. The population divergence history and variance in the many phenotypes used by the International Rice Research Institute to characterize rice life history stages are unknown in this recently reported weedy population. We show that weedy rice in California is genetically and morphologically distinct from other weedy, wild, and cultivated rice groups included in our sampling. Hybridization as the mechanism of origin is unlikely in this case due to the low level of sequence diversity, uniform haplotype grouping assignment within the California weedy group, and complete homozygosity at all loci for all individuals. Coalescent model-fitting indicates that California weedy rice diverged most recently from temperate japonica cultivars which are grown in California, possibly involving a recent regression of cultivated rice back to a weedy form since establishment in the US. The picture that emerges from our study is that, despite low diversity, weedy rice can harbor significant trait variance and be morphologically distinct from its domesticated progenitors. Understanding how and why crops turn weedy and the dynamics of feral forms in production agriculture will help ameliorate crop-weed competition,reduced yield and quality, contamination of harvested grain, and disease reservoirs due to these weeds.A collection of morpho-phenotypic traits was scored for both cultivated and weedy rice in California . Twenty seven California weedy rice plants were sampled from the four rice fields in the state reported to be infested with weedy rice. Seventy-nine once-selfedlines from the field collected mother plants were grown in the U.C. Davis outdoor facilities for phenotyping. Approximately three offspring lines were obtained from each original California weedy rice line collected from the field. Only certain traits that were applicable to field-collected“mother” plants at harvest—such as grain size—were used in the analysis to incorporate the most representative features of this emergent weed in the field. Germinated seeds were transplanted on April 18, 2007, to 22-liter pots filled with saturated soil and placed inside basins. Fertilizer was added following field recommendations. Seedlings were thinned to one per pot soon after establishment and when seedlings reached the 3- to 4-leaf stage of growth, industrial grow the basins were flooded as in a paddy field. Pots were spaced 50 cm apart and arranged in a randomized complete block design with six replicates per accession. Morphological traits evaluated in this study and measurement methods were based on rice descriptors for morpho-agronomic characterization published by the International Rice Research Institute.Twenty-seven weedy rice plants were chosen for genotyping along with 12 accessions of temperate japonica varieties that are cultivated in California. Leaf tissue from the outdoor grown plants was excised and desiccated for shipment to Clemson University for DNA extraction. DNA was extracted from desiccated leaf tissue using the Macherey-Nagel NucleoSpin 96 Plant DNA extraction kit .

Purified genomic DNA was diluted 2:1 in nuclease-free water for polymerase chain reactions . PCR was carried out using standard conditions to amplify 48 gene fragments selected by [21] from 111 sequenced tagged sites developed by [38]. PCR products were checked by gel electrophoresis and cleaned up using Exonuclease and Antarctic phosphatase treatment following the method described in [39]. Direct sequencing in both the forward and reverse directions was carried out by the Clemson University Genomics and Computational Biology Laboratory. Sequences were assembled into contiguously aligned sequence ‘contigs’ and assigned quality scores using Phred and Phrap. Contigs were aligned and inspected visually for quality and heterozygous sites in BioLign version 4.0.6.2 . Heterozygous base calls were randomly assigned to two pseudo-haplotypes, which were then phased using PHASE version 2.1. Due to low levels of heterozygosity in the data set, haplotypes were inferred with very high probabilities and consistency across five runs. All sequences have been submitted to NCBI GenBank . Phased haplotypes were aligned with sequences obtained from [21]. These additional sequences consist of the same 48 STS loci for a broad range of AA genome Oryza species including 58 weedy rice accessions sampled over a 30 year period from Arkansas, Louisiana, Mississippi, Missouri, and Texas. Also included in this dataset are sequences from the major cultivated groups from Asia and Africa , as well as wild species sampled from Asia , Africa , Central America , and Australia .Summary statistics for each STS locus including nucleotide diversity at silent sites using the Juke’s Cantor correction, Watterson’s θ at silent sites, number of segregating sites S, and Tajima’s D were calculated in DnaSP version 5.0. Arlequin version 3.5 was used to calculate pairwise FST and ФST estimates with 10,000 permutations to assess significance.Bonferroni corrections were used to determine Pvalue cutoffs. The population-mutation parameter FST is an estimate of genetic divergence within and between groups and was used to test for the extent of genetic differentiation. To better estimate divergence between California weedy rice and other rice groups, the population mutation parameter ФST was used, which is similar to FST but uses distances between haplotypes, not just haplotype frequencies. Genetic diversity was measured by computing the average nucleotide diversity , total number of segregating sites, and Watterson’s θw within each field as well as within all fields combined. Population structure was inferred using InStruct, which was designed to allow for inbreeding by not assuming Hardy-Weinberg equilibrium within populations. Using STRUCTURE for inbreeding populations results in inappropriately higher rates of inferred splitting between populations . Five permutations for each number of populations were set from 1 to 22 with 500,000 steps and a burn-in period of 100,000 steps. In Structruns were completed on the Clemson University Condor computing cluster. Log likelihoods for each run were compared to determine the best fit K value. Distruct version 1.1was used to create the graphical display from the results obtained with InStruct. Isolation with Migration modeling was used to test for best fit models of isolation-migration and simultaneously estimate effective population sizes , migration between populations , ancestral population size and time since divergence . California weedy rice was compared on a pairwise basis to California cultivated rice , strawhull weedy rice, blackhull weedy rice, O. rufipogon and O. nivara.