The Weedy Rice Complex in Costa Rica. I. Morphological Study of Relationships Between Commercial Rice Varieties,Wild <Emphasis Type="Italic">Oryza</Emphasis> Relatives and Weedy Types

Weedy rice is a complex of Oryza morphotypes widely distributed in commercial rice fields, which interfere with rice cultivation, seed production, industrial processing and commercialization of this crop in several countries. The objective of this study was to characterize the weedy rice complex of Costa Rica by comparing it with the cultivated and wild rice species found in the country. A collection of weedy rice accessions, representative of the morphotypes found in the country, was established and characterized. Their morphometric relationships were established by comparing 27 morphological traits with commercial rice cultivars, landraces and wild Oryza species and by performing a multivariate analysis. Twenty-one weedy rice morphotypes were identified among 735 weedy accessions by using a three-digit code based on seed characters. Three principal components (PCs) explained 66.25% of the variation observed. The first PC accounted for 36.21% of the variation and separated CCDD genome type Oryza latifolia and O. grandiglumis from AA genome species O. sativa, O. glumaepatula, O. rufipogon and O. glaberrima. The second (18.9%) and third (11.14%) PCs separated the weedy morphotype groups from the AA genome species O.sativa, O. glaberrima and O. rufipogon. The weedy morphotypes were scattered between the indica commercial rice varieties, the cluster landraces–glaberrima and O. rufipogon. Additionally, a group of morphotypes showed intermediate characteristics between O. sativa and O. rufipogon, suggesting that hybridization could have taken place in the past between these species. None of the morphotypes collected in Costa Rica clustered with the allotetraploids CCDD species or O. glumaepatula.     

Cross-species Transferability of Rice Microsatellites in its Wild Relatives and the Potential for Conservation Genetic Studies

Here, we investigated the transferability of 60 microsatellite markers characterized for cultivated rice Oryza sativa L. in three wild Oryza species representing different genome types: O. rufipogon Griff. (AA), O. officinalis Wall. et Watt. (CC), and O. granulate Nees et Arn. ex Watt. (G). The results indicate the 60 rice SSR loci tested produced homologous amplification products to different extents in O. rufipogon (100%), O. officinalis (90%) and O. granulata (73.3%). Proportions of polymorphism for successfully amplified loci ranged from 0.983 via 0.667 to 0.364 in O. rufipogon, O. officinalis and O. granulata, respectively. The utility of these microsatellite markers was tested for the characterization of genetic diversity in 117 genotypes of these four Oryza species. The values of genetic diversity in cultivated rice are higher than the other two wild species O. officinalis and O. granulata, suggesting microsatellites tend to have more variability in the focal species than in non-focal species to which they are applied. However, much lower levels of genetic variation were observed in rice than in its wild progenitor O. rufipogon, which indicates severe loss of genetic variation may reflect the ‘domestication bottleneck’ through which rice passed. The observation that most of the rice microsatellites are able to detect allelic polymorphisms at different extent in Oryza species suggest that rice microsatellite loci should be useful for the analysis of genetic diversity and inter- and intra-specific relationships in the genus. Therefore, high rates of successful cross-amplification of rice microsatellites among Oryza species with different genome types will offer excellent opportunities to investigate the population genetic structure of wild rice species and explore their conservation genetics.     

Identification of <Emphasis Type="Italic">SUB1A</Emphasis> alleles from wild rice <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.

Submergence stress is a major constraint to rice production in South and Southeast Asia. Most rice (Oryza sativa L.) cultivars die within a week of complete submergence, while a small number of accessions are submergence-tolerant for up to 2 weeks or more. These cultivars have the tolerant allele of the SUB1A gene, one of three ERF genes at this locus on rice chromosome 9. In all O. sativa varieties studied, the SUB1A gene is limited to a subset of indica accessions of O. sativa. Thus far, there has been no published report of the SUB1A gene in wild rice species. Here we report evidence of the SUB1A gene found in wild species of O. rufipogon Griff. accessions by the use of degenerate primers corresponding to the most highly conserved regions of the SUB1 locus. The results indicated that two SUB1A-like alleles, e.g. OrSub1A-1 and OrSub1A-2, were identified from two O. rufipogon accessions. Submergence treatment shows that both of the accessions with SUB1A-like genes were submergence-intolerant. This preliminary study provides insight into the origin and allelic variation of SUB1A, an agronomically important gene that is rapidly being introduced into widely-grown rice cultivars.     

Genetic diversity of cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) and wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) in Asia,especially in Myanmar,as revealed by organelle markers

Asian rice (Oryza sativa L.) is widely cultivated in Asia, where it has been classified into Indica and Japonica Group, the latter is further classified into Tropical and Temperate Japonica Subgroup. O. rufipogon is believed to be the closest ancestor to O. sativa, but it remains unclear whether the two groups arose from a single ancestor or different ancestors. Therefore, here, we investigated the matrilineal ancestors of O. sativa using markers for organelle (chloroplast and mitochondrial) genomes, and 119 O. sativa landraces, 10 O. glaberrima Steud., 115 O. rufipogon Griff. from Asia, and 39 accessions from other wild rice species with AA genomes. We screened 18 organelle markers developed based on polymorphic loci in the organelle genomes. In addition, we used the open reading frame 100 of a chloroplast marker. The results indicated that O. rufipogon first differentiated into two lineages and then further differentiated into Indica and Japonica Group, respectively. Accessions of O. rufipogon (R-1f and R-2d types) from Myanmar appear to be the closest ancestors of Tropical Japonica Subgroup and Indica Group, respectively. Therefore, these wild strains may have made a strong contribution to the domestication of rice landraces in Myanmar.     

Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.     

Genetic diversity associated with conservation of endangered Dongxiang wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis>)

The wild progenitor species (Oryza rufipogon) of Asian cultivated rice (O. sativa L.) is located in Dongxiang county, China which is considered its the northernmost range worldwide. Nine ex situ and three in situ populations of the Dongxiang wild rice (DXWR) and four groups of modern cultivars were genotyped using 21 SSR markers for study of population structure, conservation efficiency and genetic relationship. We demonstrated that the ex situ conservation of the DXWR failed to maintain the genetic identity and reduced genetic diversity. Therefore, in situ conservation is absolutely necessary to maintain the genetic identity, diversity and heterozygosity. Also, in situ conservation is urgently needed because natural populations in DXWR have decreased from nine to three at present due to farming activity and urban expansion. In DXWR, the three surviving in situ populations had greater expected heterozygosity than any cultivated rice, and were genetically closer to japonica than either the male-sterile maintainer or restorer lines, or indica. Japonica has the lowest genetic diversity of cultivated rice. As a result, DXWR is a rich gene pool and is especially valuable for genetic improvement of japonica rice because these O. rufipogon accessions are most closely related to the japonica as compared to O. rufipogon collected anywhere else in the world.     

Evaluation of Genetic Diversity of Wild Rice (Oryza rufipogon Griff.) in Myanmar using Simple Sequence Repeats (SSRs)

As a part of an in situ survey of wild rice (Oryza rufipogon Griff.) in Myanmar (Burma), 16 strains of wild rice were collected, and analyzed for allelic diversity over 74 loci with simple sequence repeat (SSR) markers to obtain a basic information for their conservation. Three each of indica and japonica cultivars were added for a comparison. In the six cultivars and 16 strains of wild rice, three to 15 alleles were detected per locus with an average of 7.9. The wild rice revealed a large number of unique alleles throughout their chromosomes with much wider ranges of variation than those detected in the six cultivars of O. sativa L.. The alleles found in the wild rice were classified into those specific to wild rice, common to wild rice and cultivars, and those similar to indica or japonica cultivars. According to the classification, the genotype of each of the 16 strains of wild rice was schematically depicted. The genetic variation among individual strains within a collection site was larger than the variation among the collection sites.     

Population structure of the primary gene pool of Oryza sativa in Thailand

The gene pool of cultivated Asian rice consists of wild rice (Oryza rufipogon Griff.), cultivated rice (O. sativa L.) and a weedy form (O. sativa f. spontanea). All three components are widespread in Thailand, frequently co-occurring within fields and providing the opportunity for gene flow and introgression. The purpose to this study is to understand the on-going evolutionary processes that affect the gene pool of rice by analysis of microsatellite variation. Results indicate that O. rufipogon, the wild ancestor of rice, has high levels of genetic variation both within and among populations. Moreover, the variation is structured predominantly by annual and perennial life history. High levels of variation are detected among cultivars indicating Thai cultivated rice has a broad genetic base with only a 20 % reduction in diversity from its wild ancestor. The weedy rice populations reveal varying levels of genetic variation, from nearly as high as wild rice to near zero. Weedy rice is genetically structured into 2 groups. Some populations of invasive weedy rice are the result of hybridization and gene flow between local wild rice and local cultivated rice in the regions of co-occurrence. Other populations of weedy rice are genetically nearly identical to the local cultivated rice. The diversity analysis indicates that the rice gene pool in Thailand is a dynamic genetic system. Gene flow is ongoing among its three main components, first between cultivated and wild rice resulting in weedy rice. Weedy rice in turn crosses with both cultivated varieties and wild rice.     

Genetic Diversity in AA and CC Genome Oryza Species in Southern South Asia

The CC genome of Oryza is found in nine species of Oryza that are distributed on all continents having a tropical climate. Three diploid Oryza species with CC genome are found in Africa and Asia to Papua New Guinea. In southern South Asia these three CC genome diploid species can be found, O. eichingeri and O. rhizomatis in Sri Lanka and O. officinalis in India. AA genome wild relatives of rice are also found in the same geographic region. Germplasm of both diploid CC and AA genome Oryza germplasm has recently been collected from Sri Lanka. AFLP analysis was used to compare the genetic diversity of the two Oryza genomes from a similar geographic region in southern South Asia. In addition, the diploid CC Oryza germplasm was also analyzed by RAPD and SSR methodologies and the combined results were analyzed. The results show that in southern South Asia the diploid CC genome species have a high level of genetic diversity compared to the diploid AA genome species. Molecular marker analysis revealed that populations of O. rhizomatis from northern and southeastern Sri Lanka are genetically differentiated. One accession of O. rhizomatis was aligned with O. eichingeri. This accession was collected from the site of O. rhizomatis that is the closest to a population of O. eichingeri. O. eichingeri showed lower genetic diversity than the other two diploid Oryza CC genome species. O. officinalis accessions from Assam, India, and China were genetically less diverged from O. eichingeri and O. rhizomatis than two accessions of O. officinalis from Kerala state, India. The first two authors contributed equally to this research     

Crossability patterns within and among Oryza series Sativae species from Asia and Australia

Reproductive barriers are thought to intensify with increasing genetic distance between species. To assess the extent of post-pollination reproductive isolation within and among the Asia Pacific species of Oryza series Sativae, crossing experiments using 15 accessions of O. meridionalis Ng, O. nivara Sharma et Shastry, and O. rufipogon Griff. were conducted. Intra- and interspecific crosses of the selfing species O. meridionalis and O. nivara had very low seed set and produced inviable F1 seeds indicative of strong pre- and post-zygotic barriers. Contrastingly, the outcrossing O. rufipogon exhibited high intraspecific crossability and modest compatibility with O. nivara and O. meridionalis in terms of seed set suggesting substantial pre-zygotic reproductive isolation of the species. O. rufipogon was asymmetrically compatible with O. meridionalis and symmetrically with O. nivara. The two inbreeding species manifested comparable degrees of isolation from O. rufipogon despite differences in strength of several post-zygotic barriers. Mating compatibility within and between the Asia Pacific species of Oryza series Sativae is not strongly spatially influenced, but some resistance to gene flow under sympatric conditions was observed. Intraspecific O. rufipogon F1s were more vegetatively robust and more late-flowering than their parents. Intra- and interspecific hybrids of Australasian O. rufipogon differed phenotypically from crosses with non-Australasian populations. Interspecific hybrids displayed both intermediate and parental character traits. O. nivara and O. rufipogon generated early-flowering F1s that are more similar to the former. O. meridionalis and O. rufipogon produced F1s that varied in phenology and morphology depending on the maternal and paternal species.     

Genetic Diversity of the Vigna Germplasm from Thailand and Neighboring Regions Revealed by AFLP Analysis

Thailand is a center of diversity for section Angulares of the Asian Vigna (genus Vigna subgenus Ceratotropis) and 4 Vigna species are cultivated in Thailand. Using newly collected wild and cultivated germplasm of Vigna from Thailand and outgroup accessions AFLP analysis was conducted to clarify genetic diversity and relationships. The results suggest that cultivated V. umbellata and V. mungo evolved from wild relatives in a single domestication event. Vigna umbellata is poorly differentiated from its wild and weedy relatives compared to V. mungo. Results suggest northern Thailand and the neighboring Shan state, Myanmar, is the probable center of domestication for V. umbellata as wild accessions from this area and cultivated rice bean from a wide area in Asia are not greatly diverged. Vigna minima, V. tenuicaulis and V. exilis accessions in Thailand are well differentiated with considerable intra-specific variation. Vigna hirtella consists of two well differentiated subgroups, suggesting taxonomic revision may be necessary. Close genetic relationships between V. radiata and V. grandiflora, and between V. mungo and V. trinervia are confirmed. Naturally growing V. mungo populations in northern Thailand appear to be true wild species as they are well differentiated from Indian wild and Thai cultivated populations. The origin of naturally growing cowpea in Thailand needs to be further studied using a more comprehensive set of materials. This study clarifies inter and intra-specific genetic diversity and inter species relationships of Thai Vigna species.     

Cloning of genome-specific repetitive DNA sequences in wild rice (O. rufipogon Griff.), and the development of Ty3-gypsy retrotransposon-based SSAP marker for distinguishing rice (O. sativa L.) indica and japonica subspecies

In the rice genome, insertions and eliminations of transposable elements have generated numerous transposon insertion polymorphisms (TIPs). Common wild rice (O. rufipogon Griff.), the ancestor of Asian cultivated rice (O. sativa L.), carries abundant genetic variations. To find subspecies-specific (SS) markers that can distinguish O. sativa ssp. indica and ssp. japonica, some long terminal repeat (LTR) sequences (sc1-14) of AA genome-specific RIRE retrotransposon were isolated from O. rufipogon genome. Sequences sc1 and sc12 were successfully utilized to develop the SS marker system based on retrotransposon inserted position polymorphisms. Twenty-two SS markers (ssi1-9, ssj1-13) were developed, where ssi1-9 are the indica-specific types, and ssj1-13 the japonica-specific types. The average accuracy of these markers in distinguishing the two subspecies is over 85%. SS marker ssj-10 can distinguish the two subspecies at 100% accuracy. Principal component analysis (PCA) showed that these markers could successfully distinguish indica from japonica varieties, regardless of their geographical origin.     

普通野生稻稻米加工品质和外观品质性状QTL定位

本研究利用一套以籼稻品种“特青”为遗传背景的云南元江普通野生稻（Oryza rufipogon Griff.）渗入系为材料，采用单标记回归分析和渗入片段叠代法，对出糙率、整精米率、垩白粒率、垩白度、长宽比等5个品质性状的QTL进行了分析，初步定位了16个QTL，有10个QTL来自野生稻的等位基因能改良群体的品质性状。在第5染色体RM289附近检测到了同时影响长宽比、垩白粒率QTL，来自野生稻的等位基因能增加长宽比、降低垩白粒率，贡献率也较高。在第8染色体RM152附近检测到降低垩白粒率和垩白度的QTL，其贡献率分别为14％和9%。本研究结果不仅为品质性状分子标记辅助选择提供参考，而且充分显示了利用野生稻的优异基因改良栽培稻品质性状的巨大潜力。     

Genetic diversity,population structure and differentiation of rice species from Niger and their potential for rice genetic resources conservation and enhancement

Rice genetic resources conservation and evaluation is crucial to ensure germplasm sources for further crop breeding. We conducted a wide collection of Oryza species in Niger and characterize its diversity with microsatellites (or simple sequence repeats, SSR). The aims of this research were to get a better understanding of the extent of genetic diversity, its structure and partition within rice eco-geographical zones of Niger. There were 264 accessions found in farmers’ and other fields: 173 O. sativa (Asia’s rice), 65 O. glaberrima (Africa’s rice), 25 O. barthii, and 1 O. longistaminata (weedy perennial rice), which were genotyped with 18 SSR. A total of 178 alleles were detected, with a mean of 9.89 alleles per locus. The polymorphism information content was 0.65 and heterozygosity was estimated as 0.14. Two main well-differentiate genotypic groups, which correspond to Asian and African rice species, were identified. The SSR set divided the Asia’s rice group (solely indica) into irrigated and floating rice, with rainfed lowland rice in between. The African rice species group was composed of O. glaberrima, O. longistaminata and O. barthii accessions, but without any clear genetic differentiation among them likely due admixtures within the samples of O. barthii. Five accessions that could be natural interspecific hybrids were too admixed for assigning them to any of the two well-differentiated groups. The partitioning of the overall diversity showed that maximum variation was within genotypic groups and subgroups or cropping ecologies, rather than between eco-geographical zones. The eco-geographical distribution of the diversity suggests germplasm exchange in Niger. Next-steps for conserving rice and crop wild relatives in Niger could be taken using the findings of this research.     

Exploring genetic diversity and potential novel disease resistance genes in a collection of rice (<Emphasis Type="Italic">Oryza</Emphasis> spp.) wild relatives

Wild relatives of rice (Oryza spp.) are an important source of novel resistance (R-)genes for rice improvement. Rice sheath blight, caused by Rhizotonia solani, and leaf blast, caused by Magnaportha oryzae, are major fungal diseases of rice worldwide. To identify novel R-genes, a group of Oryza spp. accessions represented by O. alta, O. australiensis, O. barthii, O. glaberrima, O. glumaepatula, O. latifolia, O. meridionolis, O. nivara, O. officinalis, and O. rufipogon, were evaluated for their reaction to leaf blast and sheath blight disease, and genotyped with 176 microsatellite (SSR) markers. Selected rice (O. sativa) accessions were included as reference. Cluster analysis performed with PowerMarker software using Rogers genetic distance and UPGMA, revealed most Oryza spp. accessions clustered with the same species or a closely related Oryza spp. Only a few Oryza spp. accessions grouped with the O. sativa accessions included as a reference. Analysis of this genotypic data in the software Structure revealed that the Oryza spp. accessions were assigned into eight different subpopulations and fit well into eight different backgrounds. Marker-trait associations between the SSR markers and disease reactions to blast and sheath blight were ascertained using the software TASSEL. Associations with blast disease were identified in ten different chromosomal regions and five of the ten were not located near known blast R-genes. Three associations were discovered with sheath blight disease and one was not near previously reported sheath blight QTL. These newly identified regions may represent novel R-genes that will be the basis future fine mapping studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Domestication and association analysis of Hd1 in Chinese mini-core collections of rice

Hd1 is one of the major photoperiod genes with high degree of polymorphisms and contributes to rice (Oryza sativa L.) flowering in different light conditions. Ninety-two rice landraces and 111 accessions of common wild rice (O. rufipogon Griff.) from the mini-core collections in China were selected and sequenced to analyze the domestication process and association of Hd1 with rice flowering. Association analysis revealed that three insertions and two deletions in the coding region of Hd1 are highly correlated with flowering time in the short-day condition. Phylogenetic analysis suggested that the polymorphisms of Hd1 in wild rice are related to the distributions. Haplotype analysis indicated that Hd1 in most O. sativa L. ssp. indica Kato and O. sativa L. ssp. japonica Kato landraces evolved from different O. rufipogon groups containing functional Hd1 and that most aus varieties were domesticated from O. rufipogon containing the long-insertions Hd1, suggesting multiple origins of Hd1. Moreover, O. rufipogon which contained the long-insertions Hd1 could only be found in Southern China, implying that Southern China might be one of the domestication centers of O. sativa. This study provides much significant information to aid further understanding of the domestication process of Hd1.     

Genetic variation and evolution of the Pit blast resistance locus in rice

Resistance (R) gene Pit in rice, encodes a protein with nucleotide binding sites–leucine rich repeat domain, prevents infections by strains of Magnaporthe oryzae in a gene for gene manner. Here, we analyzed the open reading frame (ORF) of Pit in 26 varieties including Oryza sativa L. subsp. indica Kato (Aus), indica (IND), temperate japonica (TEJ), tropical japonica (TRJ), aromatic (ARO) subgroups of Oryza sativa, and nine accessions of wild rice relatives including O. nivara and O. rufipogon from different growing areas. Twenty-one nucleotide differences were found within the ORFs of 35 accessions. Translation of these ORFs revealed a total of 11 Pit variants, seven of which were novel. Furthermore, variant H1 was identified in O. sativa and O. nivara; H2 and H10 were identified in O. sativa and O. rufipogon. H3, H4, and H11 were only found in O. sativa. H11 was the common type of TEJ and ARO; H3 and H4 was the specific type of TRJ and IND, respectively. H5, H6, and H7 were specific haplotypes of O. nivara, while, H8 and H9 were specific haplotypes of O. rufipogon. These results suggest that Pit is an ancient gene that exists before formation of rice domestication subgroups.     

Diversity of native rice (Oryza Poaceae:) species of Costa Rica

We found several populations of wild Oryza species in the lowlands of Costa Rica. The plants showed extensive morphological variation, suggesting the presence of several species. In the morphologic study, 33 traits were scored for plants of all the species. A principal component analysis revealed the significant morphological separation of the different species. The analyses indicated that there are three species, O. grandi­glumis, O. latifolia and O. glumaepatula. Two putative hybrid types were found, both significantly differing in their morphology from the known species and intermediate at several traits. O. grandi­glumis is a new record for Costa Rican flora. Its main population is located in Caño Negro Wildlife Refuge, Los Chiles, Alajuela. O. latifolia is distributed throughout the lowlands of the country and the plants of the Atlantic slope are significantly bigger in general habit than those of the Guanacaste area. During this study a population of O. glumaepatula of hundreds of thousands of plants was discovered in the Medio Queso River wetland, Los Chiles, Alajuela. This population is the most important source of genes for cultivar's improvement from the primary gene pool of rice in Costa Rica. The small ligule and the wide flag leaf characteristic of the two CCDD species separated them from the AA diploid O. glumaepatula. Seed size, ligule size, number of branches in the panicle, plant height and sterile lemma length are all bigger in O. grandi­glumis, and influenced the second factor that separated the CCDD species in two discrete clusters. The species found offer great possibilities for the improvement of rice cultivars and they should be thoroughly studied and appropriately protected.