Comparative mapping of fiber, protein, and mineral contentQTLs in two interspecific Leymus wildrye full-sib families

Steven R. Larson Æ Henry F. Mayland

Received: 20 September 2006 / Accepted: 12 March 2007 / Published online: 31 March 2007

� Springer Science+Business Media B.V. 2007

Abstract Crude protein (CP), neutral detergent

fiber (NDF), acid detergent fiber (ADF), and mineral

content are important components of forage quality in

grasses. Elevated [K]/([Ca] + [Mg]) ratios (KRAT)

substantially increase the risk of grass tetany (hypo-

magnesemia) in grazing animals, which is a serious

problem associated with some cool-season grasses.

The objectives of this study were to map and compare

QTLs controlling concentrations of CP, NDF, ADF,

Al, B, Ca, Cl, Cu, Fe, K, Mg, Mn, Na, P, S, Si, Zn,

and KRAT in two full-sib Leymus triticoides · (L.

triticoides · L. cinereus) TTC1 and TTC2 families.

Significant genetic variation and QTLs were detected

for all traits, with evidence of conserved QTLs for

ADF (LG1a, LG5Xm, LG7a), NDF (LG7a), Ca

(LG1b), CP, (LG5Xm), KRAT (LG3b, LG6b, LG7a,

LG7b), Mn (LG2b, LG3b, LG4Xm), and S (LG3a)

content in both TTC1 and TTC2 families. Moreover,

the direction of QTL effects was the same for 13 of

the 14 homologous QTLs in both families. The TTC1

and TTC2 KRAT QTLs on LG7a and LG7b were

located near markers defining homoeologous rela-

tionships between the sub-genomes of allotetraploid

Leymus, suggesting possible QTL homoeology.

Another 88 QTLs were unique to one family or the

other, but many of these clustered in genome regions

common between the two families. These results will

support development of new Leymus wildrye forages

and help characterize genes controlling mineral

uptake and fiber synthesis.

Keywords Forage � Fiber � Grass � Mineral �Protein � QTL

Abbreviations

ADF acid detergent fiber

CP crude protein

KRAT K/(Ca + Mg) ratio

NDF neutral detergent fiber

Introduction

Fiber, crude protein, and mineral content are impor-

tant criteria in forage grass breeding (Casler and

Vogel 1999; Casler 2001). Fiber content is often

measured by acid detergent fiber (ADF) and neu-

tral detergent fiber (NDF), which can limit the

digestibility, palatablity, and intake of grass forages

(Van Soest 1994; Falkner and Casler 1998). The NDF

S. R. Larson (&)

United States Department of Agriculture, Agriculture

Research Service, Forage and Range Research

Laboratory, Utah State University, Logan, UT 84322-

6300, USA

e-mail: stlarson@cc.usu.edu

H. F. Mayland

Northwest Irrigation and Soils Research Laboratory,

Agriculture Research Service, United States Department

of Agriculture, Kimberly, ID 83341, USA

123

Mol Breeding (2007) 20:331–347

DOI 10.1007/s11032-007-9095-9 1239

fraction includes cellulose, lignin, and hemicellulose;

whereas the ADF fraction is composed mainly of

cellulose and lignin without hemicellulose (Van Soest

1994). The genetic control of ADF and/or NDF

content has been examined by QTL analysis in

perennial ryegrass (Cogan et al. 2005), maize (Kra-

kowsky et al. 2005, 2006; Lubberstedt et al. 1997;

Mechin et al. 2001) and Arabidopsis (Barriere et al.

2005). Crude protein and mineral content receive less

attention by forage grass breeders, in part because

soil fertility can have relatively large effect on these

traits, because protein and mineral supplements are

commonly used, and also because crude protein is

often negatively correlated with yield (Casler and

Vogel 1999; Casler 2001). Although much of the

soluble protein is degraded to ammonia in the rumen

and eventually excreted as urea, crude protein is

highly digestible and non-degradable protein that

passes through the rumen can be efficiently utilized in

the lower digestive tract (Casler 2001). Like ADF and

NDF, crude protein concentration is relatively easy to

measure by near infrared reflectance spectroscopy

(NIRS) analysis. Thus, these traits are often evaluated

together and QTLs controlling crude protein concen-

tration were also detected in perennial ryegrass

(Cogan et al. 2005) and maize (Lubberstedt et al.

1997; Mechin et al. 2001). An inverse relationship

between crude protein and fiber content can be

expected due to phenological variation in reproduc-

tive development and the ratio of stems, leaf sheaths,

and lamina. Thus, dissection of protein and fiber

QTLs may provide some insight into the possible

ontology of the underlying genes.

Although mineral supplements are relatively inex-

pensive and effective when properly administered,

significant problems are still associated with forage

mineral deficiencies or imbalance. Forage [K+]/

([Ca++] + [Mg++]) molar charge ratios (KRAT)

greater than approximately 2.2 substantially increase

the risk of grass tetany (hypomagnesemia) in grazing

animals, which is a serious problem associated with

many cool-season grasses (Mayland 1988; Sleper

et al. 1989). Grass tetany occurs in about 1% of

grazing livestock, one-third of which likely die

(Mayland 1988). Experimental grass varieties with

high magnesium content were developed to reduce

KRAT and evaluate grass tetany potential in tall

fescue (Festuca arundinceae) (Sleper et al. 2002),

Italian ryegrass (Lolium multiflorum) (Hides and

Thomas 1981; Mosely and Baker 1991), and or-

chardgrass (Saiga et al. 1992; Saiga and Izumi 1997).

Nutritionally relevant variation in mineral concentra-

tions and KRAT have been also documented in

crested wheatgrass (Agropyron cristatum and A.

desertorum) (Asay et al. 1996; Vogel et al. 1989)

and Russian wildrye (Psathyrostachys juncea) (Asay

et al. 2001; Asay and Mayland 1990; Jefferson et al.

2001; Karn et al. 2005). In comparisons with crested

wheatgrass and Russian wildrye, Altai wildrye (Ley-

mus angustus) had especially high KRAT values

(Lawrence et al. 1982). Despite the large number of

studies of KRAT variation there has not been any

effort to dissect the genetic control of various forage

mineral concentrations at the genome level of

grasses.

The genus Leymus includes about 30 long-lived

perennial grass species distributed throughout tem-

perate regions of Europe, Asia, and the Americas.

Leymus wildryes are perhaps most abundant in the

mountains of central Asia and western North

America. These species display remarkable variation

in stature and adaptation to harsh cold, dry, and

saline environments. Basin wildrye (Leymus cinere-

us) and several other large-stature Leymus species

including Altai wildrye (L. angustus) and mammoth

wildrye (L. racemosus) have high biomass accumu-

lation potential across a wide range of high-eleva-

tion or high-latitude growing environments of

western North America (Jefferson et al. 2002;

Jensen et al. 2002; Lauriault et al. 2005), ideal for

stockpiling fall and winter forage or biofuel feed-

stocks. Leymus cinereus is the largest (up to 2 m

tall) native grass and most abundant Leymus species

in the Great Basin, Rocky Mountain, and Inter-

mountain regions of the western North America,

where grazing livestock provide major agricultural

commodities and heavily rely on natural or low-

maintenance forage production. Large stature Ley-

mus wildryes capable of producing abundant and

valuable forage on many saline/alkaline sites where

few other species are adapted. However, caespitose

L. cinereus is susceptible to damage by intense

grazing of early season and fall regrowth. Once

abundant on the floodplains of major rivers, alluvial

gullies, and other watered areas with deep, well

drained soils in the Great Basin and Intermountain

regions, L. cinereus has been eliminated from much

of its former range due to grazing, harvesting, and

332 Mol Breeding (2007) 20:331–347

123

cultivation of field crops. Cultivars of L. cinereus

are commonly used in rangeland seed mixtures in

western North America, but have limited use in

pastures or hay crops. The second most common

Leymus species in western North American is

creeping wildrye (L. triticoides). Leymus triticoides

is a shorter (0.3–0.7 m), but highly rhizomatous

grass specifically adapted to poorly drained alkaline

sites in the Great Basin, California, and other

regions of western North America. Creeping wildrye

(L. triticoides) is cultivated using vegetative prop-

agules as a saline biomass crop in California, but

poor seed production limits widespread use of this

species.

Although L. triticoides and L. cinereus are mor-

phologically divergent, both species are highly self-

sterile and hybridize with each other in nature.

Experimental families, breeding populations, and

molecular genetic maps derived from interspecific

hybrids of Leymus species L. cinereus and L.

triticoides have been developed for plant improve-

ment and genetic investigations of functionally

important traits in perennial forage grasses (Wu

et al. 2003; Hu et al. 2005; Larson et al. 2006). The

F1 hybrids are very robust plants showing a heterotic

combination of tall plant height, large stems and

leaves, prolific seed production, and relatively good

seed germination from L. cinereus with vigorous

proliferation of tillers, rhizomes, relatively good

establishment (after seed germination), regrowth

potential, and plant resiliency from L. triticoides. In

terms of applied breeding, admixed breeding popu-

lations derived from interspecific hybrids of L.

cinereus an L. triticoides show excellent potential

for high biomass production, reduced susceptibility to

grazing or harvest, and better regrowth potential. The

linkage maps include 67 cross-species anchor mark-

ers (i.e. markers mapped in other grass species) used

to identify and compare the 14 linkage groups of

allotetraploid Leymus (2n = 4x = 28) based on

synteny of corresponding markers in closely related

wheat (Triticum spp.), barley (Hordeum vulgare), and

cereal rye (Secale cereale) Triticeae cereals (Wu

et al. 2003; Larson et al. 2006). Moreover, genome

specific markers have been used to distinguish several

homoeologous linkage groups corresponding to the

Ns (Psathytrostachys) and Xm genomes of Leymus

(Wu et al. 2003). The Ns genome originates from

Psathyrostachys (Dewey 1984; Zhang and Dvorak

1991), whereas the origin of the Xm genome (Wang

et al. 1994) is less certain but seems to share

significant homology to the E genome of Lophypryum

and Thinopryum (Love 1984; Sun et al. 1995; Zhang

et al. 2006).

Our objective here was to (1) compare plant

concentrations of CP, NDF, ADF, Al, B, Ca, Cl, Cu,

Fe, K, Mg, Mn, Na, P, S, Si, Zn, and KRAT

between L. cinereus and L. triticoides, and (2)

identify and compare QTLs controlling fiber, pro-

tein, and mineral concentrations in two full-sib

mapping families, TTC1 and TTC2, derived from

interspecific hybrids of L. cinereus and L. tritico-

ides, and (3) identify strategies to improve forage

quality of interspecific breeding populations that

will overcome limitations of the parental species.

Another objective was to compare the location of

these Leymus QTLs with genomic regions control-

ling related traits in other cereal and grass species

and identify possible opportunities gene discovery

research in Leymus.

Materials and methods

Plant materials and genetic maps

The pedigree and construction of molecular genetic

maps for the TTC1 and TTC2 families were origi-

nally described by Wu et al. (2003) and updated with

additional markers by Larson et al. (2006). Briefly,

the TTC1 and TTC2 families were derived from one

L. triticoides Acc641 plant (T-tester) pollinated by

two different L. triticoides Acc:641 · L. cinereus

Acc:636 hybrid plants (TC1 and TC2) under separate

pollen exclosures producing two distinct full-sib

families (TTC1 and TTC2). The TTC1 and TTC2

families include 164 and 170 full-sib individuals,

respectively, which can also be considered half-sibs

of the T-tester plant between families. The molecular

genetic maps for the TTC1 and TTC2 families were

constructed using DNA markers that were present in

one or both hybrids, absent in the tester, and

segregating with an expected ratio of 1:1 among the

full-sib progeny (Wu et al. 2003). The TTC1 map

includes 1069 AFLP markers and 53 anchor loci in 14

linkage groups spanning 2001 cM. The TTC2 map

contained 1002 AFLP markers and 45 anchor loci in

Mol Breeding (2007) 20:331–347 333

123

14 linkage groups spanning 2066 cM. Some 488

homologous AFLP loci (i.e. AFLPs of the same size)

and 31 anchor markers have been mapped in both

families, showing similar map order. Thus, 1583

AFLP markers and 67 different anchor loci have been

mapped into 14 linkage groups, which evidently

correspond to the 14 chromosome pairs of allotetra-

ploid Leymus.

Forage quality evaluations

Ramets from each of the two mapping families

(TTC1 and TTC2) were space planted in a random-

ized complete block (RCB) design with two repli-

cates (blocks) per family at the Utah Agriculture

Experiment Station Richmond Farm (Cache Co.,

UT). Each block contained 164 TTC1 or 170 TTC2

clones plus several parental genotypes (i.e. TC1, TC2

and T-tester clones) and single-plant representatives

of the heterogeneous L. cinereus Acc:636 and L.

triticoides Acc:641 source accessions. Individual

ramets were transplanted from soil containers (4-cm

diameter) in the spring of 2001 to field plots with 2-m

row spacing and 2-m spacing within rows (2-m

centers).

Forage samples were harvested using a hand-held

sickle knife, into perforated paper bags, and subse-

quently transferred to forced-air ovens (608C) on

May 28, 2003 and May 5, 2004. Dried forage samples

were ground to pass a 1 mm screen in a Cyclotec

1093 abrasion sample mill (FOSS Tecator, Hoganas,

Sweden). For elemental analyses, the milled samples

were dry washed at 6408C, dissolved with nitric acid,

diluted with water, and analyzed using a Model 4300-

DV inductively-coupled plasma optical emission

spectrometer (ICP-OES) (PerkinElmer Life and Ana-

lytical Sciences, Inc., Wellesley, MA). The molar ion

charge ratio of K–Ca and Mg (KRAT) was calculated

as (%K)(0.0257)/[(%Ca)(0.0499) + (%Mg)(0.0823)].

Fiber (ADF and NDF) and crude protein (CP)

concentraions were estimated by scanning milled

samples using a NIRS Model 6500 (Pacific Sci.

Instruments, Silver Spring, MD). Estimates of ADF,

NDF, and CP concentrations based on NIRS scans

were calibrated using NIRSystems software with ISI

Forage Equation IS-0122FS (Infrasoft Int. LLC, Port

Matilda, PA) based on tall fescue samples as

described by Asay et al. (2002). Validation of this

NIR-based equation was performed using laboratory

assays for CP (139 assays), ADF (138 assays), and

NDF (63 assays) using Leymus samples from the

2003 and 2004 forage harvests. The LECO CHN-

2000 Series Elemental Analyzer (LECO Corp., St.

Joseph, MI) was used as a laboratory assay of N (%),

which was then multiplied by 6.25 to estimate CP

(%). The R2 values for CP validation were 0.91 in

2003, 0.90 in 2004, and 0.98 overall. The ANKOM-

200 Fiber Analyzer (ANKOM Technol. Corp., Fair-

port, NY) was used for laboratory assays of NDF and

ADF concentration according to manufacturer proto-

cols. The R2 values for ADF validation were 0.85 in

2003, 0.66 in 2004, and 0.83 overall. The R2 values

for NDF validation were 0.74 in 2003, 0.77 in 2004,

and 0.92 overall.

Data analysis

Broad-sense heritabilities were determined using

SAS code (Statistical Analysis Systems Institue

Inc., Cary, N.C.) for estimating heritability from

lines evaluated in RCB designs in multiple environ-

ments (Holland et al. 2003), modified to account for

repeated measurements on perennial plants over years

substituted for environments. All class variables (i.e.

rep, entry, and year) were treated as random effects.

Genotypic and phenotypic correlations were deter-

mined using SAS code for estimating correlations

from RCB designs in multiple environments (Holland

et al. 2003), also modified to account for repeated

measurements over years substituted for environ-

ments. The basic SAS code for estimating heritabil-

ities, genotypic correlations, and phenotypic

correlations is available at http://www4.ncsu.edu/

*jholland/heritability.html (verified 19 June, 2006).

QTL analyses were based on trait averages over

the two reps and two years determined by the

LSMEANS procedure of SAS. A sequential and

reiterative procedure of QTL detection was per-

formed using the MAPQTL 5 package (Van Ooijen

2004). Genome-wide interval mapping (IM) (Lander

and Botstein 1989; Van Ooijen 1992) was per-

formed in 1-cM increments to identify putative

QTLs and possible cofactors used in a multiple-QTL

model (MQM) (Jansen 1993, 1994; Jansen and Stam

1994). A log-likelihood ratio (LOD) threshold of 3.3

was used to identify MQM cofactors (also referred

to as primary QTLs below), whereas a LOD

threshold of 2.3 was used to identify other possible

334 Mol Breeding (2007) 20:331–347

123

secondary QTLs in the final MQM scan. The LOD

threshold of 3.3 is a close approximation of a

genome-wide 5% significance based on simulation

tables (Van Ooijen 1999) and permutation analyses

(Churchill and Doerge 1994), which seems appro-

priate for selecting cofactors used in genome-wide

MQM scans. The more relaxed LOD threshold of

2.3 corresponds to the chromosome-wide 5% sig-

nificance level as determined from permutation

analyses of each linkage group (Churchill and

Doerge 1994), which seemed reasonable for identi-

fication of other possible QTLs not used as cofactors

in the genome-wide MQM scans. A backward

elimination procedure was applied to this initial

set of cofactors using a conservative significance

level of 0.001 to ensure the independence of each

cofactor. A reiterative process of using any new

QTLs detected using MQM scans as additional

cofactors was used until no additional primary QTLs

(LOD � 3.3) were found. Where possible, all 1583

AFLP markers and 67 different anchor loci were

used for QTL analyses of the TTC1 and/or TTC2

families. However, for simplicity and comparative

purposes only the 488 homologous AFLP markers

mapped in both TTC1 and TTC2 families and 67

anchor markers are shown in the QTL graphs

(Fig. 1), which were generated by MapChart version

2.1 (Voorrips 2002).

Results and discussion

Compared with the L. triticoides Acc:641 source

accession, L. cinereus Acc:636 source accession

showed significantly greater ADF, Cu, K, and KRAT

contents and significantly lower Ca, Fe, Mg, Mn, S,

and Zn contents (Table 1). Thus, significant diver-

gence among the heterogeneous source accessions

(original source of TC1 hybrid, TC2 hybrid, and T-

tester parental clones) was apparent for these nine

traits. Elevated K, depressed Ca, and depressed Mg

concentrations all contributed to highly elevated

KRAT values in L. cinereus and relatively high

KRAT divergence between source accessions of the

interspecific TC1 and TC2 hybrid clones (Table 1).

However, the lack of divergence in other traits does

not preclude genetic variation within the heteroge-

neous source accessions, but this could not be directly

tested because the Acc:636 and Acc:641 reference

plants were not clonally replicated. It should also be

noted that the L. triticoides T-tester genotype showed

significantly less Al, Fe, and NDF content and

significantly greater K, Mg, Mn, P, S, and Si content

compared to the L. triticoides Acc:641 accession

(Table 1). The T-tester was a rogue genotype

originating from spreading rhizomes or seed of open

pollinated plants of several possible L. triticoides

accessions, including Acc:641, which may account

for its phenotypic differences from the Acc:641

accession.

Compared with the L. triticoides T-tester, the

interspecific TC1 and TC2 hybrids both showed

significantly greater ADF and KRAT contents and

significantly lower Ca, S, and Zn concentrations

(Table 1). Compared to the T-tester, the TC1 hybrid

also showed significantly greater Mn content and

significantly less Mg and P contents. The TC2 hybrid

also showed significantly less CP, B, K, Mn, Na and

Si content and significantly greater NDF and KRAT

content compared to the T-tester genotype (Table 1).

However, traits that do not show significant differ-

ences between the TC1, TC2, and T-tester parental

genotypes may still show significant genetic variation

within the TTC1 and TTC2 progeny resulting from

transgressive segregation.

Significant heritabilities and QTLs were observed

in the clonally replicated TTC1 and/or TTC2 popu-

lations for all traits (Table 2). Significant heritability

but no significant QTLs were detected for Al and B

content in the TTC2 family or Na and Si content in

the TTC1 family (Table 2). Likewise, we observed

relatively strong heritability with relatively weak

QTL effects for B and Na content in the TTC1 family

and for K content in the TTC2 family (Table 2).

However, our analysis could only detect QTLs that

were heterozygous in the TC1 and/or TC2 hybrids.

Because of the way the maps were constructed

Fig. 1 Comparison of forage quality QTLs detected in the full-

sib Leymus triticoides · (L. triticoides · L. cinereus) TTC1 and

TTC2 families, exceeding 2.3 LOD (lines) and 3.3 LOD (boxes)

significance levels as indicated in the legend. The updated

molecular genetic linkage maps include 488 homologous AFLP

markers as mapped in both TTC1 and TTC2 families and 67

anchor markers (larger bold marker text) mapped in TTC1 and/

or TTC2 families (Wu et al. 2003; Larson et al. 2006).

Annotation next to each anchor marker indicate homoeologous

groups of barley (H), wheat (ABD), cereal rye (R), and in

parentheses oat chromosome designations

c

Mol Breeding (2007) 20:331–347 335

123

0

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E36M61.254E38M49.066E36M59.167P33M47.258P33M60.360P33M47.161E37M61.306

P33M62.346TRX1.2871R

E38M49.396E38M47.293

E41M60.123a

E41M49.217

E37M49.321

E41M49.072

E41M47.148E38M60.107aE38M59.101E41M59.163

HVA.0941HE37M47.196P42M61.218P33M62.354E41M61.398E38M60.108P35M61.177P33M47.234P44M62.368E37M47.327E41M49.134E38M60.311E38M60.294P33M47.273

% 4.9F

DA

% 2.7lA

%6.8K

%0.11F

DN

%1 .6P

C%0 .6

S

%6.6n

Z

LG1a_TTC1P33M47.258

E37M61.306

E38M49.066

P33M60.360P33M47.161E36M59.167

E38M49.396

E38M47.293

P33M62.346

E41M60.123aE41M49.217

E37M49.321

E41M49.072

E41M47.148

BCD1150 1B(A)E38M60.107aE41M59.163HVA.094 1HE38M59.101

BCD1562 1BD(A)P33M62.354P42M61.218E37M47.196E41M61.398P35M61.177

GDM126.146 1DE38M60.108P44M62.368P33M47.234E41M49.134E37M47.327E38M60.294E38M60.311P35M50.127

%1.6

FD

A

%8. 1

1a

C

%4.6

S

LG1a_TTC2E36M61.336P33M47.280bP33M50.162E38M47.185

P33M60.290

E41M49.278

E38M47.222a

E36M61.191E38M49.283b

WMC336.3881ADP35M62.350E36M62.140E37M60.103P33M61.367P33M61.198P42M61.117E36M61.323E41M48.245E38M60.213E36M62.346E41M61.385E37M47.319E41M47.355E37M60.097E37M61.095

E37M49.092

E36M61.267

P33M50.246b

E38M49.241

P44M49.133E36M62.192E41M47.078

P35M60.119

%3.8a

C

LG1b_TTC1P33M47.280bP33M50.162

E36M61.336

E41M49.278

P33M60.290

E38M47.222a

E36M61.191

E38M49.283bP35M62.350E37M60.097E36M62.140E37M60.103E37M47.319E38M60.213E36M62.346E36M61.323E41M61.385E41M48.245P33M61.198P33M61.367P42M61.117E41M47.355E37M61.095E37M49.092

E36M61.267

P33M50.246b

E38M49.241

P44M49.133E36M62.192E41M47.078

E38M49.063a

%4.7

FD

A)-

(%1

.6_ a

C

% 9.0

1K

%8.2

1g

M % 9.4

FD

N%7

.5i

S

LG1b_TTC2

Positive L. cinerus QTL allele (LOD > 3.3)

Negative L. cinerus QTL allele (LOD > 3.3)

Positive L. cinerus QTL allele (2.3 < LOD < 3.3)

Negative L. cinerus QTL allele (2.3 < LOD < 3.3)

0

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P33M62.285E41M48.255P33M48.139P33M61.175P33M61.312P35M49.202E36M48.172E38M60.263E37M61.155E38M47.364P44M62.223P44M49.246P33M59.256

E38M60.056

P35M62.365

P44M62.228XANTHA.2702H

E36M50.224P33M48.212E37M60.192E36M61.361P42M61.367E37M60.342E41M62.194E41M61.188E37M62.228E36M61.297E41M49.092P33M61.216

CNL045.1542A

E37M62.252

P35M50.279E37M49.102

E36M59.222

E41M61.099

E36M48.356

P33M62.231

E38M47.076a

P33M48.126

GWM382.0952ABDP33M62.291E36M49.366E41M48.112

E38M59.074

%0.41a

C% 3.8

uC

%6.8K

%2.11T

AR

K

%1.7P

C

%8 .5S

LG2a_TTC1P33M61.175E38M47.364E36M48.172E41M48.255E38M60.263E37M61.155P33M61.312P33M48.139P35M49.202P33M59.256P44M62.223P44M49.246E38M60.056P35M62.365

P44M62.228

E36M50.224P33M48.212E37M60.192E36M61.361E36M61.297E41M61.188E41M62.194E37M60.342E37M62.228E41M49.092P42M61.367P33M61.216

E37M62.252

P35M50.279E37M49.102

E36M59.222

E41M61.099

E36M48.356

E38M47.076aP33M62.231

P33M48.126P35M49.242GWM382.095 2ABDP33M62.291E36M49.366E41M48.112

E41M48.188

%8.4

FD

A% 8

. 8a

C

%9. 4

TA

RK

%4.5

nM

%1.4

FD

N

%0.5

PC

LG2a_TTC2E36M61.248

E38M60.153

E36M59.293E41M49.131

P35M62.194P33M50.152P35M50.208

XANTHA.3952H

P35M60.227

MWG763.2772H

E37M60.188P33M60.130E37M62.272E37M60.257P35M59.253P33M48.079P35M61.364P33M61.285E37M62.183E36M59.171E37M62.136P33M59.241E37M61.141E36M61.157E37M49.147

CNL045.1722AP33M60.180

P33M59.278

E41M61.119P33M59.172

P33M50.180a

P35M60.272

E37M49.165

E37M62.269

P42M61.081E38M59.220P35M61.243P33M60.217

GWM382.1262ABDE41M47.155E38M59.095E41M60.144E37M47.356E38M59.179P42M61.140E41M59.126aE38M47.278P33M48.067

%4.8n

M%3.8

S%6.6

nZ

LG2b_TTC1E41M49.131E36M61.248E36M59.293

P33M50.152P35M62.194

P35M50.208

E38M60.153

P35M60.227E37M60.257E36M61.157E37M62.272P33M59.241E37M60.188P33M61.285P33M60.130TRX1.287 1RE37M62.183P35M61.364P33M48.079P35M59.253E36M59.171E37M61.141E37M49.147P33M60.180P33M59.278

E41M61.119

P33M59.172

E37M62.269

P33M50.180a

P35M60.272

E37M49.165

P42M61.081E38M59.220P35M61.243P33M60.217E41M47.155E38M59.095E41M60.144E37M47.356E38M59.179E41M59.126a

GWM382.126 2ABDP33M48.067E38M47.278P42M61.140

%1.6

nM

% 2.8

1P

% 7. 8

PC

%0. 6

S

LG2b_TTC2

336 Mol Breeding (2007) 20:331–347

123

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

135

140

145

150

155

160

165

170

P33M50.250

E38M47.228

P35M62.385

E38M49.084

P33M62.144

E36M48.164P42M61.232P35M62.225SIP1.3243H

GWM005.0803AE41M49.122bE41M62.223E38M60.359E41M47.071E36M61.330E38M47.076bE41M47.225E41M48.133P33M50.328P33M61.149aE41M61.290P35M59.284

E41M48.102P33M47.097P33M60.192

E41M61.220P44M62.204

VP1i5.4063ABDE37M60.083

P44M62.360E37M47.225E36M59.219

P44M49.324

P35M61.089P35M62.147E36M48.308P44M62.093P33M61.129E37M47.224

E41M62.069

P44M49.139

P33M59.118b

%4.6

aC

%3 .4 1K

% 2.5T

AR

K%8. 9

PC%5.11

SLG3a_TTC1

E41M48.390

E38M47.228

P35M62.385

E38M49.084

BCD1532 3AB(C)P33M62.144SIP1.291 3HE41M62.223E36M48.164P42M61.232X

GWM005.080 3AP35M62.225E41M49.122bE38M60.359E41M47.071E36M61.330E41M47.225E38M47.076bP33M50.328P33M61.149aE41M61.290E41M48.133P35M59.284

P33M47.097

E41M48.102P33M60.192

P44M49.324

VP1i5.406 3ABDE41M61.220E37M60.083P44M62.204P44M62.360E37M47.225E36M59.219E38M47.185

P35M61.089P35M62.147

P33M61.129P44M62.093P44M49.139E37M47.224E41M62.069

P33M59.118bE41M61.107

%6. 6

gM

%8. 4

PC

%8.8

S

LG3a_TTC2P35M49.082

P33M62.393P33M47.179P35M50.120E41M49.135E41M47.167P42M61.197

SIP1.4133HE38M49.057P33M47.072E36M62.338E41M60.166P33M47.073E37M47.386P44M49.110E38M47.374E37M49.329P33M47.167P33M62.190E36M62.173E37M61.199E37M60.339E36M61.263P33M61.153E36M59.253P44M49.329E36M61.097E37M62.082E37M60.203E36M61.141E37M49.303E41M61.131E36M49.351E38M60.242P35M49.242E36M61.234E38M60.316E38M47.140

P33M60.207

E36M62.227

E37M62.162VP1i2.3943ABDE37M47.326

E41M62.204P42M62.249

E37M49.358

CDO14014B(F)

P33M60.393

%4.5T

AR

K

%3.5n

M

%9.5n

Z

LG3b_TTC1E36M59.261P33M47.179P33M62.393

CDO460 3ABR(C)P35M49.082E41M49.135E41M47.167P42M61.197P35M50.120

SIP1.413 3HE38M49.057E41M60.166E36M62.338P33M47.072P33M47.073P33M47.167E37M47.386P44M49.110E38M47.374E37M61.199E37M60.339P33M62.190E37M49.329P33M61.153E36M62.173E36M49.351E36M59.253E36M61.097E36M61.141E37M60.203P44M49.329E37M62.082E36M61.263E41M61.131E37M49.303E36M61.234E38M47.140E38M60.242E38M60.316

P33M60.207

E36M62.227E37M62.162E37M47.326

E41M62.204

P42M62.249

E37M49.358

E37M62.075

) -(

% 4. 2

1_a

C

%3.5

TA

RK

%8.5

TA

RK

)-(

%4.1

1_n

M

% 9. 6

FD

N) -

(%9

.4_

P

%6.4

PC

) -(

% 8. 6

_ nZ

LG3b_TTC2

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

135

140

145

E38M59.239P33M59.260P35M62.140E41M61.172E41M49.283E41M49.223P42M61.073E41M49.277

E38M60.090

P44M62.317

MIPS.2844HKSU154.1374B

E38M59.346P35M61.277E41M60.069

GWM165.2024ABD

E41M62.393E41M49.143E36M49.353E38M47.103E36M61.287E41M61.084bE41M60.196E38M60.259

HVCABG.152a4HE41M59.342E36M62.319

CNL039.2293AE36M50.376E41M61.109E36M62.249

P33M62.070

E36M62.211

TCP1Ns.4304HP35M62.127

E38M49.390E36M59.247E41M61.212

%7. 51lA

% 0. 0 1e

F

%0.01n

M

LG4Ns_TTC1E41M47.169

P33M59.260E38M59.239

E41M49.223E41M49.283E41M61.172P35M62.140P42M61.073E41M49.277

P44M62.317E38M60.090

BCD1117.196a 4HDE41M60.069E38M59.346P35M61.277GWM165.202 4ABD

E41M62.393

E41M49.143E41M61.109E38M60.259E36M61.287E38M47.103E36M62.319

HVCABG.152b 4HE41M61.084bE36M50.376E41M60.196E36M49.353P33M62.070E36M62.249E41M59.342

E36M62.211

P35M62.127

TCP1Ns.430 4HE38M49.390

E36M59.247

E41M61.272

% 3.5

FD

A% 1

.8u

C

%0.1

1e

F

LG4Ns_TTC2P33M62.351E36M50.064P33M59.134

GWM006.1704BHP33M48.399

MLOH1A.3524H

P35M60.392P35M50.371P35M50.372E41M62.082P33M48.359

HVM068.1994HBCD1117.211b4HD

E41M61.381E36M62.087E41M47.150

GWM165.1724ABDE41M47.301E41M62.246

BCD250(F)E36M61.332

PRC140.4094HP33M60.110E38M60.146E36M61.306E38M47.284E41M61.317E41M59.230E36M59.113E36M49.238

KSU149.2404DE38M49.291E37M47.108E36M48.118

TCP1Xm.4594HKNOX.2394HE38M59.100aE41M47.060E41M61.269E36M49.076P35M62.276P35M62.274

P33M61.096

%7.4F

DA

%6.1 1n

M%3.5

S

LG4Xm_TTC1E36M50.064P33M62.351P33M59.134

P33M48.399

P35M50.371P35M50.372P35M60.392E41M62.082P33M48.359E41M61.381E41M47.150BCD1117.211c 4HDE41M47.301E36M62.087E41M62.246E36M61.332E41M61.317P33M60.110E38M60.146E36M61.306E38M47.284E41M59.230E36M59.113E36M49.238PRC140.409 4HKSU149.243 4DE37M47.108E38M49.291E36M48.118

KNOX.239 4HE38M59.100aE41M47.060

TCP1Xm.459 4H

E41M61.269E36M49.076

P35M62.276

P35M62.274

)-(

% 7.7

_ aC

% 6. 9

TA

RK

)-(

% 3.0

1_n

M

)-(

%5.1

1 _P

LG4Xm_TTC2

Fig. 1 continued

Mol Breeding (2007) 20:331–347 337

123

0

5

10

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25

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55

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65

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75

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115

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150

155

160

165

P33M62.133

TCP2Ns.1175H

E37M62.172

E38M60.094E37M61.149E37M47.133E41M61.091bE36M48.180P33M60.366E36M61.383E36M62.311E38M59.268

KSU171.2795BP33M50.158E37M47.378E38M60.107bE41M47.281

E36M48.254

E36M62.189

E41M62.107E37M60.105E41M61.201E41M61.256E36M62.180

P33M59.264

MWG2230.2855H

E38M47.084E41M60.244E38M59.248

P33M62.172P35M60.115

E36M59.265E41M48.056bE37M47.244P42M62.112E37M60.235P33M60.077P35M49.344VRN2.1994H-5AP33M50.292

P33M62.208

%0 .6S

%5.31n

ZLG5Ns_TTC1

P33M62.175E37M62.172TCP2Ns.117 5HP33M62.133

E38M60.094

E37M61.149

E37M47.133E36M61.383E41M61.091bE36M48.180E37M62.136E37M47.378P33M60.366

KSU171.279 5BP33M50.158E38M59.268E36M62.311E38M60.107bE41M47.281E36M48.308E36M62.189E36M48.254E41M62.107E41M61.201E41M61.256E37M60.105

E36M62.180P33M59.264

CDO504Ns.086 5ABDHRE38M47.084

E41M60.244E38M59.248P33M62.172E36M59.265P35M60.115E41M48.056bP42M62.112E37M47.244P33M60.077E37M60.235P35M49.344P33M50.292

VRN2.193 4H-5AP44M49.296

LG5Ns_TTC2

0

5

10

15

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P35M62.193

P44M49.121P33M62.219E37M61.063

MWG652.2646H

GIRi5.3516B

P33M48.131

E41M47.350

E36M62.292E41M47.385E41M62.076

MWG2264.4036HE38M49.336E36M49.310P35M62.209P42M61.099E38M59.291E36M61.170E38M47.157

E41M61.259P33M60.252E41M60.363

E41M62.185

P44M62.103E38M49.278

P35M59.214P35M50.093P35M61.205

P35M60.286

%9. 51lC

% 5.4K

% 4.6P

LG6a_TTC1P35M62.193

P44M49.121

MWG652.264 6HP33M62.219

GIRi7.394 6BE37M61.063

E41M47.350

P33M48.131

E41M47.385

P35M62.209E38M49.336MWG2264.403 6HE36M49.310E41M62.076E36M62.292P42M61.099E38M59.291E36M61.170E38M47.157E41M61.259P33M60.252E41M60.363

E41M62.185

P44M62.103E38M49.278

P35M59.214P35M50.093P35M61.205

%7.7

FD

A

%5.4

aC

) -(

%3.0

1_n

M

) +(

%1.2

1 _F

DN

%6. 4

P

)-(

%0.3

1 _P

C

) -(

%7.7

_S

)-(

% 7.8

_iS

LG6a_TTC2P35M50.213b

P33M50.323

P33M48.087P35M61.198P44M62.219P33M61.168E41M61.134E37M47.093E36M62.114P44M62.200

P35M60.093

E36M48.329

P35M62.344

P33M47.206

P35M62.183

E36M61.255E41M60.098aE37M62.190E36M61.252E38M49.315E37M49.363TRX2.2871RP35M60.149E37M61.133E41M47.099

GDM150.3657DE41M60.232P33M50.258P42M62.284P35M50.236E41M61.192P44M62.284E37M47.366P35M59.243P35M49.381P35M49.378E41M59.252E37M62.268E36M62.219

E41M60.071E36M61.367

%5.4F

DA

%2. 5lC

% .01T

AR

K

LG6b_TTC1P35M50.213bP33M50.323

P33M48.087P35M61.198P44M62.219P33M61.168E37M47.093E41M61.134E36M62.114P44M62.200

P35M60.093

E36M48.329

P35M62.344

P33M47.206

P35M62.183

E41M60.098aE36M61.255E37M62.190P35M60.149E36M61.252E37M49.363TRX2.287 1RE38M49.315E37M61.133E41M47.099P42M62.284P33M50.258GDM150.365 7DE41M60.232P35M50.236E41M61.192P35M49.378E37M62.268P35M59.243P35M49.381E37M47.366P44M62.284E36M62.219E41M60.071

%8.3

aC

% 9. 4

1T

AR

K

%0.9

gM

LG6b_TTC2

GDM101.1965BE41M59.333

E41M48.244aE36M59.308P33M59.169E41M49.094E37M60.156E41M49.120

xE38M49.136E36M50.284E38M47.147P35M50.363E36M49.284E38M60.204E38M47.313E41M60.205

E37M60.172aE38M49.245

GDM068.1145ABDP33M61.250E38M49.191P44M49.085P33M50.244E38M59.139P33M48.298

PSR128.4125ABDHRE41M48.103

E38M59.199

E37M60.092

CDO504Xm.1675ABDHR

E36M50.333

E37M61.190

P35M59.137P44M62.088bE41M49.213P44M62.089P35M60.240E41M62.122P35M62.167P35M60.100P33M47.216E37M49.100

P33M48.153

% 4.7F

DA

%9.7lC

%8.8g

M

%8.5F

DN

%0.7P

C

LG5Xm_TTC1E41M59.333E41M48.244aE36M59.308E37M60.156E41M49.094E41M49.120

GWM205.142 5ADP33M59.169GDM101.197 5BTCP2Xm.252 5HE38M60.204E38M47.147E38M49.245E38M47.313E41M60.205E37M60.172a

BCD1130 5ABD(F)BCD1707 (F)E36M50.284E36M49.284P35M50.363P33M61.250E38M59.139P44M49.085E36M62.325P33M50.244E38M49.191

GDM068.116 5ABDP33M48.298

E41M48.103

E38M59.199

CDO504Xm.167 5ABDHR

E36M50.333E37M61.190

P35M59.137E41M49.213E41M62.122P44M62.088bP44M62.089P35M60.240P35M62.167P35M60.100P33M47.216E37M49.100

E36M48.158

%2.9

FD

A

% 9.7

uC

% 1.9

FD

N% 7

. 4P

%3.7

PC

)-(

%7.7

_ nZ

LG5Xm_TTC2

Fig. 1 continued

338 Mol Breeding (2007) 20:331–347

123

(Wu et al. 2003) we did not expect to detect variation

caused by heterozygosity in the T-tester, which may

explain why some traits showed significant genetic

variation with weak or insignificant QTL effects.

Conversely, two Fe content QTLs and one Na content

QTL detected in TTC2 should be viewed skeptically

since the corresponding heritabilities were not sig-

nificant (Table 2). It is possible that experimental

noise or measurement error in one rep may have

diminished heritabilities, whereas significant QTLs

were detected on the basis of significant gene effects

in the other rep. In any case, most of the QTLs were

supported by significant heritabilities (Table 2).

We detected 83 and 89 significant genotypic

correlations among all 153 pair-wise comparisons of

the 18 traits evaluated in the TTC1 and TTC2

families, respectively (Table 3). A total of 58 pair-

wise comparisons showed significant genotypic trait

correlation in both TTC1 and TTC2 families,

including 46 comparisons that had the same sign

(positive or negative) in both families. Thus, there

was considerable overall correspondence of genetic

variation between the TTC1 and TTC2 families,

which could be attributed to correspondence of

QTLs of correlated traits in one or both families

(Fig. 1).

We detected 23 and 33 primary QTLs

(LOD � 3.3) used as cofactors (Table 4) for the

final MQM scans of the TTC1 and TTC2 families,

respectively. The final MQM scans detected 28 and

32 secondary QTLs (2.3 � LOD � 3.3) in the TTC1

and TTC2 families, respectively. Overall, 51 and 65

QTLs were detected in the TTC1 and TTC2 families,

respectively (Fig. 1). Moreover, seemingly homolo-

gous TTC1 and TTC2 QTLs (i.e. QTLs that are

overlapping by comparison of homologous markers)

were evident on LG1a (ADF), LG1b (Ca), LG2b

(Mn), LG3a (S), LG3b (Mn and KRAT), LG4Xm

(Mn), LG5Xm (ADF and CP), LG6b (KRAT), LG7a

(KRAT, ADF, and NDF), and LG7b (KRAT)

(Fig. 1). Although another 88 QTLS were unique to

one family or the other, many of these were

0

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E36M49.124P44M62.094

6-SFT.1017HE41M48.215

MWG703.1947H

E41M62.212

P44M62.197b

E38M60.140

E41M47.162MWG741.2877H

BCD4217H(D)E36M62.225E37M62.323

MWG669.4926ABDHADP.3505ABD

E38M49.297E41M48.294E36M49.380E38M60.092E41M61.112E41M60.338E37M61.340

E41M60.378P44M49.129b

E36M50.202

P44M49.130

E36M62.325

E37M61.374P35M62.166P33M48.174P35M62.280P33M60.318P33M48.178E41M47.257E41M60.185

P33M61.074

%8.01F

DA

%1.6K

%2.6T

AR

K

%2.6g

M

%8.5F

DN

LG7a_TTC1P44M62.094

6-SFT.101 7H

E41M48.215

MWG703.194 7H

E41M62.212

P44M62.197b

E38M60.140

E41M47.162

MWG741.287 7HE38M49.136E36M62.225E37M62.323E38M49.297E38M60.092E41M48.294E41M61.112E37M61.340E36M49.380E41M60.338

MWG669.492 6ABDH

E41M60.378

P44M49.129b

P44M49.130E36M50.202

E37M60.092P35M62.166E37M61.374

E41M59.252

P33M60.318P33M48.174P35M62.280P33M48.178E41M47.257E41M60.185

P33M61.074

P35M61.143

%2.3

1F

DA

%4.6

eF

% 9.5

lC

%3.5

TA

RK

)+(

% 7. 7

_F

DN

%5.5

PC

%3.6

S

%5. 6

nZ

LG7a_TTC2E41M59.215

E41M61.084aE37M62.291

6-SFT.5797HE41M59.072P33M50.284E38M47.385P33M62.071E41M60.130

E37M61.193

E41M61.091aP35M60.247E38M59.304P35M59.116E37M62.180

E38M47.096

P33M62.325

E38M59.082E41M48.111

E37M60.144

P44M62.290

E41M59.203P35M49.347P35M61.311

WMC488.1057A

P33M48.122

E41M62.229P33M60.378

E36M50.325E36M62.172E36M48.313P35M61.381

E38M49.305

%8.6B

%1 .6T

AR

K

LG7b_TTC1E41M59.215

6-SFT.579 7H

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Fig. 1 continued

Mol Breeding (2007) 20:331–347 339

123

coincident (clustered) to specific regions of the TTC1

or TTC2 linkage maps (Fig. 1).

Relatively strong positive genotypic correlations

were observed between ADF and NDF concentra-

tions (Table 3) and coincident ADF and NDF QTLs

were detected on LG1a, LG1b, LG2a, LG5Xm,

LG6a, and LG7a (Fig. 1). This correlation is expected

and observed because ADF (cellulose and hemicel-

lulose) is a sub-fraction of NDF (cellulose, lignin, and

hemicellulose). However, unique ADF or NDF

content QTLs were detected on LG3b, LG4Ns,

LG4Xm, and LG6b (Fig. 1), which might be attrib-

utable to effects related to the ratio of hemicellulose

to cellulose and lignin since ADF essentially equals

NDF plus hemicelluose. Seemingly homologous

TTC1 and TTC2 ADF and/or NDF content QTLs

were detected on LG1a, LG5Xm, and LG7a (Fig. 1)

but the majority of ADF and NDF QTLs were unique

to one family. The coincidence of relatively large

ADF and NDF content QTL effects in the centromere

region of LG7a (Fig. 1), in both families, was

particularly interesting because NDF, in vivo dry-

matter digestibility, and a cluster of lignin biosyn-

thesis genes also co-localized in the centromeric

region of LG7 in perennial ryegrass (Cogan et al.

2005). Leymus LG7 is predicted be the conserved

syntenic counterpart to perennial ryegrass LG7,

which share the telomeric 6-SFT locus (Wei et al.

2000; Lidgett et al. 2002), and other syntenic cross-

species markers (Jones et al. 2002; Wu et al. 2003).

But additional cross-species reference markers

including genetic map assignment of the lignin

biosynthesis gene ortholoci in Leymus would cer-

tainly help investigate this putative correspondence.

Both ADF and NDF content also showed negative

genotypic correlations with CP, K, S, and Zn

concentrations in both families (Table 3), which can

also be attributed to coincident QTLs in one or both

families.

Relatively strong positive correlations were de-

tected between KRAT and K content, in both TTC1

and TTC2 families (Table 3). Conversely, relatively

Table 1 Trait means ± SD for Leymus cinereus Acc:636, L. triticoides Acc:641, L. triticoides T parental genotype, interspecific TC1

and TC2 parental hybrids, and full-sib L. triticoides · (L. triticoides · L. cinereus) TTC1 and TTC2 mapping families

Trait Acc:636

(g = 18)aAcc:641

(g = 15)aT-tester

(g = 13)bTC1

(r = 14)bTC2

(r = 14)bTTC1

(c = 164)cTTC2

(c = 168)c

CP (%) 19.9 ± 2.1 19.8 ± 1.7 20.7 ± 1.2 19.9 ± 1.0 20.8 ± 1.1 21.2 ± 1.4 21.8 ± 1.5

ADF (%) 25.4 ± 1.8 24.1 ± 1.2 24.7 ± 1.2 26.4 ± 1.1 25.8 ± 0.6 24.4 ± 1.2 24.1 ± 1.4

NDF (%) 51.3 ± 1.9 51.3 ± 1.2 50.2 ± 1.6 51.2 ± 1.3 51.7 ± 1.0 49.7 ± 1.5 50.0 ± 1.5

Al (ppm) 90 ± 24 113 ± 55 77 ± 18 81 ± 21 74 ± 12 101 ± 25 80 ± 17

B (ppm) 8.5 ± 3.6 7.7 ± 3.7 9.8 ± 2.0 9.6 ± 3.6 6.8 ± 2.4 8.5 ± 3.2 9.4 ± 2.6

Ca (%) 0.235 ± 0.040 0.332 ± 0.076 0.340 ± 0.044 0.298 ± 0.022 0.267 ± 0.035 0.354 ± 0.050 0.347 ± 0.043

Cl (%) 0.75 ± 0.17 0.49 ± 0.12 0.43 ± 0.07 0.72 ± 0.17 0.44 ± 0.05 0.70 ± 0.14 0.54 ± 0.10

Cu (ppm) 22.0 ± 2.4 15.2 ± 2.8 16.7 ± 3.1 18.1 ± 2.2 18.4 ± 3.2 18.1 ± 2.5 19.7 ± 3.2

Fe (ppm) 64 ± 15 92 ± 41 68 ± 10 62 ± 16 60 ± 7 84 ± 20 72 ± 13

K (%) 3.74 ± 0.38 3.14 ± 0.37 3.53 ± 0.11 3.49 ± 0.24 3.44 ± 0.13 3.72 ± 0.28 3.65 ± 0.24

Mg (%) 0.160 ± 0.014 0.181 ± 0.025 0.215 ± 0.020 0.206 ± 0.029 0.212 ± 0.017 0.192 ± 0.024 0.198 ± 0.022

Mn (ppm) 28.4 ± 5.6 36.5 ± 8.0 45.6 ± 2.7 51.7 ± 8.5 41.1 ± 4.7 40.5 ± 6.3 40.1 ± 6.8

Na (ppm) 475 ± 189 477 ± 171 495 ± 162 532 ± 188 379 ± 118 484 ± 126 460 ± 103

P (%) 0.250 ± 0.024 0.239 ± 0.030 0.262 ± 0.012 0.240 ± 0.012 0.259 ± 0.012 0.247 ± 0.023 0.259 ± 0.022

S (%) 0.139 ± 0.016 0.181 ± 0.028 0.202 ± 0.011 0.163 ± 0.009 0.171 ± 0.009 0.194 ± 0.021 0.186 ± 0.016

Si (%) 0.054 ± 0.017 0.055 ± 0.014 0.068 ± 0.015 0.057 ± 0.014 0.049 ± 0.010 0.063 ± 0.009 0.051 ± 0.008

Zn (%) 13.4 ± 2.5 18.1 ± 3.2 17.5 ± 1.6 14.1 ± 1.8 15.4 ± 1.6 16.8 ± 1.8 17.5 ± 2.7

KRAT 3.89 ± 0.38 2.63 ± 0.45 2.66 ± 0.18 2.84 ± 0.29 2.91 ± 0.23 2.92 ± 0.38 2.85 ± 0.30

a Sample size based on measurements number of different genotypes (g) without clonal replicationb Sample size based on number of different ramets (r) for each parental genotypec Sample size based on means of two ramets from a number of different clones (c)

340 Mol Breeding (2007) 20:331–347

123

strong negative correlations were detected between

KRAT and Ca content and between KRAT and Mg

content, in both TTC1 and TTC2 families (Table 3).

These correlations are not surprising since KRAT is a

simple ratio of K–Ca and Mg concentrations. Thus,

variation in K, Ca, and Mg content should also affect

KRAT. Otherwise, K, Ca, and Mg concentrations

were largely independent of each other and KRAT

did not show genotypic correlations with any other

traits. There were a total of six and seven KRAT

QTLs, five and one K content QTLs, three and seven

Ca content QTLs, and two and three Mg content

QTLs detected in the TTC1 and TTC2 families,

respectively (Table 2). As expected from differences

between parental species (Table 1), L. cinereus

contributed all but one (TTC2 LG2a) of the positive

KRAT QTLs (positive in the sense of elevated, but

undesirable effect) (Fig. 1). Seemingly homologous

TTC1 and TTC2 QTLs were detected for KRAT on

LG3b, LG6b, LG7a, and LG7b and for Ca content on

LG1b (Fig. 1). Coincident KRAT and Ca content

QTLs on LG2a may also show homology between the

TTC1 and TTC2 families, but they show different of

effect and somewhat misaligned locations compared

across these families (Fig. 1). A number of K, Ca, and

Mg content QTLs had no significant QTL effect on

KRAT, which suggest that these effects were counter

balanced by other components of KRAT. Counter

balancing effects on KRAT components were appar-

ently significant on LG1b where increased K content

effects associated with the L. cinereus allele were

also associate with increased Ca and Mg concentra-

tions (Fig. 1). Presumably, this also explains why the

homologous Ca content QTL on TTC1 LG1b did not

have significant effects on KRAT, even though no

significant Mg and K content QTLs were detected on

TTC1 LG1b (Fig. 1). Conversely, some KRAT QTLs

(TTC1 LG3b, TTC1 LG6b, TTC2 LG7a, TTC1

LG7b, and TTC2 LG7b) evidently have significant

effects on the ratio of K–Ca and Mg concentrations

but had no major (significant) affect on these mineral

concentrations per se (Fig. 1). Interestingly, homol-

ogous TTC1 and TTC2 KRAT QTLs on LG7a and

LG7b are all located near 6-SFT marker loci and

generally seem to involve the KRAT more than K,

Ca, and Mg content per se (Fig. 1). Thus, LG7a and

LG7b KRAT QTLs may be homoeologous. In any

case, compared to other traits, KRAT showed

substantially more evidence of homology between

the TTC1 and TTC2 families. Taken together, these

Table 2 Number of QTLs detected using a restricted multiple QTL model, % variation explained by QTLs, and broad-sense

heritabilities (H) in the full-sib Leymus triticoides · (L. triticoides · L. cinereus) TTC1 and TTC2 families

Trait Number of TTC1 QTLs

(variation explained)

TTC1

H2 ± SE

Number of TTC2 QTLs

(variation explained)

TTC2 H2 ± SE

CP 4 (29.3%) 0.390 ± 11 7 (41.4%) 0.61 ± 0.07

ADF 5 (39.6%) 0.59 ± 0.08 7 (45.0%) 0.67 ± 0.06

NDF 3 (21.6%) 0.57 ± 0.06 6 (44.4%) 0.52 ± 0.09

Al 2 (22.9%) 0.47 ± 0.11 0.(0.0%) 0.35 ± 0.09

B 1 (6.8%) 0.57 ± 0.07 0.(0.0%) 0.32 ± 0.08

Ca 3 (28.4%) 0.78 ± 0.04 7 (50.5%) 0.53 ± 0.09

Cl 3 (30.8%) 0.44 ± 0.09 2 (14.4%) 0.43 ± 0.09

Cu 1 (8.3%) 0.32 ± 0.06 2 (16.4%) 0.55 ± 0.05

Fe 1 (10.0%) 0.42 ± 0.08 2 (17.3%) 0.24 ± 0.16

K 5 (38.9%) 0.57 ± 0.08 1 (10.9%) 0.55 ± 0.08

Mg 2 (15.0%) 0.67 ± 0.06 3 (25.4%) 0.64 ± 0.06

Mn 4 (35.0%) 0.55 ± 0.09 5 (38.0%) 0.62 ± 0.06

Na 0 (0.0%) 0.59 ± 0.07 1 (10.0%) 0.06 ± 0.25

P 1 (6.4%) 0.50 ± 0.10 5 (37.3%) 0.65 ± 0.07

S 6 (40.6%) 0.76 ± 0.05 5 (27.9%) 0.62 ± 0.06

Si 0 (0.0%) 0.22 ± 0.08 2 (14.4%) 0.30 ± 0.08

Zn 4 (28.1%) 0.56 ± 0.08 3 (19.5%) 0.52 ± 0.09

KRAT 6 (36.5%) 0.76 ± 0.04 7 (40.8%) 0.43 ± 0.10

Mol Breeding (2007) 20:331–347 341

123

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342 Mol Breeding (2007) 20:331–347

123

Table 4 Description of primary QTLs (LOD � 3.3) used as cofactors for restricted multiple QTL model (rMQM) scans of the

L. triticoides · (L. triticoides · L. cinereus) TTC1 and TTC2 mapping families

Trait Family-linkage group Position LOD % Explained Additive effect

of L. cinerus QTL allele

ADF TTC1-LG1a 103 4.5 9.4 0.7

ADF TTC1-LG5Xm 75 3.6 7.4 �0.9

ADF TTC1-LG7a 77 5.1 10.8 0.8

ADF TTC2-LG1b 29 4.0 8.2 0.8

ADF TTC2-LG5Xm 112 4.8 9.2 �0.8

ADF TTC2-LG6a 113 4.0 7.7 0.8

ADF TTC2-LG7a 61 6.6 13.2 1.0

AL TTC1-LG4Ns 24 6.1 15.7 18

Ca TTC1-LG1b 65 3.6 8.3 �0.029

Ca TTC1-LG2a 72 5.9 14 �0.037

Ca TTC2-LG1a 92 6.8 11.8 �0.029

Ca TTC2-LG1b 54 3.7 6.1 �0.021

Ca TTC2-LG2a 140 5.2 8.8 0.027

Ca TTC2-LG3b 106 7.1 12.4 �0.030

Ca TTC2-LG4Xm 7 4.6 7.7 �0.024

Cl TTC1-LG5Xm 19 3.6 7.9 0.08

Cl TTC1-LG6a 119 6.9 15.9 0.11

Cl TTC2-LG7b 90 3.5 10.1 �0.08

CP TTC1-LG3a 77 3.7 9.8 0.9

CP TTC2-LG2b 66 4.0 8.7 1.0

CP TTC2-LG5Xm 112 3.4 7.3 0.8

CP TTC2-LG6a 115 5.5 13 �1.1

Cu TTC2-LG4Ns 128 3.4 8.1 1.5

Cu TTC2-LG5Xm 30 3.3 7.9 �1.5

Fe TTC1-LG4Ns 24 3.8 10 11

Fe TTC2-LG4Ns 80 4.2 11 -6

K TTC1-LG1a 37 4.1 8.6 0.16

K TTC1-LG2a 142 4.1 8.6 0.17

K TTC1-LG3a 75 6.5 14.3 0.21

K TTC2-LG1b 67 4.2 10.9 �0.15

KRAT TTC1-LG2a 104 4.7 11.2 0.25

KRAT TTC1-LG6b 59 4.6 10.9 0.26

KRAT TTC2-LG4Xm 6 4.3 9.6 0.19

KRAT TTC2-LG6b 73 6.1 14.9 0.23

Mg TTC1-LG5Xm 10 3.3 8.8 0.015

Mg TTC2-LG1b 34 5.0 12.8 �0.016

Mg TTC2-LG6b 89 3.5 9 �0.013

Mn TTC1-LG2b 88 4.0 8.4 3.7

Mn TTC1-LG4Ns 38 4.7 10 4.0

Mn TTC1-LG4Xm 104 5.4 11.6 4.3

Mn TTC2-LG3b 106 5.3 11.4 �4.4

Mn TTC2-LG4Xm 56 4.5 10.3 �4.1

Mn TTC2-LG6a 84 4.6 10.3 �4.1

Mol Breeding (2007) 20:331–347 343

123

KRAT, K, Ca, and Mg data reveal excellent oppor-

tunities to modify KRAT levels in Leymus although

some component QTLs may have counter-acting

effects.

A major macronutrient, plant K concentration

showed positive genotypic correlations with CP, Cl,

Cu, P, S, Si, and Zn contents in both families and

negative genotypic correlations with NDF and ADF

content in both families (Table 3). All pair-wise tests

of genotypic correlation among ADF, NDF, CP (an

effective measure of N), P, and K concentrations

were significant (positive) in both families (Table 3).

Likewise, coincident QTL were detected among

ADF, NDF, CP, P, and K concentrations in TTC1

and/or TTC2 families (Fig. 1), but no genomic

regions had significant effects on all of these traits.

Thus, plant concentrations of the three major ma-

cronutrients N, P, and K were intercorrelated traits

and negatively correlated with ADF and NDF content

in both families (Table 3). However, weak or mostly

non-significant correlations were detected among

plant concentrations of the Na, Ca, K, Mg, P, and S

micronutrients.

The coincidence QTLs controlling plant Al, Fe,

and Mn concentrations on TTC1 LG4Ns was intrigu-

ing because these QTL peaks evidently correspond

with the BCD117 locus mapped in the TTC2 family

(Fig. 1). The BCD1117 marker is closely associated

with a major Al tolerance gene of barley (Tang et al.

2000) and other divergent Poaceae (Magalhaes et al.

2004). The mechanism of this Al tolerance gene

appears to be related to citrate secretion by roots

(Zhao et al. 2003; Ma et al. 2004), which affects

mobility and uptake of soil cations including Al, Fe,

and Mn. There is an interesting disparity in our

comparison of Leymus, wheat, and barley Al toler-

ance loci in that, like BCD117, the HVCABG marker

is also closely linked to the Alt gene in barley

(Raman et al. 2002), but these markers are 37 cM

apart in Leymus (Fig. 1). In any case, the BCD1117

and HVCABG markers, linked to Al tolerance in

other Poaceae species, account for the only primary

QTLs (i.e. LOD � 3.3) controlling plant Al and Fe

content detected in the TTC1 and TTC2 families

(Table 4; Fig. 1).

Mineral, protein, and fiber content was largely

unaffected by QTLs controlling variation in plant

height, rhizome spreading, proportion of bolting

culms (i.e. some clones show little or no reproductive

bolting), or anthesis date (Larson et al. 2006). Major

plant height QTLs were detected on homologous

regions of LG2a in both Leymus TTC1 and TTC2

families (Larson et al. 2006), but this chromosome

region does not seem to have any consistent fiber,

protein, or mineral concentrations in either family

(Fig. 1). Rhizome spreading QTLs were detected on

homologous regions of TTC1 and TTC2 LG3a and

homologous regions of TTC1 and TTC1 LG3b

(Larson et al. 2006), which approximately correspond

with homologous S content QTLs on LG3a and

Table 4 continued

Trait Family-linkage group Position LOD % Explained Additive effect

of L. cinerus QTL allele

Na TTC2-LG7b 0 3.9 10 �52

NDF TTC1-LG1a 100 4.1 11 1.0

NDF TTC2-LG3b 59 3.8 6.9 0.8

NDF TTC2-LG5Xm 112 4.9 9.1 �0.9

NDF TTC2-LG6a 110 5.6 12.1 1.0

NDF TTC2-LG7a 61 4.2 7.7 0.8

P TTC2-LG2b 70 8.0 18.2 0.019

P TTC2-LG4Xm 85 4.8 11.5 �0.014

S TTC1-LG2b 48 3.5 8.3 �0.012

S TTC1-LG3a 98 4.7 11.5 0.014

S TTC2-LG3a 121 3.4 8.8 0.009

Si TTC2-LG6a 113 3.3 8.7 �0.003

Zn TTC1-LG5Ns 154 4.4 13.5 �1.3

344 Mol Breeding (2007) 20:331–347

123

homologous Mn content QTLs on LG3b (Fig. 1).

Likewise, other rhizome spreading QTLs on TTC1

LG6a and TTC2 LG5Xm were coincident with

relatively minor mineral content QTLs (Fig. 1), but

otherwise had no major affect on mineral, protein, or

fiber content. Relatively major QTLs for anthesis date

and proportion of bolting culms were detected on

corresponding regions of the TTC2 LG4Ns linkage

group (Larson et al. 2006), which also coincide with

the Cu content QTL on LG4Ns (Fig. 1). Relatively

strong bolting and anthesis date QTLs were detected

on homologous regions of the TTC1 and TTC2

LG4Xm linkage group (Larson et al. 2006), but this

region did not show any consistent affect on fiber,

protein, or mineral concentration in both families.

Relatively minor bolting QTLs were detected in

homologous regions of TTC1 and TTC2 LG6a, but

again we did not see corresponding homologies of

mineral or fiber content QTLs in both families.

Basically, most of the fiber and mineral QTLs were

independent of plant height, growth habit, and

flowering QTLs detected in the same field evaluations

(Larson et al. 2006) and may relate to fundamentally

important regulatory steps and processes of mineral

uptake, mineral transport, and fiber synthesis. Fun-

damental components of forage quality that are

excellent applications for gene discovery research in

grasses. However, the Leymus TTC1 and TTC2

families also segregate stem diameter, leaf width,

leaf length, leaf angle, and leaf texture and other traits

that may affect forage quality. Thus, additional

forage quality and agronomic evaluations are needed

to fully ascertain the best strategies for improvement.

Nevertheless, results so far indicate that it should be

possible to select for reduced KRAT, improved

forage quality, and other desirable yield and agro-

nomic characteristics in heterotic breeding popula-

tions derived from interspecific hybrids of L. cinereus

and L. triticoides. Leymus wildryes with improved

grazing tolerance, high biomass production, and

reduced KRAT would be of great value as a low-

input feedstock for the Great Basin and Intermountain

regions of western North America and similar

ecoregions of the World.

Acknowledgements This work was supported by joint

contributions of the USDA Agriculture Research Service,

Utah Agriculture Experiment Station, Department of Defense

Strategic Environmental Research and Development Program

CS1103 project, and US Army BT25-EC-B09 project (Genetic

Characterization of Native Plants in Cold Regions). Trade

names are included for the benefit of the reader, and imply no

endorsement or preferential treatment of the products listed by

the USDA.

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