栽培エンマーコムギと野生エンマーコムギの戻し交雑自殖系統を用いた穎果の形態と重量の評価

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Acccepted:April 18, 2017

Corresponding author: Naoki Mori ([email protected])

Evaluation of grain dimension and weight using backcross recombinant inbred lines between

wild and domesticated emmer wheat

Yuki Miyazaki

1）

, Pham M. Ngoc

1）

, Katie L. Liberatore

2）

, Shahryar F. Kianian

2）

, Cristian I. Vladutu

1）

, Naoki Mori

1）

1）_{Graduate School of Agricultural Science, Kobe University（1-1 Rokkoudai-cho, Nada-ku, 657-8501, Japan）} 2）_{Cereal Disease Laboratory, USDA, ARS（1551 Lindig Avenue, St. Paul, MN 55108, USA）} Summary: Emmer wheat (Triticum turgidum ssp. dicoccum) represents the primitive situation in the

domestication of AABB tetraploid wheat. As one of the earliest domesticated grain species, it was a principal crop in the development and spread of Neolithic agriculture in the Old World. Grain weight and dimension (size and shape) have been major targets of selection since the beginning of agriculture. To clarify the genetic mechanism (s) affecting grain weight and dimension, we utilized 92 backcross recombinant inbred lines (BRILs) derived from a cross between a domesticated emmer wheat and a wild emmer wheat (T. turgidum ssp. dicoccoides). Weight, grain dimensions (width, length, and height), ratios of dimensions (shape), and correlations between traits were evaluated for two consecutive years, 2015 and 2016. All grain dimension components showed strong positive correlation with grain weight. Among them, the highest correlation coefﬁ cient (r = 0.822) was observed between the grain weight and width, suggesting that grain width was a main target of selection for increasing grain weight during emmer wheat domestication. In addition, both the grain length/width ratio and the length/height ratio showed negative correlation with grain weight. These results indicate that a transition from slender to round grain shape was advantageous in the early stage of wheat domestication.

Key words: emmer wheat, backcross recombinant inbred lines, domestication, grain dimension, grain weight

Introduction

Emmer wheat (Triticum turgidum ssp. dicoccum (Schrank ex Schubler) Thel.), a Neolithic founder crop, originated in the Fertile Crescent about 10,000 years ago (Lev-Yadun et al. 2000, Zohary and Hopf 2000). It has primitive features such as non free-threshing habit and a relatively fragile rachis. Based on archaeological and biological evidence, emmer wheat is considered to be the earliest domesticated tetraploid wheat with an AABB genome combination.

Selection pressure associated with cultivating the wild emmer wheat (T. turgidum ssp. dicoccoides (Körn. ex Asch. & Graebner) Thel.) in the incipient phases of domestication triggered changes in a set of traits, e.g. rachis fragility, ﬂ owering time, plant architecture, inflorescence architecture, and grain characteristics. This process is referred to as the domestication syndrome (Hammer 1984). Among them, grain weight and dimension were major targets of selection since the early phase of domestication. Archaeobotanical studies indicate that grain size of wheat and barley started to increase in the Pre-Pottery Neolithic A and early Pre-Pottery Neolithic B eras (Willcox 2004, Fuller 2007). Most of the traits related to grain weight,

size and shape are quantitatively inherited and importantly, quantitative trait loci (QTLs) affecting these traits have been reported on almost all chromosomes in wheat (Sun et al. 2009, Gegas et al. 2010, Peleg et al. 2011, Okamoto et al. 2012, Williams and Sorrels 2013, Russo et al. 2014, Kumar et al. 2016). However, the genetic mechanism underlying the early phase of wheat domestication is still unclear since most previous studies were designed using common wheat or modern cultivated durum wheat.

To understand the genetic modiﬁ cations in the early stage of wheat domestication we have been studying the domestication related traits in the predecessor to modern cultivated durum wheat, emmer wheat (Thanh et al. 2013). The aim of the present study is to gain deeper insight into the extent of selection on grain weight and dimension in the early phase of wheat domestication. We evaluated weight and dimension (length, width and height) of grains using 92 backcross recombinant inbred lines (BRILs) derived from a cross between a domesticated emmer wheat and a wild emmer wheat.

Materials and methods

Plant materials

To develop backcross recombinant inbred lines, we used an

Research Article

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accession of domesticated emmer wheat, T. turgidum ssp. dicoccum, collected in Ethiopia (original accession no. KU7309, hereafter designated Dcm1001) and a wild emmer wheat, T. turgidum ssp. dicoccoides, collected in Iraq (KU8736A, hereafter Dcc63) as parental lines. These two accessions were provided by National BioResource Project (NBRP), Japan (http://www.shigen.nig.ac.jp/wheat/komugi/top/top.jsp). The F1 plant derived from a cross between Dcm1001 and Dcc63 was backcrossed with Dcm1001 and 29 BC1F1 plants were produced. These BC1F1 plants were backcrossed once again with Dcm1001 and BC2F1 plants were obtained. Using these BC2F1 plants, a total of 92 BRILs (29 families, BC2F12 and BC2F13 generation) were produced by single seed descent method. Theoretically these BRILs contain 12.5% of the genome of wild emmer wheat (Dcc63) in a genetic background of domesticated emmer wheat (Dcm1001).

Trait evaluation

Grain dimension and grain weight were examined using 92 BRILs of BC2F12 and BC2F13 generations in two consecutive years, 2015 and 2016, respectively. These lines were grown in a randomized block design with two replicates in a glass house at Kobe University, Kobe City, Japan. Grain length, width, and height (Fig. 1) were measured for 15 grains in each replication using a digital venire caliper (Mitutoyo Corp., Japan). Grain weight was measured for six spikes in each replication using an analytical balance. For further evaluation of grain dimension, the following three ratios, length/width, length/height and width/ height were calculated. For the comparison of the grain traits between the parental lines t-test was performed using Stat View ver. 5.0 (SAS Institute Inc., USA). To examine the correlation between grain weight and grain dimensions, Pearson s correlation coefficients (r) were computed using Stat View ver.5.0.

Fig. 1 Grains of wild emmer wheat (Dcc63) and domesticated emmer wheat (Dcm1001). The grain length, grain width, and grain height components measured in this study are shown with arrows.

Results and discussion

Domesticated emmer wheat grains are heavier and rounder than wild emmer grains

Grain dimension of the parental lines were evaluated by measuring the grain height, grain length and grain width. Based on the results, three ratios, length/width, length/height, and width/height were computed (Table 1). All traits except grain length were significantly different between domesticated (Dcm1001) and wild (Dcc63) emmer wheat. Domesticated emmer grain weight was about three times higher than that of wild emmer wheat, suggesting a strong positive selection for weight in the domestication of emmer wheat. Domesticated emmer wheat also showed larger grain width and height. These results are consistent with archaeobotanical evidence that suggest grain size increased in the early stage of domestication (Willcox 2004, Fuller 2007). In addition, the length/width and length/height ratios decreased in domesticated emmer wheat compared to the wild species. This indicates that overall grain shape of the domesticated emmer wheat is rounder than that of wild emmer wheat.

Table 1 Average grain weight and grain dimensions in domesticated (Dcm1001) and wild (Dcc63) emmer wheat

7UDLWV 'FPVWGY 'FFVWGY 㼠㻙㼠㼑㼟㼠㻝㻕 :HLJKWJ 㻖㻖 /HQJWKPP 㼚㻚㼟㻚 :LGWKPP 㻖㻖 +HLJKWPP 㻖㻖 /HQJWKZLGWK 㻖㻖 /HQJWKKHLJKW 㻖㻖 :LGWKKHLJKW 㻖㻖 1) ** indicates a signiﬁ cant difference between Dcm1001 and Dcc63 at 0.01 level while n.s. indicates not signiﬁ cant at 0.05 level determined by t-test. Standard deviation (stdv) is indicated in parentheses.

Emmer BRIL grain trait distributions are not bound by parental phenotypes

To investigate the genetic factors underlying the change in emmer grain weight and shape during domestication, segregation of grain traits were examined in 92 BRILs. Frequency distributions of grain weight, three grain dimensions and three grain ratios among the BRILs in 2016 are shown in Figs. 2 and 3. Similar distributions were observed for all seven traits in 2015 (data not shown). All traits showed a continuous distribution suggesting that a number of genetic factors as well as environmental effects underlie each trait. Transgressive variation was also observed for all traits. These results suggest that polygenic inheritance and epistatic interactions affect the observed phenotypic variation in emmer wheat grain dimension and grain weight.

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0 10 20 30 40 50 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05 0.055 0.06 0.065 No. of lines Grainweight (g) Dcm1001 F1 Dcc63

$

0 10 20 30 40 50 1.67 1.84 2.01 2.18 2.35 2.52 2.69 2.86 3.03 3.2 3.37 No. of lines Grainwidth (mm) Dcc63 F1 Dcm1001

&

0 10 20 30 40 50 7.8 8.1 8.4 8.7 9 9.3 9.6 No. of lines 㻳㼞㼍㼕㼚㻌㼘㼑㼚㼓㼠㼔㻌㻔㼙㼙㻕 Dcm1001 F1 Dcc63

%

0 10 20 30 40 50 1.95 2.12 2.29 2.46 2.63 2.8 2.97 3.14 3.31 3.48 3.65 3.82 No. of lines Grainheight (mm) Dcm1001 F1 Dcc63

'

Fig. 2 Frequency distribution of the four traits measured in the backcross recombinant inbred lines between domesticated emmer wheat (Dcm1001) and wild emmer wheat (Dcc63). (A) grain weight; (B) grain length; (C) grain width and (D) grain height. Open arrows indicate the average values of each parental line and the F1 (labeled accordingly). The horizontal bar

indicates the mean value of the 92 BRILs.

0 10 20 30 40 50 2.84 3.1 3.36 3.62 3.88 4.14 4.4 4.66 4.92 5.18 5.44 No . of line s

Grain length / Grain width

Dcm1001 F1 Dcc63

㻔㻭㻕㻌

0 10 20 30 40 50 2.42 2.6 2.78 2.96 3.14 3.32 3.5 3.68 3.86 4.04 4.22 4.4 4.58 No . of lin es

Grain length / Grain height

Dcm1001 F1 Dcc63

㻔㻮㻕㻌

0 10 20 30 40 50 0.8 0.828 0.856 0.884 0.912 0.94 0.968 0.996 1.024 No . of lin e s 㻳㼞㼍㼕㼚㻌㼣㼕㼐㼠㼔㻌㻛㻌㻳㼞㼍㼕㼚㻌㼔㼑㼕㼓㼔㼠 Dcm1001 F1 Dcc63

㻔㻯㻕㻌

Fig. 3 Frequency distribution of the three ratios computed in the backcross recombinant inbred lines between domesticated emmer wheat (Dcm1001) and wild emmer wheat (Dcc63). (A) grain length / grain width; (B) grain length / grain height and (C) grain width / grain height. Open arrows indicate the average values of each parental line and the F1 (labeled

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Increased grain weight is correlated with rounder grain shape

Grain weight and grain dimension may or may not be controlled by independent mechanisms. To investigate this question, correlations between traits were examined. Analysis revealed significant positive or negative correlation between grain dimension components and grain weight (Table 2). All three components of grain size (length, width, and height) were positively correlated with grain weight. Of these, grain width showed the highest correlation (r = 0.822) with grain weight (Table 2, Fig. 4A). This result suggests that a positive selection

for larger grain width contributed signiﬁ cantly to the increase of grain weight during emmer wheat domestication. In addition, to evaluate the effect of domestication on the grain shape we examined grain length/width, length/height, and width/height dimension ratios. Among these, length/width (r = -0.533) and length/height (r = -0.413) were negatively correlated with the grain weight (Table 2, Figs. 4B and 4C). Since both of these ratios indicate the roundness of the grain, negative correlation of these ratios with grain weight suggests that evolution of spikelets with rounder grains contributed to increased grain weight.

Table 2 Correlation coeﬃ cients (r) among the traits of grain weight and dimension components in BRILs 7UDLW :HLJKW /HQJWK :LGWK +HLJKW /HQJWKZLGWK /HQJWKKHLJKW /HQJWK :LGWK +HLJKW /HQJWKZLGWK /HQJWKKHLJKW :LGWKKHLJKW 1) * and ** indicate signiﬁ cant differences at 0.05 and 0.01 levels, respectively.

Fig. 4 Correlation analysis between grain weight and grain dimension. (A) correlation between grain weight and grain width; (B) correlation between grain weight and grain length/grain width and (C) correlation between grain weight and grain length/grain height. Dashed circles indicate the average values of parental lines and F1 plants.

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Millet (1986) measured the volume of floret cavities in 10 common wheat and three durum wheat accessions, and suggested that grain weight is partly determined by the volume of the ﬂ oret cavity. Taking this in account, we could propose a hypothesis that a set of morphological changes in glumes toward rounder shape of the spikelet occurred during the domestication of emmer wheat.

Toward the identiﬁ cation of genes that contributed to grain weight and dimension in emmer wheat domestication

A number of studies have aimed to identify the key genes that control wheat grain morphology and weight (Sun et al. 2009, Gegas et al. 2010, Peleg et al. 2011, Okamoto et al. 2012, Williams and Sorrels 2013, Russo et al. 2014, Kumar et al. 2016). These studies showed a wide distribution of QTLs for grain weight, size, and shape across the wheat genome. Of these, Peleg et al. (2011) identiﬁ ed 12 loci associated with kernel weight using RILs between durum wheat and wild emmer wheat. Kumar et al. (2016) identiﬁ ed QTLs for grain shape and size on 17 chromosomes in common wheat using a high density SNP linkage map. They suggested that a QTL on chromosome 4B plays an important role in grain weight and shape and the QTL might be an ortholog of GS3 or qGL3 in rice (Wan et al. 2005, Fan et al. 2006, Mao et al. 2010).

Based on two-dimensional measurements of grain dimension using multiple mapping populations, Gegas et al. (2010) suggested that grain size and shape are largely independent traits and that these traits are under the control of a limited number of genetic components. While ancestral wheat grains and multiple modern wheat mapping populations were characterized in their study, early genetic variation contributing to grain morphology and weight may have been missed. The strength of the population used in the present study is a close assessment of the variation among wild and domestic emmer wheat to pinpoint early changes and their contributions to grain weight and morphology. Whether or not the positive correlations between grain weight and dimension components presented here are the result of independent genetic mechanism (s) remains unclear. To test this hypothesis, it is necessary to further refine QTL and isolate genetic factors underlying grain size, shape, and weight. A large-scale QTL analysis using a high density SNP map is underway to identify the genetic factors that determine grain size, shape, and weight, and to gain deeper insight into the genetic mechanism (s) underlying the early phase of wheat domestication.

Acknowledgements

We are grateful to NBRP-Wheat, Japan for supplying the wheat accessions used in this study.

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