Genetic Mapping of Major-Effect Seed Dormancy ...

Published November 6, 2015

Research

Genetic Mapping of Major-Effect Seed Dormancy Quantitative Trait Loci on Chromosome 2B using Recombinant Substitution Lines in Tetraploid Wheat Shiaoman Chao,* Elias Elias, David Benscher, Goro Ishikawa, Yung-Fen Huang, Mika Saito, Toshiki Nakamura, Steven Xu, Justin Faris, and Mark Sorrells

Abstract Durum wheat (Triticum turgidum L.) cultivars can benefit from having some level of seed dormancy to help reduce seed damage and lower grain quality caused by preharvest sprouting (PHS) occurring during wet harvesting conditions. Previously, a single chromosome substitution line carrying chromosome 2B of wild emmer [Triticum turgidum L. subsp. dicoccoides (Körn. ex Asch. & Graebn.) Thell.] in the durum cultivar Langdon background was found to have higher levels of seed dormancy and PHS tolerance. In this study, a population of recombinant substitution lines was developed and used to construct a single nucleotide polymorphism (SNP)-based high-density genetic linkage map. Seed germination tests were used to evaluate seed dormancy levels. Multiple interval mapping analysis revealed four quantitative trait loci (QTL) regions affecting seed dormancy. Two regions containing major-effect QTL contributed by wild emmer were consistently expressed in four environments and explained 5.89 to 11.14% of the phenotypic variation. One QTL region was located near the centromere and the other on the long arm of chromosome 2B in Bayes credible intervals of 4 and 7 cM, respectively. Recombinant inbred lines (RILs) carrying both QTL had an average of 35% reduced rate of germination measured by weighted germination index compared with RILs carrying neither QTL. The two QTL regions identified in this study should be useful for improving PHS tolerance in wheat. Efforts to transfer the two QTL into elite durum cultivars are in progress to examine the effects of genetic background and environment on QTL expression and to evaluate the performance of other agronomic traits in the presence of the QTL.

S. Chao, S. Xu, J. Faris, USDA–ARS, Cereal Crops Research Unit, Fargo, ND 58102, USA; E. Elias, Dep. of Plant Sciences, North Dakota State Univ., Fargo, ND 58108, USA; D. Benscher, M. Sorrells, Dep. of Plant Breeding and Genetics, Cornell Univ., Ithaca, NY 14853, USA; G. Ishikawa, M. Saito, T. Nakamura, Tohoku Agricultural Research Center, National Agriculture and Food Research Organization, Morioka, Iwate 020-0198, Japan; Y.F. Huang, Agriculture and AgriFood Canada, Ottawa, Ontario, K1A 0C6, Canada. Y.F. Huang, Current address: Department of Agronomy, National Taiwan Univ., Taipei, Taiwan. Received 22 May 2015. Accepted 13 Aug. 2015. *Correspon ding author ([email protected]). Abbreviations: CI, credible interval; DArT, diversity array technology; DIC, wild emmer; EST, expressed sequence tag; GI, germination index; GR, germination rate; LDN, Langdon; LOD, logarithm of odds; OPA, oligo pool assay; PCR, polymerase chain reaction; PHS, preharvest sprouting; PLUG, polymerase-chain-reaction-based landmark unique gene; QTL, quantitative trait loci; SI, support interval; SNP, single nucleotide polymorphism; SSR, simple-sequence repeat.

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n nature, seed dormancy provides a survival mechanism allowing plants to escape unfavorable growing conditions. Domestication and plant breeding may have eliminated some of the seed dormancy mechanisms present in the wild ancestors (Bewley, 1997). Cultivars, however, can benefit from having some level of seed dormancy to help reduce the seed damage detrimental to end-use grain quality caused by PHS occurring during wet harvesting conditions. Although it is well established that red wheat (T. aestivum L.) grains tend to have elevated levels of seed dormancy (Gfeller and Svejda, 1960), equivalent or significantly better PHS tolerance has also been identified in white genotypes and in tetraploid durum wheat [T. turgidum L. ssp. durum (Desf.) Husn.] (McCaig and DePauw, 1992; Clarke et al., 1994). Pleiotropic gene effects attributable to the close relationship between red

Published in Crop Sci. 55:1–14 (2015). doi: 10.2135/cropsci2015.05.0315 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved.

crop science, vol. 56, january– february 2016

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kernel color and seed dormancy was strongly implicated by mutagenesis experiments (Warner et al., 2000). In rice (Oryza sativa L.), a genetic dissection study has revealed such a pleiotropy located on chromosome 7 that influenced a gene network involving biosynthesis pathways of both abscisic acid for promoting seed dormancy, and flavonoid for producing red pericarp pigment (Gu et al., 2011). Previous linkage mapping studies in wheat based on a wide variety of biparental crosses have indicated that seed dormancy is a genetically complex trait controlled by multiple genes (Anderson et al., 1993; Flintham et al., 2002; Munkvold et al., 2009), and the trait expression is also strongly influenced by environmental factors such as drought and high temperature (Reddy et al., 1985, Biddulph et al., 2005, Biddulph et al., 2008). In common wheat, depending on the resistance sources used, significant QTL affecting PHS tolerance and strong seed dormancy have been reported on most of the chromosomes. Apart from QTL found on group 3 chromosomes where the R genes reside (Mori et al., 2005, Imtiaz et al., 2008; Mohan et al., 2009), others have been identified on chromosomes 1A (Anderson et al., 1993), 1B (Flintham et al., 2002), 2A (Mares et al., 2002; Miura et al., 2002), 2B (Liu et al., 2008; Munkvold et al., 2009), 2D (Mares et al., 2002), 4A (Mares et al., 2005; Mori et al., 2005; Chen et al., 2008; Cabral et al., 2014), 4B (Mori et al., 2005; Rasul et al., 2009), 5A (Groos et al., 2002), 5B (Tan et al., 2006), 5D (Fofana et al., 2009), 6A (Zanetti et al., 2000), 6B (Roy et al., 1999), 7B (Zanetti et al., 2000), and 7D (Rasul et al., 2009). Results from association mapping analyses using breeding lines and germplasm collections further demonstrated that many of the candidate DNA markers significantly associated with seed dormancy and PHS tolerance appeared to coincide with previously identified QTL (Kulwal et al., 2012; Rehman Arif et al., 2012). However, it remains to be determined if the two mapping approaches identified the exact same QTL. Despite complex inheritance, a gene with major effects on PHS tolerance on the short arm of chromosome 3A has recently been cloned facilitated by the use of comparative mapping strategies (Liu et al., 2013). This gene, a wheat homolog of a MOTHER OF FLOWERING TIME (TaMFT-A1), was the same gene previously identified using microarray approach from spring wheat (Nakamura et al., 2011), and was also found to play an important role in regulating seed dormancy in winter wheat (Lei et al., 2013). In contrast to extensive studies reported in common wheat, fewer genetic studies have been conducted in durum wheat. Nationally, North Dakota ranks first in durum wheat production accounting for over 50% of total durum wheat production in the United States (USDA, 2014). The economic impact on the state wheat industry hinges on successive crops with high grain quality. Severe sprouting may affect pasta processing such as semolina 2

speck count, higher cooking loss, spaghetti firmness, spaghetti color characteristics, and shelf stability (Matsuo et al., 1982; Soper et al., 1989; Fu et al., 2014). Although the effect of sprouting on pasta quality appeared to be less severe than the grave effect on bread making quality, sprout-damaged durum generally is highly undesirable and is discounted in the market (Knox et al., 2012), which leads to reduced income for producers. Therefore, developing durum cultivars with improved PHS tolerance has been one of the breeding objectives for the durum breeding programs in the Northern Plains region. Thus far, genetic mapping studies in durum wheat have revealed the QTL for seed dormancy and PHS tolerance at genomic regions similar to those found in common wheat on chromosomes 1A, 1B, 2A, 2B, 4A, 5B, 6B, 7A, and 7B (Knox et al., 2005, 2012; Singh et al., 2014). In addition to durum wheat cultivars, sources of sprouting resistance were also reported in other tetraploid subspecies including wild emmer accessions (Clarke et al., 1994; Hucl et al., 1996). From evaluating three sets of singlechromosome substitution lines developed by transferring wild emmer wheat chromosomes into the background of a durum wheat cultivar Langdon ( Joppa and Cantrell, 1990; Xu et al., 2004), we and others have reported that genes controlling both strong seed dormancy and PHS tolerance were present on at least five chromosomes (2A, 2B, 3A, 4A, and 7B) of wild emmer origin (Watanabe and Ikebata, 2000; Chao et al., 2010). Further studies, however, are needed to confirm if the QTL found in tetraploid wheat are the same as those detected in hexaploid wheat. Of the five wild emmer chromosomes we previously identified, the substitution line involving chromosome 3A conferred the highest level of seed dormancy and was coincident with the red kernel phenotype (Chao et al., 2010). In the breeding efforts on improving PHS tolerance, red durum is unacceptable to the pasta industry (Soper et al., 1989); the presumed pleiotropic gene effect associated with the R genes renders the use of this gene as a source for enhancing PHS tolerance in durum wheat impossible. In this study, we focused on a substitution line carrying chromosome 2B, which had a level of seed dormancy comparable to the chromosome 3A substitution line (Chao et al., 2010). The aim was to develop a population of RILs, construct a high-density SNP-based genetic map of chromosome 2B, and to identify the QTL controlling seed dormancy.

Materials and Methods Plant Materials A RIL population was developed from a cross between Langdon (LDN) as the male parent and LDN(521-2B) as the female parent using the single-seed decent method. LDN(521-2B) is a single-chromosome substitution line developed by substituting LDN 2B chromosome with its counterpart from the wild

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emmer accession PI 481521 in the LDN background (Xu et al., 2004). The resulting RIL population allows genetic dissection of quantitative trait inheritance controlled by genes on chromosome 2B to be conducted in a homogenous background from LDN. After eliminating lines with poor seed setting, off types, and those having excess amount of heterozygotes at the F5:7 generation based on marker data, the final RIL population used in this study consisted of 150 progeny. This population was recently used to identify genetic loci on chromosome 2B associated with domestication and the tenacious glume trait (Faris et al., 2014b).

Field Experiments All 150 RILs were grown in a randomized complete block field design at two locations including Prosper, ND, and Ithaca, NY, between 2009 and 2013. Because of limited seed source for LDN(521-2B), both parents were only included in the North Dakota locations. With the exception of the 2010 North Dakota experiment, where three replicates were evaluated, the other four environments—New York 2009 and 2013 and North Dakota 2012 and 2013—were all evaluated using materials grown in two replicates. Weather conditions recorded for Cass County, ND, where all the field trials were conducted, showed that the mean maximum temperature in July during grain fill was 28.3C (82.9F) in 2010, 31.5C (88.7F) in 2012, and 28.6C (83.5F) in 2013. The number of days with temperature 32.2C (90F) was recorded as 13 in July 2012, while 3 and 6 d were reported in July 2011 and 2013, respectively (http://www.ncdc.noaa.gov). Thus, the year 2012 was much hotter during seed maturation than the other two seasons. Field planting and spike harvesting followed the same practice as described previously (Chao et al., 2010). Briefly, at physiological maturity, when the glumes on the spikes and peduncles of the tillers lost green coloration, 20 spikes were harvested in the North Dakota environments. Five heads were harvested in New York environments and sent to Fargo, ND, for seed germination tests. After drying at ambient temperature for 5 d, spikes were stored in a −20C freezer.

Seed Germination Germination tests were performed by germinating 100 handthreshed and healthy seeds at 20C with 100% relative humidity following the previously described protocol (Chao et al., 2010). Because LDN tended to maintain some level of dormancy at physiological maturity, after-ripening period, the period of dry storage at room temperature allowing seeds to release dormancy, was adjusted by evaluating the percentage germination rate (GR) of the two parents before seed germination tests were performed for the progeny. Both GR and weighted germination index (GI) (Reddy et al., 1985, Walker-Simmons, 1987) estimates were used for QTL mapping analysis.

DNA Markers, Genetic Map Construction, and Quantitative Trait Loci Analysis To identify polymorphic DNA markers, the parents were initially genotyped with 1200 genome-wide wheat simplesequence repeat (SSR) markers and a wheat iSelect SNP array containing 9000 wheat SNP markers (Cavanagh et al., 2013). Among the polymorphic SSR markers identified, 36 known to crop science, vol. 56, january– february 2016

detect loci on chromosome 2B (Somers et al., 2004) were used to genotype the RIL population, and 12 of them producing no missing data were selected to anchor the 2B linkage map (Supplemental Table S1). Additional wheat expressed sequence tag (EST)-based DNA markers, known as polymerase chain reaction (PCR)-based landmark unique gene (PLUG) markers (Ishikawa et al., 2007), were developed by comparing wheat UniGene sequences with the single copy rice gene sequences. Exon–exon junctions were predicted from highly homologous regions between wheat and rice allowing PCR primers amplifying the intron regions to be designed for marker analysis. We first developed and evaluated 120 PLUG primer pairs, which amplified fragments with sizes >400 bp by PCR, using PCR– restriction fragment length polymorphism (RFLP) on agarose gels as described by Ishikawa et al. (2009). An additional 272 PLUG primers amplifying fragments with sizes 0.05), likely a result of the strong influence of environmental conditions (Fig. 1). Results from Levene’s test for homogeneity indicated variances of both GR and GI were not homogeneous (p < 0.001) across the five environments. Therefore, subsequent QTL analyses were performed using both GI and GR values obtained from each of the five environments separately. The ANOVA results showed that environment had a significant influence (p < 0.001) crop science, vol. 56, january– february 2016

Table 2. ANOVA table with mean squares of germination test data measured using both germination rate (GR) and germination index (GI). Source

df

GR

Genotype (G) Environment (E)

149 4

673.438*** 47426.452***

GE Error

596 747

204.968*** 117.656

GI 0.045*** 3.007*** 0.012 0.011

*** Significant at the 0.001 probability level (F-test).

on seed dormancy measured by both GR and GI (Table 2). While G  E was highly significant (p < 0.001) for GR values, it was not for GIs, indicating that G  E interaction did not contribute much to the variations in magnitude of the rate of germination among RILs measured by weighted germination index, GI (Table 2).

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Figure 2. (a) Physical and (b, c) genetic linkage maps of tetraploid wheat chromosome 2B. (a) The deletion bin-based physical map of chromosome 2B. Deletion bin designations are shown on the left, and the deletion breakpoints are indicated by arrows. (b) A genetic linkage map of the entire chromosome 2B generated with a subset of the 254 markers. The red bar represents the location of the centromere. The dotted lines connecting the physical and genetic linkage maps indicate the likely breakpoint positions on the linkage map. (c) A genetic linkage map of the subregion of chromosome 2B showing the locations of 95% Bayes credible intervals for the two major-effect quantitative trait loci indicated in red and in shaded areas. The centromere is depicted as the black oval.  indicates the portion of the 95% Bayes credible intervals not shown.

Genetic and Physical Map of Chromosome 2B A high-density genetic linkage map was developed for chromosome 2B from tetraploid wheat that spanned a genetic distance of 136.4 cM consisting of a total 254 markers including 234 SNPs, 12 SSRs, seven PLUG markers denoted as TNAC loci on the map (Table 1), and a wheat seed dormancy gene, TtSdr-B1 (Fig. 2; Supplemental Table S1). On average, the marker density was one marker per 0.5 cM, ranging from 0 to 16 cM, with two gaps of 9 and 16 cM located at the distal end of the short and long arms, respectively. The centromere was placed unambiguously between TNAC9311 and IWA6723 based on the BLASTn results obtained from querying the survey sequences of wheat chromosome arms 2BS and 2BL (Fig. 2; Supplemental Table S1). Altogether 73.2% of the markers (186 out of 254) were mapped at the same location as at least one other marker. Regions flanking the centromere, in particular, had a large number of cosegregating markers. Three 6

prominent clusters of cosegregating markers were located at 58.4 cM with 16 markers on the short arm, at 64.7 cM with 48 markers, and at 65.0 cM with 26 markers on the long arm. It was noted, however, that many of the SNPs clustered at the 65.0 cM position had a match to the same wheat survey sequence contigs, indicating that those SNPs though discovered from different ESTs were from different portions of the same gene regions. This was apparently the case for other cosegregating SNPs on the map as well (Table 3; Supplemental Table S1). Three regions, all on the long arm, had segregating markers that significantly (p < 0.05) deviated from the expected 1:1 ratio. One region was at 70.5 to 75.0 cM and involved 14 markers, the second was at 90.7 to 104.1 cM and included 23 markers, and the third was at 106.5 to 113.5 cM and involved four markers. Altogether, the markers with distorted segregation ratios accounted for 16% (41 out of 254) of all the markers mapped on chromosome 2B (Supplemental Table S1).

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Table 3. A list of 50 single nucleotide polymorphism (SNP) markers with their match to the wheat 2B chromosome arm survey sequences, their genetic map position in cM, deletion bin location, and their match to the expressed sequence tag (EST) accession.

Marker

cM

Wheat chromosome survey sequences

IWA3605 IWA379 IWA1359 IWA546 IWA608 IWA2556 IWA8046 IWA6838 IWA2624 IWA1010 IWA10 IWA2674 IWA2151 IWA439 IWA3428 TNAC9313 IWA6209 IWA207 IWA4136 IWA5927 IWA31 IWA7420 IWA7499 IWA8454 IWA6000 IWA6215 IWA7204 IWA6016 IWA772 IWA1215 IWA1217 IWA780 IWA779 IWA2253 IWA243† IWA244† IWA242† IWA1489 IWA5506 wmc175‡ IWA4097‡ IWA3176 IWA2873 IWA2872 IWA6561 IWA2874 IWA8534 IWA8589 TNAC3164 IWA5093

14.1 14.8 34.7 36.0 47.4 48.4 48.7 50.1 50.1 51.8 54.6 56.4 57.4 58.4 58.4 60.2 61.9 64.7 65.0 65.0 65.0 65.0 65.0 65.0 65.0 65.7 65.7 66.3 66.3 66.3 66.3 69.5 69.5 70.2 71.5 71.5 71.5 72.9 77.1 83.2 90.7 94.5 94.5 94.5 94.5 94.5 104.7 113.5 113.5 131.5

5164161_2BS 5204409_2BS 6950482_2BS 3514149_2BS 5179254_2BS 5155171_2BS 5231013_2BS 5242701_2BS 5242701_2BS 5246217_2BS 1363796_2BS 5206547_2BS 5244571_2BS 5233319_2BS 5215879_2BS 5188369_2BS 5185181_2BS 8087671_2BL 8080499_2BL 8080499_2BL 8080499_2BL 7935750_2BL 7935750_2BL 7935750_2BL 8006961_2BL 8072412_2BL 8072412_2BL 2215627_2BL 8013239_2BL 8013239_2BL 8013239_2BL 8005258_2BL 8005258_2BL 8013384_2BL 8019012_2BL 8019012_2BL 8019012_2BL 8089357_2BL 7936677_2BL – 8083682_2BL 8002985_2BL 8002985_2BL 8002985_2BL 8002985_2BL 8002985_2BL 6669028_2BL 8085192_2BL 7989214_2BL 8057859_2BL

2B deletion bin

EST accession

2BS3-0.84-1.00 2BS3-0.84-1.00 2BS3-0.84-1.00 2BS4-0.75-0.84 2BS1-0.53-0.75 2BS1-0.53-0.75 2BS1-0.53-0.75 2BS1-0.53-0.75 2BS1-0.53-0.75 2BS1-0.53-0.75 2BS1-0.53-0.75 C-2BS1-0.53 C-2BS1-0.53 C-2BS1-0.53 C-2BS1-0.53 C-2BS1-0.53 C-2BS1-0.53 C-2BL2-0.36 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL2-0.36-0.50 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL2-0.36-0.50 2BL4-0.50-0.89 2BL4-0.50-0.89 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00 2BL6-0.89-1.00

BE499523 BE591604 BE518440 BG275030 BM140364 BE445628 BG607608 BE499478 BE499478 BF202681 BE399688 BE404601 BG607045 BF291736 BF202287 BF473259 BG262632 BE488220 BE403506 BE403506 BE403506 BE424076 BE424076 BE424076 BF202975 BF483237 BF483237 BG608232 BE592008 BE592008 BE592008 BE443191 BE443191 BF475068 BE490763 BE490763 BE490763 BF202596 BF473744 – CK209589 BE500307 BE500307 BE500307 BE500307 BE500307 BF291977 BI479150 BE446530 BE426818

†

SNPs showed discrepancy between the genetic map and the physical map locations.

‡

Markers assigned to bin locations from other studies.


Of the 254 markers mapped on chromosome 2B, 50 were assigned to eight deletion bins, including 47 SNPs with one of which, IWA4097, previously assigned by Ishikawa et al. (2009), two PLUG markers, and one SSR (wmc175), which was assigned by Sourdille et al. (2004) (Table 3). Each of the three clusters of cosegregating markers was most likely confined to a single deletion bin, where the C-2BS1-0.53 bin contained the cluster mapped to 58.4 cM, the C-2BL2-0.36 bin had the cluster mapped at 64.7 cM position, and the 2BL2-0.36-0.50 bin included the markers clustered at 65.0 cM (Fig. 2; Table 3). With the exception of the marker clusters, in which the precise marker order among cosegregating markers could not be resolved, the order of the markers elsewhere on the 2B map generally agreed well with those in common on the consensus maps of both tetraploid (Maccaferri et al., 2014) and hexaploid wheat (Cavanagh et al., 2013). When comparing the genetic map with the physical map, discrepancies for marker orders between the two maps were observed for three SNPs. The three markers were closely linked to markers located in the 2BL2-0.36-0.50 bin. However, the EST BE490763, from which the three SNPs were derived, was previously placed in the 2BL6-0.89-1.00 bin (Table 3).

Quantitative Trait Loci Analysis and Quantitative Trait Loci Allele Effects Multiple interval mapping analysis was performed using all 254 makers mapped and the phenotype data obtained from the five environments separately for GI and GR values. Similar results were observed between the two measures, thus only the results based on the analysis using the GI values were reported here. In total, 15 QTL affecting seed dormancy were detected at the 5% significance threshold ( = 0.05) from all environments that explained 3.2 to 11.1% phenotypic variation (Table 4). The 95% Bayes CI ranging from 2 to 71.4 cM represented four regions containing genes influencing seed dormancy (Supplemental Fig. S2). No epistatic interactions were found, suggesting epistasis was minimal for this chromosome. Two regions, one located near 65 cM adjacent to the centromere and the other near 90 cM on the long arm, contained majoreffect QTL that were expressed consistently in at least four of the five environments evaluated (Fig. 2). The QTL peak positions in each of these two regions were shifted depending on the environments between 64.6 and 66.3 cM and between 85.9 and 94.5 cM (Table 4). The intervals of 61 to 65 and of 87 to 94 cM were repeatedly found to contain the QTL expressed in multiple environments, and both were contributed by the LDN(521-2B) parent. Therefore, these intervals of 4 and 7 cM in size, respectively, likely represent the credible regions harboring the genes controlling seed dormancy derived from wild emmer chromosome 2B. They were denoted as QPhs.fcu2B.1, for the one near the centromere, and QPhs.fcu-2B.2

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Table 4. Quantitative trait loci (QTL) detected on chromosome 2B based on germination index value with logarithm of odds (LOD) score, position, percentage variation explained, additive effect, and marker intervals for the QTL detected. The Bayes credible intervals (CI) in italics are those containing two major-effect QTL denoted as QPhs.fcu-2B.1 located in the interval of 61 to 65 cM, and QPhs.fcu-2B.2 located in the interval of 87 to 94 cM. QTL

Peak position

LOD

Variation

Additive effect‡

LOD-1 support interval

95% Bayes CI

Marker intervals TNAC9071, IWA6317

GI_NY09 ( = 0.05) GI_NY09_model

64.6 91.4† –

2.7

% 6.4

−0.04

57.7–64.6

cM 58–73

4.6 –

11.1 26.8

−0.05 –

89–91.4 –

89–97 –

GI_ND10 ( = 0.05)

51.5 65.3 85.9† 99† –

3.0 7.7 6.5 5.3 –

2.6 7.1 5.9 4.7 73.2

−0.02 −0.03 −0.03 −0.02 –

49.1–61 61–65.3 84–90.7 96–103 –

50–61.6 61–65.7 85–89 97–103 –

IWA2624, IWA169 TtSdr-B1, gwm630 TNAC3119, IWA8295 IWA2459, wmc627

2.9 3.6 2.1 –

7.3 9.2 5.1 22.1

0.03 −0.03 −0.03 –

24–29.5 60.2–66.3 90.1–94.5 –

9–49.1 59–77.1 68–136.4 –

IWA7545, barc55 TtSdr-B1, IWA5506 IWA1689, IWA6164

GI_ND12_model

28.9 66.3† 94.5 –

GI_ND13 ( = 0.05)

22† 64.6†

3.2 8.3 3.9 10.0 69.5

0.02 −0.03 −0.03 −0.04 –

19–47 60.2–64.6 76.4–80 89–91.4 –

14.8–47.7 61–66 72.9–79 90–92 –

IWA379, IWA7030 TtSdr-B1, gwm630 IWA1489, IWA469 IWA8295, dupw207

GI_ND13_model

77.1† 91.4† –

3.3 7.8 4.0 9.3 –

GI_NY13 ( = 0.05) GI_NY13_model

94.5† 131.5† –

4.0 3.4 –

10.9 9.2 16.6

−0.04 0.03 –

93.1–94.5 129.1–131.5 –

90–99 125–135 –

IWA8295, IWA8029 IWA1535, IWA6164

GI_ND10_model GI_ND12 ( = 0.05)

†

Peak positions were also significant at a threshold of  = 0.01.

‡

Negative and positive additive values indicate that the favorable alleles contributed by LDN(521-2B) and LDN, respectively.

for the one on the long arm. The amount of phenotypic variation explained by each main-effect QTL ranged from 6.4 to 9.2% for QPhs.fcu-2B.1 and from 5.1 to 11.1% for QPhs.fcu-2B.2 (Table 4). To further examine the genetic effects of the DIC alleles on dormancy levels at the two regions containing major-effect QTL, we compared RILs carrying the LDN alleles with those carrying either one or both DIC alleles at the QTL (Table 5). Results showed that the lines carrying the LDN alleles were significantly less dormant (p < 0.05) than those carrying the DIC alleles at either one of the two QTL regions in four of the environments studied with the exception of the ND12 environment. In general, the effects of the DIC alleles did not differ (p > 0.05) between the two QTL regions. However, for lines grown in the NY13 environment, a significant difference (p < 0.05) between the LDN and the DIC allele was found only at the QPhs.fcu-2B.2 locus because that was the only QTL expressed in that environment. Results further showed that lines carrying both QTL were generally more dormant than those having a single QTL. The exception being the NY09 environment where the lines expressing QPhs.fcu-2B.2 provided similar levels of dormancy as those having both QTL expressed (Table 5).

8

IWA8295, IWA2459

Table 5. Allele effects on dormancy levels based on germination index measure. DIC.1 and DIC.2 denoted as recombinant inbred lines (RILs) carrying DIC allele at QPhs.fcu-2B.1 and QPhs.fcu-2B.2 locus, respectively. DIC denoted as RILs carrying DIC alleles at both quantitative trait loci (QTL). LDN denoted as RILs carrying LDN allele at both QTL. QTL No. of allele RIL† DIC DIC.1 DIC.2 LDN

38 25 17 60

NY09‡

ND10

ND12

ND13

NY13

0.231a 0.298b 0.275a,b 0.395c

0.424a 0.510b 0.516b 0.604c

0.352a 0.425b 0.428b 0.449b

0.185a 0.285b 0.280b 0.370c

0.337a 0.405b 0.321a 0.378b

†

Ten RILs had recombination events in the QTL regions and were excluded from the analysis.

‡

Numbers followed by the same letter in the same column are not significantly different at the 0.05 level of probability.

Physical Maps and Synteny Comparisons of Two Regions Containing Major-Effect Quantitative Trait Loci Physically, QPhs.fcu-2B.1, located in the 61 to 65 cM interval, encompassed at least three deletion bins flanking the centromere, including C-2BS10-.53, C-2BL2-0.36, and 2BL2-0.36-0.50, and involved 82 markers, of which 74 cosegregated in two marker clusters. The inability to narrow this region to a single physical bin location was primarily due to the lack of recombination events near

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Table 6. Syntenic comparisons with the genomes of barley, Brachypodium distachyon, and rice at the region containing 2B.2 quantitative trait loci. Letters in italics indicate regions with perfect marker colinearity. Tetraploid_2B Marker

Barley

Rice

Brachypodium

cM

Chr_Hv Accession_Hv Position

IWA3988

87.2

6H

MLOC_44276.3

Mb 538.665

IWA1707 IWA8295 IWA4098 IWA7371 IWA4097 IWA4096 IWA4095 TNAC9215 IWA5051 IWA3176 IWA2873 IWA2872 IWA6561 IWA2874

87.2 90.1 90.7 90.7 90.7 90.7 90.7 91.4 93.1 94.5 94.5 94.5 94.5 94.5

2H 2H 2H 2H 2H – – 2H 2H 2H 2H 2H 2H 2H

MLOC_54759.1 MLOC_57847.2 MLOC_65568.1 MLOC_65568.1 MLOC_65568.1 MLOC_52101.1 MLOC_61657.1 AK373696 MLOC_9892.1 MLOC_55003.1 MLOC_55003.1 MLOC_55003.1 MLOC_55003.1 MLOC_55003.1

584.655 584.655 585.079 585.079 585.079 – – 588.005 587.859 590.542 590.542 590.542 590.542 590.542

Chr_Bd

Locus_Bd

End

Chr_Os

Locus_Os

Start

End

Bd5

Bradi5g22187

chr11

Os11g0485900

Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5 Bd5

Bradi5g22310 24.719 24.727 Bradi5g22420 24.795 24.801 Bradi5g22700 24.961 24.969 Bradi5g22700 24.961 24.969 Bradi5g22700 24.961 24.969 Bradi5g22700 24.961 24.969 Bradi5g13150 16.690 16.693 Bradi5g22830 25.037 25.039 Bradi5g25390 26.726 26.736 Bradi5g25090 26.537 26.543 Bradi5g25090 26.537 26.543 Bradi5g25090 26.537 26.543 Bradi5g25090 26.537 26.543 Bradi5g25090 26.537 26.543

chr04 chr04 chr04 chr04 chr04 chr04 chr01 chr04 chr04 chr04 chr04 chr04 chr04 chr11

Os04g0624600 31.751 31.759 Os04g0625900 31.824 31.828 Os04g0628600 31.982 31.992 Os04g0628600 31.982 31.992 Os04g0628600 31.982 31.992 Os04g0628600 31.982 31.992 Os01g0500100 17.230 17.231 Os04g0630800 32.085 32.088 Os04g0666900 34.040 34.048 Os04g0663100 33.840 33.845 Os04g0663100 33.840 33.845 Os04g0663100 33.840 33.845 Os04g0663100 33.840 33.845 Os11g0263000 8.883 8.899

the centromere. Syntenic comparisons of this QTL region with genome sequences of rice, barley, and B. distachyon also indicated poor colinearity (Supplemental Table S3). In contrast, the 87 to 94 cM interval containing QPhs.fcu-2B.2 is likely confined to the 2BL6-0.89-1.00 bin. Comparative analysis revealed that a 3-cM portion (between 87.2 and 90.7 cM) of the QTL-containing region in wheat was highly conserved with a 424-Kb region on barley chromosome 2H (584,655–585,079 Kb; Table 6). Perfect colinearity was also found at the same region between wheat and a 384-Kb region on B. distachyon chromosome 5 (24,655–25,037 Kb) and a 334-Kb region on rice chromosome 4 (31,751–32,085 Kb; Table 6). The remaining QTL region between 90.7 and 94.5 cM was less conserved as a result of an inversion relative to barley, rice, and B. distachyon. Synteny was also not perfect among multiple SNPs mapped at the same positions in wheat (Table 6). For example, both IWA3988 and IWA1707 were mapped to the 87.2-cM position, but IWA3988 was similar to sequences on barley chromosome 6H and rice chromosome 11, as opposed to barley chromosome 2H and rice chromosome 4 to which IWA1707 was similar. Another example was four of the five SNPs located at the 90.7cM position in wheat matched to a locus Bradi5 g22700 on B. distachyon chromosome 5, whereas IWA4095 matched to a different locus 8 Mb from the rest of four SNPs on the same B. distachyon chromosome (Table 6). IWA4095 also shared sequence similarity with rice chromosome 1 rather than chromosome 4, like the other four SNPs. Similarly, the five SNPs that mapped to the 94.5-cM region in wheat all had similarity to a single locus in barley chromosome 2H and B. distachyon chromosome 5, but only four of the five had similarity to a locus on rice chromosome 4, while IWA2874 was similar to a locus on chromosome crop science, vol. 56, january– february 2016

Start

——— Mb ——— 24.655 24.661

——— Mb ——— 17.126 17.131

11 of rice (Table 6). Taken together, despite inversions and rearrangements, a portion of the 7-cM region containing QPhs.fcu-2B.2 shared high levels of synteny between wheat and three other grass species.

Discussion

Genetic and Physical Maps of Tetraploid Wheat Chromosome 2B In this study, a high-density genetic map for chromosome 2B was constructed using a cross made between LDN and a single chromosome substitution line involving wild emmer as the donor parent for chromosome 2B. On the map, we observed marker clusters flanking the centromere, particularly those at proximal regions of the long arm of the chromosome where recombination was absent among 48 markers mapped in the C-2BL20.36 bin (Fig. 2). It is well established that in wheat and many other organisms, the physical distribution of recombination rate is highly variable across the chromosomes (Dvorak and Chen, 1984; Faris et al., 2000). Thus, the low recombination rate observed near the centromere poses a complication for fine mapping the QTL region such as the one found in this study. We detected an inconsistency in the order of markers on the genetic linkage map compared with the physical map for three SNP markers located on the long arm of chromosome 2B (Table 3). This discrepancy has not been reported in durum wheat maps constructed based on durum crosses (Mantovani et al., 2008; Gadaleta et al., 2009) or in the consensus tetraploid wheat map developed involving a durum  wild emmer cross (Marone et al., 2012). Frequent chromosome rearrangements on the B genome chromosomes were observed for a high proportion (70%) of the wild emmer accessions from Israel

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( Joppa et al., 1995), which is where PI 481521 originated. However, because the relative map orders between the three markers in question and their flanking markers matched well with those on the tetraploid SNP-based consensus map involving crosses between durum and wild emmer or cultivated emmer (Maccaferri et al., 2014), it is highly unlikely that the chromosome rearrangement was the cause of the discrepancy. Rather, from observing the Southern blotting image using BE490763 as a probe, multiple fragments on chromosome 2B were detected, but not all of which could be resolved for the deletion bin mapping study because of the comigration of restriction fragments (http://wheat.pw.usda.gov/cgi-bin/westsql/map_ image.cgi?i=UNL100BE490763.jpg). Therefore, a more plausible explanation for the discrepancy is that the three SNPs mapped to the 2BL2-0.36-0.50 bin in this study represent the loci that were not resolved in the previous deletion bin mapping study. Segregation distortion has been frequently reported in the progeny of intraspecific and interspecific hybrids in plants, including tetraploid wheat (Blanco et al., 1998; Peng et al., 2000; Nachit et al., 2001; Mantovani et al., 2008; Peleg et al., 2008; Faris et al., 2014a). Genetic loci associated with segregation distortion identified on chromosome 5B in tetraploid wheat have been shown to cause the preferential transmission alleles through male gametes (Kumar et al., 2007). Segregation distortion systems depending on gamete competition seem to be more likely to be found in males of monogamous species, where gamete wastage has the smallest impact on fecundity (Lyttle, 1991). In the current study, we used LDN as the male parent to produce the F1 hybrids, and the distorted loci all had excess LDN alleles compared with 521-2B alleles. Other studies based on durum  wild emmer crosses showed the wild alleles of the male parent had higher tendency for distorted segregation than the durum alleles of female parents (Peleg et al., 2008). The opposite findings were reported in favor of the female LDN alleles over the male DIC alleles (Peng et al., 2000). Also observed was the uneven distribution of distorted loci along the chromosome arms. In this study, all the distorted markers were located on the long arm of the chromosome, whereas others reported that distorted markers were found mostly on the short arm (Peleg et al., 2008) or on both arms of chromosome 2B (Faris et al., 2014a). It is clear that the mechanisms underlying the causes for segregation distortion are complex, particularly the interplay among various mechanisms that could occur during the multiple selfing generations in producing RIL populations (Peleg et al., 2008). Of the 16 markers mapped in the interval between 87.2 and 94.5 cM containing QPhs.fcu-2B.2, 13 located in the 90.7 to 94.5 cM region had distorted segregation in favor of LDN alleles. Distorted loci can bias genetic distance estimates when constructing linkage maps and 10

thus become problematic for genetic analyses (Liu et al., 2010). However, previous simulation studies have indicated that segregation distortion was not always detrimental to the power of detecting QTL with additive effects, in particular when the marker density is high and marker data are complete (Xu, 2008). In this study, QPhs.fcu-2B.2 was consistently detected in four of the five environments with the wild emmer parent contributing the additive genetic variance, suggesting the distorted markers had little, if any, impact on the power of detecting the QTL.

Quantitative Trait Loci Associated with Seed Dormancy on Chromosome 2B The RIL population developed for this study segregated for only chromosome 2B in an otherwise homozygous LDN background. We detected four QTL intervals at a threshold of  = 0.05 affecting the seed dormancy trait. Two QTL regions containing major-effect QTL contributed by the wild emmer parent were identified. The interval containing QPhs.fcu-2B.1 encompassed the pericentromeric region flanked by the durum wheat TtSdr-B1 gene on the short arm and a SNP marker, IWA4106, on the long arm (Fig. 2). The wheat TaSdr-B1 gene, an ortholog of rice Sdr4 gene, was previously shown to be closely associated with higher seed dormancy measured by GI in common wheat (Zhang et al., 2014). In our population, progeny having the wild emmer allele at this gene were more dormant than those carrying the LDN allele; the same was also true for the other 80 markers mapped in the same interval. Therefore, further studies will be necessary to determine if the TtSdr-B1 gene would be the causal gene responsible for higher levels of dormancy in this tetraploid wheat population. Major QTL affecting seed dormancy and PHS located on the short arm of chromosome 2B that were near QPhs. fcu-2B.1 have been reported in several studies. For example, Munkvold et al. (2009) used a white wheat cross to locate an interval containing a major QTL for PHS flanked by SSR markers barc55 and wmc474, which later was found to contain two QTL linked in coupling (Somyong et al., 2014). Both of these markers mapped distal to the TtSdrB1 gene on our map, suggesting that the QTL found in our study may be different from theirs. Results from an association mapping study based on a set of 96 European winter wheat germplasm placed two diversity array technology (DArT) markers associated with dormancy and PHS in the deletion bins flanking the centromeric region, namely C-2BS1-0.53 and C-2BL2-0.36 (Rehman Arif et al., 2012), which was similar to our findings. Using a durum wheat cross, Knox et al. (2012) observed a minor 2B QTL expressed in one of the environments linked to an SSR marker, wmc592, which was closely linked to wmc245 on the SSR-based consensus map (Somers et al., 2004). We placed wmc245 near the centromere, and

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therefore, the QTL described by Knox et al. (2012) was probably in the same QTL interval as the one we detected. For QPhs.fcu-2B.2, two SNP markers flanking the 7-cM interval were IWA3988 and IWA2874 (Table 6). An association mapping analysis based on a set of 208 elite US soft white winter wheat lines showed a significant association between PHS and a DArT marker (Kulwal et al., 2012), which was ~27 cM proximal to the SSR marker wmc361 based on a 2B genetic map containing both DArT and SSR markers (Yu et al., 2014). On our map, wmc361 is about 40 cM distal to QPhs.fcu-2B.2. Similarly, a minor QTL detected by Liu et al. (2008) was mapped to a region ~30 cM distal to wmc361. Therefore, the QTL detected by these authors were more distal and likely different from QPhs.fcu-2B.2 discovered in this study. Mohan et al. (2009) detected an epistatic QTL on 2BL located between SSR markers gwm501 and wmc332, which was likely in a region overlapping with QPhs.fcu-2B.2 found in this study. However, we didn’t detect epistasis involving this region. In summary, the lack of recombination in the interval containing QPhs.fcu-2B.1 prohibited us from placing this QTL in a more precise region on either 2BS or 2BL, making it difficult to conclude if QPhs.fcu-2B.1 is in fact the same or different from previously reported QTL. QPhs.fcu-2B.2 is likely novel, and high resolution mapping is required to further narrow the region and eventually identify the gene governing seed dormancy.

Quantitative Trait Loci Validation The impact of fluctuating environmental conditions on changing the levels of seed dormancy developed during seed maturation has been well documented. Temperature, in particular, is one of the most influential factors affecting the induction of seed dormancy during seed formation (Reddy et al., 1985) and may have contributed to the lowered dormancy level observed in the LDN(521-2B) parent grown in the ND12 environment. ANOVA results further reflected that seed dormancy measured, based on both GR and GI, was highly influenced by environment (Table 2). Genotype  environment interaction was highly significant (p < 0.001) for GR, which measured the rate of germination, but not for GI, which differentiated the rate of germination by adding more weight to the genotypes that germinated early. Spearman’s rank correlations among five environments were significant (p < 0.05) for both GR and GI (data not shown), indicating that while the magnitudes varied, a high degree of correspondence for rankings among RILs was observed across environments. This generally agreed with previous studies based on either breeding populations (Graybosch et al., 2013) or a set of wheat germplasm accessions (Biddulph et al., 2008; Rasul et al., 2012), suggesting that despite complex inheritance, PHS tolerance can be manipulated genetically. We identified two QTL with major effects that crop science, vol. 56, january– february 2016

were consistently expressed in multiple environments. A recent report based on transcriptome analysis showed that the candidate genes for a major seed dormancy QTL located on wheat chromosome 4A were highly expressed during grain maturation, and that the gene expression was strongly suppressed by increasing temperature (Barrero et al., 2015). We, therefore, speculate that the temperature effect on reducing QTL expression might explain why the effects between the DIC allele and the LDN allele at either of the two QTL did not differ significantly for the materials grown in the ND12 environment (Table 5). Nonetheless, lines expressing both QTL were on average more dormant and provided adequate dormancy levels in that environment. Therefore, the two major-effect QTL identified in this study should be useful for improving PHS tolerance in wheat. However, the intervals containing the QTL are still large physically, particularly for QPhs.fcu-2B.1 near the centromere. Therefore, further work to reduce the size of the QTL segment and eliminate potentially deleterious alleles resulting from linkage drag will be needed. The availability of mapped SNP markers from high-density wheat SNP arrays, such as the iSelect 90K array (Wang et al., 2014), the wheat chromosome arm survey sequences (IWGSC, 2014), and the collinear regions with other grass species provide ample resources of new markers to saturate the QTL region. Efforts on transferring the two QTL to elite durum cultivars with low dormancy levels are under way to examine the effects of genetic background and environment on QTL expression and to evaluate the performance of other agronomic traits in the presence of the QTL. Acknowledgments The authors thank Stan Stancyk for field assistance at Prosper, ND, and Dawn Feltus and Mary Osenga for technical assistance. This research was supported by USDA CRIS Project No. 3060-520-037-00D. We also acknowledge support from Hatch project 149-430 and from USDA–NIFA–AFRI grants, award numbers 2009-65300-05661. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.

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