Analysis was con‐ ducted with correlated allele frequencies and an admixture model for K values ranging from 1 to 20. Each run was conducted with a burn‐in period of 100,000 steps followed by 100,000 Monte Carlo Markov Chain replicates. To further assess the relationships of rice samples without assumptions of specific relationship or population models, a principal component analysis of all samples was conducted in DARwin software using 99 genetic markers. In order to examine the level of differences or genetic relatedness among weedy rice biotypes and other rice groups as a whole, we looked at the pairwise fixation index among clusters of wild rice, cultivated, and weedy rice biotypes.The 99 markers used in this study covered the 12 chromosomes of rice with an average of 8 markers per chromosome and a mean interval distance of 4.43 Mb between markers. The markers showed high polymorphism with an average of 5 alleles and a mean polymorphism information content value of 0.61 per marker. In total, 508 different alleles were scored among 96 rice genotypes using the 99 markers . The presence or ab‐ sence of a 14‐basepair deletion at the Rc gene correlated with red or white pericarp in rice individuals . All weedy rice individuals had the wild‐type allele lacking the deletion, demonstrating the effectiveness of this marker for genetic identification of red pericarp in California weedy rice . In the neighbor‐joining phylogenetic analysis, individuals largely clustered by rice type . While bootstrap support for many basal branches of the tree is low, the grouping of most rice individuals into clusters by rice type is well‐supported. The cultivated japonica rice varieties were separated from indica and all other rice groups with the exception of one red‐pericarped temperate japonica variety, bud curing indicating the effectiveness of SSR markers in differentiating the two main subspecies of rice. Moreover, the tropical and temperate japonica rice varieties are well separated.
The indica rice samples, however, were not placed together, with IR29, IR64, and Milagrosa clustered together separately from red‐pericarped Pokkali landrace rice. Basmati rice, which is an aromatic rice be‐ longing to group V rice, clustered with aus rice varieties with high bootstrap support. The wild rice species samples were scattered throughout the tree as expected by the wide diversity in their genomes. The wild rice O. officinalis samples, which have a CC genome type, clustered together. The southern United States weedy rice individuals were grouped into two separate clusters, with strawhull weedy rice clustered together with high bootstrap support, while blackhull weedy rice grouped together with less support. The California weedy rice samples were grouped into four clusters, which correspond to five distinct biotypes categorized by hull color, grain type, and presence of awn . The first cluster grouped all the short grain , strawhull, awn‐ less individuals . While high bootstrap values support the grouping of Type 1 individuals, the cluster is grouped near an O. nivara individual and one temperate japonica variety as well as aus and Basmati rice with low statistical sup‐ port. The second California weedy rice cluster included all the medium grain , bronzehull, and awnless weedy rice individuals , placed near southern SH weedy rice and some wild rice. A third cluster grouped all the MG, strawhull with long awn . The single SG blackhull with long awn individual closely grouped together with the strawhull Type 3 weedy rice. The fourth weedy rice cluster grouped together some MG and long grain strawhull weedy rice accessions with variable awn length . Type 5 weedy rice was placed with high bootstrap support near the japonica rice varieties, and these two groups were placed with low support near Type 3 and Type 4 weedy rice.
The clustering of California weedy rice by grain attributes validates the division of weedy rice samples by phenotypic similarities. Two noncertified introduced cultivated red‐pericarped specialty rice varieties grown in California, , clustered with California weedy rice. These red‐pericarped cultivated rice varieties have not gone through California’s third‐party variety certification and inspection process and have been previously implicated in rice contamination . RR125 clustered within Type 2 weedy rice and RR126 clustered within MG Type 5 weedy rice, indicating that these red‐peri‐ carped specialty rice varieties are related to California weedy rices. It is unclear from this analysis, however, whether the California weedy rice could be derived directly from these noncertified varieties or whether their relationship is the result of gene flow from these varieties or their ancestors into another population. Since California weedy rice individuals clustered into distinct biotypes, genetic differences among groups of weedy rice were examined in more detail. Analysis of molecular variance indicated that California weedy rice collections are very diverse, with the majority of the variation due to differences among groups while 40% is due to variation among individuals, and differences within group or biotype account for only 5% of genetic variation . Each weedy rice biotype is genetically distinct from the others with an overall FST value of 0.548 among biotypes. Comparison of genetic diversity patterns among the four major biotypes indicate that Type 5 is the most diverse group with the highest number of alleles detected per locus , highest percentage of poly‐ morphicloci , and most heterozygous alleles . In contrast, Type 3 weedy rice has the lowest number of alleles detected per locus , lowest Shannon diversity index within group , and lowest number of heterozygotes. Type 2, which was found in four counties , is also diverse but has the highest inbreeding coefficient estimate of 0.90, indicating homozygosity of individuals in this group .
Overall, California weedy rice biotypes are genetically diverse but with a high frequency of homozygous alleles at 99 loci as indicated by high mean FIS estimate for each group or biotype as well as the overall estimates of FIS and FIT , as would be expected for a species such as rice that reproduces primarily by self‐fertilization. To investigate the relationships among rice individuals while allowing for gene flow and admixture, unlike phylogenetic analysis, STRUCTURE analysis was used to assign each individual’s genotype to genetic clusters or populations. The largest increase in data probability was observed at K = 6 , and this model distinguishes the major biotype groups fairly well . The STRUCTURE grouping of California weedy rice individuals and all other rice samples is consistent with their group membership from phylogenetic analysis . The majority of individuals assign to a single cluster with high probability, and most individuals of the same biotype assign to the same genetic cluster . However, the majority of indica rice and wild rice individuals assign to multiple clusters, indicating higher background genetic diversity or admixture between clusters. Some weedy rice individuals also assign to multiple clusters, indicating hybridization with or evo‐ lutionary origin from other rice groups. The cluster that all Type 1 individuals assign to also has minor genetic contributions from O. nivara, one indica rice variety, and some Type 2 weedy rice individuals. Some Type 2 individuals show admixture with strawhull weedy rice from the southern United States, indica rice, or wild rice species. Type 3 and Type 4 rice individuals all assign highly to a cluster that also has minor contributions from wild rice. A principal component analysis was used to assess genetic similarities among individuals without assuming spe‐ cific relationship or population models . The first three axes account for 22.9%, 11.6%, curing weed and 10.2% of genetic variation present. As in previous analyses, most rice individuals cluster together by rice type, and were spatially well differentiated on the first two axes . Type 1 rice clustered closely with aus rice, Basmati, the single temperate japonica individual that clustered separately from the others in phylogenetic analysis , and BH southern weedy rice. Type 2 weedy rice individuals clustered with SH southern weedy rice and indica rice. Type 3 and Type 4 rice clustered closely together, well differentiated on axis 2 from all other rice samples. Type 5 clus‐ tered together with temperate and tropical japonica rices. The wild rice samples did not cluster together closely, but were distributed mostly in the lower right corner. Genetic differentiation between biotypes was assessed for all weedy, wild, and culti‐ vated rice biotypes. Most estimates of pairwise FST were high, ranging from 0.177 to 0.696 . The very high pairwise FST values between the single Type 4 individual and all other groups indicate genetic differentiation but are likely artificially high due to the sample size of 1. The majority of California weedy rice biotypes, with the exception of Type 5, show high genetic differentiation from the temperate japonica rice cultivars grown in California .
In contrast, low pairwise FST between a weedy rice biotype and another rice type can indicate more shared genetic content. For example, Type 2 shows low differentiation from indica cultivars and from wild rice , indicating less differentiation between these groups and possible relatedness.The increasing spread of weedy rice in California and the recent report of weedy rice originating from cultivated California rice varieties raised questions about the origin of California weedy rice and its management. For this reason, we conducted a genetic study to understand the relationships be‐ tween existing weedy rice in California and to investigate their possible origins. In the phylogenetic analysis, weedy rice individu‐ als clustered together by biotype, indicating that for California weedy rice biotypes, samples can be easily classified by phenotype into groups that are biologically and genetically meaningful . The five biotypes of California weedy rice clustered within multiple larger genetic groups of weedy, wild, and cultivated rice . This division of weedy rice into separate clusters most likely indicates at least four separate evolutionary origins of California weedy rice from diverse lineages of cultivated, weedy, and wild rice. In fact, the four major groups of weedy rice are quite divergent from each other based on principal component analysis . Population structure analysis gives more insight into relationships of individuals and biotypes, revealing close correspondence between genetic populations and rice types . However, some rice groups, especially wild rice and indica rice, are more genetically heterogeneous, with genotypes assigning to multiple genetic clusters. STRUCTURE analysis also identified admixed individuals, indicating hybridization of weedy rice both with other weedy rice biotypes and with wild and cultivated rice , despite the fact that rice is primarily self‐fertilizing with generally low outcrossing rates . Individual and biotype differentiation analyses provide insights into the relationships of California weedy rice biotypes. The high pairwise FST values between most California weedy rice biotypes, with the exception of Type 5, and the temperate japonica cultivars widely grown in California, indicates high genetic differentiation between California weedy rice and California cultivated rice and their relatively low shared genetic content , suggesting that most weedy rice did not evolve from the cultivated rice varieties widely grown in California. The observed FST levels do not necessarily exclude the possibility of infrequent hybridization with cultivated rice within California. One Type 1 individual and one Type 2 individual showed over 10% genetic assignment to the genetic cluster containing Type 5 and japonica rices in STRUCTURE analysis . However, the majority of California weedy rice biotypes have a high inbreeding coefficient and low level of hetero‐ zygosity at 99 loci . Therefore, it is likely that hybridization between rice groups happened many years or generations ago. Type 5 weedy rice was shown in phylogenetic, STRUCTURE, and PCA analyses to be closely related to japonica cultivars, raising questions of whether it is derived directly from the temperate japonica cultivars grown in California or from tropical japonica cultivars outside California and imported. The high inbreeding coefficient of Type 5 weedy rice and moderate genetic differentiation from temperate japonica rice make it likely that its evolutionary origin significantly predates its recent detection, although it is possible that a small weedy rice population could have been present unnoticed for some time prior to detection. Another possibility for the origin and spread of California weedy rice is from the cultivation of red‐pericarped specialty rice varieties. While the majority of rice‐growing acreage in California is devoted to non-colored pericarp rice production, some specialty colored pericarp rice varieties are also grown at a commercial scale.