Development of an integrated linkage map of einkorn ... - Springer Link

Theor Appl Genet (2017) 130:53–70 DOI 10.1007/s00122-016-2791-2

ORIGINAL ARTICLE

Development of an integrated linkage map of einkorn wheat and its application for QTL mapping and genome sequence anchoring Kang Yu1,3 · Dongcheng Liu1 · Wenying Wu1 · Wenlong Yang1 · Jiazhu Sun1 · Xin Li1 · Kehui Zhan2 · Dangqun Cui2 · Hongqing Ling1 · Chunming Liu3 · Aimin Zhang1,2

Received: 26 May 2016 / Accepted: 12 September 2016 / Published online: 22 September 2016 © Springer-Verlag Berlin Heidelberg 2016

Abstract Key message An integrated genetic map was constructed for einkorn wheat A genome and provided valuable information for QTL mapping and genome sequence anchoring. Abstract Wheat is one of the most widely grown food grain crops in the world. The construction of a genetic map is a key step to organize biologically or agronomically important traits along the chromosomes. In the present study, an integrated linkage map of einkorn wheat

Communicated by J. Dubcovsky. Kang Yu and Dongcheng Liu have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00122-016-2791-2) contains supplementary material, which is available to authorized users. * Aimin Zhang [email protected] Kang Yu [email protected] Dongcheng Liu [email protected] Wenying Wu [email protected]

was developed using 109 recombinant inbred lines (RILs) derived from an inter sub-specific cross, KT1-1 (T. monococcum ssp. boeoticum) × KT3-5 (T. monococcum ssp. monococcum). The map contains 926 molecular markers assigned to seven linkage groups, and covers 1,377 cM with an average marker interval of 1.5 cM. A quantitative trait locus (QTL) analysis of five agronomic traits identified 16 stable QTL on all seven chromosomes, except 6A. The total phenotypic variance explained by these stable QTL using multiple regressions varied across environments from 8.8 to 87.1 % for days to heading, 24.4–63.0 % for spike length, 48.2–79.6 % for spikelet number per spike, 13.1–48.1 % for plant architecture, and 12.2–26.5 % for plant height, revealing that much of the RIL phenotypic variation had been genetically dissected. Co-localizations of closely linked QTL for different traits were frequently observed, especially on 3A and 7A. The QTL on 3A, 5A

Dangqun Cui [email protected] Hongqing Ling [email protected] Chunming Liu [email protected] 1

State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, People’s Republic of China

2

Collaborative Innovation Center for Grain Crops in Henan, Henan Agricultural University, No. 95 Wenhua Road, Zhengzhou, Henan 450002, People’s Republic of China

3

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Fragrant Hill, Beijing 100093, People’s Republic of China

Wenlong Yang [email protected] Jiazhu Sun [email protected] Xin Li [email protected] Kehui Zhan [email protected]

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and 7A were closely associated with Eps-Am3, Vrn1 and Vrn3 loci, respectively. Furthermore, this genetic map facilitated the anchoring of 237 T. urartu scaffolds onto seven chromosomes with a physical length of 26.15 Mb. This map and the QTL data provide valuable genetic information to dissect important agronomic and developmental traits in diploid wheat and contribute to the genetic ordering of the genome assembly. Abbreviations AFLP Amplified fragment length polymorphism ANOVA Analysis of variance BAC Bacterial artificial chromosome CIM Composite interval mapping cM CentiMorgan CTAB Hexadecyltrimethylammonium bromide DArT Diversity arrays technology EST Expressed sequence tag HD Days to heading LOD Logarithm of the odds PA Plant architecture PH Plant height PVE Phenotypic variance QTL Quantitative trait locus or loci RIL Recombinant inbred line RFLP Restriction fragment length polymorphism SNP Single nucleotide polymorphism SPL Spike length SPLN Spikelet number per spike SSR Simple sequence repeat STS Sequence-tagged site

Introduction Hexaploid wheat (Triticum aestivum L., AABBDD) was derived from two successive natural hybridization events of three progenitors, T. urartu (AuAu, 2n = 2x = 14), an Aegilops species of the Sitopsis section (SS, 2n = 2x = 14) and Aegilops tauschii (DD, 2n = 2x = 14) (Petersen et al. 2006). Einkorn wheat, T. monococcum ssp. monococcum L. (AmAm, 2n = 2x = 14) is very closely related to T. urartu and the A genome of hexaploid wheat (Johnson and Dhaliwal 1976) and is the only cultivated diploid wheat (Hori et al. 2007). This species was domesticated from its wild form T. monococcum ssp. boeoticum (AbAb, 2n = 2x = 14) in the Middle East (Harlan 1980). The presence of adaptive cultivated characters makes this species an interesting subject for the genetic dissection of domestication traits (Dubcovsky and Dvorak 2007; Sood et al. 2009). As a wild relative of the A genome of hexaploid wheat, T. monococcum harbors useful traits and genes (Stein et al. 2000; Feuillet et al. 2003; Tiwari et al. 2009; Zaharieva and Monneveux 2014; Alvarez et al. 2016).

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Theor Appl Genet (2017) 130:53–70

Molecular linkage maps (genetic maps) serve as a basic tool for dissecting agronomically important traits (Tanksley et al. 1992), and have been broadly used over the past two decades in wheat (Blanco et al. 1998; Röder et al. 1998; Semagn et al. 2006) and other plant species (Paterson et al. 1988; Gardiner et al. 1993; Jacobs et al. 1995; Harushima et al. 1998; Chen et al. 2002; Menz et al. 2002; Reddy et al. 2011). Recently, along with achievements in sequencing technology, an ultra-high density linkage map with 270,820 high-quality single nucleotide polymorphism (SNP) markers was constructed and used to call quantitative trait loci (QTL) for yield-related traits in rice, providing useful markers for genetic analyses and breeding applications (Xie et al. 2010; Yu et al. 2011). However, only a few linkage maps in einkorn wheat have been constructed with different types of markers, e.g., restriction fragment length polymorphism (RFLP) markers (Dubcovsky et al. 1996), amplified fragment length polymorphism (AFLP) markers (Taenzler et al. 2002; Hori et al. 2007), and simple sequence repeat (SSR) markers (Singh et al. 2007). The first linkage map was constructed using a 74 F2 population derived from T. monococcum ssp. monococcum (DV92) and T. monococcum ssp. boeoticum (G3116), which contained 335 markers, including 328 RFLP markers, and spanned a genetic distance of 1,067 cM (Dubcovsky et al. 1996). Recently, barley ESTs were used to enhance map saturation in einkorn wheat, resulting in 242 EST markers on the genetic linkage map of recombinant inbred lines (RILs) derived from a cross KT1-1 (T. monococcum ssp. boeoticum) × KT3-5 (T. monococcum ssp. monococcum) (Hori et al. 2007). In addition, a map using 274 DArT and 82 SSR markers is also available (Jing et al. 2009). Linkage maps have been widely exploited for map-based cloning of genes underlying QTL for important agronomic traits in wheat (Stein et al. 2000; Faris et al. 2003; Feuillet et al. 2003; Yan et al. 2003, 2004; Simons et al. 2006; Uauy et al. 2006) and other plant species (Yano et al. 2000; Takahashi et al. 2001; Taenzler et al. 2002; Huang et al. 2003; Liu et al. 2015). Map-based cloning in the complex hexaploid wheat genome is an arduous task and, when possible, genes have been cloned using diploid relatives (Stein et al. 2000; Faris et al. 2003; Feuillet et al. 2003; Yan et al. 2003, 2004; Simons et al. 2006; Alvarez et al. 2016). The diploid nature of T. monococcum and the high levels of gene conservation and collinearity with other Triticeae species make it an attractive model for gene discovery in wheat (Singh et al. 2007; Zaharieva and Monneveux 2014). Moreover, genetic maps of diploid wheat have been used to identify multiple QTL (Shindo et al. 2002; Taenzler et al. 2002; Hori et al. 2007; Nakamura et al. 2007), and can be basis for map-based cloning of underlying genes. Once identified in diploid wheat the homologous genes can be easily identified in hexaploid wheat (Tiwari et al. 2009; Zaharieva and Monneveux 2014).


Genetic maps are also widely used to anchor genomic sequences onto linkage groups or chromosomes for genome assembly (Huang et al. 2009, 2013; Jia et al. 2013; Xu et al. 2013; Qin et al. 2014; Li et al. 2015; Zhang et al. 2015). For example, the assembly of Ae. tauschii genome sequences has benefited from a genetic map anchoring 1.277 Gb (30.19 % total length) with SNP markers, and 0.44 Gb with 838 SSR (422 scaffolds) and other markers (16,193 scaffolds) from published maps (Jia et al. 2013). Anchored genome sequences can then be used as a source for the development of additional markers, such as SSR and SNP markers (Ling et al. 2013), and provide a valuable tool for fine-mapping and map-based cloning. Unlike T. monococcum ssp. boeoticum KT1-1, T. monococcum ssp. monococcum KT3-1 has several interesting agronomic traits, such as large grains and long spikes. It also carries several developmental and domestication traits that might be useful in wheat breeding (Hori et al. 2007). In the present study, we used a RIL population derived from a cross between T. monococcum ssp. monococcum and T. monococcum ssp. boeoticum to construct an integrated genetic linkage map with 926 loci and different types of markers. These markers cover 1,377 cM map distance with an average marker interval of 1.5 cM. This map was used to identify QTL for agronomically important traits and to anchor a few T. urartu scaffold sequences.

Materials and methods Plant material T. monococcum ssp. boeoticum KT1-1 is winter-type wild einkorn wheat with blue aleurone layer and leaf hairiness, while T. monococcum ssp. monococcum KT3-5 is springtype cultivated einkorn with transparent aleurone layer and no leaf hairiness. KT3-5 is an early flowering mutant generated by X-ray irradiation of KT3-1 (Shindo and Sasakuma 2001). KT3-5 has shoot apical meristem transitions to the double-ridge stage 10 days earlier than KT3-1 and the differences are modulated by temperature (Gawron´ski et al. 2014). This line shows a deletion of the circadian clock LUX, which has been postulated as a candidate gene for the early flowering of KT3-5 (Gawron´ski et al. 2014). The 109 RILs (F10) of KT1-1/KT3-5 were selected for integrated linkage map construction and QTL mapping. This population segregates for plant height (PH), days to heading (HD), plant architecture (PA), and yield components (Hori et al. 2007). The RIL population and its parents were kindly provided by the KOMUGI Wheat Genetic Resources Databases of Japan (Shindo and Sasakuma 2001). A total of 341 markers have been previously mapped in the same population (Hori et al. 2007).

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Field experiment and phenotyping The RILs and their parents were grown with two replicates in a completely randomized block design at the experimental station of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing (40°5′56″N and 116°25′8″E) in four successive years (2011, 2012, 2013, and 2014), and at the experimental station of Henan Agricultural University, Zhengzhou (34°51′52″N, 113°35′45″E) in 2 years (2013 and 2014). These environments will be designated E1, E2, E3, E4, E5, and E6, respectively. All RILs and their parents were planted in single 2-m rows with 40 cm between rows and 20 cm between individuals. The PA, PH, spike length (SPL), spikelet number per spike (SPLN), and HD were scored on five randomly selected guarded plants from each line and replicate. We divided the PA of RILs into four levels, from one for a fully prostrate type (resembling KT1-1) to four for a fully erect type (resembling KT3-5), based on the angles of the tiller with the ground level, while all the other traits were measured on the primary tiller. The HD was calculated as days from sowing to heading, and the date of heading was subsequently recorded for all plants in each line as the date when half of the spikes emerged from the flag leaf. Aleuronic layer color, leaf hairiness and growth habit were mapped as simple Mendelian traits given their qualitative and simple segregating pattern. The former two were scored in the field, while growth habit was recorded by the presence (spring habit) or absence (winter habit) of the flag leaf in the greenhouse under growing conditions of 16 h light and 25 °C and 8 h darkness and 15 °C (no vernalization). Genotyping DNA extraction: DNA from the parents and RILs was extracted from the leaves of young seedlings using the previously described hexadecyltrimethylammonium bromide (CTAB) protocol (Saghai-Maroof et al. 1984). SSR markers: Wheat genomic SSR markers (Table S1) were retrieved from GrainGenes2.0 (http://wheat.pw.usda. gov/GG2/index.shtml). An additional set of SSR markers developed from the draft genome sequences of T. urartu (Ling et al. 2013), was also used (Table S2), particularly those anchored to the syntenic region of the earliness per se-Am3 (Eps-Am3) locus (Gawron´ski and Schnurbusch 2012). The amplification of SSR markers and the separation of PCR products were performed following the methods of Ling et al. (2013). Briefly, the PCR mixture consisted of 1 × Taq Master Mix (CWBIO), 0.02 μM M13 tailed-forward primer, 0.10 μM reverse primer, 0.10 μM universal fluorescent-labeled M13 primer (FAM (6-carboxy-fluorescein),

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VIC, NED or PET), 30 ng DNA, and ddH2O in a 15 μL reaction volume. Amplification was performed with the following parameters: 1 cycle at 94 °C for 5 min; 15 cycles of 94 °C for 30 s, Ta (annealing temperature determined experimentally for each SSR primer combination) for 30 s, and 72 °C for 30 s; 25 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s; and 1 cycle of 72 °C for 10 min for the final extension. A standardized multi-pooling procedure was used to prepare the SSR products for electrophoresis (Schuelke 2000), and the pooled products were purified using the standard sequencing procedure (Applied Biosystems). Briefly, diluted PCR products labeled with different fluorescent dyes, were pooled at a ratio of 1:1:2:2 for VIC:FAM:NED:PET tagged products, and 5 μL pooled samples were precipitated using 37.5 μL ethanol, 5 μL 37.5 mM EDTA (pH 8.0) and 5 μL 0.9 M NaAc (pH 5.2). Purified PCR products were resuspended in 7 μL deionized formamide containing 0.7 μL GeneScan500 LIZ size standard, and were separated through capillary electrophoresis on ABI3730 lx Genetic Analyzer (Applied Biosystems) following manufacturer’s standard procedures. Genotypes for each marker were scored using GeneMapper (ver. 4.0; Applied Biosystems). DArT markers: The DArT markers were screened using Wheat DArT Array (ver. 3.0; Wenzl et al. 2004; Akbari et al. 2006) on 90 RIL lines and two parents at Diversity Arrays Technology Pty. Ltd, Yarralumla, Australia (DArT Pty Ltd, http://www.diversityarrays.com/). The presence (1) or absence (0) of each marker was determined on the basis of signals from labeling and image analysis (Akbari et al. 2006). The DArT marker genotyping resulted in 691 high quality markers. Markers with ≥20 % missing values were removed. Gene and protein markers: Three vernalization requirement genes (Vrn1-AY188331, Vrn2 (ZCCT1)- AY485644, and Vrn3 (FT1)-DQ890163) (Yan et al. 2003, 2004, 2006) were cloned and sequenced in the parental lines, KT1-1 and KT3-5, respectively. Polymorphic markers were designed based on the detected sites of variation of these genes between the two parents. These SNP or insertion/deletion (indel) markers were used to genotype the 109 RILs (Table S2). SDS–PAGE was also used to genotype the glutenin subunits across the RIL populations, following the method of Dong et al. (2010). Linkage map construction Linkage maps included previous RFLP (Shindo et al. 2002) and STS (Gawron´ski and Schnurbusch 2012) data. Distorted markers (Chi-squared test P 90 % and percentage of identity >98 % for markers with sequence length >100 bp. For SSR markers without sequence information, hits were retained if both primers had a 100 % identity and a 30 cM on 4A (Shindo et al. 2002; Hori et al. 2007) was partitioned into three smaller gaps by the addition of nine DArT markers. The other two gaps with a total length of 34.28 cM were on the distal region of chromosome 7AL, which was extended

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by newly mapped DArT markers (Fig. 1). Most gaps in the previous RFLP map (Shindo et al. 2002) and the integrated EST map (Hori et al. 2007) were filled by DArT markers. For example, gaps of ~30 cM on 2A, 6A and 7A were filled

0.00 1.69 2.24 2.79

3.90 6.21 7.34 7.89 12.68 13.23 13.79 17.99 19.11 19.69 20.81 28.49 32.79

Chr.1A

TmGlu-A3* XwPt-470301 XwPt-0595 XwPt-470158 XwPt-8320 XwPt-470330 XwPt-6269 XwPt-0196 XwPt-469981 XwPt-2527 XwPt-1782 XwPt-9752 XwPt-861659 XwPt-377173 XwPt-861059 XwPt-377159 XwPt-861261 XwPt-376649 XwPt-4735 XwPt-469684 XwPt-470598 XwPt-860736 XwPt-861894 XwPt-3560 XwPt-861936 Xcfd21 CHS-H** XtPt-8831

59.34 59.89 62.98 64.91 67.09 68.21

69.27

Xwec80* XwPt-469317** XwPt-376659** CASA0189* CASA0958** XwPt-860845** TmGlu-A1* XwPt-3358* Xcfa2129*

111.89 114.71

131.30

XwPt-469677** Xbcd265a** XwPt-860934* XwPt-377113** XwPt-377022* XwPt-861286* XwPt-861458*

135.45 137.16 138.28

XwPt-862151 Xgdm126 XwPt-0314

144.11 147.83

XwPt-5935 XwPt-5907 XwPt-4129 XwPt-4532 XwPt-2526 Xwec92 XwPt-0690 XwPt-861808 XwPt-469997 XwPt-3208 Xbcd307 XwPt-376486* TmAGPL-1A XwPt-2312 XwPt-469544* XwPt-862074* XwPt-0432* XwPt-861793* XwPt-861096* XwPt-470347* XwPt-860819* XwPt-469623* XwPt-1685* CASA0770

123.84 125.01 126.17 127.31 129.02

qSPL-1A.1@E1

XwPt-860875* Xcfa2099**

106.84 110.28 111.34

qSPL-1A.1@E4

102.44 103.56 108.57

Xpsr609 Xwec32 XwPt-1480

70.57 79.63 88.09 88.60 92.19 94.45 95.56 97.25 98.35 98.91 101.67 103.78 110.83 111.38

140.52

150.11 150.67 151.22 152.91 154.64 158.43 163.43 166.94 167.49 173.72 174.27 174.83

141.07 143.35 145.69

75.66 76.72

77.25 77.77 80.00 80.60 81.82 85.81 86.71 88.92 91.71 92.24 93.35 95.04 96.73 98.37 100.24 101.16 101.62 102.17 103.86 104.41 108.55 109.10 109.65 111.93 112.49 115.45 124.26 125.99 130.41 134.98 140.42 143.12 144.18 146.34 148.32 150.96 151.90 152.85 153.78 156.17 156.63 158.03 162.52 168.51 171.21 172.51 172.98

161.40 161.94 166.29 169.14 170.26 171.38 171.94 172.50 173.05 175.33 178.84

Xwms311 XwPt-862214 XwPt-862135 CASA0342 Xwms265 XwPt-469559 XwPt-470463 XwPt-6662 XwPt-469527 XwPt-3281 XwPt-469139 XwPt-470583 XwPt-7739 XwPt-0718 Xbarc163

175.37 178.29 179.89 183.85 187.36 191.39 191.85 192.31 193.75 194.21

195.33

197.01 199.90 201.52 210.93 218.53 220.81 221.95 222.49 223.04 223.62 224.19 226.47 227.54 229.70 233.61 235.21 236.34 237.40 238.47 238.97 239.48

239.93

Xpsr131b Xpsr131a

36.34

XwPt-470231**

40.64

XwPt-376008**

61.95

XwPt-862019** XwPt-860850** XwPt-5749* XwPt-6214* XwPt-0105* XwPt-9833* XwPt-4645

92.11 93.34 96.23 96.79 98.17 101.76 102.87 103.37

105.08

106.13 107.25 108.38 112.63 117.91 123.38 126.20 128.15 128.67 130.95

132.06 134.95 136.64 138.33 139.44 142.95 143.50 144.05 146.33 146.88 148.57 152.08 155.03 157.37 159.65 160.77 162.50 163.59 167.02 169.90 170.45 179.03 186.94

Germin XwPt-375894 XwPt-5663 Xwg876 Xbcd265b Xbcd808-c Xbcd808-a XwPt-469729 XwPt-9298 XwPt-470659 XwPt-376433* Xbcd1051 Xrz141 Xwec87b Xrz261 CASA0916 CASA0296 CASA0587 CASA0900 CASA0712 CASA0447 XwPt-8576 CASA0160 CASA0260 XwPt-469848 CASA0048 Tu3AL5083.3 Tu3AL5005.3 XwPt-470550 XwPt-861910 XwPt-862216 Blue.al** CASA0552* XwPt-862017 Xbarc70 Xwms192 Xwms192-2 XwPt-470305 XwPt-376419 XwPt-861141 XwPt-470091 XwPt-376653 XwPt-6514 XwPt-861218 XwPt-470207 Xcfa2173 XwPt-376652 XwPt-860843 XwPt-377177 XwPt-376995 XwPt-860755 Xpsr1051 XwPt-861528 XwPt-9276 XwPt-9168 XwPt-861717 XwPt-3058 XwPt-861787 XwPt-861562 XwPt-8989 XwPt-470389 XwPt-470031 XwPt-470470 XwPt-470274 Xabc310 XwPt-860944 XwPt-469181 Xwec78 XwPt-470495 XwPt-469328 XwPt-470377 Xwms118 XwPt-3435 XwPt-470020 XwPt-861912 XwPt-469532 XwPt-861367 XwPt-1311 XwPt-470515 XwPt-376910

PA

13

18.90

81.78 85.51 87.22 88.91

SPLN

Fig. 1 Genetic map showing ten genomic regions harboring quantitative trait loci (QTL) for five traits in an einkorn wheat recombinant inbred line (RIL) population of T. monococcum ssp. boeoticum (KT11) and T. monococcum ssp. monococcum (KT3-5). The genetic distances are in cM (calculated using Kosambi). Markers and trait QTL are denoted with different font colors. The asterisks “*” and “**” that show segregation distortion (SD) P = 0.05 and 0.01, respectively. The QTL detected from each environment were shown at the respec-

Xwg622

91.06

HD

243.88 244.93 245.44 246.38 251.42 256.88

XwPt-861273 XwPt-862233 XwPt-861939 XwPt-8650 XwPt-861748 XwPt-469673 XwPt-469555 XwPt-862092

11.87

62.52

SPL

243.01

6.89

qSPL-4A.2@E3

152.66 153.71 155.59

64.10 64.67

75.11

5.77

qSPL-4A.2@E2

147.91

Tu3AL5260.1 XwPt-469944 Xrz590 CHS-RV XwPt-470452 XwPt-861005 XwPt-469883 XwPt-469561 XwPt-861719 XwPt-470172 Xwms526 Xgdm93

61.33 62.41

70.96

2.89

qSPL-4A.1@E4

133.20 133.75 134.93 137.63 138.83

60.80

58.49

64.73 68.51

0.00

qSPL-4A.1@E1

Xcfd2-1 Xbcd135 XwPt-470520

60.28

56.21

qPH-2A.1@E6

122.43 123.50 124.07

48.69 53.55 55.26 56.38 56.92 58.00

50.69 53.45 53.97 54.52

qPH-2A.1@E5

113.69

XwPt-860799 XwPt-0015 Tu3AL5152.2 XwPt-6711 CASA0097 XwPt-375843 XwPt-5498 XwPt-470074 XwPt-470261 XwPt-861112 XwPt-470187* XwPt-0460** XwPt-860874** Xwec54** CASA0368** CASA0653* Tu3AL5230.1** CASA0227* Xrz753** CASA0232* CASA0796* XwPt-861397* XwPt-376266* XwPt-8826* XwPt-861176* XwPt-470413* XwPt-6527* XwPt-376451* XwPt-861874* XwPt-470100* XwPt-469745* XwPt-861964* XwPt-376688* XwPt-862032* XwPt-861590* CASA0388** XwPt-861138** XwPt-861209** XwPt-377055** XwPt-469773** XwPt-7547** XwPt-2128** XwPt-4244** Xrz740** Xpsr102** Xpsr1196** XwPt-0088* CASA0912** XwPt-376273** Xpsr901(63)** XwPt-375900** Tu3AL5175.2** Xcfd73* CASA0180-1* XwPt-2430 XwPt-861896 XwPt-469753 XwPt-470061 XwPt-470363

qPH-2A.1@E3

CASA0866 XwPt-8573

40.32 40.87

qPH-2A.1@E4

30.85 31.97

qPH-2A.1@E2

XwPt-470244 Xwec50b Xwec50a XwPt-469570

qPH-2A.1@E1

23.61 25.91 26.38 27.46

qPA-2A.1@E4

XwPt-861359

15.34 18.10 20.10 20.65 21.20 21.77 27.33 33.99 36.36 37.47 40.98 41.53 42.65 43.67 45.16 48.01 48.55 49.61

qPA-2A.1@E5

17.44

48.13

8.81 13.67

Chr.4A XwPt-7486** XwPt-469485** XwPt-469719** XwPt-860737** XwPt-6012* CASA0269 XwPt-469252 XwPt-861271 Xcfd2-3* Xbarc57* XwPt-470073* XwPt-861746 XwPt-861772 Xbarc321-2 Xbarc12 XwPt-861379 XwPt-376271 XwPt-861439 XwPt-1410 Xcdo460 Xcfd79 Xwms369 CASA0038 Xbarc321-1 XwPt-1468 XwPt-4407 Xcdo395 XwPt-860997 Tu3AL5128.1* CASA0668 XwPt-376635 XwPt-470334 XwPt-1806 XwPt-2624 XwPt-6604 CASA0753 XrPt-3056 XwPt-469796 XwPt-469782 XwPt-470183 Xcdo407 Xwec53* XwPt-376327** XwPt-861753** XwPt-862035** XwPt-469859* XwPt-470263* Tu3AL5014.1** CASA0194* XwPt-861564 XwPt-469947 XwPt-470062 XwPt-0820 INDEL_111_112-0* Xwms30 Xwms608 CASA0001 XwPt-470477 XwPt-470409 CASA0362 Xwec102 INDEL_105_106-4* AS_PCR_153_154-4* XwPt-861272 Xcfa2134 XwPt-860841 Xabc176* XwPt-469542** Tu3AL5230.2* AS_PCR_149_150-10.5* AS_PCR_69_70-14.3 AS_PCR_147_148-12.9 TP_71_72-12.7* XwPt-469339* XwPt-470128 XwPt-377130 Xpsr578 XwPt-469227 XwPt-469535 Xrz444 XwPt-861913 XwPt-469174 XwPt-860802 XwPt-860862 XwPt-861007 XwPt-376819 XwPt-376751 XwPt-470276 Tu3AL5035.2 XwPt-376626 XwPt-860735 CASA0491 Xwec97a Xwec97b XksuH15 Tu3AL5002.1 XwPt-860851 Xcfd152 Xbarc69 Tu3AL5018.1 XwPt-1092 Xcfa2193 Tu3AL5033.1 Tu3AL5032.1 Tu3AL5034.1 Tu3AL5030.1 Tu3AL5038.1 Tu3AL5044.1 Tu3AL5037.1 Tu3AL5046 Tu3AL5047 Tu3AL5052.1 Tu3AL5051.1 Tu3AL5066.1 Xabc174 XwPt-376701 Tu3AL5058.1 XwPt-2266 XwPt-376043 Xcfa2170 XwPt-469957 XwPt-9018 Tu3AL5035.1 Tu3AL5068.1 Xpsr1205a INDEL_245_246-69.7 Tu3AL5083.1 Tu3AL5091.1 Xpsr1205b Tu3AL5100.1 Tu3AL5099.1 XwPt-1085 XwPt-7188 XwPt-469586 XwPt-470163 XwPt-7470 XwPt-0530 XwPt-470400 XwPt-860960 CASA0033 INDEL_243_244-79.4* Tu3AL5124.1 Tu3AL5125.1 XwPt-5295* XwPt-375883 XwPt-861296 XwPt-861538 Tu3AL5173 Tu3AL5127.1* Tu3AL5172* XwPt-9238* XwPt-4398* XwPt-1694* Xwec57* XwPt-6826 PAV_185_186-93.2* XwPt-469774* AS_PCR_117_118-92.2** Tu3AL5204 Tu3AL5205* AS_PCR_205_206i-98.1** INDEL_161_162-98.1** Tu3AL5245* PAV_269b_270-102.5** Tu3AL5250* Tu3AL5251* PAV_295_296-102.9** INDEL_201_202-103.4** AS_PCR_17_18-103.4** AS_PCR_315_316-103.4** TP_13_14-103.4** Tu3AL5142.1** Tu3AL5294* Tu3AL5291* XwPt-861370* XwPt-6234* XwPt-469816* Tu3AL5290* Tu3AL5070.1* Tu3AL5071.2* XwPt-469676 XwPt-861322 XwPt-469195 Tu3AL5163.1* XwPt-861203 Tu3AL5168.1* PAV_177_178-109.7* JIP Xabc172

qPH-4A.1@E4

72.85

XwPt-8135

45.36 45.91 47.02

0.00

Xbcd348

13.94

43.76

Chr.3A

XwPt-6053 XwPt-2147 XtPt-4602 XwPt-862117 CASA0913 Xcdo456

qPH-4A.1@E2

69.79 70.87

Chr.2A

qPA-2A.1@E1

XwPt-3016 XwPt-861678

58.22

7.95

qPH-1A.1@E4

93.86 95.69

55.74

1.66 5.26

qPH-1A.1@E5

73.28 76.10 79.24 84.35

XwPt-862167* Xbcd98 XwPt-469235 Xbarc119 XwPt-861599 Xgdm36 Xrz166* Xcdo580* Xrz251* Xbarc148 XwPt-469147 XwPt-469585 XwPt-860753 XwPt-470634 XwPt-376999 Xwec47** Xbcd454** XwPt-469405** CASA0506* Tu3AL5015.2* Tu3AL5005.2* Tu3AL5005.1* CASA0374* XwPt-861648 XwPt-469442 XwPt-470156 Tu3AL5230.3 CASA0367 Xcfd59 Xcfd83 Xcfd65 Xwms55 XwPt-861475 XwPt-470153 Tu3AL5083.2

39.93 43.66 46.76 49.75 50.82 53.11 54.78

0.00 0.55

by 76 DArT markers, resulting in marker intervals less than 11 cM (Fig. 1). We also located a set of protein and phenotypic markers on this linkage map, including high and low

tive positions with confidence interval (CI). On 3A, the QTL regions of each trait are defined by the ranges of all CIs from the identified environments. Detailed information of QTL is available in Table 2. The yellow shaded portions of the bars are the most probable centromere positions as inferred from published records. The dark red shaded portions of the bars are the QTL regions with CIs. HD days to heading, SPL spike length, SPLN spikelet number per spike, PA plant architecture, PH plant height


59

Chr.5A 0.00 0.56 7.61 14.94 16.10 16.63 17.18 18.29 19.40 20.52 22.21 24.62 26.93 27.48 28.03 29.08 29.60 30.12

30.64

31.16 31.67 32.79

141.76 146.00 149.51 150.64 151.20 152.41 159.72 161.20 164.08 165.79 168.68 169.81 170.36 172.05 172.59 173.75 180.00 181.67 184.55 185.67 186.22 189.10 189.66 191.37 192.48 193.59 195.19 196.25 197.81 198.32 198.78 207.45 209.16 209.71 211.48

81.24 84.19 85.30 88.69 89.22 94.70 96.41 99.79 103.94 106.86 107.42 108.57 109.12 111.35 116.70 122.01 123.78 127.33 128.52 133.69 136.61 140.81

142.52

143.63 148.43

CASA0242 XwPt-7794 XwPt-861492 XwPt-470355 XwPt-469293

199.54

219.37 219.93

XwPt-470538** XwPt-860857**

71.01 71.56 72.12 75.01 77.89 82.41 82.98 85.29 86.34 93.21 94.85 96.43 96.95 97.99 99.89 100.90 101.97 102.49 103.03 103.55 104.07 106.35 109.86 112.17 112.72 113.27 113.82 116.78 117.34 118.45 119.50 122.42 124.13 124.71 126.48 127.03 128.72 133.52 134.07 135.20 137.64 138.77 139.88

140.43 140.98 141.54 143.25 147.40 151.54 152.65 153.77 159.93 160.48 161.03 165.83 166.90 172.85 173.97 183.89 198.33

qPA-7A.1@E3

136.84

78.87

52.86 56.69 59.40 61.40 63.58 64.13 66.44 69.39

qPA-7A.1@E4

122.22 124.58 129.05 133.48 136.27

63.16 63.69 64.19 64.76 67.15 68.42 70.11 72.60 75.11 76.59

43.54 47.74 48.88 49.44 51.75

qSPLN-7A.1@E6

117.08 117.63 118.18 121.10 121.66

62.64

35.47 37.77 39.50 41.84

qSPLN-7A.1@E4

116.15

61.02 62.10

31.52 32.07 34.35

qSPLN-7A.1@E5

115.22

60.47

qSPL-5A.1@E6

109.02 111.20

54.66 55.21 56.33

qHD-5A.1@E4

108.48

50.08 50.65 52.36

qHD-5A.1@E1

98.14 100.56 102.12 104.79 107.33

49.53

qHD-5A.1@E2

76.85 79.18 84.72 87.05 87.62 90.68 91.20 93.56 95.25

47.82

qSPL-5A.1@E4

76.32

45.54

20.16

21.27 22.98 23.53 24.64 25.20 26.93 28.64

qSPLN-7A.1@E3

75.77

39.45 44.99

19.05

20.71

qSPLN-7A.1@E1

74.08

38.34

6.38 7.49 9.80

qHD-7A.1@E4

54.18 55.16 60.77 65.75 69.88

35.45

4.69

qHD-7A.1@E3

52.43

34.90

Chr.7A

Tu3AL5061.1** XwPt-861924** XwPt-860943** XwPt-861284** Xabg704** XwPt-469296** CASA0522** XwPt-469305** XwPt-469700** XwPt-862099** XwPt-469824** XwPt-3572** XwPt-860797** XwPt-861282** XwPt-4617** XwPt-861133** XwPt-861194** XwPt-861335** XwPt-862142** XwPt-3876** XwPt-4601** XwPt-861056** XwPt-3648** XwPt-3135** Xwms635-1** XwPt-470475** XwPt-861587** XwPt-9299** XwPt-0040** CASA0407** XwPt-376185** XwPt-376665** Xwms132** XwPt-469953** XwPt-0714** XwPt-469973** XwPt-470111** CASA0906** XwPt-469892** XwPt-469650** XwPt-8726** XwPt-469675** XwPt-1579** Xwms60** Xwms130* Xbarc154* Xwms328.2* XtPt-6195** XwPt-861626** Xabc158** Xbarc219** FT1-Vrn3** XwPt-470566** XwPt-861377** XwPt-861757** XwPt-861918 XwPt-470359* XwPt-0969* XwPt-3381** Xbarc174* XwPt-6651 Xbarc174-2 Xcfa2174 XwPt-469159 Tu3AL5152.1 Tu3AL5236 XwPt-470397 XwPt-860800 XwPt-861877 Xwec79 Xpsr103 Xwms554 Xbarc346 Xwms72 CASA0540 XwPt-9172 XwPt-861971 XrPt-0950 XwPt-469488 XwPt-469434 XwPt-470526 CASA0829 XwPt-469455 CASA0827 CASA0670 XwPt-861498 XwPt-861765 XwPt-469984 XwPt-470497 XwPt-376691 XwPt-469192 XwPt-376993 XwPt-861012 XwPt-861289 XwPt-376685 XwPt-4346 XwPt-469845 XwPt-469609 XwPt-469522 XwPt-861710 HKT1 XwPt-469390 XwPt-861656 Xwec14-2 CASA0845 XwPt-470432 XwPt-4352 CASA0775 XwPt-861533 Xabc305 XwPt-861820 XwPt-861253 XwPt-862072 XwPt-861198 XwPt-0961 XwPt-469968 XwPt-469198 XwPt-4553 Xwec42 Xabg461 XwPt-861027 XwPt-860881 XwPt-861993 XwPt-9215 XwPt-0194 XwPt-4342 XwPt-5547 XwPt-470169 XwPt-861890 XwPt-5280 XwPt-2207 XtPt-3608 Xrz682 XwPt-470056 XwPt-469772 XwPt-861053 XwPt-861522 XwPt-860898 XwPt-470075 XwPt-375859 XwPt-861526 XwPt-861928 XwPt-9501 XwPt-469410 Xwms344 XwPt-469882 XwPt-377084 XwPt-470210 Tu3AL5322** XwPt-861106* XwPt-469606** XwPt-860826**

0.00 3.55

qHD-7A.1@E1

43.21

19.75 21.46 22.01 27.48 29.17 33.69

XwPt-1170 Xpsr899 XwPt-5234 XwPt-4836 XwPt-7938 XwPt-9075 XwPt-470424 XwPt-376054 XwPt-470486 XwPt-469856 XwPt-469669 XwPt-469665 XwPt-377033 XwPt-469679 XwPt-3326 XwPt-5732 XtPt-4515 XwPt-861949 XwPt-470529 XwPt-9584 XwPt-2822 XwPt-862223 XwPt-862060 XwPt-861234* XwPt-862086 XtPt-6278 XwPt-470272 XwPt-375828 XwPt-8833 XwPt-377125 XwPt-8331 XwPt-3091 XwPt-470586 XwPt-0959 XwPt-470051 XwPt-469347 XwPt-469707 XwPt-861152 XwPt-469613 XwPt-9068 XwPt-375917 XwPt-376184 XwPt-469637 XwPt-7136 XwPt-862243 CASA0400 XwPt-377170 XwPt-861022 XwPt-376574 XwPt-375818 XwPt-376145 XwPt-376068 XwPt-469751 Tu3AL5015.1 XwPt-470194 CASA0175 CASA0396 CASA0077 CASA0515 CASA0358 Xcfd80 Xcfd80-2 XwPt-469976 XwPt-9578 Xrz599* Xwec71-1 Xwec52-2 Xpsr371 Xrz567 XwPt-861938 CASA0693 Xwms533 XwPt-375872 XwPt-470226 XwPt-861643 XwPt-470256 XtPt-8440 XwPt-7682 Xbarc334 XwPt-860760 XwPt-862127 Tu3AL5203 Xabc154 XwPt-469141 XwPt-7541 XwPt-469971 XwPt-861804 CASA0263 XwPt-375820 XwPt-469768 Tu3AL5175.1 XwPt-860810 XwPt-469155 Xwms219 Xrz446 XwPt-375969 Xwms617 XwPt-469179 Xpsr546 XwPt-9474 XwPt-9058 XwPt-5696 XwPt-5572 XwPt-469916 XwPt-1642 XrPt-9324 XrPt-6189 XwPt-1375 XwPt-6995 XwPt-860718 XwPt-9000 XwPt-861638 Xbcd876

qHD-7A.1@E2

40.29

44.98 46.13 47.86 48.42 50.15

XwPt-377038** XwPt-470666** XwPt-3999** Xwms205** Tu3AL5019.1** XwPt-8008** XwPt-469793** XwPt-8002** XwPt-8506** XwPt-861952** Xwms120** XwPt-862173** XwPt-470365** Tu3AL5071.3** XwPt-469779** Xbcd1871** XwPt-470381** XwPt-376292** XwPt-377328** Xcdo749** CASA0938** Xcdo677** XwPt-1557** XwPt-469290** XwPt-377093** Xbarc117** Xpsr360** Tu3AL5071.1** CASA0111** Xbarc1** XwPt-469490** XwPt-470482** XwPt-469361** XwPt-469648** Xbarc180** Xgdm68** Xrz273** XwPt-469479** XwPt-470167** XwPt-861864** Xwms328.1** Xcdo1326-c** Xcdo348-2** XwPt-469486** XwPt-5883** XwPt-469222** XwPt-861440** XwPt-469531** Xcfa2121** XwPt-376409* XwPt-861470* XwPt-861469* Xbcd926* XwPt-470580 XwPt-861609 XwPt-860984 XwPt-469253 Xwec9-3* Xwec103* Xwec-9-3** Xrz387 XtPt-9852 XwPt-861830 XwPt-861374 XwPt-862153 XwPt-470219 XwPt-469577 XwPt-470605 CASA0028 Xwms639 Xwms639-2 XwPt-861661 XwPt-5949 XwPt-861531 XwPt-376161 Xbcd9** Xbarc151** Xgdm130 XwPt-469185 XwPt-470296 XwPt-9069 Xwec70* Xrz404 Xrz474 Xbcd1030* XwPt-470096* XwPt-376180* Xwms271* Xabg003* Xcdo504-1** Xcdo400** Xcdo504-2** Xwg644** Vrn1** Tu3AL5117.1* XwPt-860872 XwPt-470652 CASA0879 XwPt-861783 XwPt-469634 XwPt-469725 XwPt-861771 Xpsr426* Xbcd808-b Xcdo1326-a* Xcfd2-2 XwPt-860725 XwPt-0935 XwPt-9205 XwPt-8094 Xcdo584-1 Xcdo584 XwPt-469962 XwPt-470190 XwPt-469467 XwPt-469404 XwPt-861794 Xrz421 Xabg391 Xpsr370 XwPt-2490 Xwg114 Xcfd39 XwPt-861004 XwPt-861500 XwPt-376570 XwPt-376789 Xcdo20 XwPt-376253 XwPt-470451 Xwms6 XwPt-469658 Hair_leaf XwPt-862154 XwPt-860900 XwPt-470407 XwPt-376529 XwPt-469153 Xwms635-2 XwPt-469600 Xwec87a Xcfd47 Tu3AL5152.3 Xgdm127 ZCCT1-Vrn2 Vrn

0.00 7.70 11.25 12.96 15.24 16.35 18.63

qSPLN-7A.1@E2

35.67 36.22 38.73 39.71

Chr.6A CASA0190* XwPt-2041

Fig. 1 continued

molecular-weight glutenin subunits (HMW-GSs and LMWGSs), aleurone color, vernalization requirement, and leaf hairiness (Fig. 1). HMW-GSs (TmGlu-A1) and LMW-GSs (TmGlu-A3) were located at 111.89 cM and 0.00 cM on chromosome 1A, respectively, which match the locations of the homologous genes of Glu-A1 and Glu-A3 in T. aestivum (Dong et al. 2010), and is also comparable with a previous report (Dubcovsky et al. 1996). The vernalization requirement (Vrn) was mapped onto chromosome 5A at a position of 198.78 cM (Fig. 1). Since Vrn genes have been cloned in common wheat, several Vrn genes were also located on this linkage map using STS markers specifically developed for these gene sequences. The Vrn1 gene mapped at 116.15 cM on the 5AL, flanked by an RFLP marker (Xwg644) and a DArT marker (XwPt-860872) in a 2-cM region. The Vrn2

(ZCCT1-Vrn2 loci) gene was closely linked with the locus for growth habit (Vrn2 locus) at the 5AL distal end. The Vrn3 (FT-Vrn3 loci), indel variation in promoter region, co-segregated with an SSR marker Xbarc219 on 7AS. Leaf hairiness (Hairleaf) was also mapped on 5A (184.55 cM), with a proximal distance of 14.23 cM from the Vrn2 locus on the same chromosome. The aleurone color (Blue.al) mapped at the centromeric region of 4A (112.6 cM, Fig. 1). Overall, the mapped positions of these loci for protein and morphological traits were consistent with previous data (Dubcovsky et al. 1996; Yan et al. 2003, 2006; Hori et al. 2007). A major QTL for earliness per se (Eps-Am3) was previously located on chromosome 3A (Gawron´ski and Schnurbusch 2012; Gawron´ski et al. 2014). T. urartu genome

13

60


Fig. 2 The segregation distortion profile of markers on seven chromosomes on the einkorn wheat genetic map. The P value of the Chisquared distribution and parental preference markers are shown with blue and orange lines, respectively. The vertical dashed lines denote

the separations between chromosomes, shaded for 1A, 3A, 5A and 7A, and unshaded for 2A, 4A and 6A. The green and red dashed lines show the SD at P = 0.05 and 0.01. A/B represents the frequency ratio of the parent A (KT1-1) and B (KT3-5) alleles

sequences (Ling et al. 2013) were used to develop SSR markers to saturate the genetic map of chromosome 3A. Out of 322 SSR markers designed for chromosome 3A (the marker name started with Tu3AL5), 43 markers were successfully mapped on chromosome 3A, and the other 23 markers were mapped on the remaining chromosomes. Of the newly mapped SSR markers on 3A, twelve were located in the Eps-Am3 region (226.47–251.42 cM), which is closely linked or co-localized with the STS markers developed by Gawron´ski and Schnurbusch (2012) (Fig. 1). Moreover, nine SSR markers (names starting with CASA) were also localized on chromosome 3A, which were developed using T. urartu whole-genome sequences (Fig. 1). Overall, 52 newly developed SSR markers extended map length by 29.0 cM (accounting for 11.3 % map length) and increased the marker density significantly, especially in the Eps-Am3 region. Our linkage map was compared with published genetic maps of hexaploid wheat (wheat-Composite2004, http:// wheat.pw.usda.gov), tetraploid wheat (Marone et al. 2012), and other einkorn wheat (Jing et al. 2009) for 181 shared markers (Table S3). Of the 114 shared markers, ~75 % had collinear positions with those in respective homologous groups on the wheat composite map (Fig. S1, http:// wheat.pw.usda.gov), except markers located on 4AL and 5AL (Fig. S2 and Table S3), where translocation events happened during wheat’s evolutionary history (Devos et al. 1995). A large number of markers in our map were shared with those on the T. durum consensus map (84 %;

Fig. S3; Marone et al. 2012) and the Tm DArT map (Jing et al. 2009). Three inconsistent markers in the Tm DArT map (Jing et al. 2009), XwPt-3876, XwPt-3648, and XwPt3135 on the linkage group 4A_2, were mapped on chromosome 7A because of their close linkage with other markers (Table S3, Fig. S4), implying that 4A_2 reported by Jing et al. (2009) might belong to chromosome 7A. In total, 82.87 % (150/181) of the markers are in the same homologous groups as other maps and many of them have co-linearity between einkorn and polyploid wheat (http://wheat. pw.usda.gov; Jing et al. 2009; Marone et al. 2012), which provides support to the marker order presented in this study. Among the detected genotypes of 926 markers, 48.72 % were inherited from KT1-1, 51.16 % from KT3-5, and 0.12 % were heterozygous in the RIL population, indicating there were no significant differences between parental allele ratios. However, 298 had significant segregation distortion (P