Euphytica (2006)

DOI: 10.1007/s10681-005-9077-0 C© Springer 2006

Bringing wild relatives back into the family: recovering genetic diversity inCIMMYT improved wheat germplasm

M.L. Warburton1,∗, J. Crossa1, J. Franco2, M. Kazi1, R. Trethowan1, S. Rajaram3, W. Pfeiffer1,P. Zhang1, S. Dreisigacker1 & M. van Ginkel1

1The International Maize and Wheat Improvement Center (CIMMYT, Int.). Apdo Postal 6-641 06600 Mexico D.F.,Mexico; 2Facultad de Agronomia, Universidad de la Republica, Ave. Garzon 780, CP 12900, Montevideo, Uruguay;3International Center for Agricultural Research in the Dry Areas (ICARDA) P.O. Box 5466, Aleppo, Syrian ArabRepublic (∗author for correspondence: e-mail: mwarburton@cgiar.org)

Received 1 June 2005; accepted 14 December 2005

Key words: microsatellite, simple sequence repeat (SSR), wheat, genetic diversity, genetic uniformity, synthetichexaploid wheat

Summary

The dangers of a narrow genetic base of the world’s major domesticated food crops have become a great globalconcern in recent decades. The efforts of the International Maize and Wheat Improvement Center (CIMMYT) tobreed common wheat cultivars for resource poor farmers in the developing world (known as the Green Revolutionwheats) has met with notable success in terms of improved yield, yield stability, increased disease resistanceand utilization efficiency of agricultural inputs. However, much of the success was bought at the cost of an overallreduction in genetic diversity in the species; average Modified Roger’s distances (MRD) within groups of germplasmfell from 0.64 in the landraces to a low of 0.58 in the improved lines in the 1980s. Recent efforts by CIMMYTbreeders to expand the genetic base of common wheat has included the use of landraces, materials from otherbreeding programs, and synthetic wheats derived from wild species in the pedigrees of new advanced materials.The result, measured using SSR molecular markers, is a highly significant increase in the latent genetic diversityof recently developed CIMMYT breeding lines and cultivars compared to the original Green Revolution wheats(average MRD of the latest materials (0.63) is not significantly different from that of the landraces, as tested usingconfidence intervals). At the same time, yield and resistance to biotic and abiotic stresses, and end-use qualitycontinue to increase, indicating that the Green Revolution continues to this day.

Abbreviations: CIMMYT, International Maize and Wheat Improvement Center; CML, CIMMYT maize inbred line;MRD, modified Roger’s distance; SH, Shannon’s diversity index; SHW, synthetic hexaploid wheat; SSR, simplesequence repeat

Introduction

The dangers of a narrow genetic base of the world’smajor domesticated food crops have been well doc-umented and dramatically evidenced by potato lateblight in the Great Irish Famine in 1845–47, south-ern corn leaf blight in 1970, and other lesser knownepidemics throughout history. Genetic erosion causedby diminished use of traditional farmers’ cultivars

(landraces) has been a cause for concern since it wasformally recognized by Harlan and Frankel in the1970s (and suggested by Vavilov many decades earlier(Vavilov, 1997). Apprehension about genetic erosionand a narrowing genetic base is well warranted. Whenmany farmers grow genetically related cultivars on avery large scale, they are deploying genotypes thatcarry similar genetic mechanisms of resistance to vari-ous plant diseases. Should a pathogen or pest overcome

this genetic resistance, the entire crop becomes vul-nerable to the disease or insect, eventually reachingepidemic levels and endangering food supplies (Smaleet al., 2002).

Domestication has caused bottlenecks in many cropspecies (Tanksley et al., 1997; Reif et al., 2005). Inmany cereals, the bottleneck was minimized by theexistence of large and diverse populations. However,in polyploids such as common wheat (Triticum aes-tivum L.), the bottleneck was exacerbated because theinterspecific crosses that formed the foundation ofhexaploid common wheat probably occurred only afew times. Therefore only a very small number of in-dividual plants within each of the three grass specieswere involved. When the polyploid plants underwentselection and improvement by early farmers, new andpotentially diverse plants viewed as undesirable wouldlikely have been rejected and thus would have beenexcluded from the gene pool of the new species (Cox,1998; Ladizinsky, 1984).

Early modern plant breeding may also have inad-vertently reduced diversity (Nat. Res. Council, 1972;Harlan, 1972) as improved cultivars emanating fromadvancing scientific knowledge replaced the landracesin great swaths across the world. Such was the casewith the introduction of the Green Revolution wheatsdeveloped in the 1950s and 1960s by the InternationalMaize and Wheat Improvement Center (CIMMYT)and subsequently adopted by many national wheatimprovement programs. CIMMYT-derived wheats aregrown widely in the developing world (Smale et al.,2002) and have also contributed significantly to thewheat industries of developed countries such asAustralia (Brennan & Fox, 1998) and the USA (vanBeuningen & Busch, 1997). Private sector and devel-oped country contributions to wheat breeding havebeen minimal in the developing world (Evenson &Gollin, 2003). Therefore, the impact of CIMMYTwheats on global wheat diversity in recent decades hasbeen very large, particularly in the developing world.If CIMMYT wheats are genetically uniform, the vul-nerability of global wheat production to a devastat-ing new disease or insect pest outbreak is high. In-creased genetic diversity provides a buffer against suchrisks.

By its very nature, breeding cultivars for release tofarmers derived from a cross between complementaryparents goes hand-in-hand with a drastic reduction indiversity, particularly in the early segregating breedinggenerations. The final selection of a few advanced linesfor release to the farmers reflects a process of reduction

in diversity that is not negative in terms of final utility.Individuals that are susceptible to diseases and abioticstresses are discarded, as are those too tall or too short,too early or too late, or with low yield and quality.

Furthermore, many recent plant breeding effortsshow a relative increase in diversity in many crops,including common wheat and durum wheat (Almanza-Pinzon et al., 2003; Christiansen et al., 2002; Smaleet al., 2002; Maccaferri et al., 2003; van Beuningen& Busch, 1997). At the same time, yield gains havebeen advancing at an increasing rate for many cropsbred by the CGIAR centres, including wheat (Evenson& Gollin, 2003). Widening the genetic base may beaccomplished by using materials from other breed-ing programs, landraces, mutation stocks, transloca-tion lines, and exotic species (Zohary et al., 1969).This includes the use of synthetic wheats, which aremade by repeating the interspecific crosses that orig-inally created common wheat. A synthetic wheat in-volves a cross between a tetraploid (genome AABB)such as durum wheat (T. turgidum) and Ae. tauschii(DD).The hybrid usually requires embryo rescue andartificial chromosome doubling. Synthetic wheats haveprovided new diversity from both the tetraploid and thediploid parental species for many specific traits (Coxet al., 1998 for review) and this diversity is evidentat the DNA sequence level as well (Lage et al., 2003;Zhang et al., 2005). In some cases, unknown suppres-sion and epistatic networks may complicate the inher-itance of traits in intraspecific crosses. What is clearis that levels of resistance/tolerance are noted in syn-thetic wheats and synthetic derivatives that have neverbeen observed before in common wheat. These newsynthetic derived wheat cultivars are just now becom-ing available to farmers via release through nationalprograms.

The objectives of this study are: (1) to measure themolecular diversity in a representative sample of themajor wheat cultivars grown in the developing worldover time; (2) to compare this diversity to that of thelandraces they replaced; and (3) to measure the effectsof new synthetic derived wheat germplasm on the di-versity levels of CIMMYT wheats.

Materials and methods

Definition and Selection of germplasm

Three types of wheat germplasm were compared(a full list of germplasm and descriptions of the

entries is provided in Table 1):

1. Landraces: those wheat stocks that were grown byfarmers in the past (commercially or for subsistence)and are not products of the application of post-Mendelian genetic breeding schemes. Most likelythese landraces were derived from farmer selectionsof seed from superior individuals for planting inthe next cycle. Little if any information is gener-ally known on the origin or domestication processof such landraces. The sample of landraces includedin this study is a random sample chosen from thou-sands that were grown in the past and of which alarge number are maintained in the CIMMYT genebank.

2. Modern wheats: these wheat cultivars reflect theapplication of post-Mendelian genetic breedingschemes. Some may have resulted from line selec-tion within diverse populations, but most will havebeen the result of intentional crossing of agronom-ically different individuals. The first such wheatsappeared in the late 19th Century. By the secondhalf of the 20th Century they had replaced thelandraces in 90% of the world’s wheat growingareas.

3. Synthetic derivatives: These are CIMMYT lines de-veloped by crossing common hexaploid wheats atleast once to a synthetic (SHW). Many of the newestCIMMYT-derived breeding lines currently underevaluation by the national breeding programs ofmany developing countries for release as new culti-vars are synthetic derivatives.

Germplasm chosen for the study was selected torepresent different Year Groups (1–7, Table 1). EachYear Group is composed of approximately the samenumber of entries in order to balance the sample sizes,so differences in levels of genetic diversity wouldbe due solely to genetic composition. Year Group 1included landrace cultivars that were grown by farmersin the developing world before the advent of the GreenRevolution and introduction of other improved wheatcultivars. A larger study of landraces was conducted byDreisigacker et al. (2005); 25 landraces from that studywere chosen at random to represent genetic diversitycompared to other year groups in this study. The levelsof diversity as measured by gene diversity within thelarger sample of landraces (which included 45 landraceaccessions) were not statistically different from thatof the 25 landraces included in this study (data notshown).

Year Group 2 included the first Green Revolutionwheats developed by CIMMYT, plus some other majorwheat cultivars of non-CIMMYT origin grown aroundthe world. Year Groups 3–6 are wheat cultivars cho-sen on the basis of their popularity among develop-ing world farmers or importance as major progenitorsof other cultivars. Each of these Year Groups spans 7or 8 years, except for Year Group 2, which spans 16years, because fewer improved cultivars were releasedin the 1950s. Year Group 7 contains CIMMYT breed-ing lines currently in international trials; they are thebest lines currently available and it is felt by the au-thors that these lines will be representative of the nextwave of cultivars to be released to developing worldfarmers. Unfortunately, the lag phase between devel-opment of breeding lines and the release of cultivarsstemming from those breeding lines makes it impossi-ble to be completely sure that the lines in Year Group7 will be the same as those that will become cultivars.However, the lines in Year Group 7 (and therefore thediversity levels in the group) are sure to be representa-tive of the cultivars grown by farmers if they continueto sample CIMMYT germplasm in a fashion similarto that seen in the past four decades. By 2003, amongthe lines in international trials targeted towards irri-gated and dryland conditions, 25% contained syntheticwheats in their pedigree. Therefore 25% of the lines inYear Group 7 were also chosen from synthetic derivedbackcross lines.

Marker assays

Genomic DNA was extracted from bulked leavesharvested from 7–10 young plants according toSaghai-Maroof et al. (1984) and modified accordingto CIMMYT protocols (http://www.cimmyt.cgiar.org/ABC/Protocols/manualABC.html). Quality and quan-tity of the extracted DNA was determined on 1%agarose gels by visually comparing extracted DNAbands to a known concentration of a standard lambdaDNA cut with HindIII. Forty SSR markers coveringall chromosomes with between 1–4 markers per chro-mosome (except 3A and 6D) were used in the study.The majority of the markers were EST derived mark-ers in order to try to characterize diversity changeswithin actual genes (Table 2). SSR information was ob-tained from IPK (Gatersleben, “wms” series primers)and DuPont (Wilmington, “dp” series primers).

PCR reactions for SSR analysis were performedaccording to Dreisigacker et al. (2005). Briefly, reac-tions containing a final volume of 20 ul were amplified

Table 1. Genotypes used in the study, including the name and source of the genotype and the identification system used at CIMMYT (Cross

ID plus Selection ID (CID and SID). The year of release is indicated; landrace varieties (which do not have a year of release) are indicated

with the abbreviation LR

Genotype name CID SID Year of release Germplasm source∗ Country of origin

Year Group 1 (Landraces)

84TK520.001.01 123050 0 LR No C-d Turkey

84TK523.006.02 123052 0 LR No C-d Turkey

84TK538.002.02 204323 0 LR No C-d Turkey

86PK1271 200902 0 LR No C-d Pakistan

ABYSSINIA 1 298326 0 LR No C-d Ethiopia

AK BUGDAY 223 0 LR No C-d Turkey

AK702 244693 0 LR No C-d Turkey

ALKANA 166420 0 LR No C-d Chad

BARBELA 0248 290316 0 LR No C-d Spain

BARBON 196959 0 LR No C-d Mexico

COLOGGNE ABASTARDO 11660 209702 0 LR No C-d Spain

CRILLO GTM NATIONAL V 378118 0 LR No C-d Guatemala

DIKWA 1 224511 1 LR No C-d Nigeria

GENTIL BIANCO 192274 0 LR No C-d Italy

KHARKOVSKAYA 2 139237 0 LR No C-d Russia

KUBANKA 2894 1 LR No C-d Russia

PILLON 196933 0 LR No C-d Mexico

PISSI KHAWRI 211035 0 LR No C-d India

QUARITITO 197005 0 LR No C-d Mexico

SHOREWAKI 152405 0 LR No C-d Pakistan

TCHERE 166419 0 LR No C-d Chad

TRIGO BLANCO 88348 0 LR No C-d Chile

V2084-13 50518 1 LR No C-d Israel

YAYLA305 5801 1 LR No C-d Turkey

YILMAZ1 77277 0 LR No C-d Turkey

Year Group 2 (1950–1966)

YAQUI 50 6455 2 1950 C Mexico

ANDES 55 221376 0 1956 C Columbia

NARINO 59 6239 5 1959 C Columbia

OROFEN 60 217176 0 1960 C Chile

GABO 60 3469 12 1960 C Mexico

NAINARI 60 3469 13 1960 C Mexico

PITIC 62 6674 7 1962 C Mexico

PENJAMO 62 6665 4 1962 C Turkey

KNOX 62 2815 1 1962 No C-d USA/Canada

NADADORES M 63 6879 5 1963 C Mexico

CRESPO 63 1318 1 1963 C Columbia

MIRONOVSKAYA 808 87691 0 1963 No C-d USSR

SONORA 64 6681 5 1964 C Mexico

TRIUMPH 64 5370 0 1964 No C-d USA

LERMA ROJO 64 6846 0 1964 C Mexico

MEXIPAK 65 72426 0 1965 C Pakistan

C306/SUJATA 709 1 1965 No C-d India

KIRAC 66 2702 1 1966 No C-d Turkey

AGENT 5933 1 1966 No C-d USA

INIA F 66 7124 5 1966 C Mexico

Year Group 3 (1967–1974)

SONALIKA 6977 3 1967 C Mexico/India

BJ67 6348 13 1967 Unk. India

SCOUT 66 4685 1 1967 No C-d USA

SAFED LERMA 6911 2 1967 C India

KALYANSONA 6831 1 1967 C Mexico/India

CALIDAD 6548 10 1968 C Argentina

Table 1. Continued

Genotype name CID SID Year of release Germplasm source∗ Country of origin

UP301 7124 14 1968 C India

RED RIVER 68 6348 8 1968 C USA

YAMHILL 5774 1 1969 No C-d USA

MEXIPAK-69 6831 83 1969 C Pakistan

POTAM S70 7259 5 1970 C Mexico

BEZOSTAJA 516 0 1970 No C-d USSR

HIRA 122580 0 1970 C-d India

BLUEBIRD 15 6980 221 1970 C Pakistan

SAVA 4667 1 1970 No C-d Yugoslavia

TANORI F 71 7310 11 1971 C Mexico

MARCOS JUAROS INTA 6861 39 1971 C Argentina

BLUEBOY II 592 1 1971 No C-d Mexico

ARZ 7117 3 1973 C Libya

ANZA 6733 19 1973 C USA

ARVAND 6016 1 1973 C-d Iran

JUPATECO F 73 7538 4 1973 C Mexico

CUMHURIYET 75 6761 1 1974 C Turkey

LIMPOPO 3033 1 1974 C-d Zimbabwe

MAYA 74 6789 27 1974 C Guatemala

Year Group 4 (1975–1982)

SAMWHIT 5 6831 0 1975 C Nigeria

YECORA ROJO 6980 32 1975 C USA

SALAMANCA 75 7050 13 1975 C Mexico

SAKHA 8 6980 36 1976 C Egypt

LIESBECK 7178 1 1976 C South Africa

VEERY 7691 619 1977 C Mexico

CHIVITO 50784 0 1977 C Guatemala

HERMOSILLO M 77 7558 5 1977 C Mexico

UP 262 5435 1 1978 C Nepal

ABU GHRAIB#3 6861 11 1978 C Iraq

GEREK 79 1897 0 1979 No C-d Turkey

HD2189 2256 3 1979 C-d India

ITAPUA 25 7132 4 1979 C Paraguay

BAHAWALPUR 79 415 −1 1979 C Pakistan

ZAMINDAR 80 7797 7 1980 C Pakistan

SAKHA 69 4608 8 1980 C Egypt

OSLO 3848 1 1980 C-d USA

KENYA KONGONI 2648 1 1981 C-d Kenya

ATA 81 6976 6 1981 C-d Turkey

LOK 1 139180 0 1981 C-d India

PAKISTAN 81 7691 268 1981 C Pakistan

CORDILLERA 3 7691 293 1982 C Paraguay

KENYA NYANGUMI 139164 0 1982 C-d Kenya

MILLALEAU INIA 7691 295 1982 C Chile

Year Group 5 (1983–1989)

KANCHAN 2565 3 1983 No C-d Bangladesh

HARTOG 7624 17 1983 C Australia

DASHEN 7691 299 1984 C Ethiopia

HUW 234 84629 2 1984 No C-d India

VASKAR 7401 9 1984 C Nepal

HYBRID DELHI 2327 23141 0 1985 No C-d India

PIRSABAK 85 7691 90 1985 C Pakistan

DEBEIRA 1382 0 1985 C-d Sudan

ITAPUA 30 7794 5 1985 C Paraguay

HD2285 0 0 1985 No C-d India

GONEN 1974 1 1986 C Turkey

Continued

Table 1. Continued

Genotype name CID SID Year of release Germplasm source∗ Country of origin

BR 18 6960 51 1986 C Brazil

SITTICH 7908 7 1986 C Mexico

CHAM 4 7831 4 1986 C Syria

BUCK PONCHO 61920 1 1987 No C-d Argentina

GRANERO INTA 7506 46 1987 C Argentina

MAQUI INIA 50956 3 1987 C-d Chile

KENYA KWALE 7541 7 1987 C Kenya

IAN 8 PIRAPO 7414 243 1987 C Paraguay

ACHTAR 7819 0 1988 C Morocco

PUNJAB 88 8407 0 1988 C Pakistan

RAYON F 89 8195 5 1989 C Mexico

PROINTA FEDERAL 7414 205 1989 C Argentina

BUCK CHARRUA 88223 −1 1989 No C-d Argentina

PROINTA OASIS 22974 7 1989 C Argentina

Year Group 6 (1990–1997)

NESSER 24762 7 1990 C Jordan

ESTANZUELA PELON 90 124424 6 1990 C Uruguay

ICA YACUANQUER 42165 36 1991 C Columbia

TINAMOU I1 7919 191 1991 C Mexico

INQALAB 91 113388 5 1991 C-d Pakistan

CHAM6 24762 30 1991 C Libya

TIA.1 9292 17 1992 C Mexico

ARIVECHI M 92 19652 474 1992 C Mexico

ITAPUA 40-OBLIGADO 8050 89 1992 C Paraguay

KLEIN DRAGON 8050 73 1992 C Argentina

PARWAZ-94 113390 3 1993 C-d Pakistan

SARIAB-92 7795 23 1993 C Pakistan

UP 2338 162515 1 1994 C-d India

LUMAI 1 140658 0 1994 No C-d China

LONGMAI 8 139842 0 1994 No C-d China

BORLAUG M 9 8109 206 1995 C Mexico

PBW343 8890 1549 1995 C India

BAW898 20570 301 1996 C Bangladesh

TOBARITO M 14143 179 1997 C Mexico

INIFAP M 97 49434 556 1997 C Mexico

SAN CAYETAN 7896 358 1997 C Mexico

Year Group 7 (Breeding lines, 2002–2003)

ALUCAN/DUCULA 43142 1942 2002 C CIMMYT

PRINIA/WEAVER//STAR/3/WEAVER 101518 87 2002 C CIMMYT

RABE/2∗MO88 91288 76 2002 C CIMMYT∗∗CNDO/R143//ENTE/MEXI 2/3/AEGILOPS

SQUARROSA (TAUS)/4/WEAVER/5/2∗KAUZ

135029 258 2002 C CIMMYT

∗∗CHEN/AEGILOPS SQUARROSA 135045 91 2002 C CIMMYT

(TAUS)//FCT/3/2∗WEAVER

TNMU/TUI 43217 778 2002 C CIMMYT

CMH80A.542/CNO79 25640 6 2002 C CIMMYT

IAS58/4/KAL/BB//CJ71/3/ALD/5/CNR/6/THB/CEP7780 48520 366 2002 C CIMMYT

TNMU/MILAN 82306 245 2002 C CIMMYT

PASTOR//MUNIA/ALTAR 84 133503 145 2002 C CIMMYT

MUNIA/ALTAR 84//AMSEL 133891 539 2002 C CIMMYT

TNMU/MUNIA 134257 218 2002 C CIMMYT

PFAU/WEAVER 58955 271 2002 C CIMMYT

SW89-5124∗2/FASAN 74933 107 2002 C CIMMYT∗∗CROC 1/AE.SQUARROSA (205)//2∗BCN 91513 432 2002 C CIMMYT

BR14∗2/SUM3//TNMU 82341 614 2002 C CIMMYT

PFAU/MILAN 92628 356 2002 C CIMMYT

TAM200/TUI 68815 259 2002 C CIMMYT

Table 1. Continued

Genotype name CID SID Year of release Germplasm source∗ Country of origin

∗∗SABUF/7/ALTAR 84/AE.SQUARROSA

(224)//YACO/6/CROC 1/AE.SQUARROSA

(205)/5/BR12∗3/4/IAS55∗4/CI14123/3/IAS55∗4/EG,

AUS//IAS55∗4/ALD 167180 453 2002 C CIMMYT∗∗CROC 1/AE.SQUARROSA (224)//OPATA 72726 530 2002 C CIMMYT

LAJ3302/3/GZ156/NAC//PSN/URES/4/WEAVER 101209 116 2002 C CIMMYT

KA/NAC 8198 10 2002 C CIMMYT∗∗ALTAR 84/AE.SQ//2∗OPATA 58561 34 2002 C CIMMYT

WEEBILL1 260140 84 2002 C CIMMYT

KAUZ//ALTAR 84/AOS/3/KAUZ 53995 0 2002 C CIMMYT∗∗SERI//ALTAR 84/AE.SQ (219) 91509 988 2003 C CIMMYT∗∗BORL94∗2//ALTAR 84/AE.SQ (221) 153713 100 2003 C CIMMYT∗∗BCN//CETA/AE.SQ (895) 215902 105 2003 C CIMMYT∗∗BCN//68.111/RGB-U//WARD/3/AE.SQ (325) 265032 8 2003 C CIMMYT∗∗BCN∗2//CHEN/AE.SQ (429) 53353 130 2003 C CIMMYT∗∗BCN//DVERD 2/AE.SQ (214) 91520 716 2003 C CIMMYT∗∗OPATA//SORA/AE.SQ (323) 215943 51 2003 C CIMMYT∗∗YACO∗2//ALTAR 84/AE.SQ (191) 154297 0 2003 C CIMMYT

∗Germplasm sources include CIMMYT breeding lines in development or released directly by a National Program as a cultivar with a local

name (C); cultivars released by a National Program or breeding company that has a CIMMYT breeding line as one or both of the parents of

the new cultivar (C-d, or CIMMYT derived); cultivars that do not contain CIMMYT germplasm in their pedigrees (No C-d, Non CIMMYT

derived); and cultivars with no known pedigree (Unk.).∗Breeding line with a synthetic in its pedigree.

in a 96-well Peltier thermal cycler (MJ Research, Inc.,Watertown, MA) following a standard temperature pro-file: 29 cycles consisting of 1 min denaturation at 94 ◦C,2 min annealing using temperatures between 50 and64 ◦C (depending on primer combination), and 2 minextension at 72 ◦C. Primers were labeled with 6-FAM,HEX or TET fluorescent dyes. PCR products were am-plified separately and run on an ABI Prism 377 DNASequencer (Perkin Elmer/Applied Biosystems). Whenpossible, multi-loading was applied to increase effi-ciency, with two to six loci with non-overlapping allelesizes loaded together in one well of the gel. Two con-trol genotypes were loaded on every gel. Fragmentswere sized using Genescan 3.1 and assigned to allelecategories using the software package Genotyper 2.1(Perkin Elmer/Applied Biosystems Biotechnologies,Foster City, USA).

Statistical analysis

The diversity index provides information about howcommon or rare the alleles are in the populations. Ge-netic distances also give information on allelic diversityin populations. The measures of diversity used in thisstudy are:

Shannon’s Diversity Index (SH)This index is commonly used to characterize diversityand it accounts for abundance and evenness of the al-leles. The Shannon’s diversity index (Shannon, 1948;Pardo et al., 1997) is represented by:

SH = −A∑j

p j ln(p j ),

where ln stands for natural logarithm, j = 1, 2, . . . , Adenotes the number of alleles, and p j is the relativefrequency of the j th allele on the whole data set suchthat:

p j = n j

N,

A∑j=1

p j = 1,

where n j is the frequency of the j th allele, and N =∑Aj=1 n j .The estimated variance of SH is calculated as:

S2SH =

∑Aj=1 n j ln(n j )

2 −(∑A

j=1 n j ln(n j ))2

/N

N 2

+ A − 1

2N 2

Table 2. Simple Sequence Repeat (SSR) markers used for PCR ampli-

fication of the wheat lines in this study. Information includes chromo-

somal location, repeat type and annealing temperature (Ann. Temp.)

for each marker. Polymorphic Information Content (PIC) and num-

ber of alleles per SSR are also included

Marker Chromosome Repeat type Ann. Temp. PIC # alleles

dp023 4B GCT 60 0.06 3

dp038 1A GCG 60 0.45 7

dp041 4D TATG 60 0.24 7

dp042 5D CATA/TA 60 0.54 9

dp099 4A AAG 50 0.29 2

dp115 5B ACG/ACCG 60 0.55 6

dp122 3D ACGGC 47 0.31 2

dp137 6A AGC 60 0.54 7

dp138 5D AGC 60 0.03 2

dp167 6A AAGC/AT 60 0.77 11

dp188 1A AAC 65 0.09 2

dp190 4A AAC 60 0.59 8

dp205 5B AAG 60 0.48 8

dp226 7A AAG 60 0.50 13

dp253 2B AC 55 0.36 5

dp276 7B ACAT 60 0.34 4

dp278 4D ACAT 60 0.31 7

dp287 4B ACC 60 0.34 6

dp318 7B AGGC 60 0.16 3

dp328 4A ACT 60 0.38 4

dp330 1A ACT 50 0.05 2

dp344 2B AG 60 0.71 7

dp378 6B AG 60 0.21 5

dp395 5A AGAT 60 0.18 5

dp427 2A AGC 55 0.52 4

dp431 1D AGC 60 0.05 3

dp438 3B AGC 55 0.16 6

dp450 7B AGC 60 0.46 5

dp532 1A ATCC 55 0.48 6

dp533 7B ATCC 60 0.14 4

dp535 4D ATCC 60 0.12 3

dp565 2D CCG 60 0.07 3

gwm002 5A CA 55 0.67 10

gwm018 1BS CA/GA/TA 60 0.50 10

gwm095 2AS AC 55 0.72 11

gwm261 2D CT 60 0.60 12

gwm295 7D GA 60 0.79 9

gwm415 5AS GA 60 0.40 4

gwm513 4BL GT/TCAT/GT 60 0.64 6

gwm631 7A GT 60 0.56 4

Modified Roger’s Distance (MRD)For i = 1, 2, . . . , L loci and j = 1, 2, . . . , ni number ofalleles per locus the Modified Roger’s Distance be-tween individual P and P ′ is given by

MRD = 1

(2L)1/2

(∑i

∑j

(Pi j − P ′i j )

2

)1/2

,

where (∑

i

∑j (Pi j − P ′

i j )2)1/2 is the Euclidean dis-

tance between individuals. The variance of the MRDis the variance of the n(n − 1)/2 pairwise distances ofthe lines within each Year Group.

Polynomial regressionA polynomial regression of second degree was ad-justed to the response variable (Y) represented by theShannon’s diversity index and the Modified Roger’sDistance in the predictor variable year groups groupof years (X ) such that Y = β0 + β1 X + β2 X2. Theresponse variable (Y ) was standardized in order toeliminate scale differences between different diversitymeasurements.

Results and discussion

Graphs of molecular diversity in the wheats studied arepresented in Figures 1–2. Average Modified Roger’sdistances (MRD) within groups of germplasm in thestudy fell from 0.64 in the landraces to a low of 0.58 inthe improved lines in the 1980s and rose again in the lat-est CIMMYT wheat breeding lines to levels not signif-icantly different from that of the landraces(0.63) (datanot shown), as tested using confidence intervals. Wheninterpreting the results against the time line, it must berealized that the time between the crossing/selectionprocess during breeding and the final large-scale pro-duction of resulting cultivars can be anywhere between5 and 15 years.

Average worldwide yield for wheat in developingcountries is shown in Figure 3. This figure is repro-duced using yield data from developing countries fromthree different sources. The first data point from Evans(1993) is yield of landraces in developing countries be-fore the adoption of the Green Revolution wheats. Thisis by necessity an average over years and locations,because yield of landraces varies widely dependingon landrace, farmer, climate, and a host of other oftenunknown factors. Wheat yields in the developing worldfrom 1961 to 2000 were taken from the FAO web-site at http://faostat.fao.org/faostat/form?collection=Production.Crops.Primary&Domain=Production&servlet=1&language=EN&hostname=apps.fao.org&version=default. Globally representative yields ofCIMMYT breeding lines from 2000–2005 cannot yetbe determined since they have only recently startedto be distributed worldwide and sufficient data arenot yet available. However, yield data from recentCIMMYT trials indicate that the newest breeding lines

Figure 1. Plot of the quadratic response of the Shannon diversity index over time (measured for each Year Group, see text for explanation).

Each observation has ± standard error.

will have an average yield advantage of 3.5% overcultivars released in 2000 (Byerlee & Moya, 1993).Furthermore, CIMMYT impact studies indicate thatfarmers see a proportional gain in yield over oldervarieties when the newly released varieties derivedfrom CIMMYT advanced lines are grown under theirown conditions (Trethowan et al., 2001, 2003).

From 1944 until the mid 1960s, the breeders fromCIMMYT’s predecessor organization, the Mexican Of-fice of Special Studies, worked to create what were latercalled Green Revolution wheats. The wheat lines wereinitially targeted toward the irrigated valleys in north-west and central Mexico. The breeders establishedthe first shuttle breeding program between two very

contrasting locations (Braun et al., 1996). The firstlocation, Cd. Obregon, was irrigated and used high-input agronomical measures where selection empha-sized yield potential, uniform semi-dwarf height, earlymaturity, lodging tolerance, and durable disease re-sistance (the major diseases being stem rust (causedby Puccinia graminis) and leaf rust (P. triticina). Thesecond location, Toluca, was a high rainfall environ-ment 2,640 masl, where the major stresses were striperust (P. striiformis), Septoria tritici blotch, Fusariumhead scab and waterlogging. This latter cool summersite was not a commercial production location, but se-lection was conducted against susceptibility to preva-lent stresses in this highly disease-prone location. This

Figure 2. Plot of the quadratic response of the Modified Roger’s Distance over time (measured for each Year Group, see text for explanation).

Each observation has ± standard error.

shuttle approach requires material to adapt to two dis-tinct agroecological zones, and has been shown to resultin advanced lines with broad adaptation.

The result was a narrowing of genetic diversitycoming out of the breeding pipeline relative to thatgoing in, due to selection against unwanted formsof the targeted traits and genomic sequences linkedto them. This reduction can be seen in Figures 1 &2, which track two different measures of diversitywithin each Year Group. The selection process wassuccessful when measured by increased yield overthe landrace cultivars; this yield increase has contin-ued since the 1950s (Figure 3). From the late 1950suntil the end of the 1960s it became increasinglyclear that CIMMYT wheats, initially bred for irri-gated conditions in Mexico, also showed adaptation to

irrigated conditions in parts of Asia, the Mediterraneanregion, South Africa, and parts of the Southern Coneof Latin America, and as a result were widely adoptedby farmers in those regions. Such wide-scale adoptionof the new wheat cultivars led to fears of genetic uni-formity and increased danger of epidemics (Nat. Res.Council, 1972), stemming from the southern corn leafblight epidemic in North America in 1970.

From the mid-1960s onwards, the CIMMYT breed-ing program expanded its use of germplasm intro-ductions from around the world (including landraces,winter wheats, Chinese and Latin American materials,and some translocation stocks [carrying the 1BL/1RStranslocation with a chromosome fragment from rye])(Rajaram & van Ginkel, 2001; Rajaram et al., 2002). Atthe same time, the program increased selection pressure

Figure 3. Change in yield (in tonness per hectare) for wheats from

developing world countries over the time period for which data have

been reported in the FAO website (webpage reported in the text) and

average yield of the landraces which were grown prior to the 1960s.

Data from 2001–2005 are projected estimates (see text).

for resistance to biotic and abiotic stresses at the Tolucasite to better address the needs of what had grown tobe a global wheat program, now also needing to targetnon-irrigated regions experiencing rainfed productionconditions and the associated disease spectrum. De-spite the more diverse gene pool overall, this stringentselection probably decreased diversity as unwanted al-leles for low yield, stress susceptibility, and poor qual-ity were discarded.

To address and expand the international perspec-tive, an international global testing system was estab-lished with collaborators in the 1970’s. The informa-tion generated on local adaptation of the germplasmto diverse locations was used in the crossing programbased in Mexico. This multifaceted approach wouldbe expected to have varied effects on genetic diver-sity as there was increasingly strong selection pres-sure for uniform height, a narrowed maturity window,lodging tolerance, disease resistance (foliar, spike, andto a lesser extent, root diseases), tolerance to abioticstresses (e.g., drought, heat, sprouting), and qualitytraits (e.g., plump grain, grain colour, protein quan-tity and quality). Diversity fluctuates in Figures 1 and2 over the next four Year Groups, maintaining approx-imately the same overall level. A similar study on Eu-ropean wheats released between 1840 and 2000 showsthat allelic diversity as measured by SSR markers re-mained stable until the 1960s, at which time a decreasein allelic diversity began and continued throughout theremainder of the period currently covered (Rousselet al., 2005). This decrease is more pronounced in somegeographical areas than others, but reflects the contin-uous selection of wheats specialized for each region,and the agriculture policies present in each country.

As ever-wider adoption of CIMMYT wheats oc-curred due to increasing yield benefits and enhanceddisease resistance, fears of genetic uniformity and theassociated threat of disease epidemics increased. Thisled to a new emphasis on so-called horizontal resis-tance (Van der Plank, 1963), now often interpreted asdurable or adult-plant resistance (Singh & Rajaram,2002). CIMMYT adopted the phrase ‘slow-rusting’from Dr. Ralph Caldwell to describe this type of re-sistance to the rust pathogens. Rather than seek resis-tance from outside sources, CIMMYT scientists recog-nized that broad-based CIMMYT germplasm alreadycarried a considerable number of minor or non-racespecific genes for rust resistance. During the 1970sand 1980s, alleles for resistance, mostly found withinCIMMYT germplasm, were pyramided into CIMMYTlines and cultivars contributing to increasing yields aswell (Figure 3). However, latent (overall) diversity re-mained fairly stable and low, and the danger of epi-demics, especially to new pathogens, was still present.

The first attempts to use synthetic hexaploid wheatsin a breeding program were prompted by the need todevelop germplasm resistant to Karnal bunt (Tilletiaindica), a disease originating in South Asia, but withthe potential to spread beyond the region and whichhas now been noted in north-western Mexico, southernUSA and South Africa. It was, however, soon real-ized that this work would greatly expand overall diver-sity in the breeding program. In the early 1990s, thesynthetic hexaploid wheats were backcrossed to manyCIMMYT and global elite breeding lines. CIMMYT’ssynthetics introduced new variation for morphologi-cal and agronomic traits, (Villareal et al., 1994a, b);resistance to biotic stresses (Kema et al., 1995, Lageet al., 2002, 2004; Ma et al., 1995; Mujeeb-Kazi et al.,2001; Cox, 1998) and abiotic stresses (Rajaram & vanGinkel, 2001; Trethowan et al., 2005, Villareal et al.,2001). The synthetics were subsequently also shownto be very diverse at the molecular level, and genet-ically distinct from cultivated wheats (Villareal et al.,1994b; Zhang et al., 2005). A total of 1,000 spring habitsynthetics have been derived at CIMMYT from 460 T.tauschii accessions to date (Mujeeb-Kazi et al., 2001).

Useful traits displayed in the synthetics have beensuccessfully transferred to CIMMYT elite breedinglines and are selected for in the synthetic derivedbackcross lines. Some new synthetic crosses are be-ing planned at CIMMYT in order to incorporate newsequence and trait diversity from unrepresented species(e.g. T. dicoccoides and T. dicoccum) into the commonand durum wheat breeding pools, but emphasis will

be placed on evaluating and utilizing the syntheticsalready available. The advanced backcross lines arepopular with breeders worldwide due to their excellentlevels of resistance to biotic and abiotic stresses, and insome cases, high grain quality. The latent diversity inthese newest CIMMYT breeding lines is the highest ithas been in 50 years, and has reached levels not signif-icantly different from those in landraces (Figures 1 &2; confidence intervals not shown). The successful in-corporation and re-mixing of genetic diversity fromwheat’s wild relatives has created wheats containingmore variation than has ever been available to farmersand breeders, possibly since hexaploid wheat first ap-peared 8,000 years ago. The potential risk of epidemicsdue to genetic uniformity has certainly been greatly re-duced with these materials, while yield potential, yieldstability, and tolerance to stresses has continued toincrease.

Conclusions

The data presented here, for the period from the mid20th century to its conclusion, indicate an initial drop ininherent diversity from the level measured in landraces,followed by a period of equilibrium. This is under-standable as increasingly diverse common wheats fromaround the world were introduced into the CIMMYTcrossing programs, while at the same time fixed lineswere selected that required very precise adaptation tospecific wheat growing environments around the globe,particularly in developing countries. During this pro-cess, the use of durable resistance mitigated the effectsof some diseases, nevertheless new pathogens or bio-types would find a relatively uniform genome in thehost crop as a result of earlier narrowing of genetic di-versity. However, the use of synthetic hexaploid wheatsin crosses during the past 10 years has dramatically al-tered the balance of genetic diversity. The inherent di-versity of the new synthetic derivatives is comparable tothat of the landraces; however, they express improvedyield, disease resistance, abiotic stress tolerance andbetter end-use quality.

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