Genetic Architecture of Seed Dormancy in US Weedy ...

RESEARCH

Genetic Architecture of Seed Dormancy in U.S. Weedy Rice in Different Genetic Backgrounds Prasanta K. Subudhi,* Arnold Parco, Pradeep K. Singh, Teresa DeLeon, Ratna Karan, Hanamareddy Biradar, Marc A. Cohn, Darshan S. Brar, and Takuji Sasaki

ABSTRACT Seed dormancy (SD) is a key domestication trait closely related to preharvest sprouting tolerance. Wild and weedy rices (Oryza spp.) exhibit higher degrees of seed dormancy compared to the cultivated rice. Red rice (Oryza sativa L.), a major weed in the rice growing areas of the southern United States, was used to elucidate the genetic architecture of SD. Quantitative trait loci (QTL) analysis conducted in two recombinant inbred line (RIL) populations developed from the crosses involving two rice cultivars (Bengal and Cypress) and a red rice accession (PSRR-1) revealed six to seven QTL for seed dormancy, which accounted for 49 to 52% of the total phenotypic variance. The magnitude of the QTL contribution to phenotypic variance was influenced by genetic backgrounds. The majority of QTL had minor effects, except the QTL linked to Rc and Sdr4. The genetic architecture for seed dormancy in U.S. red rice was distinct compared with the earlier reported weedy accessions. Four QTL were mapped onto similar positions in both populations. Both cultivars and red rice contributed alleles for increased SD. Most of the digenic epistatic interactions involved loci other than the QTL with main effects. The nucleotide polymorphisms at the Sdr4 locus could not explain the phenotypic variation for seed dormancy in our materials. The variation in SD among the rice cultivars could be attributed to segregation of minor QTL, which may be exploited to improve preharvest sprouting tolerance.

P.K. Subudhi, A. Parco, P.K. Singh, T. DeLeon, R. Karan, and H. Biradar, School of Plant, Environmental, and Soil Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803; M.A. Cohn, Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803; D.S. Brar, International Rice Research Institute, Philippines; T. Sasaki, National Institute of Agrobiological Sciences, Tsukuba, Japan. Received 10 Apr 2012. *Corresponding author ([email protected]). Abbreviations: ABA, abscisic acid; BR-RIL, recombinant inbred line population developed from the cross Bengal × PSRR-1; CIM, composite interval mapping; CR-RIL, recombinant inbred line population developed from the cross Cypress × PSRR-1; LOD, logarithm of the odds; ORF, open reading frame; PCR, polymerase chain reaction; QTL, quantitative trait loci; RIL, recombinant inbred line; SD, seed dormancy; SNP, single nucleotide polymorphism; SSR, simple sequence repeat.

S

eed dormancy (SD) refers to the temporary failure of viable seed germination under favorable conditions (Bewley, 1997). It is an important attribute that is targeted for manipulation by plant breeders to improve preharvest sprouting tolerance in rice and other cereals. Intense seed dormancy is an adaptive attribute for successful expansion and persistence of all wild and weedy species, but it poses a serious challenge for the farmers to grow a successful crop. On the other hand, nondormant cultivars have reduced seed longevity and are prone to preharvest sprouting during hot and humid harvesting seasons, resulting in reduced grain yield and quality (Ringlund, 1993). Limited SD is a signature trait of domestication that distinguishes crop cultivars from their wild ancestors. The study of the evolutionary genetic changes associated with the domestication process would offer new insights Published in Crop Sci. 52:2564–2575 (2012). doi: 10.2135/cropsci2012.04.0228 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

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into the past human intervention as well as new clues for crop improvement. Seed dormancy is a complex attribute controlled by multiple genes that are under strong influence of environmental conditions (Gu et al., 2004). Because of strong interaction with environmental factors, seed dormancy is poorly understood at the molecular level. Inherent differences in SD among wild, weedy, and cultivated accessions have been exploited to investigate this trait by the quantitative trait loci (QTL) approach in weedy rice (Gu et al., 2004; Jing et al., 2008; Ye et al., 2010), wild species of rice (Oryza rufipogon Griff. [Cai and Morishima, 2000, 2002; Thomson et al., 2003; Lee et al., 2005] and Oryza nivara S. D. Sharma & Shastry [Li et al., 2006]), and rice cultivars (Lin et al., 1998; Hori et al., 2010; Li et al., 2011; Lu et al., 2011; Marzougui et al., 2012). A seed dormancy QTL Sdr4 on chromosome 7 (Sugimoto et al., 2010), which encodes a protein with no similarity to proteins with known functions, explained the functional difference between ‘Nipponbare’ and ‘Kasalath’ at the Sdr4 locus by a cluster of nucleotide polymorphisms corresponding to two indels and three amino acid substitutions. The Sdr4 is suggested as an intermediate regulator of SD whose expression in turn is regulated by OsVP1, a global regulator of seed maturation. Abscisic acid (ABA) is believed to be the major signaling molecule involved in induction of SD (Finkelstein et al., 2008). Gu et al. (2010) delimited the major QTL qSD12 from red rice and identified three candidate genes. Although the near-isogenic dormant line for the qSD12 accumulated much higher ABA in early stages of seed development than the dormant line, none of the candidate genes was involved in ABA biosynthetic pathways. Red pericarp (Rc), a distinguishing characteristic of weedy red rice, is believed to be closely associated with SD and has been selected against during rice domestication (Sweeney et al., 2007). The white pericarp in most cultivated varieties is attributable to a 14-bp deletion within exon 6 of the Rc gene, which encodes a bHLH (basic helix-loop-helix) protein (Sweeney et al., 2006). Association between SD and red pigmentation is caused by the pleiotropic locus qSD7-1/Rc, which not only increased ABA accumulation but also activated a conserved network of eight genes in the flavonoid biosynthetic pathway (Gu et al., 2011). This observation contrasts with the earlier report of Gianinetti and Vernieri (2007), who found no correlation between ABA content and the dormancy status of dry or imbibed seeds of weedy red rice. Therefore, identification and cloning for additional QTL in a wide range of genetic materials is required for an unambiguous understanding of the molecular mechanism of seed dormancy. Weedy rice, widely known as red rice in the United States, is an annual conspecific weed relative of cultivated rice (Oryza sativa). It is characterized by high genetic variability and phenotypic plasticity (Oka, 1988) and is prevalent in CROP SCIENCE, VOL. 52, NOVEMBER– DECEMBER 2012

rice growing areas in southern United States, Europe, and Central and South America. It poses significant constraints to rice productivity because of its aggressive growth habit, and removal from the rice field using herbicides can be difficult because of its resemblance to cultivated rice. Losses from red rice in the United States can be as high as 80% (Estorninos et al., 2005). The ability of red rice to easily hybridize with closely related cultivated rice (Langevin et al., 1990) facilitates synthesis of unique genetic materials to study the genetics of domestication traits. Because of its small and diploid genome, the availability of a high density genetic map, and whole genome sequence (Goff et al., 2002; IRGSP, 2005), it is an ideal model for genetic studies. The origin of red rice in many geographic regions of the world, where no wild relatives of rice occur, continues to be a genetic puzzle. Molecular analysis indicated that the red rices were more related to O. sativa subsp. japonica Kato or subsp. indica Kato types (Cho et al., 1995; Suh et al., 1997). But the red rice collections of the southern United States were highly diverse, and some were closely related to O. nivara or O. rufipogon (Vaughan et al., 2001; Gealy et al., 2002). It is worthwhile to test two likely hypotheses of reversion from cultivars or hybridization with O. rufipogon, which will require precise localization and isolation of the domestication QTL. Genomics tools and approaches, particularly the QTL mapping and comparative mapping, which have proven their potential to dissect historical genetic consequences in crop evolution, offer new possibilities for crop improvement (Paterson, 2002). More specific outcomes are the unraveling of genetic architecture of domestication-related traits with emphasis on number, chromosomal location, phenotypic effects, and genetic interactions (Tanksley, 1993). In rice, the genetic changes associated with the evolution of cultivated rice from wild species are not yet known. A limited number of studies (Gu et al., 2004; Jing et al., 2008) revealed that weedy rice largely differs from the cultivated rice with respect to only a few QTL. The overall goal of the present study is to determine the QTL architecture for seed dormancy using two recombinant inbred line (RIL) populations from crosses between two rice cultivars and a red rice accession. The variation and consistency of these QTL in two different genetic backgrounds as well as with the reported QTL in wild, weedy, and cultivated rice were assessed. We also determined if the sequence variation of cloned genes Sdr4 and Rc can explain the phenotypic variability for this trait in our plant materials.

MATERIALS AND METHODS Mapping Population Development Two RIL populations were developed from crosses involving two high yielding rice cultivars (Bengal and Cypress) and a weedy

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red rice accession PSRR-1 using the single seed descent method. Bengal is an early maturing, high yielding, medium-grain cultivar that originated from the cross ‘MARS’//‘M201’/MARS (Linscombe et al., 1993a). Cypress is a high-yielding, early-maturing long-grain cultivar developed from the cross ‘L-202’/‘Lemont’ (Linscombe et al., 1993b). Both rice cultivars were released by the Rice Research Station, Crowley, Louisiana Agricultural Experiment Station. The red rice accession PSRR-1 was purified by single plant selection for two generations from a mixture of red rice seeds collected from the Rice Research Station at Crowley, LA. PSRR-1 has light green leaves, vigorous growth, long auricles and ligules, straw-hulled medium grains, lax open panicles, and pubescent leaves. These plants are extremely susceptible to shattering and have a high intensity of both hull and pericarp dormancy compared to Bengal and Cypress. The F1 plants in each cross were selfed to produce F2 seeds. Starting with F2 generation, single seed from each plant was used to produce the next generation. The same procedure was followed for additional generations depending on the population until the seed of each RIL was increased for this study. The RIL population developed from the cross Bengal × PSRR-1 (BR-RIL) consisted of 198 individuals in the F7:8 generation whereas the RIL population developed from the cross Cypress × PSRR-1 (CR-RIL) included 174 individuals in the F8:9 generation.

Evaluation of Seed Dormancy Both RIL populations along with parents were grown at the Louisiana State University AgCenter Central Research Station in Baton Rouge, LA, during spring 2009. Each line was sown into one row at a density of 20 plants m–1 with a total row length of 1.5 m and row to row spacing of 20 cm. Standard cultural practices were followed throughout the season (Linscombe et al., 1999). Five plants were randomly sampled from each line for the SD assay. Because there was a great deal of variation in days to flowering in both RIL populations, flowering date for each RIL was recorded to ensure proper timing of harvest. Mean temperatures between heading date and harvesting ranged from 23 to 34°C. Plants were considered to reach physiological maturity 30 to 35 d after flowering. Seeds were then collected in coin envelopes, dried for 4 d at ambient temperature to reduce the moisture content to approximately 12%, and stored at –15°C until the germination test was performed. Intact seeds with the hulls were used for each germination test. From each plant, three sets of 30 seeds each were used for a germination test. Seeds were placed in petri plates lined with one layer of seed germination paper (Anchor Paper Co.) wetted with sterile distilled water and were placed in darkness in an incubator at 30°C. Observations were taken at 7 and 14 d after imbibition. Splitting of hull by the emerging radicle was used as the criterion for visible germination. The number of seeds germinated in each replication of each RIL and parent was expressed as a percentage. Germination percent was arcsine transformed to improve normality for statistical analysis and QTL mapping. Only one qualitative trait, pericarp color, was evaluated in both populations for presence and absence of the red color.

Genotyping and Linkage Map Construction Leaf tissues were collected at the early vegetative stage, and DNA was isolated from each RIL and parents following the procedure of Tai and Tanksley (1990). From each linkage group, 30 2566

to 40 simple sequence repeat (SSR) markers were chosen for a polymorphism survey between parents. Two hundred twelve and 189 polymorphic SSR markers uniformly distributed across the whole rice genome were selected for genotyping in the BR-RIL and CR-RIL, respectively. Primers were synthesized by Alpha DNA using the available primer information (McCouch et al., 2002; http://www.gramene.org/markers/microsat/ssr.html [accessed 28 June 2012]), and SSR analysis was conducted following Chen et al. (1997). The thermal profi le was as follows: an initial denaturation step of 4 min at 94°C followed by 40 cycles of 1 min denaturation at 94°C, 1 min annealing at 50°, 55°, or 60°C (depending on the individual SSR marker), and a 2 min extension at 72°C. After cycling, a final extension time of 10 min at 72°C was followed. The polymerase chain reaction (PCR) products were separated on a 4.5% superfine resolution agarose (Amresco) gel and visualized under ultraviolet light after ethidium bromide staining. Linkage maps were constructed with MAPMAKER/EXP using the Kosambi function (Lander et al., 1987). The ordering of markers was verified by manual examination of the three-point data. In the BR-RIL 3.39% of data points and in the CR-RIL 3.14% data points were missing.

QTL Mapping, QTL Nomenclature, and Statistical Analysis Quantitative trait loci mapping was conducted using the composite interval mapping (CIM) procedure implemented in QTL Cartographer version 2.5 (Wang et al., 2011). In the CIM procedure, a forward–backward regression procedure was followed with walk in speed of 1.0 cM. Threshold logarithm of the odds (LOD) values for CIM based on 1000 permutations (P < 0.01) were 3.39 and 3.55 for the BR-RIL and CR-RIL, respectively. In addition to the significant QTL, the QTL identified at LOD 2.5 were included as suggestive QTL. The total phenotypic variation explained by all putative QTL was estimated by fitting a model in the multiple interval mapping procedure of QTL Cartographer. The identified QTL were compared with the results from single marker analysis. The QTL were named following the nomenclature of McCouch et al. (1997) with some modifications to distinguish the QTL detected for the same trait in both RIL populations. A superscript of “BR” or “CR” was added after the QTL to indicate a QTL identified in the BR-RIL or CR-RIL, respectively. For example, qSD1BR and qSD1CR indicated SD QTL located on chromosome 1 but detected in the BR-RIL and CR-RIL, respectively. Analysis of variance was conducted using the transformed data on germination percent collected separately on five plants in each RIL. Broad-sense heritability was calculated as H = σ2g/(σ2g + σ2e), in which H is broad-sense heritability, σ2g is the genetic variance, and σ2e is the error variance from the trial. Pairwise tests among all marker loci on the linkage map were performed to detect epistatic interactions (P < 0.001) using the EPISTACY program (Holland, 1998). Because the RILs were homozygous, the estimated interaction variance is solely composed of additive by additive epistasis resulting from interactions among homozygous genotypes at the loci involved. The following general linear model was used to detect digenic epistasis between markers in a two-way analysis of variance implemented in EPISTACY program: Yijm = μ + α i + α j + Ψij + ε ijm ,

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in which Yijm is the trait value of the mth individual with the digenic genotype at marker loci i and j, μ is the population mean, α i and α j are the main effects (the additive effects) associated with the marker loci i and j, respectively, Ψij is the effect arising from interactions between the alleles at marker loci i and j (additive × additive), and εijm is the residual effect including the genetic effect unexplained by the two loci in the model plus the experimental error. Frequency distributions were generated using Microsoft Office Excel 2007 (Microsoft, 2007) and correlation coefficient between pericarp color and germination percentage was determined using SAS software (SAS Institute, 1999).

Sequencing of Rc and Sdr4 Sequences of the targeted regions of Rc and Sdr4 responsible for the phenotypic variability were determined from the genomic DNA of Bengal, Cypress, or PSRR-1 through PCR amplification using Phusion High Fidelity DNA Polymerase (New England Biolab Inc.) and cloning in the pGEM-T Easy vector system I (Promega Corp.) followed by sequencing. Primers were designed based on the available sequences in the database. Primers used for amplification of the sequences of the genes Sdr4 and Rc are listed below:

on chromosome 7 in both populations (Sweeney et al., 2006). Overall, the order of the molecular markers in both linkage maps was largely similar to those published earlier (McCouch et al., 2002) with following exceptions. In BR-RIL map, RM8250 (chromosome 6) (Temnykh et al., 2001; McCouch et al., 2002) and RM235 (chromosome 12) were mapped to chromosomes 9 and 8, respectively. RM1208 (chromosome 11) (McCouch et al., 2002) was placed on the top of chromosome 12 in both populations. The average PSRR-1 genome content was 52% in both populations, but it varied a great deal in the BR-RIL and CR-RIL with a range of 27 to 80% and 34 to 74%, respectively. Thirty-one percent of markers had distorted segregation ratios in the BR-RIL, but it was 40% in the CR-RIL. These segregation distorted markers were spread across all chromosomes except 2, 4, and 5 in the BR-RIL; in the CR-RIL, only chromosome 9 did not exhibit unbalanced segregation (Fig. 1). All markers on chromosomes 3, 7, 10, and 11 had disproportionately higher frequencies of the PSRR-1 genotypes.

SDR4FNEW: 5′-GGGCCCCAACGCATCCGTTC-3′

Phenotypic Variation

SDR4R1: 5′-TCACGCGTCGCCGGCGGC-3′

Both rice cultivars, Bengal and Cypress, differed significantly from PSRR-1 with respect to SD (Fig. 2). Bengal was extremely nondormant (92% germination), Cypress was partially dormant (65% germination), and PSRR-1 showed a high degree of seed dormancy (2% germination). The intensity of SD in hybrids of both Bengal × PSRR-1 and Cypress × PSRR-1 crosses was similar to PSRR-1. The mean germination percentages were 18 and 41 in the BR-RIL and CR-RIL, respectively. For SD, the BR-RIL was skewed toward red rice phenotype, but the CR-RIL exhibited a whole range of variability with some transgressive segregants with less dormancy than Cypress. The broad-sense heritabilities for seed dormancy were 0.74 and 0.76 in the BR-RIL and CR-RIL, respectively. Seventy-five percent of RILs in both populations had red pericarp. Because it is widely thought that red pericarp and SD are linked, the relationship between these traits was analyzed in both populations (Fig. 3). The majority of RILs with red pericarp showed seed dormancy in the BR-RIL, but in the CR-RIL, RILs with both red and white pericarp showed a wide range of variation in SD. In both populations, white pericarp with intense SD, like red rice, was observed. Overall, red pericarp was significantly correlated with seed dormancy in both populations, and the correlations accounted for 22 and 10% of the phenotypic variance in the BR-RIL and CR-RIL, respectively.

RC15F: 5′-CTGAAGGAAGTGATGACAACAAGACC-3′ RC15.2R: TAAGTATGACTTATATTTTACATATTTGCAC-3′. The available sequence information for Bengal, Cypress, and other accessions was downloaded from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov [accessed 29 May 2012]) for comparison. In the case of Rc, the genomic region containing the 14 bp deletion was sequenced. Although primers were designed to amplify the complete open reading frame (ORF) of the Sdr4 gene, a few bases toward the end of the ORF were not correctly determined because of the presence of a repeat sequence. The DNA sequencing was performed in an ABI Prism 3130 sequencer (Applied Biosystems) at the Gene Lab of the School of Veterinary Medicine, Louisiana State University. The DNA sequences were aligned using BioEdit software (Hall, 1999) and edited using Jalview (Waterhouse et al., 2009). The partial nucleotide sequences identified in this study were deposited in GenBank under accession numbers JN014835 (Rc-PSRR-1), JN014836 (Sdr4-PSRR-1), JN014837 (Sdr4-Cypress), and JN014838 (Sdr4-Bengal).

RESULTS Linkage Map Construction, Genome Composition, and Segregation Distortion The BR-RIL linkage map consisted of 212 SSR markers and Rc (the pericarp color locus), which were distributed across all 12 chromosomes with a total distance of 1410 cM. The CR-RIL map consisted of 189 SSRs and Rc with a total distance of 1574 cM. Chromosome length ranged from 89.7 cM (chromosome 10) to 186.2 (chromosome 1). The Rc locus was mapped to the expected location CROP SCIENCE, VOL. 52, NOVEMBER– DECEMBER 2012

QTL Analysis In the BR-RIL, composite interval mapping detected seven QTL distributed across chromosomes 1, 3, 4, 7, and 11 (Table 1). There were three QTL on chromosome 7. Based on the peak map positions, qSD7-1BR was 38 cM apart from

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Figure 1. Genome-wide segregation distortion from the expected 1:1 ratio in the recombinant inbred line population developed from the cross Bengal × PSRR-1 (BR-RIL) (A) and recombinant inbred line population developed from the cross Cypress × PSRR-1 (CR-RIL) (B). Ch, chromosome.

qSD7-2BR whereas distance between qSD7-2BR and qSD7-3BR was 23 cM. A wide range of variation was observed with respect to the magnitude of additive effects and percentage of the phenotypic variation explained by these QTL. Additive effect was the highest for the qSD7-2BR followed by qSD7-3BR and qSD7-1BR. The largest QTL qSD7-2BR explained 25% of the phenotypic variance, and the rest of the QTL accounted for 2 (qSD3BR) to 8% (qSD7-3BR) phenotypic variation. The Rc locus was the closest marker to the largest seed dormancy QTL. All seven QTL accounted for 52% of the phenotypic variation. Red rice alleles of five QTL were responsible for increased SD whereas Bengal alleles enhanced SD in the rest. In the CR-RIL, six QTL were detected, accounting for a total phenotypic variation of 49%, (Table 2). The 2568

QTL were mapped to chromosomes 3, 4, 7, 10, and 12. The contribution of each QTL ranged from 4 to 12%. Cypress was the donor for increased SD in three QTL, and red rice allele increased SD at three QTL. There were two QTL on chromosome 7. The linkage groups of each RIL population were aligned using common markers and comparison of the QTL positions in both populations revealed four consistent QTL in spite of the differences in contribution to phenotypic variation (Fig. 4); one QTL was on chromosome 3 and two QTL were on chromosome 7, with the red rice allele enhancing SD. On the other hand, the cultivated rice allele of the QTL on chromosome 4 was responsible for enhanced dormancy. The loci qSD7-1CR and qSD7-2CR

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Figure 2. Frequency distribution of germination percentage (arcsine transformed) in the recombinant inbred line population developed from the cross Bengal × PSRR-1 (BR-RIL) (A) and the recombinant inbred line population developed from the cross Cypress × PSRR-1 (CR-RIL) (B). Phenotypic values of parents Bengal, Cypress, and PSRR-1 are indicated by arrows.

Figure. 3. Variability in dormancy among recombinant inbred lines (RILs) with red and white pericarp in the RIL population developed from the cross Bengal × PSRR-1 (BR-RIL) (A) and the RIL population developed from the cross Cypress × PSRR-1 (CR-RIL) (B).

were 46 cM apart on chromosome 7. The magnitude of the consistent QTL qSD7-2BR was smaller in the CR-RIL but the magnitude of the QTL corresponding to the qSD7-3BR was higher in the CR-RIL. The additive effects were also higher for the alleles of the chromosome 7 QTL. Twenty-seven digenic epistatic interactions were detected for SD in the BR-RIL (Supplemental Table S1). There was only one significant interaction between qSD1BR and qSD4BR whereas two other QTL, qSD7-3BR and qSD11BR, interacted with a segment on chromosomes 1 and 8, respectively. The majority of interactions involved loci not linked to the detected QTL and accounted for 6 to 10% of the total genotypic variation after accounting for the main effects of two marker loci. Overall, clusters of closely linked markers on four chromosomes (chromosomes 1, 7, 11, and 12) were involved in majority of interactions. In the CROP SCIENCE, VOL. 52, NOVEMBER– DECEMBER 2012

CR-RIL, 24 marker loci were responsible for 17 digenic interactions controlling SD (Supplemental Table S2). Most interactions involved loci not linked to the detected QTL and only qSD10CR was involved in interaction.

Sequence Variation in Rc and Sdr4 Comparison of sequences of Rc revealed that the 14 bp in the exon 6 responsible for red pericarp was present in PSRR-1, but it was deleted in Cypress, Bengal, and ‘Jefferson’ (all white pericarp) as expected (Supplemental Fig. S1). Comparison of Sdr4 nucleotide and amino acid sequences of PSRR-1, Bengal, and Cypress with those of Nipponbare and Kasalath revealed several single nucleotide polymorphisms (SNPs) and indels resulting in amino acid changes (Fig. 5; Supplemental Fig. S2 and S3). The Cypress haplotype resembled Nipponbare with differences

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Table 1. Quantitative trait loci (QTL) for seed dormancy in the BR-RIL† detected using composite interval mapping procedure. QTL

Flanking markers

Position‡

RM5362–RM3362 RM5819–RM5480 RM3839–RM3288 RM6652–RM6663 RM7121–Rc RM5508–RM351 RM206–RM254

159.1 25.4 64.7 3.0 41.4 74.1 91.5

BR

qSD1 qSD3BR qSD4BR qSD7-1BR qSD7-2BR qSD7-3BR qSD11BR †

3.32 2.31 2.80 3.28 12.92 4.41 2.71

AE¶

Percent PVE#

DPE††

3.631 3.162 –3.334 3.853 8.589 4.250 –3.505 Total

4.5 2.4 3.7 3.8 24.7 8.2 4.9 52.2‡‡

R R B R R R B

BR-RIL, recombinant inbred line population developed from the cross Bengal × PSRR-1.

‡

QTL peak position on the linkage map.

§

LOD, logarithm of the odds.

¶ #

LOD§

AE, additive effects of Bengal allele.

PVE, Phenotypic variation explained by each QTL.

††

DPE, direction of phenotypic effect. B and R denote Bengal and PSRR-1 alleles increasing the phenotypic values, respectively.

‡‡

Estimate of the total phenotypic variation explained by the QTL from a multiple QTL model ﬁt in QTL Cartographer (Wang et al., 2011).

Table 2. Quantitative trait loci (QTL) for seed dormancy in the CR-RIL† detected using composite interval mapping procedure. QTL CR

qSD3 qSD4CR qSD7-1CR qSD7-2CR qSD10CR qSD12CR †

Flanking markers RM22–RM5819 RM6250–RM5503 RM2006–RM7121 RM455–RM351 RM216–RM2504 RM7315–RM6296

LOD§

AE¶

Percent PVE#

DPE††

2.42 3.95 4.92 5.34 2.97 2.89

5.151 –6.528 8.087 7.197 –5.145 –6.466 Total

6.6 7.4 11.2 12.3 4.0 7.3 49.2‡‡

R C R R C C

8.8 65.3 22.4 68.5 10.3 21.2

CR-RIL, recombinant inbred line population developed from the cross Cypress × PSRR-1.

‡

QTL peak position on the linkage map.

§

LOD, logarithm of the odds.

¶ #

Position‡

AE, additive effects of Cypress allele.

PVE, Phenotypic variation explained by each QTL.

††

DPE, direction of phenotypic effect. C and R denote Cypress and PSRR-1 alleles increasing the phenotypic values, respectively.

‡‡

Estimate of the total phenotypic variation explained by the QTL from a multiple QTL model ﬁt in QTL Cartographer (Wang et al., 2011).

at three SNPs: 591, 596, and 642 nucleotide positions. PSRR-1 and Bengal haplotypes are very close to each other with differences at five SNPs. Kasalath differed from PSRR-1 and Bengal at the 103 to 105 nucleotide indel position and seven SNPs (Fig. 5A). The 18-bp direct repeat of Nipponbare was found in only Cypress but not in Bengal and PSRR-1, resulting in clustered changes in DNA sequences and amino acids (Fig. 5B).

DISCUSSION Elucidation of genomic differences between weedy rice and cultivated rice can provide insights into the origin and evolution of weedy rice as well as opportunities for crop improvement. To date, the genetics of seed dormancy has been studied in two weedy rice accessions (Gu et al., 2004; Jing et al., 2008), and none of the important domestication traits, such as SD, seed shattering, and awning, has been investigated in the U.S. red rice. This study reports the development and utilization of two RIL populations involving a U.S. red rice accession for the first time to determine the genetic basis of a key domestication trait. Both populations are unique genetic resources because 2570

of the involvement of a red rice accession that differed from the weedy rice used in earlier studies (a subsp. indica weedy rice from Thailand [Gu et al., 2004] and a weedy rice from China [ Jing et al., 2008]) with respect to many domestication traits, such as awn length, hull color, and photosensitivity, to name a few.

Segregation Distortion and Selective Advantage of Red Rice Alleles The overall percentage of markers with skewed segregation was comparable or lower than that previously reported for crosses involving O. rufipogon (Cai and Morishima, 2000; Thomson et al., 2003; Lee et al., 2005) but higher than that for the intraspecific crosses (Xu et al., 1997; Harushima et al., 2002) and a cross involving temperate subsp. japonica weedy rice (Bres-Patry et al., 2001). In both populations, segregation distortion was observed for a majority of markers on chromosomes 3, 7, 10, and 11 in both populations (Fig. 1), which is consistent with the observation of Xu et al. (1997) and Bres-Patry et al. (2001). Segregation distortion could have been caused by factors for gametic competition or abortion of zygotes

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Figure 4. Map location of consistent seed dormancy quantitative trait loci (QTL) on chromosomes 3, 4, and 7 in the recombinant inbred line population developed from the cross Bengal × PSRR-1 (BR-RIL) and recombinant inbred line population developed from the cross Cypress × PSRR-1 (CR-RIL). Coincidence of QTL was inferred from common markers and physical position of markers on the Nipponbare genome sequence. BR and CR indicate the BR-RIL and CR-RIL, respectively. The location of Sdr4 (Sugimoto et al., 2010) is shown on the map. B (Bengal), C (Cypress), and R (PSRR-1, red rice) indicate direction of phenotypic effect (gray circles). Dashed circles indicate common QTL between the BR-RIL and CR-RIL. The QTL consistent with earlier reports are denoted by numbers: 1, Lee et al. (2005); 2, Thomson et al. (2003); 3, Gu et al. (2004); 4, Ye et al. (2010); 5, Li et al. (2011). Chr, chromosome.

or male gametes or female gametes (Sano, 1990; Lyttle, 1991). Sterility during generation advance might have also contributed to the segregation distortion in some chromosomal regions. Despite easy crossability between rice and red rice, occurrence of sterility in segregating generations suggested a significant role of epistatic interaction among genes from cultivars and red rice. Our study demonstrated that the red rice accession PSRR-1 had the ancestral functional allele with a 14-bp sequence, like O. rufipogon, but it was deleted in both cultivars used in this study (Supplemental Fig. S1). In both RIL populations, 75% of the RILs had red pericarp instead of the expected 50% (Fig. 3), suggesting selective advantage of the red pericarp over white pericarp. This observation did not agree with the previous report of Gu et al. (2004), who observed simple Mendelian segregation for the pericarp color. Besides the pericarp color, red rice alleles were observed in higher frequency for a majority of marker loci, which is in sharp contrast to the general notion of CROP SCIENCE, VOL. 52, NOVEMBER– DECEMBER 2012

underrepresentation of wild alleles in wide crosses (Cai and Morishima, 2000). The majority of QTL were localized to these chromosome regions, showing segregation distortion as in earlier studies (Cai and Morishima, 2000; Bres-Patry et al., 2001). Red pericarp is ubiquitous in U.S. weedy rice, but dormant, weedy rice with a white pericarp is nearly absent in natural field environments, despite occurrence of outcrossing between red rice and cultivated rice (Shivrain et al., 2007). Further evidence was provided by Gross et al. (2010) who reported consistent presence of a functional Rc haplotype in divergent red rice populations. Association of Rc with a major QTL for an adaptive trait such as seed dormancy may be responsible for genetic eradication of recessive phenotypes with white pericarp. Alternatively, genes closely linked to the Rc might provide selective advantage. The preponderance of red rice alleles, including Rc, needs further genetic investigation to understand the molecular mechanisms associated with increased fitness and persistence of red rice.

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Figure 5. Comparison of indels, single nucleotide polymorphisms (SNPs), and amino acid sequence of Sdr4 among PSRR-1, Bengal, Cypress, Kasalath, and Nipponbare. (A) Haplotype differences with respect to the two indels and 17 SNPs. Amino acid changes are noted within parenthesis. (B) The 18-bp direct repeats are indicated by arrows and the resulting amino acid changes are shown. Nipponbare and Kasalath sequences were obtained from the earlier study (Sugimoto et al., 2010).

Molecular Basis of Seed Dormancy at the Sdr4 locus Using the available rice genome information, we concluded the correspondence of the consistent qSD7-3BR (qSD7-2CR in the CR-RIL) with the Sdr4 (Sugimoto et al., 2010) (Fig. 4). Functional differences between the Sdr4 allele of both parents, Nipponbare and Kasalath, were explained by a cluster of functional nucleotide polymorphisms corresponding to two indels and three amino acid substitutions (Fig. 5). Based on the two indels and 17 SNPs analyzed in this study, these polymorphisms could not be associated with the variation in seed dormancy. Bengal and PSRR-1 had the Sdr4-k′ haplotype, but they differed significantly with respect to seed dormancy. Similarly, it is not consistent with the finding of Sugimoto et al. (2010) because Cypress and Nipponbare differed in seed dormancy yet had identical Sdr4-n haplotypes. The epistatic interactions involving qSD7-3BR (corresponding to Sdr4 linked markers) supported the observation of Sugimoto et al. (2010) that Sdr4 was transcriptionally regulated by OsVP1 (a global regulator of seed maturation) (Hattori et al., 1994; Bailey et al., 1999). Based on our results, the sequence differences in the Sdr4 might not be sufficient to explain the phenotypic variation. 2572

Therefore, sequence variation in upstream genes, such as OsVP1 or involvement of other seed dormancy loci and their interaction should be investigated to shed light on the molecular mechanism.

Genetics of Seed Dormancy and Crop Improvement Contrary to the results of classical studies supporting simpler genetic models (Seshu and Sorrells, 1986; Takahashi, 1997), the present study revealed complex genetic control with involvement of both major and minor QTL whose expressions were influenced by epistasis (Gu et al., 2004) and genetic backgrounds (Onishi et al., 2007). Based on the single-marker analysis, markers nearest to the peaks of all QTL were associated with seed dormancy with the exception of only qSD12CR. The red rice accession differed from cultivated rice with respect to at least four QTL regions that were consistent in both RIL populations (Fig. 4). Other QTL present in either population could be responsible for the phenotypic differences between red rice and cultivated rice, but the variation in expression of these QTL could be because of differences in the genetic background of the rice cultivars (Linscombe et al., 1993a, 1993b).

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Comparison of QTL positions with those from earlier studies involving weedy rice (Gu et al., 2004; Jing et al., 2008) revealed clear differences in their genetic makeup. Seed dormancy in our study showed complete dominance in contrast to the studies of Gu et al. (2004), who observed incomplete dominance. However, broadsense heritabilities for SD in both populations in this study were comparable to those of Gu et al. (2004), which varied between 0.68 and 0.81. Despite the high intensity of SD, the genetic architecture was quite distinct for each of these weedy accessions. For example, there was no overlapping of the QTL in the Chinese weedy rice (Jing et al., 2008). Although the consistent QTL on chromosomes 4 and 7 corresponded well to the QTL identified in subsp. indica weedy rice SS18-2 (Gu et al., 2004; Ye et al., 2010), the cultivar-derived allele at chromosome 4 QTL in this study enhanced SD in both crosses instead of weedy rice (Fig. 4). Moreover, the qSD12 with the largest effect (Gu et al., 2004), which promotes abscisic acid accumulation in early developing seeds to induce primary dormancy (Gu et al., 2010), was not detected in either populations, indicating genetic differences between SS18-2 and PSRR-1. These observations suggest that the high variability in the genetic composition of weedy rice may be dependent on the geographical region. It was interesting to note that none of the earlier studies involving O. rufipogon (Cai and Morishima, 2000; Thomson et al., 2003; Lee et al., 2005) and O. nivara (Li et al., 2006) reported the major QTL on chromosome 7 identified in this study, which has pleiotropic effects on both SD and red pericarp color (Gu et al., 2011). In the CR-RIL, this QTL was detected with a slightly lower LOD value. The interference of other factors could be responsible for reducing the magnitude of this major QTL. This is further supported by our observations that many of the RILs with red kernel had higher germination percentages in the CR-RIL (Fig. 3), which could be attributed to recombination of QTL from other parts of the genome (Cai and Morishima, 2000). Comparison with the studies involving cultivated rice revealed the QTL corresponding to Sdr4 in both populations coincided with the QTL reported by Li et al. (2011). Contrary to earlier weedy rice QTL studies, both rice cultivars in this study harbored alleles for enhancing SD. Cypress exhibited a higher degree of seed dormancy compared to Bengal but lower than that for PSRR-1. For QTL mapping, selection of parents with different alleles is crucial to develop the populations that would show a range of variation resulting from recombination and epistatic interaction among several parental genes. Although the study was conducted for only one season, the QTL identified in both populations explained ~50% of the phenotypic variation and four QTL were congruent in both populations, suggesting reliability of the data. The QTL with largest effect were CROP SCIENCE, VOL. 52, NOVEMBER– DECEMBER 2012

located on chromosome 7, but QTL with SD enhancing alleles from cultivated rice were with minor effects. Differences in QTL architecture observed in both populations indicated that only minor QTL were the main contributing factors for the difference in SD between the two cultivars. Another possibility could be attributed to the fact that in populations where large effect QTL segregate, epistatic QTL are difficult to detect, even if their actual effects are large (Yamamoto et al., 2000), particularly when the population size is small (Tanksley, 1993). A QTL × environment interaction has also been reported to influence QTL detection and their effects (Prada et al., 2004). Therefore, it will be interesting to determine the interaction of the major QTL (corresponding to Rc) with different genetic backgrounds, which will be useful in predicting the effect of genes from wild or weedy parents. Our study suggested that even without involvement of major genes, a higher level of SD might result, possibly because of accumulation of QTL with minor effects as in Cypress. In most modern rice cultivars, seed dormancy has been eliminated because of the combined effects of domestication and breeding for efficient crop production. Seed dormancy needs to be optimized to reduce crop loss caused by preharvest sprouting while ensuring rapid and uniform germination and ease in harvesting for successful crop production. A major concern in using weedy rice in breeding is the linkage drag with undesirable attributes, such as red pericarp, black hull, awn, photosensitivity, and shattering. Particularly, clustering of weedy trait QTL (Cai and Morishima, 2002) and QTL with undesirable pleiotropic effects (Gu et al., 2005) could hinder the progress in a breeding program. However, the novel seed dormancy QTL from cultivated rice identified in this study could be useful for improving preharvest sprouting tolerance.

Supplemental Information Available Supplemental material is available at http://www.crops. org/publications/cs. Acknowledgments We thank Chris Roider and Dr. Pat Bollich for their help in field experiments. This research was supported by United States Department of Agriculture-National Research Initiative Competitive Grants Program (Grant No. 2006-35320-16555). This manuscript is approved for publication by the Director of Louisiana Agricultural Experiment Station as manuscript number 2012-306-7411.

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