© 2008 Science From Israel / LPPLtd., Jerusalem

IsraelJournalofPlantSciences Vol.55 2007 pp.267–276

*Author to whom correspondence should be addressed.E-mail: ceoloni@unitus.it

Pyramiding different alien chromosome segments in durum wheat: Feasibility and breeding potential

AndreA GennAro,a PAolA Forte,a robertA CArozzA,a MAriA l. SAvo SArdAro,a dAnielA Ferri,a AleSSAndrA bitti,a GrAziA M. borrelli,b MAriA G. d’eGidio,c And CArlA Ceolonia,*

aDepartmentofAgrobiologyandAgrochemistry,UniversityofTuscia,Viterbo01100,ItalybC.R.A.—CerealResearchCentre,Foggia71100,Italy

cC.R.A.—ResearchUnitforCerealQuality,Rome00191,Italy

(Received7August2007;acceptedinrevisedform7February2008)

ABSTRACT

Wheat chromosome engineering, i.e., the transfer of alien chromosome segments from various Triticeae species into cultivated wheats, is greatly benefiting from the recent advancements in molecular genetics, cytogenetics, and genomics. Powerful tools are currently available that make selection of desired genotypes far more pre-cise and effective than in the past, thus giving this transfer strategy considerable po-tential for meaningful practical achievements. Such tools were successfully applied to engineer the durum wheat genome with small alien segments containing genes for disease resistance and quality traits, including Lr19 (leaf rust resistance) and Yp (yellow endosperm pigmentation) from Thinopyrumponticum, Pm13 (powdery mildew resistance) from Aegilops longissima, and the Glu-D3 and Glu-D1 glutenin subunit genes from Triticumaestivum. Targeted segments with such genes, all being of minimal size, were first separately incorporated into durum wheat by ph1-induced homoeologous recombination. The positive performance of selected recombinant lines from each transfer project encouraged attempts to develop multiple alien seg-ment combinations. The results obtained with double and also triple recombinants indicate a good tolerance of the durum wheat genome even toward such complex manipulations, which thus appear to offer good prospects for simultaneously enrich-ing durum wheat germplasm with several valuable traits from related Triticeae.

Keywords: chromosome engineering, multiple alien transfers, disease resistance, quality, Triticum spp.

INTRODUCTION

Manipulation of multiple genes to achieve a multifac-eted improvement has always been the ultimate goal of any breeding strategy, from conventional intraspecific crossing activities, aimed at combining the best features of divergent parental varieties, to gene stacking via transformation technology. The former approach, par-ticularly for species such as the Triticum spp., finds an inherent impediment for further and diversified goals in the currently restricted genetic variation of the cultivat-ed gene pool, with many potentially useful genes “left

behind” in the course of domestication and of intensive selection during the last century’s “Green Revolutions”. On the other hand, progress toward genetically modified crops with numerous novel genes can only be expected once the bottleneck of developing technologies for the coordinated manipulation of multiple genes or traits is removed (Halpin, 2005).

Lying somewhere between traditional breeding and genetic engineering, chromosome engineering allows

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the transfer, in principle genome-wide, of genes from the wild or anyway crossable gene pool of the crop rela-tives via meiotic recombination between corresponding chromosomes of the recipient and donor species.

Crop species of paramount importance to mankind, such as the polyploid wheats, not only benefit from a wealth of natural genetic resources of alien Triticeae, but also a unique richness of cytogenetic materials that have greatly facilitated knowledge of the wheat and related Triticeae genomes and hence their controlled manipulation via chromosome engineering (Feldman and Sears, 1981). It is mainly thanks to the mind and work of E.R. Sears that such materials were conceived and developed, and that the main procedures for wheat chromosome engineering, i.e., the “transferring of seg-ments of alien chromosomes carrying particular desired genes to wheat chromosomes” (Sears, 1972), were originally delineated. In this framework, a refinement to the “toolkit” of wheat chromosome engineers came from isolation of mutants for the Ph1 gene, both in com-mon and in durum (reviewed in Ceoloni et al., 2005b; Qi et al., 2007). By removal of normally occurring suppression of recombination between wheat and ho-moeologous Triticeae chromosomes, largely due to the action of the wheat Ph1 gene, the way has been opened to transfers also from distantly related gene pools. These, although never as frequently obtained as when homologous exchanges are involved, result in largely balanced products in which the size of the alien seg-ment, and consequent linkage drag, can be conveniently minimized (e.g., Ceoloni et al., 2005a).

Until recently, these propitious conditions have been somewhat in contrast with the relatively modest success of chromosome engineering in making alien genetic variation effectively available to wheat breed-ers. Luckily, concomitantly with the increased need for enlarging the crop genetic basis that is pervading the breeding perspectives of this millennium (see, e.g., McCouch, 2004), the genomics revolution is offering unrivalled opportunities for understanding structure and function and hence for targeted manipulation of even complex plant genomes. No doubt it is greatly impacting all breeding strategies, including chromo-some engineering. In fact, molecular marker technol-ogy, as well as molecular cytogenetic techniques, such non-radioactive in situ hybridization (e.g., FISH and GISH), effectively complementing classical methods of analysis, are making detection and characterization of wheat–alien introgression products much more efficient and accurate than ever (see, e.g., Ceoloni and Jauhar, 2006, for a review). Such a “smart breeding” strategy is thus expected to accelerate the development of breeder-friendly germplasm, even in the form of more ambitious

products to be delivered, such as those where different valuable alien traits are pyramided. Here we will il-lustrate how this goal has been successfully pursued in durum wheat.

CHROMOSOME ENGINEERING AT THE DURUM WHEAT LEVEL: AN OVERVIEW

OF THE PERFORMANCE OF SINGLE ALIEN TRANSFERS

Durum wheat (Triticum turgidum subsp. durum, 2n = 4x = 28, AABB) is a species of traditional economic importance for Italy and other world areas with similar environmental conditions. We have exerted a major effort in its chromosome manipulation with the aim of stably introducing novel attributes enhancing the crop’s response to relevant diseases, as well as its qualitative performance. Our early attempts (Ceoloni et al., 1996) clearly confirmed the lower buffering ability of the T.durum genome as compared to that of T. aestivum (2n = 6x = 42, AABBDD) toward genic and chromo-somal alterations (reviewed in Ceoloni et al., 2005b), and hence the greater difficulties inherently associated with its manipulation (Ceoloni and Jauhar, 2006). None-theless, by taking advantage of currently available diag-nostic and selection tools, we succeeded in fulfilling an essential requisite for stability and consequent practical usefulness of introgression products, particularly those involving durum wheat, i.e., to obtain lines that incorpo-rate minimal amounts of alien chromatin. Materials of this kind could be precisely characterized and selected by molecular linkage maps (Donini et al., 1995; Cenci et al., 1999), meiotic pairing analyses (Donini et al., 1995; Carozza et al., 2003), and FISH/GISH-based physical maps (e.g., Biagetti et al., 1999; Carozza et al., 2003; Ceoloni et al., 2005a; see also Fig. 1) of the wheat–alien chromosomes resulting from each specific transfer pro-cess. Such integrated cytogenetic maps allowed marker and gene assignment to wheat and alien target chromo-some regions of defined length and position, as well as precise line ranking.

This could be achieved for each of three different transfer projects, through which the Pm13 gene from the diploid Aegilops longissima, the Lr19+Yp genes from the decaploid Thinopyrum ponticum, and the Glu-D1 or Gli-D1/Glu-D3 genes from T.aestivum were separately introduced into durum wheat. In all cases, lines could be isolated in which the size of the target alien segment did not exceed 23% of the engineered arm (Table 1). Even at pre-breeding stages such transfers appeared to be genetically well compensated, as indeed expected since they were obtained by resorting to the manipu-lation of homoeologous recombination. In particular,

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free seasons (Gennaro et al., 2003, and unpublished). Yield stability over years and different environments, accompanied by the high carotenoid pigment content determined by the alien Yp gene (Table 1; Gennaro et al., 2003), and by good values of other relevant grain and semolina traits (Gennaro et al., 2003, and unpublished), led to the recent decision to submit one RC line for reg-istration to the Italian National Variety List.

Similarly advanced breeding work has not been performed for recombinant lines OR, PS, and PL (see Table 1), whose novel traits, however, may have consider-able breeding value. On the one hand, the Pm13 gene has been able so far to confer a complete resistance to pow-dery mildew pathotypes spread in Italy, as well as in other European countries (Chelkowski, pers. comm.) and in China (Zhou et al., 2005). On the other hand, introduction of the high- (HMW) and low-molecular-weight (LMW) glutenin subunit genes from common wheat chromosome 1D (Glu-D1d in line PL and Glu-D3 in line PS, Table 1), known to have a major impact on bread-making proper-ties, in place of their durum wheat 1A orthologs, signifi-cantly affects gluten properties and indicates the actual possibility to widen the spectrum of potential end uses of the recipient species (Ceoloni et al., 2000).

The observations reported above encouraged at-tempts aimed at simultaneously enriching a durum wheat genotype with different valuable traits of alien or-igin, and these have been undertaken adopting a strategy of “segment pyramiding”. In fact, while a few examples of stable stacking of multiple alien translocations with breeding potential exist in common wheat (e.g., Singh et al., 1998), no case history is available to our knowledge in durum wheat.

transfer of the Pm13 gene was first obtained at the 6´ level (Ceoloni et al., 1992) by use of the ph1b Chinese Spring mutant (Sears, 1977) and then moved into a 4´ background by homologous recombination (Ceoloni et al., 1996), while for the other transfers illustrated in Table 1 (Ceoloni et al., 1996, 2005a; Vitellozzi et al., 1997), wheat–alien chromosome recombination was induced exploiting ph1c durum wheat mutants (Giorgi, 1983; Ceoloni et al., 2005b).

The selected recombinant chromosomes (Table 1), named RC (Lr19+Yp), OR (Pm13), PS (Gli-D1/Glu-D3), and PL (Glu-D1d), were proved to be normally transmitted through both germlines (Ceoloni et al., 1996, 2005a; Vitellozzi et al., 1997), and not to impair relevant agronomic parameters of the recipient lines, as shown by evaluations of F3 progeny of the different recombinant lines following 2–3 backcrosses to adapted varieties (Ceoloni et al., 2000).

Also in later generations, particularly remarkable was the overall performance of one of the durum wheat–Th.ponticum recombinant lines, named R5-2-10, pos-sessing both the Lr19 and Yp genes in a 7AgL segment distally located on 7AL and spanning 23% of the arm (Ceoloni et al., 2005a). Starting from BC2F4, derivatives from the cross of R5-2-10 with the leaf-rust-susceptible Italian variety Simeto (named RC lines, Table 1) were subjected for several years to multi-location field tests. These provided consistent evidence of the efficacy of the Lr19-dependent leaf-rust-resistant response, as well as a remarkably positive performance of RC lines. They in fact yielded significantly more than varietal controls following heavy leaf-rust epidemics, but also confirmed their intrinsically good yield potential in virtually rust-

Table 1Characteristics of selected durum wheat recombinant lines carrying alien chromosome segments transferred from

wild and cultivated relatives

Rec. Recombinant Donor Alien segment Target genes Effectsline chromosome species (% arm)

RC Thinopyrum 23 Lr19+Yp Res. leaf rust + ponticum > carotenoid pigment content

OR Aegilops 20 Pm13 Res. powdery mildew longissima

PS Triticumaestivum 17 Gli-D1/ Glu-D3 Enhanced gluten qualityPL Triticumaestivum 16 Glu-D1d

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A STEP FORWARD IN MANIPULATING THE DURUM WHEAT GENOME: ALIEN

CHROMOSOME SEGMENT PYRAMIDING

In order to combine in the same durum wheat geno-type different alien chromosome segments with genes affecting different relevant traits (i.e., a chromosome engineering-based approach of gene pyramiding), suc-cessful attempts were carried out that resulted in the production of stable lines with two (Fig. 1) and even three wheat–alien recombinant chromosome pairs. This was achieved by inter-crossing homozygous plants for the recombinant chromosomes described in Table 1, i.e., RC, OR, PS, and PL. For genotyping of F2s and sub-sequent populations obtained from the double hetero-zygotes, marker systems with progressively improved efficiency have been set up in the course of the work.

For RC selection, a dominant STS marker, which had turned out to be associated with Th. ponticum Lr19-containing 7AgL segments introgressed into com-mon wheat lines (Prins et al., 2001), was initially used, followed by a GISH-based screening to discriminate

homozygous or heterozygous carriers (Micali et al., 2003). Subsequently, enrichment of the 7AL and 7AgL distal regions with a variety of markers (Gennaro et al., in preparation) further facilitated RC selection in segre-gating progeny, particularly through the development of a PCR-based, codominant assay for Lr19 as a result of NBS profiling of durum wheat recombinant lines carry-ing Lr19 (Gennaro et al., 2006).

Presence/absence of the selected 3BS/3SlS recombi-nant chromosome harboring the Pm13 gene (OR) was established by means of a dominantly inherited, Ae.longissima-specific STS marker derived from a RAPD amplicon (Cenci et al., 1999). Homozygous vs. hetero-zygous condition was in this case ascertained by assess-ing one of the wheat SSR loci located in the exchanged 3BS segment (Ceoloni, unpublished).

As to the 1D transfers, segregation of theGlu-3 1AS and 1DS alleles (PS line) was monitored by means of a primer pair, designed on Glu-3 (LMW) glutenin gene sequences, which amplifies two 1DS-specific and two 1A-specific fragments (D’Ovidio et al., 1997). For screening of materials segregating for the PL recom-

Fig. 1. Pre-annealing GISH, i.e., GISH (Anamthawat-Jónsson and Reader, 1995) using differently labeled total DNA probes of Ae.tauschii (D-genome; FITC, green fluorescence) and durum wheat (AB genomes; Cy3, orange-red fluorescence) onto somatic metaphase chromosomes of a RC+/PS+ double recombinant line, allowed detection of the terminal 1DS segment on the 1A chromosome pair (arrowed), as well as, due to partial homology between D- and Th.ponticum genomic DNAs, of the terminal 7AgL segment on 7A chromosomes (arrowed).

Gennaroetal./Pyramidingalienchromosomesegmentsinwheat

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binant, two primer pairs, one specific for the 1D ´ 5 component at the Glu-D1-1d locus and the other specific for the Glu-A1-2a (=1Ay) allele, were initially employed (Micali et al., 2003). More recently, development of detailed molecular maps of the PS and PL recombinants (Carozza et al., 2003; Ceoloni et al., in preparation), and, as a result of this, use of the WMC93 codominant SSR marker (Somers et al., 2004), which turned out to be associated with the exchanged 1AL vs. 1DL seg-ments containing Glu-1 loci, allowed their discrimina-tion through one PCR assay only.

Marker-driven analysis of F2 progeny of double heterozygous combinations (in OR/PS/RC combination the RC chromosome was homozygous) indicated simul-taneous transmission of the two recombinant chromo-somes to be normal through both germlines in all cases (Micali et al., 2003, and data not shown).

EVALUATION OF AGRONOMIC AND GRAIN QUALITY CHARACTERISTICS OF MULTIPLE

RECOMBINANT LINES

Agronomic traits

To test the effect of the multiple combinations on parameters of agronomic relevance, small-scale field tests were carried out for two years (2003–2004 and 2004–2005) in the same locality (Viterbo, central Italy) on F3 and F4 progeny of genotypes homozygous for ei-ther one or for all (2 or 3) recombinant chromosomes, or for their absence (Table 2). A mild leaf-rust attack was observed in both years; thus, a possible selective advan-tage for RC carriers (Lr19 gene) could be considered negligible. Characters including number of grains/ear (G/E), number of grains/spikelet (G/S), and 1000 grain weight (TGW) were evaluated, and data of the various genotypes within each combination were compared by ANOVA (Table 2). For all combinations, though with some between-years differences, no major difference was observed for ear fertility parameters, except for a significant increase of G/E shown in the second year by RC homozygotes compared to the other genotypes of the RC/OR F4 progeny. Also noticeable, albeit not sta-tistically significant, are the higher G/E and G/S values of OR/PS/RC triple homozygotes, which were consis-tently detected over the two generations and were not at the expense of their TGW (Table 2). Since both grain size and grain number are important yield-contributing traits, this evidence gives good hopes for considerable yield potential of such a complex genotype, as indeed initial yield trials seem to highlight (see ahead and Table 3).

Also for TGW most inter-genotype comparisons re-

sulted in nonsignificant differences. However, remark-ably higher values were associated with the presence of the RC chromosome, either alone or in combination with PS, in F3 progeny of the RC+PS combination, though among F4 genotypes only minor differences were ob-served. On the other hand, significantly lower TGW was detected in RC+/PL+ F3 plants and in their RC–/PL+ sibs, but the difference became nonsignificant when F4 genotypes of the RC/PL combination were compared. Reduced TGW was also observed in 1AS.1AL-1DL translocation lines carrying the same Glu-D1d allele (HMW glutenin subunits 5 + 10) as present in our PL line (Klindworth et al., 2005), although contained in a longer 1DL segment (Carozza et al., 2003; Ceoloni et al., 2005a; Xu et al., 2005). Major and minor QTLs for grain yield and yield components, including TGW, are widely distributed around the wheat genome, but no major QTL specifically related to TGW appears to be associated with distal 1AL or 1DL chromosome regions (Quarrie et al., 2005). Nonetheless, also hypothesizing that a weak genetic factor was involved in the different 1AL-1DL exchanges, compensation for the depressing effect could be exerted by positive alleles at other TGW-related QTLs present in variable genetic backgrounds, resulting in inconsistent and, probably, environmentally dependent results.

This possibility is in line with variable TGW values shown by F5 progeny of RC/PL lines subjected to yield trials in two rather different Italian environments (Ta-ble 3). In one of them (Po Valley = PV), the RC+/PL+ plots showed a lower TGW compared to control variet-ies, while the RC–/PL– materials had a higher value; on the other hand, the reverse was observed in the Adriatic Coast (AC) location, where the RC+/PL+ genotype per-formed better than the commercial varieties, whereas the opposite was true for the RC–/PL– genotype. As to the RC/PS combination, in both AC and PV locations RC+/PS+ progeny had higher TGW than control variet-ies, while the RC–/PS– sib progeny outperformed the control varieties in one location (PV) but had a lower value in the other (AC).

As evidently offset by other yield-contributing fac-tors, the observed variations in TGW did not seem to have a direct correspondence with yield performances (Table 3). In this regard, it has to be underlined that in the 2005–2006 season in both PV and AC environments a mild leaf-rust attack occurred; therefore, the inher-ent yield potential of contrasting genotypes (sib lines carrying or lacking the RC chromosome pair with the Lr19gene) could be assessed and compared to similarly rust-free (but otherwise susceptible) control varieties. Overall, very good yield performance was exhibited by both RC+/PS+ and RC+/PL+ recombinant genotypes,

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Tabl

e 2

Mea

ns a

nd A

NO

VA p

val

ues o

f yie

ld-r

elat

ed tr

aits

of d

iffer

ent g

enot

ypes

sele

cted

from

var

ious

reco

mbi

nant

com

bina

tions

. Dat

a de

rive

from

5–1

0 sp

ikes

/row

(trip

licat

e, ra

ndom

-iz

ed, 1

.2-m

-long

row

s/ge

noty

pe) o

f F3 (

uppe

r lin

e) a

nd F

4 (lo

wer

line

) pla

nts (

2–5

prog

eny/

geno

type

)R

ecom

b.

Gra

ins/

spik

elet

G

rain

s/ea

r 10

00 g

rain

wei

ght

com

bina

tion1

Gen

otyp

es2

Gen

otyp

es2

Gen

otyp

es2

+/

+ +/

– –/

+ –/

– p

+/+

+/–

–/+

–/–

p +/

+ +/

– –/

+ –/

– p

RC

/ PS

3.

7 3.

7 3.

9 3.

8 0.

17

63.6

64

.2

64.5

68

.4

0.14

56

.4

57.8

52

.9

53.5

0.

03

3.5

3.4

3.5

3.4

0.57

68

.9

65.1

66

.1

66.3

0.

24

45.7

46

.9

45.1

45

.5

0.82

RC

/ PL

3.

5 3.

7 3.

8 3.

7 0.

07

62.0

69

.3

70.1

67

.2

0.36

41

.0

53.9

41

.6

51.6

0.

01

3.5

3.6

3.6

3.7

0.15

66

.3

69.1

66

.4

70.8

0.

19

42.0

44

.8

45.3

46

.2

0.06

RC

/ O

R

2.5

2.9

2.7

2.9

0.13

41

.4

49.2

47

.7

49.5

0.

48

52.0

48

.1

48.6

46

.5

0.25

3.

4 3.

7 3.

4 3.

4 0.

06

65.4

73

.5

65.4

68

.4

0.03

43

.6

44.2

46

.1

43.7

0.

51O

R /

PS /

RC

3 3.

8 —

3.

5 3.

6 0.

33

77.0

-

70.2

71

.9

0.08

42

.8

—

43.8

43

.1

0.90

3.

6 —

3.

5 3.

3 0.

17

73.0

-

72.1

67

.5

0.14

42

.7

—

41.6

41

.7

0.41

1 See

Tab

le 1

for a

bbre

viat

ions

and

des

crip

tion

of re

com

bina

nt c

hrom

osom

es. 2 “

+” a

nd “

–” in

dica

te h

omoz

ygos

ity fo

r pre

senc

e an

d ab

senc

e, re

spec

tivel

y, o

f the

firs

t and

seco

nd

reco

mbi

nant

chr

omos

ome

as in

the

sequ

ence

indi

cate

d in

the

first

col

umn.

3 The

RC

chr

omos

ome

is a

lway

s hom

ozyg

ous i

n th

is tr

iple

com

bina

tion.

Tabl

e 3

Prel

imin

ary

field

eva

luat

ions

of F

5 dur

um w

heat

mul

tiple

reco

mbi

nant

line

s and

thei

r sib

s (2

prog

eny

per

geno

type

) com

pare

d to

ada

pted

var

ietie

s use

d as

con

trols

(2–3

per

loca

lity;

con

trol m

eans

= 1

00) i

n di

f-fe

rent

Ital

ian

envi

ronm

ents

(AC

= A

dria

tic S

ea C

oast

, cen

tral-e

ast;

PV=

Po

Valle

y, c

entra

l-nor

th; T

C=

Ty

rrhe

nian

Sea

Coa

st, c

entra

l-wes

t)1,2

Line

Te

st w

eigh

t 10

00 g

rain

wei

ght

Yie

ld

AC

PV

TC

AC

PV

AC

PV

TCR

C+

/ PS+

10

1 99

—

10

9 10

5 98

10

3 —

RC

– / P

S–

102

97

—

95

110

104

104

—R

C+

/ PL+

10

3 99

—

10

3 87

* 10

3 10

0 —

RC

– / P

L–

101

95

—

95

109*

91

10

3 —

OR

+ / P

S+ /

RC

+ —

—

11

1*

—

—

—

—

160*

*O

R–

/ PS–

/ R

C+

—

—

110*

—

—

—

—

17

2**

1 Lea

f-ru

st a

ttack

was

ver

y m

ild i

n AC

and

PV

loca

litie

s (2

005–

2006

sea

son)

and

ver

y se

vere

in

TC

(200

6–20

07 se

ason

). 2

repl

icat

es o

f 7.2

m2 pl

ots i

n AC

and

PV;

3 re

plic

ates

of 4

.2 m

2 plo

ts in

TC

.2 *, **

Indi

cate

sig

nific

ant d

iffer

ence

s vs

. con

trol m

eans

at p

= 0

.05

and

0.01

, res

pect

ivel

y, a

ccor

ding

to

Stud

ent’s

t-te

st. A

ll ot

her d

iffer

ence

s wer

e no

nsig

nific

ant.

Gennaroetal./Pyramidingalienchromosomesegmentsinwheat

273

which in both areas had a seed production close to or even better than the controls. On the other side, presence of the Lr19 gene determined a tremendous increase in yield (average 66%) of OR/PS/RC recombinant geno-types over the three susceptible control varieties in a field test carried out in 2006–2007 near the Tyrrhenian Sea Coast (TC, Table 3). This is an area where durum wheat is widely and traditionally cultivated, but where the crop is consistently and severely damaged by the leaf-rust disease. Thus, materials of this kind may well provide an effective alternative to conventional, largely susceptible varieties, currently representing the majority of the cultivated durum wheat germplasm.

Quality characteristics

The overall good prospects that the multiple recombi-nant lines we developed seem to hold in terms of ag-ronomic performance are further substantiated by their quality attributes. As previously shown for the separate recombinants (Ceoloni et al., 2000; Gennaro et al., 2003), in double and triple combinations carrying the alien genes Yp (RC chromosome, Table 1), Glu-D1, and Glu-D3 (PL and PS chromosome, respectively), such genes determined considerable increases of indexes used to measure quality characteristics that, besides protein content, are of major interest for durum wheat improvement, i.e., gluten quality and color.

Gluten quality, as expressed by SDS-sedimentation and Gluten Index, was appreciably affected by the pres-ence of 1D-encoded proteins in RC+/PL+, RC+/PS+, and OR+/PS+/RC+ lines (Table 4). Compared to the controls, increments in SDS values were around 30% in double recombinants with either PL or PS lines chromo-some pairs, and about 15% in the triple combination.

Differences in Gluten Index were smaller (around 15% for PS- and PL-containing double recombinants and very minor in the triple one). As expected, quality parameters were not genotype-dependent in the RC/OR combina-tion. Quantitative variations in the effect of 1D-encoded proteins even among recombinants carrying the same 1D transfer (i.e., RC+/PS+ and OR+/PS+/RC+) may be due to residual differences in their background seed storage protein composition. In fact, the two to three backcrosses to the same durum wheat cultivar ‘Simeto’, performed with all separate recombinants prior to their use as parents of multiple combinations, are not expected to homogenize their background genotypes, although they seem to possess the same major protein determinants of good pasta quality, including an LMW-2 type of pattern and the Glu-B1-encoded 7+8 HMW glutenin subunits, as the recurrent variety (not shown). Preliminary results from alveograph measurements on F4 progeny of multiple recombinants with either PS or PL segments indicate that semolina rheological proper-ties are considerably but differently influenced by Gli-D1/Glu-D3- and Glu-D1d-encoded proteins, the former mainly determining a substantial increment in dough strength, the latter having a stronger increasing effect on tenacity.

Semolina of the same recombinant combinations was also used to assess color parameters (Table 4). As a whole, the observed values confirmed earlier obser-vations on RC-type recombinant lines carrying the Yp gene from Th.ponticum (Gennaro et al., 2003). In terms of Yellow Index, RC+ F3 plants exhibited a 10–16% increment with respect to RC–. Spectrophotometric determination of total carotenoid pigments (Borrelli et al., 1999) and evaluation of their lutein and b-carotene

Table 4Quality parameters of durum wheat multiple recombinant lines (mean values of 2–4 F3 progeny plants per genotype)

Genotype1 SDS Gluten Yellow Carotenoid pigments LOX (mm) index index Total Lutein b-carotene (EU/g) (μg/g) (μg/g) (μg/g)RC+ / PS+ 47 98 23.4 9.3 4.4 0.10 1.9RC– / PS– 36 84 21.3 7.8 2.7 0.08 2.3RC+ / PL+ 55 100 24.3 11.0 5.9 0.10 2.3RC– / PL– 43 87 21.6 8.5 4.0 0.08 2.1RC+ / OR+ 38 72 24.1 9.9 4.5 0.15 1.2RC– / OR– 37 90 20.8 6.8 2.6 0.09 1.3OR+ / PS+ / RC+ 55 99 24.4 9.3 4.4 0.14 1.1OR– / PS– / RC+ 48 96 23.6 8.9 4.2 0.13 1.91See Table 1 for abbreviations and description of recombinant chromosomes. “+” and “–” indicate homozygosity for presence or absence, respectively, of the given recombinant chromosome.2LOX = lipoxygenase activity.

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fractions by high-performance liquid chromatography (Adom et al., 2003) showed more clear-cut differences. In particular, total pigment content was about 20%, 30%, and over 40% higher in RC+/PS+, RC+/PL+, and RC+/OR+, respectively, than in their sib lines lacking the alien Yp gene. As expected, the main contribution to these increments was due to lutein, the major com-ponent of total pigments in the wheat grain (Adom et al., 2003): lutein was almost 50% more abundant in RC+/PL+ lines as compared to RC–/PL– controls, and the increases were over 60% and 70%, respectively, in the case of RC/PS and RC/OR combinations. Accord-ing to expectation, comparison between the OR/PS/RC triple recombinant genotypes, all being homozygous for the presence of the RC chromosome, revealed no ap-preciable difference in total pigment content, nor in that of lutein and b-carotene (Table 4). On the other hand, amounts of the latter compound were considerably high-er (+66%) in RC+/OR+ plants than in RC–/OR– con-trols, while in both RC+/PS+ and RC+/PL+ genotypes a 25% increment over the corresponding –/– genotypes of the same progeny was detected. In semolina extracted from the various recombinant combinations, lipoxygen-ase (LOX) activity, the main determinant of oxidative degradation of pigments during processing, was also assessed. In line with the known independent location of wheat LOX genes (Hart and Langston, 1977) and of major QTLs affecting endosperm color pigments (e.g., Parker et al., 1998), no genotype-dependent relation be-tween LOX activity and pigment content was detected (Table 4). Indeed, among RC+ genotypes, a number were found to express relatively low LOX activity, and these are expected to cause a minimum reduction of color in pasta products (Borrelli et al., 1999).

CONCLUDING REMARKS

In the breeding perspective of this millennium, we are assisting the fortunate transition of what was until re-cently just a largely shared conviction, i.e., that of the need for widening the crop genetic basis as a key to meaningful breeding advancements, to its translation into the actual possibility to develop effectively im-proved varieties, capable of meeting an array of constant as well as newly arisen socioeconomic and environmen-tal challenges.

Our results demonstrate how this definitely applies to the “smart” or “precision” chromosome engineer-ing-based breeding approach, even when the target is pyramiding multiple alien segments, and the crop species, as in the durum wheat case, has a genome relatively “sensitive” to extensive manipulations. We have in fact succeeded in stably assembling into durum

wheat chromosomal segments from up to three related genomes, two from the wild gene pool (Ae. longis-sima and Th.ponticum) and one from common wheat, contributing to the simultaneous enhancement of traits that can positively affect environment safety and pro-duction incomes and costs, but also the technological and nutritional quality of the end product. The multiple recombinant lines have so far quite brilliantly passed the most critical test for a breeding application, i.e., yield performance, as seems to be particularly the case for RC (Lr19+Yp genes)-containing lines. Moreover, their quality attributes represent an important added value. Actually, the feasible combination of high carotenoid pigment content and low LOX activity mentioned above can enhance the aesthetic appeal and also the health benefits of food products derived from RC genotypes (Adom et al., 2003). Also, a possible expansion of the durum wheat market can be envisaged if a “dual-purpose” gluten can be realized, and good prospects seem to be particularly associated with lines bearing the PS (Gli-D1/Glu-D3 genes) recombinant chromosome (Ceoloni et al., 2000, and in preparation).

While larger-scale field and quality tests are currently underway to better evaluate the overall potential of the materials described here, they are being employed in marker-assisted breeding to simultaneously introduce the novel traits into an array of adapted varieties. This will allow the development of novel germplasm in which the effects of the segment introgressions can be conveniently optimized, in view of suiting dif-ferent environments and market appreciation. In the meantime, additional alien segment combinations are being developed, in which, for example, various alien resistance genes toward the same or different pathogens are brought together, or where both PS and PL transfers are introduced into the same 1A chromosome. The lat-ter “reshuffling”, whether practically useful or not, will allow one to delineate a more complete picture of the effects of 1D gene products on durum wheat gluten properties.

Although, as earlier recalled, the case history is very limited for wheat (for other crop species see, e.g., Mc-Couch, 2004, and references therein), it is likely that the successful chromosome segment pyramiding described here will be reproducible with a variety of alien trans-fers, and it will certainly be more amenable if addressed to the hexaploid genome of common wheat.

However, the recent accomplishments with either single or multiple introgressions already provide ample evidence of the actual possibility of a finely-tuned and breeding-responsive makeup of the wheat genome through a “smart” chromosome engineering approach (see, e.g., Ceoloni et al., 2005a; Ceoloni and Jauhar,

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2006; Qi et al., 2007, and references therein). This should be considered in the present and future perspec-tives of invigorating cultivars with “new blood”, for which a policy should be judiciously followed that contemplates the resort, at proper times, to all efficiently working strategies, refraining from roughly-made eval-uations (Jacobsen and Schouten, 2007), but just aspiring to effectively achievable benefits.

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