The qSD12 locus controls offspring tissue-imposed seed dormancy in rice

Seed component structures were grouped into maternal and offspring (embryo and endosperm) tissues to characterize a dormancy quantitative trait locus (QTL) for tissue-specific function using a marker-assisted genetic approach. The approach was devised to test if genotypic/allelic frequencies of a marker tightly linked to the QTL deviate from Mendelian expectations in germinated and nongerminated subpopulations derived from a segregation population of partially after-ripened seeds and was applied to the dormancy QTL qSD12 and qSD7-1 in a nearly isogenic background of rice. Experimental results unambiguously demonstrated that qSD12 functions in the offspring tissue(s) and suggested that qSD7-1 may control dormancy through the maternal tissues. These experiments also provide the first solid evidence that an offspring tissue-imposed dormancy gene contributes to the segregation distortion in a mapping population developed from partially after-ripened seeds and, in part, to the germination heterogeneity of seeds from hybrid plants. Offspring and maternal tissue-imposed dormancy genes express in very early and late stages of the life cycle, respectively, and interact to provide the species with complementary adaptation strategies. The qSD12 locus was narrowed to the region of approximately 600 kbp on a high-resolution map to facilitate cloning and marker-assisted selection of the major dormancy gene.     

Dormancy genes from weedy rice respond divergently to seed development environments

Genes interacting with seed developmental environments control primary dormancy. To understand how a multigenic system evolved to adapt to the changing environments in weedy rice, we evaluated genetic components of three dormancy QTL in a synchronized nondormant genetic background. Two genetically identical populations segregating for qSD1, qSD7-1, and qSD12 were grown under greenhouse and natural conditions differing in temperature, relative humidity, and light intensity during seed development. Low temperatures tended to enhance dormancy in both conditions. However, genotypes responded to the environments divergently so that two populations displayed similar distributions for germination. Additive and/or dominance effects of the three loci explained approximately 90% of genetic variances and their epistases accounted for the remainder in each environment. The qSD1 and qSD7-1 main effects were increased, while the qSD12 additive effect was decreased by relatively low temperatures. Both gene main and epistatic effects were involved in G x E interactions, which in magnitude were greater than environmental main effect. The divergent responses of dormancy genes observed in this simple multigenic system presumably have selective advantages in natural populations adapted to changing environments and hence represent a genetic mechanism stabilizing the dormancy level of weedy rice ripened in different seasons or temperature regimes.     

Multiple loci and epistases control genetic variation for seed dormancy in weedy rice (Oryza sativa)

Weedy rice has much stronger seed dormancy than cultivated rice. A wild-like weedy strain SS18-2 was selected to investigate the genetic architecture underlying seed dormancy, a critical adaptive trait in plants. A framework genetic map covering the rice genome was constructed on the basis of 156 BC(1) [EM93-1 (nondormant breeding line)//EM93-1/SS18-2] individuals. The mapping population was replicated using a split-tiller technique to control and better estimate the environmental variation. Dormancy was determined by germination of seeds after 1, 11, and 21 days of after-ripening (DAR). Six dormancy QTL, designated as qSD(S)-4, -6, -7-1, -7-2, -8, and -12, were identified. The locus qSD(S)-7-1 was tightly linked to the red pericarp color gene Rc. A QTL x DAR interaction was detected for qSD(S)-12, the locus with the largest main effect at 1, 11, and 21 DAR (R(2) = 0.14, 0.24, and 0.20, respectively). Two, three, and four orders of epistases were detected with four, six, and six QTL, respectively. The higher-order epistases strongly suggest the presence of genetically complex networks in the regulation of variation for seed dormancy in natural populations and make it critical to select for a favorable combination of alleles at multiple loci in positional cloning of a target dormancy gene.     

Phenotypic selection for dormancy introduced a set of adaptive haplotypes from weedy into cultivated rice

Association of seed dormancy with shattering, awn, and black hull and red pericarp colors enhances survival of wild and weedy species, but challenges the use of dormancy genes in breeding varieties resistant to preharvest sprouting. A phenotypic selection and recurrent backcrossing technique was used to introduce dormancy genes from a wild-like weedy rice to a breeding line to determine their effects and linkage with the other traits. Five generations of phenotypic selection alone for low germination extremes simultaneously retained dormancy alleles at five independent QTL, including qSD12 (R(2) > 50%), as determined by genome-wide scanning for their main and/or epistatic effects in two BC(4)F(2) populations. Four dormancy loci with moderate to small effects colocated with QTL/genes for one to three of the associated traits. Multilocus response to the selection suggests that these dormancy genes are cumulative in effect, as well as networked by epistases, and that the network may have played a "sheltering" role in maintaining intact adaptive haplotypes during the evolution of weeds. Tight linkage may prevent the dormancy genes from being used in breeding programs. The major effect of qSD12 makes it an ideal target for map-based cloning and the best candidate for imparting resistance to preharvest sprouting.     

Isolation of three dormancy QTLs as Mendelian factors in rice

Seed dormancy is a key adaptive trait under polygenic control in many plants. We introduced the chromosomal regions containing the dormancy QTLs qSD1, qSD7-1, and qSD12 from an accession of weedy rice into a nondormant genetic background to examine component genetic effects and their interactions with time of afterripening (DAR). A BC4F2 plant, which was heterozygous for the three loci, was selected to develop the BC4F3 population. Single point analysis detected only qSD7-1 and qSD12 (R2 = 38-72%) at 10, 30, and 50 DAR in the population. However, multiple linear regression analysis detected genetic effects of the three QTLs and their trigenic epistasis, an environmental effect of DAR (E), and interactions of E with qSD12 and with the qSD1 x qSD7-1 and qSD7-1 x qSD12 epistases. The linear model demonstrates that QTL main effects varied with DAR, and that some epistasis or epistasis-by-DAR interactions partially counteract the main effects. The three QTLs were isolated as single Mendelian factors from the BC4F3 population and estimated for component genic effects based on the BC4F4 populations. Isolation improved estimation of the qSD1 effect and confirmed the major effect of qSD12. The qSD1 and qSD12 loci displayed a gene-additive effect. The qSD7-1, which was further narrowed to a chromosomal region encompassing the red pericarp color gene Rc, displayed gene additive and dominant effects.     

Genetic analysis of adaptive syndromes interrelated with seed dormancy in weedy rice (Oryza sativa)

Seed dormancy in rice interrelates to the weedy characteristics shattering, awn, black hull color, and red pericarp color. A cross between the weedy strain SS18-2 and the breeding line EM93-1 was developed to investigate the genetic basis and adaptive significance of these interrelationships. These characteristics or their components differed in average degree of dominance from –0.8 to 1.5, in heritability from 0.5 to 0.96, and in their contribution to phenotypic or genotypic variation in dormancy by up to 25%. Five dormancy, four shattering, and three awn-length quantitative trait loci (QTLs) were detected in the BC₁ population replicated in 2 years. Two QTLs for hull color were identified, and the SS18-2-derived and EM93-1-derived alleles increased the intensity of black, and red or yellow pigmentations, respectively. The only QTL for pericarp color co-located with the red pericarp gene Rc, with the SS18-2-derived allele increasing the intensity of black and red pigmentations. Four of the five dormancy QTLs were flanked or bracketed by one to four QTLs for the interrelated characteristics. The QTL organization pattern indicates the central role of seed dormancy in adaptive syndromes for non-domesticated plants, implies that the elimination of dormancy from cultivars could arise from the selections against multiple interrelated characteristics, and challenges the use of dormancy genes at these loci in breeding varieties for resistance to pre-harvest sprouting (PHS). However, another QTL (qSD12) provides candidate gene(s) for PHS resistance because it has a large effect in the population and it is independent of the loci for interrelated characteristics.     

Dynamic quantitative trait locus analysis of seed dormancy at three development stages in rice

In this study, a rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of seed dormancy (SD) at 4 (early), 5 (middle) and 6 (late) weeks after heading stages. Dynamic analysis showed that the indica IR28 variety tended to have deeper dormancy than the japonica Daguandao at the middle and late development stages. The level of SD decreased with the process of seed development. The significant interaction between heading date (HD) and SD occurred only in those seeds collected at the early development stage. A total of nine additive quantitative trait loci (QTLs) and eight epistatic QTLs for SD were identified at three seed development stages. Of them, one additive and four epistatic QTLs were identified for the early stage, six additive and one epistatic QTL for the middle stage and two additive and three epistatic QTLs for the late stage. The phenotypic variation explained by each additive and epistatic QTL ranged from 5.8 to 30.6 % and from 3.8 to 13.1 %, respectively. Compared with the additive QTLs, epistatic interactions were much more important for SD at the early and late development stages. Two major additive QTLs, qSD3.1 and qSD4.1, were identified; each QTL could explain more than 20 % of the total phenotypic variance and each dormancy-enhancing allele could decrease the germination percentage by about 10 %. By comparing the chromosomal positions of these additive QTLs with those previously identified, five additive QTLs, qSD1.2, qSD2.1, qSD3.2, qSD4.1 and qSD9.1, might represent novel genes. One QTL identified here, qHD1, and nine QTLs identified in previous studies for HD were co-located with our QTLs for SD, which indicated that the significant correlation between SD and HD might be due to the linkage of QTLs for SD and HD. Four RILs with deep dormancy at development stages but non-dormancy after post-ripening under different germination conditions were selected. Using the selected RILs, three cross combinations of SD for the development of RIL populations were predicted. The selected RILs and the identified QTLs might be applicable for the improvement of pre-harvest sprouting tolerance by marker-assisted selection in rice.     

Comparative genetic analysis of a wheat seed dormancy QTL with rice and <Emphasis Type="Italic">Brachypodium</Emphasis> identifies candidate genes for ABA perception and calcium signaling

Wheat preharvest sprouting (PHS) occurs when seed germinates on the plant before harvest resulting in reduced grain quality. In wheat, PHS susceptibility is correlated with low levels of seed dormancy. A previous mapping of quantitative trait loci (QTL) revealed a major PHS/seed dormancy QTL, QPhs.cnl-2B.1, located on wheat chromosome 2B. A comparative genetic study with the related grass species rice (Oryza sativa L.) and Brachypodium distachyon at the homologous region to the QPhs.cnl-2B.1 interval was used to identify the candidate genes for marker development and subsequent fine mapping. Expressed sequence tags and a comparative mapping were used to design 278 primer pairs, of which 22 produced polymorphic amplicons that mapped to the group 2 chromosomes. Fourteen mapped to chromosome 2B, and ten were located in the QTL interval. A comparative analysis revealed good macrocollinearity between the PHS interval and 3 million base pair (mb) region on rice chromosomes 7 and 3, and a 2.7-mb region on Brachypodium Bd1. The comparative intervals in rice were found to contain three previously identified rice seed dormancy QTL. Further analyses of the interval in rice identified genes that are known to play a role in seed dormancy, including a homologue for the putative Arabidopsis ABA receptor ABAR/GUN5. Additional candidate genes involved in calcium signaling were identified and were placed in a functional protein association network that includes additional proteins critical for ABA signaling and germination. This study provides promising candidate genes for seed dormancy in both wheat and rice as well as excellent molecular markers for further comparative and fine mapping.     

Identification of QTLs with additive,epistatic and QTL × development interaction effects for seed dormancy in rice

Seed dormancy (SD) is an important agronomic trait affecting crop yield and quality. In this study, one rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of SD at the early (4 weeks after heading), middle (5 weeks after heading) and late (6 weeks after heading) development stages. The level of SD decreased with the process of seed development, and the SD was significantly affected by the heading date (HD) and temperature at the early and middle development stages. A total of eight additive quantitative trait loci (QTLs) for SD were identified at three development stages, and more QTLs were expressed at the early and late development stages. Among them, four, one and three additive QTLs were identified at the early, middle and late development stages, respectively. Epistatic QTLs and QTL-by-development interactions were detected by the joint analysis of multi-development phenotypic values, and one additive and two epistatic QTLs were identified. The phenotypic variation of SD explained by each additive, epistatic QTL and QTL × development interaction ranged from 8.0 to 13.5 %, 0.7 to 3.9 % and 1.3 to 2.8 %, respectively. One major QTL qSD7.1 for SD was tightly linked to the major QTL qHD7.4 for HD, which might be applied to reveal the relationship of SD and HD. By comparing chromosomal positions of these additive QTLs with those previously identified, five additive QTLs qSD1.1, qSD2.1, qSD2.2, qSD4.1 and qSD4.2 might represent novel genes. The best three cross combinations for the development of RIL populations were predicted to improve SD. The selected RILs and the identified QTLs might be applicable to improve the rice pre-harvest sprouting tolerance by the marker-assisted selection approach.     

Substitution mapping of QTLs controlling seed dormancy using single segment substitution lines derived from multiple cultivated rice donors in seven cropping seasons

Key message

A permanent advanced population containing 388 SSSLs was used for genetic analysis of seed dormancy; 25 QTLs including eight stable, six major and five new were identified.

Abstract

Seed dormancy (SD) is not only a complex biological phenomenon, but also a key practical problem in agricultural production closely related with pre-harvest sprouting (PHS). However, the genetic mechanisms of SD remain elusive. Here, we report the genetic dissection of SD in rice using 388 single segment substitution lines (SSSLs) derived from 16 donor parents. Continuous variation and positive correlations in seed germination percentages were observed in seven seasons. Genetic analysis revealed the narrow sense heritability in different seasons varied from 31.4 to 82.2% with an average value of 56.8%. In addition, 49 SSSLs exhibited significant difference to recipient parent HJX74 on SD in at least two seasons, and 12 of them were stably identified with putative QTLs in all of their corresponding cropping seasons. Based on substitution mapping, a total of 25 dormancy QTLs were detected on 11 chromosomes except the chromosome 5 with an interval length of 1.1 to 31.3 cM. The additive effects of these QTLs changed from ?0.31 to ?0.13, and the additive effect contributions ranged from 16.7 to 41.4%. Six QTLs, qSD3-2, qSD4-1, qSD7-1, qSD7-2, qSD7-3 and qSD11-2, showed large additive effect contributions (≥30%). Five QTLs, qSD3-3, qSD7-1, qSD7-4, qSD9-1 and qSD10-1, may represent novel ones. Furthermore, linkage and recombinant analysis delimited qSD7-1 to a locus 1.5 cM away from marker Oi2 and a 355-kb fragment flanked by RM1134 and Ui159, respectively. Taken together, this work conducts a comprehensive genetic dissection of SD and will provide more selections for breeding elite PHS-resistant rice varieties.

     

Molecular dissection of QTL governing grain size traits employing association and linkage mapping in Basmati rice

Grain size is one of the key traits that determines the quality of Basmati rice from the consumers’ as well as the traders’ point of view. Though many genes governing grain size have been identified in indica and japonica, little work has been done in Basmati rice. The present study aims at dissection of a QTL region governing grain size traits in Basmati employing association and linkage mapping approaches. Association mapping revealed that three markers, i.e., RM 6024 (grain breadth), RM1237 and RM18582 (grain length-breadth ratio), which cover 889 kb in the targeted QTL region have been significantly associated with grain size traits. Using linkage mapping, the targeted QTL region has been further delimited to a physical distance of 268 kb that comprises 24 annotated genes. The gene expression analysis of parents, revealed 19 genes differentially expressing within the QTL. Of them, 15 genes showed high expression in Basmati370, while four were expressed in Jaya, and whereas five genes did not show any differential expression between parents. Among differentially expressed genes, a highly expressed gene in Basmati370, Os05g0374200 (Cytokinin dehydrogenase 1 precursor) seems to be involved in accumulation of cytokinins, thus affecting the grain size. Therefore, our findings demonstrated that by complimenting association and linkage mapping, it is likely to dissect a QTL governing grain size traits in Basmati rice and also the QTL could be a potential target for marker-assisted breeding and map-based cloning studies.     

Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta 2 using a highly representative rice BAC library

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta ² (closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2?±?1.3 and 8.0?±?2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta ² region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a “two-step walking” method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta ² . The ratio of physical to genetic distances (>?1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta ² region showed various cross-hybridizing signals near the centromeric regions of all chromosomes.     

Soil burial induced dormancy in weedy rice seeds through hormone level changes: Implications in adaptive evolution and weed control

Seed dormancy plays a key role in preventing seeds of higher plants from random germination under adverse environmental conditions. Previous studies suggested that a critical temperature could regulate germination of weedy rice seeds without primary dormancy at seed dispersion. However, what will happen to the non-dormant seeds after shattering in the soil seed banks when temperature fluctuates to exceed the critical temperature remains an interesting question to be investigated. To determine whether or not soil burial can change the status of dormancy in weedy rice seeds, we examined germination ratios of weedy rice seeds after soil-burial treatments. In addition, we compared hormone levels in the untreated seeds and viable but ungerminated seeds after soil burial. Results showed that soil burial induced a proportion of 41%–72% dormant seeds in the initially non-dormant weedy rice seeds. Also, the induction of seed dormancy is associated with the change of hormone levels in the seeds treated by soil burial, suggesting that soil burial can significantly activate the control of hormone production in seeds. Together, the previously reported mechanism of critical temperature-inhibited seed germination and the newly found phenomenon of soil burial-induced seed dormancy provide a “double-security” strategy to ensure germination of weedy rice seeds under a favorable condition in agricultural ecosystems. The findings not only reveal the important role of rapid evolution of adaptive functions in weeds, such as weedy rice, in adapting to changing agricultural environments, but also facilitate the design of strategies for effective weedy rice control practices.     

Seed-Mediated Gene Flow Promotes Genetic Diversity of Weedy Rice within Populations: Implications for Weed Management

Increased infestation of weedy rice—a noxious agricultural pest has caused significant reduction of grain yield of cultivated rice (Oryza sativa) worldwide. Knowledge on genetic diversity and structure of weedy rice populations will facilitate the design of effective methods to control this weed by tracing its origins and dispersal patterns in a given region. To generate such knowledge, we studied genetic diversity and structure of 21 weedy rice populations from Sri Lanka based on 23 selected microsatellite (SSR) loci. Results indicated an exceptionally high level of within-population genetic diversity (H_e = 0.62) and limited among-population differentiation (F_st = 0.17) for this predominantly self-pollinating weed. UPGMA analysis showed a loose genetic affinity of the weedy rice populations in relation to their geographical locations, and no obvious genetic structure among populations across the country. This phenomenon was associated with the considerable amount of gene flow between populations. Limited admixture from STRUCTURE analyses suggested a very low level of hybridization (pollen-mediated gene flow) between populations. The abundant within-population genetic diversity coupled with limited population genetic structure and differentiation is likely caused by the considerable seed-mediated gene flow of weedy rice along with the long-distance exchange of farmer-saved rice seeds between weedy-rice contaminated regions in Sri Lanka. In addition to other effective weed management strategies, promoting the application of certified rice seeds with no weedy rice contamination should be the immediate action to significantly reduce the proliferation and infestation of this weed in rice ecosystems in countries with similar rice farming styles as in Sri Lanka.     

A novel QTL <Emphasis Type="Italic">qSPP2.2</Emphasis> controlling spikelet per panicle identified from <Emphasis Type="Italic">Oryza longistaminata</Emphasis> (A. Chev. et Roehr.), mapped and transferred to <Emphasis Type="Italic">Oryza sativa</Emphasis> (L.)

Increasing the rice productivity from the current 10 to 12 tons/ha to meet the demand of estimated 8.8 billion people in 2035 is posing a major challenge. Wild relatives of rice contain some novel genes which can help in improving rice yield. Spikelet per panicle (SPP) is a valuable trait for determining yield potential in rice. In this study, a major QTL for increasing SPP has been identified, mapped, and transferred from African wild rice O. longistaminata to O. sativa (L.). The QTL was mapped on the long arm of chromosome 2 in a 167.1 kb region flanked by SSR markers RM13743 and RM13750, which are 1.0 cM apart, and is designated as qSPP2.2. The QTL explained up to 30% of phenotypic variance in different generations/seasons and showed positive additive effect of allele contributed by O. longistaminata. In addition, O. longistaminata allele in qSPP2.2 contributed to increase in grains per panicle, but decrease in the tillers per plant. The 167.1 kb region contains 23 predicted genes. Based on the functional annotation, three genes, LOC_Os02g44860, LOC_Os02g44990, and LOC_Os02g45010, were selected as putative candidates for characterization. Sequence analysis of the three genes revealed functional variations between the parental lines for LOC_Os02g44990 and a variation in 5′UTR for LOC_Os02g45010 which will help further to identify putative candidate gene(s). This is the first yield component QTL to be identified, mapped, and transferred from O. longistaminata.