Diversity Assessment of Turkish Maize Landraces Basedon Fluorescent Labelled SSR Markers

Gönül Cömertpay & Faheem S. Baloch &

Benjamin Kilian & Ahmet C. Ülger & Hakan Özkan

Published online: 15 July 2011# Springer-Verlag 2011

Abstract Landraces of maize represent a valuable geneticresource for breeding and genetic studies. Since 1970,landraces have been collected from all over Turkey, but thegenetic diversity represented in this collection is still largelyunknown. In this study, a sample of 98 landraces sampledfrom 45 provinces of Turkey was assessed genotypically at28 simple sequence repeat (SSR) loci and phenotypicallyfor 19 morphological traits. The landraces varied signifi-cantly for all the latter traits. A total of 172 SSR alleleswere detected, giving a mean of 6.21 alleles per locus. Thegenetic distance between pairs of landraces ranged from0.18 to 0.63, with a mean of 0.35. Positive and negativecorrelation exists among different morphological andagronomic traits. Positive association among different traitsshowed that improvement of one character may simulta-

neously improve the other desired trait. Based on UPGMAdendrogram and Neighbor-Net (NNET) analyses from bothmorphological traits and SSR data, respectively, it isobvious that maize landraces from the same geographicalregion were often placed in different clusters, indicatingthat grouping based on genetic parameters was not closelyrelated to the geographic origin. The wide diversity presentin Turkish maize landraces could be used as geneticresource in designing maize breeding program for devel-oping new cultivars adapted to different geographic andclimatic conditions, and may also contribute to worldwidebreeding programs.

Keywords Maize . Landraces . Diversity . Morphologicaltraits . SSR . Turkey

Introduction

Maize was first brought to the Mediterranean region fromMexico by Spanish explorers (Kün 1985). Maize wasintroduced to Ottoman agriculture via Egypt and Syriamore than four centuries ago (Kırtok 1998; Kün 1985; Ögel2000). Leng et al. (1962) reported that maize wasintroduced into the Balkans during the expansion of theOttoman Empire; similarly, Brandolini and Brandolini(2001) mentioned that Italian farmers received maizegermplasm from Spain, Portugal and later from theOttoman Empire. In Italy and Central Europe, the crop isknown as ‘Grano de Turco’ or ‘Türkische Weizen’, whichimplies that it was introduced in Central Europe andBalkans by Ottoman traders (Ögel 2000). Janick andCaneva (2005) explained that name was given because thesilk resembled the bread of a Turk or simply to accentuateits foreign nature.

This article is dedicated to our dear colleague, the late Prof. Dr. AhmetCan Ülger.

Electronic supplementary material The online version of this article(doi:10.1007/s11105-011-0332-3) contains supplementary material,which is available to authorized users.

G. Cömertpay : F. S. Baloch :A. C. Ülger :H. Özkan (*)Department of Field Crops, Faculty of Agriculture,University of Çukurova,01330 Adana, Turkeye-mail: hozkan@mail.cu.edu.tr

B. KilianLeibniz Institute of Plant Genetics and Crop Plant Research (IPK)Genebank/Genome Diversity,Corrensstrasse 3,06466 Gatersleben, Germany

Present Address:G. CömertpayDepartment of Field Crops, Adiyaman University,Kahta Vocational School,Adiyaman, Turkey

Plant Mol Biol Rep (2012) 30:261–274DOI 10.1007/s11105-011-0332-3

Since their introduction, maize landraces have been ofrelevant socio-economic importance for family farmingsystems and are still cultivated throughout different regionsof Turkey. Maize landraces are open-pollinated varieties(OPVs), and therefore they underwent long-term naturaland artificial selection in the past centuries. A large numberof maize landraces have arisen over time, selected for theiradaptation to local environmental conditions by farmers.

Maize was of marginal importance for the Turkishagriculture until 1970s; the crop was cultivated only insmall scales (Kün 1994) for human nutrition and animalfeed. Maize landraces have long been a significantcomponent of small farm systems in Turkey, but their usecontinue to persist in some small farmer communities.Introduction and production of hybrid maize, in the late1970s, led to the rapid spread of maize cultivation. Today,most of cultivated maize in Turkey is of hybrid nature;however, traditional maize landraces still grow in smallfarmer communities in different parts of Turkey, especiallyin the Black Sea region. Maize is one of the most importantcrops in Turkish agriculture, after wheat and barley. It isextensively cultivated in Black Sea region (37%), followedby the Mediterranean (29%) and Marmara (16%) regions(Taşdan 2005). According to the Statistical database ofFood and Agricultural organization of the world (FAOSTAT2009), 592,000 ha of land (about 35% of areas undercultivation in Turkey) produce 4.25 million tons of maizegrains per year. In Turkey, 64% of maize is used for foragepurposes and 36% for food and industrial products (Egeand Karahocağil 2001).

Modern plant breeding has succeeded spectacularly inraising crop productivity in line with the rising humanpopulation. However, its reliance on a small number of elitecultivars has eroded the genetic base of crops, and inparticular, endangers the continued existence of landraces,which represent a repository of allelic variation which maybe needed to achieve further genetic advance for yield andquality. International surveys have clearly shown the urgentneed to save and manage local landraces, since thesematerials contain valuable genes for future cultivationpractices and future breeding for higher yield and betterquality.

Both variation in phenotype and genotype have beenexploited to characterize and manage genetic diversity ingermplasm collection of various plant species. Morpholog-ical evaluations are the strongest determinant of agronomicvalue and have been of great value in studies of cropevolution, germplasm evolution and in revealing differ-ences between varieties. Compared with other methods,morphological markers are direct, inexpensive and easy.However, phenotypic markers suffer from low polymor-phism, poor heritability, impracticality of early screening,

and limited number, and are affected by environmentalconditions (Smith and Smith 1992). Morphological traitsand molecular markers are frequently used to study geneticdiversity, to develop conservation strategies, to facilitatetheir management and to development plant geneticresources (Rao 2004; Sharma et al. 2011; Saeed et al.2011).Among maize landraces, genetic variability has beenprimarily characterized by morphological traits (Ilarslan etal. 2001; Brandolini and Brandolini 2001; Pressoir andBerhaud 2004; Beyene et al. 2005; Hartings et al. 2008),isozymes and seed storage protein. One the other hand,molecular markers provide a direct measure of geneticdiversity and go beyond indirect diversity measures, basedon morphological traits or geographical origin. Genotypicassays are more robust and practical than phenotypic ones.Different markers systems such Amplified fragment lengthpolymorphism (AFLP), random amplified polymorphicDNA (RAPD), inter simple sequence repeat (ISSR),sequence related amplified polymorphism (SRAP), singlenucleotide polymorphism (SNP), etc., are currently avail-able for monitoring the genetic diversity (Baraket et al.2011). Among various DNA markers, microsatellites orsimple sequence repeat (SSR) markers are widely preferredin diversity analysis owing to their co-dominant nature,abundance in the genome, high polymorphism, highinformativeness, repeatability and reliability (Legesse etal. 2007; Bourguiba et al. 2010). SSR analysis usingfluorescent dye-labeled markers through a DNA sequencerfurther enhances the efficiency and precision of genotyping.Due to their high allelic diversity and genetically co-dominant nature, SSR loci have been used successfully forgenetic diversity studies and population structure of maize(Senior et al. 1998; Xia et al. 2004) In addition to this, the“bulk DNA fingerprinting” method, using fluorescent dye-labeled SSR markers, has been recently optimized formaize leading to better resolution of alleles in geneticallyheterogeneous landraces (Eschholz et al. 2008).

Throughout history, Turkey has been a junction ofcultures between Europe, Asia and Africa. Subsequently,Turkey played an important role in spreading maizelandraces (Ögel 2000; Brandolini and Brandolini 2001;Leng et al. 1962). Turkish landrace germplasm could beuseful in increasing genetic diversity of maize because thelong term adaptation of landraces to a wide range of diverseenvironmental and geographic conditions as well as todifferent agro-ecological systems led to the creation ofabundant genetic variation. Since the 1970s, the AegeanAgricultural Research Institute has been sampling andpreserving maize landraces from all over Turkey, but thecollection has scarcely been characterized to date. A set of32 accessions was genotyped using isozyme markers byIlarslan et al. (2001), and this was followed by a

262 Plant Mol Biol Rep (2012) 30:261–274

classification based on various morphological and agro-nomic traits (Ilarslan et al. 2002). More recently, thediversity present in 17 maize landraces from NorthernTurkey was characterized using RAPD markers (Okumus2007). The indications from these studies were that theTurkish maize landrace collection harbored a substantialamount of genetic diversity. However, the landracesevaluated so far represent only a small subset of availableresources; furthermore, they come from few geographicregions and do not allow the study of the genetic structureof maize landraces in Turkey. Therefore, our aim was tocharacterize large samples of landraces, covering almost allmaize growing regions of Turkey, representing diverseclimate, geography and topography using morphologicaland agronomical traits as well as SSR markers.

Material and Methods

Plant Material, Field Experiment and Morphological Traits

The seeds of the landraces were kindly obtained fromMenemen gene bank of the Aegean Agricultural ResearchInstitute, Izmir, Turkey. Identification numbers and loca-tions are presented in Table 1, while their collection sitesare illustrated in Fig. 1. Field experiment was carried out in2004 at the University of Çukurova, Adana (37°00′56″N,35°21′29″E), a location which experiences a typicalMediterranean climate of hot, dry summers, and thereforerequired irrigation. The clay soil had a pH of 7.1. Cropmanagement followed local agronomic practice. Eachlandrace plot consisted of two 5-m rows comprising 25plants per row and separated by 70 cm. The experiment wasconducted without replications due to limited seed quantity.Seed of nine accessions (Adana, Adapazarı1, Adapazarı2,Adıyaman1, Adıyaman2, Afyon1, Artvin3, Manisa1, Zon-guldak2) germinated too poorly; therefore, these landraceswere eliminated during evaluation of morphological traits.Twenty plants from each landrace were randomly selectedand labeled, and 19 morphological and agronomicalimportant traits were recorded. Observations on 19 traitswere taken based on the CIMMYT/IBPGRI (1991) maizedescriptor and Rebourg et al. (2001). The 19 traits were:days to tasseling (DTS), plant height (PHT), ear height(EHT), stem diameter (SDI), angle of the uppermost leaf(AUL), number of leaves (NL), leaf area (LFA), number ofprimary branches in the tassel (PBNT), ear length (ELG),diameter (EDI), peduncle length (EPLG), weight (EWE)and row number (ERN), number of kernels per row (NKR)and per ear (TNKE), individual plant yield (PYL), andkernel length (KLG), kernel width (KWD) and kernelthickness (KT) (Table 2). The total number of kernels per

ear was measured by multiplying the ear rows number withthe number of kernels per row. The leaf area was computedas: LA=leaf length×leaf width×0.75. Kernel length, kernelwidth and kernel thickness were measured by selecting tenkernels from the middle of the ear of each of the 20 plants.A total of 200 kernels were measured from each landrace.

Molecular Analysis

Genomic DNA was isolated from each of all landracesusing the population bulk strategy, as described by Dubreuilet al. (1999) and Rebourg et al. (2001). Each bulk wasprepared by pooling an equal amount of leaf material from15 individuals in a population except one landrace,‘Amasya3’ that was bulk with 14 individuals. Althoughthe strategy does not permit estimation of the degree ofheterozygosity within a population, it facilitates large-scaleanalyses of diverse maize populations including estimationof within-population allele frequencies (Dubreuil et al.2006). DNA was extracted according to method describedby Doyle and Doyle (1990), with some modifications(Özkan et al. 2005). Eight landraces (Amasya4, Artvin2,Çanakale3, Edirne3, Eskişehir1, Kütahya1, Muğla1, Sam-sun3) were excluded from the SSR analysis, because ofinsufficient and poor DNA quality. The three well-characterized US inbreds B52, B73 and Mo17 wereincluded for comparative purposes. First of all, DNA fromeach of 15 plants per population were amplified with fiveSSR primers (control), and peak value of each allele wereused to estimate the expected allele frequencies in bulkDNA of each population as described in the protocol ofRebourg et al. (2001). The eight bulk landraces wereinitially genotyped with a set of 42 fluorescent dye-labeledSSR markers according to Sharma et al. (2010). Based onthis screening, 28 SSR were selected based on their knowninformativeness and their distribution over the genome(Reif et al. 2005; Dubreuil et al. 2006; www.maizegdb.org).

M13 tailed-primer PCR amplification of SSR wasperformed according to Schuelke (2000) in 12 μl PCRcontained 1× buffer, 0.125 mM dNTP, 0.4 pmol M13sequence tailed forward primer, 0.3 pmol reverse primer,3.0 pmol universal M13 primer labeled with one of the fourfluorescent dyes 6-FAM, VIC, NED and PET, 0.12 U/μl TaqDNA polymerase, and ~50 ng genomic DNA, andconsisted of 35 cycles of 95°C/1 min, 55°C/2 min and72°C/2 min, followed by five cycles of 94°C/30 s, 53°C/1 min and 72°C/1 min, with a final extension for 72°C/10 min. A set of four PCR products (0.75 μl of each), eachlabeled with a different dye, was combined with 0.14 μlGeneScan-500 LIZ® size standards (Applied Biosystems)and 6.86 μl Hi-Di™ formamide (Applied Biosystems),denatured at 94°C for 5 min, chilled on ice and separated

Plant Mol Biol Rep (2012) 30:261–274 263

Table 1 Origin, collection sites and altitude of 98 open pollinated Turkish maize populations used in this study

GenBank identificationnumber

No inFig. 1

Geographicalprovince

Collection site Kernelype

Latitude Longitude

TR 51484 2 Adanaa Kozan, Gaziköy Flint/Dent 37°26′46.76″ 35°49′36.75″

TR 51540 3 Adapazarı1a Karasu Flint/Dent 41°05′56.01″ 30°41′30.61″

TR 51620 4 Adapazarı2a Karapınar Flint/Dent 41°03′24.56″ 30°37′34.02″

TR 51719 5 Adıyaman1a Gölbaşı Flint/Dent 37°47′00.21″ 37°38′41.70″

TR 37985 6 Adıyaman2a Samsat, Balcılar vil. Dent 37°34′44.34″ 38°29′02.92″

TR 37996 7 Afyon1a İşçehisar Flint/Dent 38°51′46.81″ 30°44′58.14″

TR 37998 8 Afyon2 Dinar Flint/Dent 38°04′00.84″ 30°10′07.68″

TR 38008 9 Ağrı1 Yığıntepe vil. Flint/Dent 39°46′09.48″ 42°56′11.40″

TR 38147 10 Ağrı2 Tutak, Yoğunhisar vil. Flint/Dent 39°38′28.78″ 43°00′02.01″

TR 38150 11 Amasya1 Taşova Flint/Dent 40° 45′10.80″ 36°19′43.68″

TR 38201 12 Amasya2 Evince Flint/Dent 40°45′10.80″ 36°19′43.68″

TR 38035 13 Amasya3 Bekdemir Flint/Dent 40°29′16.41″ 35°35′07.37″

TR 38036 101 Amasya4b Göynücek Flint/Dent 40°26′28.15″ 35°32′05.22″

TR 38039 14 Artvin1 Erhavi Flint/Dent 41°21′10.85″ 41°18′37.50″

TR 38243 102 Artvin2b Borçka Flint/Dent 41°21′48.05″ 41°40′44.76″

TR 38272 16 Artvin3a 4 Km E Orus, Şenköy vil. Flint/Dent 41°10′48.47″ 41°49′23.29″

TR 37484 17 Artvin4 Şavşat Flint/Dent 41°14′35.24″ 42°21′50.12″

TR 37490 18 Aydın1 Bozdoğan, Kılavuzlar vil. Flint/Dent 37°40′26.52″ 28°18′19.35″

TR 37499 19 Aydın2 Sultanhisar, Uzunlar vil. Flint/Dent 37°56′59.76″ 28°12′12.10″

TR 37500 20 Balıkesir1 Gönen, Tütüncüler vil. Dent 40°06′14.75″ 27°39′16.51″

TR 38375 21 Balıkesir2 Manyas, Süleymanlı vil. Flint/Dent 40°02′34.92″ 27°50′23.41″

TR 38411 22 Balıkesir3 Bigadiç, Kadıköy Flint/Dent 39°27′06.84″ 28°05′11.04″

TR 38422 23 Bolu1 Göynük, Karacalar vil. Flint/Dent 40°22′48.36″ 30°38′02.40″

TR 38437 24 Bolu2 Düzce, Döngelli vil. Flint/Dent 41°03′49.73″ 31°11′08.00″

TR 37543 25 Burdur1 Yeşilova Flint/Dent 37°30′28.11″ 29°45′12.94″

TR 38471 26 Burdur2 Tefenni, Çaylıkköyü vil. Flint/Dent 37°18′45.93″ 29°46′34.57″

TR 37605 27 Bursa1 Orhangazi, Çeltikli vil. Flint/Dent 40°29′16.03″ 29°18′35.12″

TR 37630 28 Bursa2 Demirtaş vil. Flint/Dent 40°16′20.64″ 29°05′35.16″

TR 37644 29 Çanakkale1 Çan Flint/Dent 40°01′24.24″ 27°02′43.63″

TR 37780 30 Çanakkale2 Çan Flint/Dent 40°01′24.24″ 27°02′43.63″

TR 37876 103 Çanakkale3b Lapseki, Ada tepe vil. Flint/Dent 40°20′41.25″ 26°41′15.85″

TR 55545 32 Çorum1 Ortaköy Flint/Dent 40°16′20.36″ 35°14′40.52″

TR 55463 33 Çorum2 Sungurlu Flint/Dent 40°09′54.10″ 34°22′28.82″

TR 55469 34 Denizli1 Acıpayam, Gölcük vil. Flint/Dent 37°10′13.08″ 29°14′26.52″

TR 49312 35 Denizli2 Kayhan vil. Flint/Dent 37°45′06.26″ 29°07′15.41″

TR 57657 36 Denizli3 Tavas, Solmaz vil. Dent 37°29′10.32″ 28°57′49.32″

TR 57661 37 Diyarbakır Çermik, Pamuklu vil. Flint/Dent 38°02′40.47″ 39°17′31.29″

TR 55515 38 Edirne1 İpsala Flint/Dent 40°55′48.72″ 26°23′42.00″

TR 44446 39 Edirne2 Havsa Dent 41°33′01.47″ 26°49′14.33″

TR 44469 104 Edirne3b Karaağaç Flint/Dent 41°03′51.12″ 26°31′46.92″

TR 44519 40 Edirne 4 Keşan Dent 40°51′29.52″ 26°37′48.72″

TR 36977 41 Erzurum1 Horasan, Esence vil. Flint/Dent 40°02′49.20″ 42°10′1560″

TR 37006 42 Erzurum2 Tortum, Pehlivanlı vil. Flint/Dent 40°17′57.16″ 41°32′56.40″

TR 37010 105 Eskişehir1b Sivrihisar Flint/Dent 39°27′04.68″ 31°32′16.08″

TR 37013 43 Eskişehir2 Sivrihisar Flint/Dent 39°27′04.68″ 31°32′16.08″

TR 37056 44 Gaziantep1 Nizip, Belkız, Kavunlu vil. Flint/Dent 37°00′50.04″ 37°47′28.32″

TR 37105 45 Gaziantep2 Nizip, Aşağıçardaklı Fındıklı Flint/Dent 37°00′50.04″ 37°47′28.32″

TR 50558 46 Giresun1 Doğakent, Demirci vil. Flint/Dent 40°55′03.00″ 38°23′25.08″

TR 50550 47 Giresun2 Barça vil. Flint/Dent 40°53′12.56″ 38°26′32.15″

TR 50541 48 Hakkari Uludere Flint/Dent 37°25′45.06″ 42°52′59.27″

TR 50548 49 Isparta1 Keçiboru, Aydoğmuş vil. Flint/Dent 37°58′15.24” 30°14′42.72″

TR 50537 50 Isparta2 Keçiborlu, Gümüşgün vil. Flint/Dent 37°58′15.24” 30°14′42.72″

TR 50540 51 İstanbul1 Silivri, Danamandıra Flint/Dent 41°18′42.12″ 28°14′41.28″

TR 50527 52 İstanbul2 Çatalca, Karaca köy vil. Flint/Dent 41°08′32.28″ 28°27′51.12″

264 Plant Mol Biol Rep (2012) 30:261–274

on an ABI 3130xl Genetic Analyzer (Applied Biosystems).The amplicon fragments were analyzed using GeneMappersoftware v3.7 (Applied Biosystems), as described in theuser manual.

Statistical Analysis

Standard one-way analyses of variance were performed foreach trait, using the JUMP statistical software package

Table 1 (continued)

GenBank identificationnumber

No inFig.1

Geographicalprovince

Collection site Kernelype

Latitude Longitude

TR 50511 53 İzmir1 Bozdağ Flint/Dent 38°20′30.12″ 28°04′36.12″

TR 50587 54 İzmir2 Bergama, Çakırlı vil. Flint/Dent 39°07′21.72″ 27°10′41.88″

TR 50565 55 İzmir3 Torbalı, Karaot vil. Dent 38°10′23.70″ 27°21′17.91″

TR 50563 56 K.maraş1 Andırın Flint/Dent 37°34′35.04″ 36°21′02.06″

TR 50564 57 K.maraş2 Türkoğlu Flint/Dent 37°30′12.13″ 36°50′58.93″

TR 50654 58 Kars Kötek Flint/Dent 40°13′15.24″ 43°00′54.00″

TR 50667 59 Kastamonu1 Araç, Yeniceköy vil., Köseler Flint/Dent 41°14′43.08″ 33°19′48.36″

TR 50674 60 Kastamonu2 Emirler vil. Flint/Dent 41°43′51.60″ 32°46′21.00″

TR 50677 61 Kırklareli1 Lüleburgaz Flint/Dent 41°24′19.45″ 27°20′54.97″

TR 53245 62 Kırklareli2 Çakıllı Flint/Dent 41°44′16.08″ 27°13′42.60″

TR 50643 63 Kocaeli Kandıra, Akçaova Flint/Dent 41°04′18.48″ 30°09′06.84″

TR 47889 64 Konya Beyşehir, Damlapınar vil. Flint/Dent 37°50′32.64″ 31°55′01.20″

TR 45270 106 Kütahya1b Tavşanlı, Üyücek vil. Flint/Dent 39°32′49.20″ 29°29′29.04″

TR 39563 65 Kütahya2 Saphane, Gaipler vil. Flint/Dent 39°01′33.60″ 29°13′24.24″

TR 54176 66 Manisa1a Saruhanlı, Bedeller vil. Flint/Dent 38°44′50.01″ 27°34′08.04″

TR 54214 67 Manisa2 Yurtdağı Flint/Dent 38°37′19.92″ 27°25′46.92″

TR 54189 107 Muğla1b Marmaris, Gökçe vil. Flint/Dent 36°51′57.62″ 28°14′28.19″

TR 54191 68 Muğla2 Köyceğiz, Beyobası vil. Dent 36°550′7.32″ 28°42′56.82″

TR 54199 69 Muğla3 Köyceğiz, Beyobası vil. Flint/Dent 36°550′7.32″ 28°42′56.82″

TR 54712 70 Ordu1 Perşembe Flint/Dent 41°03′55.95″ 37°47′48.06″

TR 48470 71 Ordu2 Mesudiye, Güzle vil. Flint/Dent 40°27′44.28″ 37°46′14.16″

TR 48477 72 Rize1 Çamlıhemşin Flint/Dent 41°02′42.72″ 41°00′16.56″

TR 48479 73 Rize2 Çayeli Flint/Dent 41°05′24.00″ 40°43′41.16″

TR 50136 74 Rize3 İkizdere yolu, İskender vil Dent 40°47′01.32″ 40°33′09.72″

TR 50161 75 Sakarya1 Küçükhatatlı vil. Flint/Dent 40°47′51.16″ 30°23′11.24″

TR 48452 76 Sakarya2 Hendek, Kazımiye vil. Flint/Dent 40°47′34.20″ 30°43′30.73″

TR 48454 77 Samsun1 Bafra, Altınkaya Dam Flint/Dent 41°34′06.60″ 35°54′27.00″

TR 48461 78 Samsun2 Tekkeköy, Kurtlukent vil. Flint/Dent 41°12′47.16″ 36°27′29.88″

TR 42703 108 Samsun3b 19 Mayıs, Karaköy vil. Dent 41°17′22.98″ 36°20′16.23″

TR 42712 79 Sinop1 Köprübaşı Flint/Dent 42°00′54.98″ 35°05′06.75″

TR 42719 80 Sinop2 Gerze, Çalboğaz vil. Flint/Dent 41°48′14.76″ 35°12′06.48″

TR 42725 81 Ş.urfa Hilvan, Uğra vil. Dent 37°35′21.12″ 38°57′17.28″

TR 42750 82 Tekirdağ1 Güngörmez Flint/Dent 41°29′37.15″ 27°59′18.81″

TR 42803 83 Tekirdağ2 Saray Flint/Dent 41°26′31.20″ 27°55′11.28″

TR 42856 84 Tokat1 Reşadiye, Soğukpınar vil. Flint/Dent 41°12′21.60″ 35°40′56.29″

TR 42949 85 Tokat2 Niksar, Kıraç vil. Flint/Dent 40°35′26.52″ 36°56′59.64″

TR 42958 86 Trabzon1 Tonya Flint/Dent 40°53′07.44″ 39°17′21.48″

TR 42985 87 Trabzon2 Soğuksu vil. Dent 40°59′24.82″ 39°43′28.89″

TR 42614 88 Trabzon3 Akyaz vil. Flint/Dent 41°00′05.08″ 39°43′33.69″

TR 49202 89 Trabzon4 Akçaabat, Düzköy vil. Flint/Dent 41°01′18.03″ 39°34′14.08″

TR 49214 90 Uşak1 Dumlupınar Flint/Dent 38°51′18.72″ 29°58′34.32″

TR 49234 91 Uşak2 Banaz, Güllüçam vil. Flint/Dent 38°45′57.24″ 29°45′11.88″

TR 49309 92 Zongukdak1 Ereğli Flint/Dent 41°16′56.64″ 31°25′27.84″

TR 45513 93 Zonguldak2a Bartın Flint/Dent 41°38′04.03″ 32°20′57.45″

a There is no morphological data for these landraces=9 individualsb There is no SSRs data for these landraces=8 individuals

Plant Mol Biol Rep (2012) 30:261–274 265

(SAS Institute 2002). Inter-trait correlations were alsocalculated using this software. Standardized trait meanvalues were used to perform principal component (PCA)and cluster analyses using NTSYS-pc (Rohlf 2004). Togroup the landraces based on their morphological dissim-

ilarity, a cluster analysis and PCAwere conducted based onEuclidean distances and applying the Unweighted PairGroup Method with Arithmetic mean (UPGMA) (Rohlf2004). The polymorphism information content (PIC) ofeach SSR was calculated following Botstein et al. (1980).

Fig. 1 Collection sites of 98 Turkish maize landraces

Table 2 List of the 19 morphological descriptors utilized in this study with variety adjusted means associated, standard deviations (SD) andranges

Morphological descriptor Abbreviations Mean SD Range

Min Max

Days to tasseling DTS 62.21 5.86 50.30 74.80

Plant height (cm) PHT 169.20 44.12 81.39 260.40

Ear height (cm) EHT 82.65 33.57 19.63 172.40

Stem diameter (cm) SDI 18.30 2.76 11.63 25.00

Angle of uppermost leaf (°) AUL 36.01 7.97 16.60 57.00

Number of leaves NL 10.46 1.88 5.70 14.00

Leaf area (cm2) LFA 353.25 63.85 179.18 526.20

Primary branches number of tassel PBNT 14.81 3.26 6.75 22.85

Ear length (cm) ELG 15.95 2.76 9.38 20.46

Ear diameter (cm) EDI 36.20 3.85 26.10 44.50

Ear rows number ERN 11.35 2.10 7.90 17.16

Number of kernels per row NKR 29.22 5.98 11.00 43.40

Total number of kernels per ear TNKE 336.32 106.13 124.40 645.5

Ear peduncle length (cm) EPLG 10.04 3.82 1.46 17.74

Ear weight (g) EWE 110.68 36.44 44.00 227.35

Individual plant yield (g) PYL 104.84 33.45 40.00 199.50

Kernel length (mm) KLG 8.97 1.08 7.08 13.56

Kernel width (mm) KWD 8.64 0.91 6.83 10.93

Kernel thickness (mm) KT 4.52 0.35 3.69 5.25

266 Plant Mol Biol Rep (2012) 30:261–274

NeighborNet (NNet) planar graphs of SSR DICE distances(Dice 1945) between individuals were constructed usingSplitsTree v4.11 (Huson and Bryant 2006) software.

Results

Phenotpic Variation

The 89 landraces differed significantly from one anotherwith respect to all of the morphological and agronomictraits, and showed a considerable level of phenotypicvariations (Tables S1, S2 and S3). Information on thevariables analyzed in these 89 landraces including popula-tion’s means, standard deviations, and maximum andminimum values are presented in Table 2.

Days to tasseling ranged from 50.30 for landrace Ağrı1to 74.84 for Samsun2. Maximum plant height and earheight (260.40 and 172.40 cm, respectively) were recordedfrom landraces of Edirne1, whereas the minimum plant andear height (81.39 and 19.63 cm, respectively) wereobserved from Muğla2 and Sakaraya1 landraces, respec-tively. Stem diameter varied from 11.63 to 25 cm (forlandraces Muğla2 and Rize1). The angle of the uppermostleaf varied from 16.60° to 57.0° (for landraces of Rize2 andSakaraya1). The number of leaves per plant ranged from5.70 to 14.0 (for Manisa2 and Samsun2), respectively. Theleaf area ranged between 179.18 cm2 for Sakarya1 and526.20 cm2 for landrace Kahramanmaraş1 (Table S1).

For primary branches, the number of tassels varied from6.75 to 22.85 for landraces Rize2 and Afyon2, respectively.Trabzon4 landraces showed the largest ear length of20.46 cm, whereas, Kütahya1 landraces showed theminimum length (9.38 cm). The maximum ear diameter of44.50 cm and the maximum number of rows per ear of17.16 were seen for landraces Uşak1 and Amasya2,respectively. The average number of kernels per row andtotal number of kernels per ear ranged from 11.00 to 43.40and from 124.40 to 645.50 for Diyarbakir and Samsun2landraces, respectively (Table S2).

The largest ear peduncle length was found in landraceÇorum2 (17.74 cm), and the shortest were detected forSakaraya1 (1.46 cm), while the maximum ear weight(227.35 g) was found for Samsun2 and the minimum earweight (44 g) for Muğla2. The average yield per plant was104.84 g, while the highest one (199.50 g) was seen inlandraces from Samsun2. Muğla2 landraces showed thelowest yield (40 g). The maximum kernel length of11.92 mm and the maximum kernel width of 10.93 mmwere recorded for landraces Samsun2 and Ordu1, respec-tively, whereas the minimum values were observed forBursa2 and Gaziantep2 (6.83 and 6.82 mm, respectively).Landraces from Sinop1 showed the lowest value for kernel

thickness (3.69 mm), whereas landraces from Denizli2showed the largest value (5.25 mm) (Table S3).

Statistical comparisons of relationships among 19 agro-nomical and morphological traits for 89 landraces areshown in Table 3. Correlation coefficients showed thatsignificant positive as well as negative correlations existamong different traits studied. The large number ofobservations raised the test power, giving significance tomost of the correlations: hence, only values above 0.6 arediscussed here. Days to heading was highly positivelycorrelated with PHT, EHT, SDI, NL, LFA, ELG, NKR,TNKE, EWE and PYL. Plant height was highly correlatedwith EHT, SDI, NL, LFA, ELG, NKR, EWE and PYL.Plant yield was highly positively correlated with DTS, PHT,SDI, NL, LFA, ELG, EDI, NKR, TNKE and EWE. Kernellength (KLG) was highly correlated with EDI, EWE andPYL.

Using a PCA based on the correlation matrix, it appearedthat the first four principal components accounted for 79.26%of the total variance (Table 4). The traits contributing to thefirst principal component (PC1), which explained 54% of thetotal variance, were DTS, PTH, EHT, SDI, NL, LFA, ELG,NKR, TNKE, EWE and PYL. PC2 accounted for 11% of thevariance and was heavily dependent on ERN, TNKE, KWDand KT. The third component (PC3) was built from EHT,BPNT, EDI, EPLG, EWE, PYL, KLG, KWD, KY andaccounted for 7.8% of the variance.

Cluster analysis was used to reveal the relationshipamong landraces. Phenotypic similarity was calculatedfrom 19 morphological traits by UPGMA cluster analysisbased on Euclidian distance coefficients. The analysis splitthe 89 maize landraces into two clusters: cluster A,containing 11 maize landraces, and cluster B containing78 landraces (Fig. 2)

SSR Polymorphism and Allelic Diversity

With 28 SSR loci distributed uniformly over all ten maizechromosomes, in total 172 alleles with an average of 6.21alleles per locus were detected among 90 landraces inbulked DNA samples. The number of alleles observed ateach locus varied from 2 to 11 (Table 5). The averagepolymorphism information content (PIC) value was 0.717.The highest PIC value of 0.889 was obtained for primerpair phi089, whereas the lowest was 0.327 for primerphi062. The length of amplified fragments ranged from 72to 83 bp for primer ‘phi046’ to 329–347 bp for primersphi227562. The mean DICE genetic distance between allpairs of population was 0.347. The lowest DICE distancewas obtained for the pair of populations Gaziantep1–Kastamonu1 (D=0.178), whereas the highest value wasobtained for populations Amasya2–Adapazari2 (D=0.628)(data not shown).

Plant Mol Biol Rep (2012) 30:261–274 267

NeighborNet (NNet) planar graph computed from DICEgenetic distances is shown in Fig. 3. The analysis separatesthe 90 Turkish maize landraces into different groups. Thefirst split is highlighted in red and separates one group of41 landraces (lower left site in Fig. 3) from the remaining52 lines. A second larger split highlighted in blue separates25 lines including the three US inbred lines (Fig. 3) fromall other landraces. The green split separates three Turkishlandraces that originate from the Black Sea region (Rize1,Rize2, Samsun2) together with the inbred lines—from allother Turkish landraces. Finally, split four, highlighted inbrown color, groups Rize2 and B73 to the exclusion of allother lines investigated. Maize landraces from the sameprovince clustered mostly in different groups, indicatingthat grouping in maize based on SSR markers does notreveal the geographic origin.

Discussion

Landraces are defined as geographically or ecologicallydistinct crop populations developed under the influence ofT

able

3Correlatio

ncoefficientam

ongdifferentmorph

olog

ical

andagrono

mical

characteristicsforTurkish

maize

land

races

PHT

EHT

SDI

AUL

NL

LFA

PBNT

ELG

EDI

ERN

NKR

TNKE

EPLG

EWE

PYL

KLG

KWD

KT

DTS

0.854

0.812

0.774

−0.534

0.887

0.815

0.164

0.803

0.598

0.418

0.645

0.663

0.481

0.775

0.719

0.547

0.001

−0.119

PHT

0.971

0.720

−0.492

0.890

0.820

0.238

0.792

0.494

0.239

0.655

0.554

0.565

0.701

0.685

0.497

0.074

−0.046

EHT

0.678

−0.466

0.844

0.778

0.277

0.705

0.419

0.242

0.581

0.509

0.542

0.592

0.588

0.380

−0.017

−0.016

SDI

−0.589

0.797

0.802

0.325

0.770

0.641

0.464

0.628

0.679

0.496

0.747

0.709

0.505

−0.067

−0.152

AUL

−0.535

−0.585

−0.067

−0.426

−0.377

−0.192

−0.316

−0.324

−0.375

−0.423

−0.403

−0.383

−0.083

−0.012

NL

0.799

0.260

0.813

0.668

0.419

0.697

0.699

0.513

0.817

0.780

0.580

0.014

−0.067

LFA

0.354

0.834

0.600

0.363

0.646

0.614

0.571

0.717

0.700

0.508

0.086

−0.068

PBNT

0.304

0.220

0.208

0.262

0.252

0.194

0.104

0.174

−0.078

−0.070

−0.114

ELG

0.672

0.383

0.777

0.708

0.503

0.856

0.836

0.573

0.104

−0.112

EDI

0.576

0.544

0.692

0.226

0.841

0.732

0.630

0.077

−0.114

ERN

0.309

0.798

0.109

0.496

0.438

0.236

-0.655

−0.260

NKR

0.811

0.483

0.769

0.759

0.560

0.010

−0.340

TNKE

0.368

0.794

0.743

0.516

−0.388

−0.357

EPLG

0.399

0.411

0.313

0.027

−0.090

EWE

0.937

0.746

0.056

−0.203

PYL

0.703

0.074

−0.213

KLG

0.324

−0.223

KWD

0.277

a,bCorrelatio

ncoefficientvaluegreaterthan

0.20

5and0.26

2issign

ificantat

P<0.05

andP<0.01

prob

ability

level,respectiv

ely

Table 4 Eigenvectors, eigenvalues, individual and cumulative per-centages of variation explained by the first six principal components(PC) after assessing morphological traits in Turkish maize landraces

Eigenvectors

Variables PC1 PC2 PC3 PC4

DTS 0.28 0.07 −0.09 −0.12PTH 0.27 0.18 −0.21 0.01

EHT 0.25 0.16 −0.32 −0.01SDI 0.27 −0.01 −0.11 −0.07AUL −0.17 −0.16 0.10 0.32

NL 0.29 0.07 −0.09 −0.09LFA 0.27 0.11 −0.15 −0.03PBNT 0.09 −0.10 −0.40 0.41

ELG 0.28 0.06 0.01 0.08

EDI 0.24 −0.09 0.28 −0.18ERN 0.16 −0.52 −0.03 −0.31NKR 0.25 −0.05 0.10 0.35

TNKE 0.26 −0.34 0.07 0.00

EPLG 0.17 0.14 −0.24 0.26

EWE 0.28 −0.03 0.27 −0.05PYL 0.27 −0.01 0.23 0.05

KLG 0.21 0.11 0.47 0.03

KWD 0.00 0.58 0.32 0.19

KT −0.06 0.35 −0.17 -0.58

Eigenvalue 10.43 2.12 1.47 1.04

Percent 54.90 11.15 7.75 5.46

Cumulative percentages 54.90 66.05 73.80 79.26

268 Plant Mol Biol Rep (2012) 30:261–274

regionally prevailing conditions of climate, soil andmanagement. Landraces are conspicuously diverse in theirgenetic composition both between populations and withinthem (Brown 1978; Angioi et al. 2011). Landraces mightcontain novel favorable alleles for agronomic traits such asyield, pest resistance and abiotic stress tolerance (Harlan1975; Zeven 1998). Knowing the extent and nature of

available diversity of crop landraces is of paramountimportance for their potential utilization in crop improve-ment and in conservation programs. Besides investigatingpolymorphisms at the DNA level, evaluation of phenotypicvariations for agronomical important traits is a prerequisitefor effective utilization of maize genetic resources (Sharmaet al. 2010).

In this work we have reported a diversity analysis of acollection of Turkish maize landraces, both at the genotypicand the phenotypic level. All landraces were grown underthe same environmental conditions in order to eliminategenotype×environment interaction. The results of our studyare in good agreement with those of previous studies,underlining the inherent heterogeneity of maize. Agronom-ical and morphological variation in maize landraces havebeen studied previously by researchers from differentcountries such as India (Sharma et al. 2010), Korea (Huhand Moon 2001), Italy (Brandolini and Brandolini 2001;Hartings et al. 2008), Ethiopia (Beyene et al. 2005), Spain(Ruiz de Galarreta and Alvarez 2001), Canada (Azar et al.1997), Portugal (Angelo et al. 2008), France (Gouesnard etal. 1997), Zimbabwe, Zambia, Malawi (Magorokosho2006) as well as Turkey (Ilarslan et al. 2002). The widediversity present in the Turkish maize landraces intended tocomplement the ongoing efforts for maize improvementprograms. In the present study, the cluster analysis based onagro-morphological data could not effectively discriminatelandraces originating from different regions of Turkey. Thisreinforces the limitation of such data for revealing patternsof genetic structure among heterogeneous maize popula-tions (Rebourg et al. 2001; Sharma et al. 2010). Neverthe-less, phenotypic characterization is an important aspect ofsampling strategy when germplasm is being collected froma diverse range of environments (Bogyo et al. 1990).Morphological variation can of course be of directapplication in a breeding context—for example, the widevariation in flowering time (50–74 days) in these landracesindicates a wealth of allelic variation of relevance to thedevelopment of cultivars adapted to regions of Turkeywhich have short growing seasons. Further multilocationtrials conducted over several years will be needed toevaluate this germplasm in more detail for selecting thesuitable genotype, specifically with a view to understandingthe contribution of G×E to phenotypic diversity, and tostudy the association between different characteristics.

Relationships between traits were investigated usingcorrelation coefficient estimations and PCA. Correlationbetween different traits is generally due to the presence oflinked genes and the epistatic effect of different genes.Environment plays an important role in correlation. In somecases, environment affects both the traits simultaneously in thesame direction or sometimes in different directions (Yücel etal. 2009). Selection for right character is also important

Fig. 2 Association among 89 Turkish maize landraces as revealed byUPGMA cluster analyses based on 19 morphological and agronomictraits

Plant Mol Biol Rep (2012) 30:261–274 269

because of correlation among different traits. For example,positive correlation among yield and kernel physical traitssuch as kernel width, kernel length and kernel thicknessshowed that improvement in kernel physical properties alsoresults in improvement in plant yield. Negative associationof days to tasseling with plant and ear height could be usedfor developing early maturing and short statured cultivarswith thicker grains. Similarly, several morphological oragronomic traits were positively correlated with each other,possibly pointing to common pathways. Positive associationamong different traits showed that improvement of onecharacter may simultaneously improve the other desired trait.Therefore, selection for right character is also importantbecause of correlation among different traits. However,results require careful verification by testing the germplasmunder different agro-climatic conditions

Molecular markers have greatly aided the assessment ofgenetic diversity in a number of crop species (Baloch et al.2010; Bourguiba et al. 2010; Uzun et al. 2011; Bang et al.2011). Microsatellites have proven to be particularly good

for this purpose, given their informativeness, co-dominance, ubiquity in the genome and assay simplicity(Reif et al. 2005; Beyene et al. 2006; Hartings et al. 2008).When attempting to genotype populations rather thanindividuals, various sampling strategies are possible, andhere we adopted a pooling strategy, the pros and cons ofwhich have been widely debated (Rebourg et al. 2001; Reifet al. 2005; Eschholz et al. 2008; Liu et al. 2005). Anexample of its use in maize has been provided by Eschholzet al. (2008), who suggested on cost-effectiveness groundsthat the size of each pool should be 15 individuals, whichwas the size chosen here. Dubreuil et al. (2006) alsoreported that bulk sampling method is highly repeatable andreliable, and allele frequencies estimated within the poolrepresenting a given population were very similar withindividuals. Despite some significant advantages, DNAbulk analysis also presents some technical limitations, dueto PCR artefacts, that induce genotyping errors and mis-estimation of allele frequencies such as preferential ampli-fication of some alleles, allele dropout or unclear SSR

SSR locus Repeat unit Bin no. N Size range PIC

phi339017 AGG 1.03 5 161–180 0.720

phi227562 ACC 1.11 5 329–347 0.731

phi083 AGCT 2.04 6 139–155 0.703

phi127 AGAC 2.08 5 117–142 0.575

phi029 AG/AGCG 3.04 6 157–180 0.649

phi053 ATAC 3.05 8 173–212 0.840

umc1399 (CTAG)5 3.07 6 124–141 0.805

phi046 ACGC 3.08 4 72–83 0.623

phi006 CCT 4.11 6 97–111 0.602

umc1143 AAAAT 6.00 8 82–101 0.796

phi423796 AGATG 6.01 6 132–157 0.664

phi123 AAAG 6.07 6 153–166 0.763

phi089 ATGC 6.08 11 89–145 0.889

umc1545 (AAGA)4 7.00 7 85–100 0.811

phi034 CCT 7.02 7 131–134 0.707

phi114 GCCT 7.03 5 153–188 0.690

phi328175 AGG 7.04 5 135–151 0.600

phi121 CCG 8.03 4 111–119 0.800

umc1161 (GCTGGG)5 8.06 8 148–172 0.875

phi233376 CCG 8.09 10 113–173 0.820

umc1279 (CCT)6 9.00 7 97–116 0.768

phi065 CACTT 9.03 5 151–171 0.655

phi108411 AGCT 9.05 6 127–142 0.785

phi448880 AAG 9.06–9.07 7 189–209 0.785

phi050 AAGC 10.03 4 92–102 0.674

phi062 ACG 10.04 4 176–179 0.327

umc1061 (TCG)6 10.06 6 109–126 0.709

umc1196 CACACG 10.07 7 147–178 0.723

Mean 174/6.21 0.717

Table 5 Information for 28 SSRmarker loci used in this study,including name, repeat units, binlocation (Bin no.), number ofalleles (N), size range in bp andPIC values for 90 Turkish maizelandraces

270 Plant Mol Biol Rep (2012) 30:261–274

bands on acrylamide gel (Dubreuil et al. 2006; Yao et al.2007). However, we observed that genotyping of singlebulk of 15 plants per landrace with fluorescent dye-labeledSSR markers using ABI DNA Analyzer was sufficientlyaccurate. Our results also suggested that bulk DNAsampling analysis is rapid, efficient, labor saving, andinexpensive, and can successfully assess genetic diversityand genetic relationship among maize landraces.

One measure of genetic diversity at the genotypic levelis the number of alleles present at each locus. Here, themean number of alleles per SSR was very similar to thosereported Qi-Lun et al. (2008), who found 6.4 alleles perlocus using 45 SSRs in maize landraces from Wulingmountain region in China. Yao et al. (2007) detected anaverage of 6.1 alleles per locus for 42 SSRs among 54maize landraces from southwest China. Warburton et al.(2002) found an average of 6.3 alleles using 85 SSRsamong seven CIMMYT populations, while Reif et al.

(2005) reported a mean of 5.9 alleles in European flintmaize populations. Average number of allele per locus inthis study was lower than the findings of Dubreuil et al.(2006), who discovered 7.8 alleles per SSR locus among aset of 144 North American, South American and Caribbeanpopulation. In comparison with our results, lower valueshave been found for landraces from Northeastern Argentina(4.78; Bracco et al. 2009), for 38 waxy maize landracesfrom Yunan and Guizhou provinces of China (4.1; Liu et al.2005), highland Ethiopian maize accessions (4.9; Beyene etal. 2006), African maize inbred lines (3.85; Legesse et al.2007), maize inbreds from Eastern Croatia (4.2; Jambrovicet al. 2008), tropical maize germplasm (5.2; Laborda et al.2005), Mexico and Venezuela landraces (5.6; Matsuoka etal. 2002), maize inbred lines from China (4.1; Xie et al.2008). Wu et al. (2004) reported an average of 5.4 alleles inChinese popcorn landraces based on 61 SSR primers. Ahigh number of alleles per locus, as was uncovered in the

Fig. 3 Genetic diversity among Turkish maize landraces. Neighbor Net (NNet) planar graph of 90 Turkish landraces and three inbred lines basedon 172 polymorphic SSR marker

Plant Mol Biol Rep (2012) 30:261–274 271

present landraces, suggests a plentiful level of diversity,which presumably reflects and could be explained by earlyintroduction of maize in Turkish agriculture. However, thispresumption needs to be treated with some caution as theset of SSRs was specifically chosen based on a high levelof informativeness. Previous studies have shown that maizecontains an abundant amount of SSRs, and these markersare highly polymorphic in maize populations and even ininbred corn (Enoki et al. 2002; Warburton et al. 2002; Xiaet al. 2004)

PIC provides a somewhat better estimator of diversitythan the raw number of alleles, because it takes account ofthe relative frequencies of each allele present (Laborda etal. 2005). Here, the mean PIC was 0.72, with 26 of the 28SSR loci having a PIC >0.6. For comparison, average PICvalue obtained in Turkish maize landraces were larger thanmaize landraces from Japan (0.69) using 60 SSRs (Enoki etal. 2002), India (0.60) using 42 SSRs (Sharma et al. 2010),Ethiopia (0.61) using 20 SSRs (Beyene et al. 2006) and USmaize germplasm (0.59) using 70 SSR (Senior et al. 1998).Lower PIC values have been found, e.g., for Iranian inbredlines (0.54) using 46 SSR loci (Choukan et al. 2006). Thissuggests that SSR markers can be successfully applied inevaluation of maize germplasm diversity studies.

The cluster analyses based on both the genotypic andphenotypic data failed to cluster landraces on the basis oftheir provenance (Figs. 2 and 3), unlike the experience ofYao et al. (2007) in an analysis of Chinese maize landraces.Reasons for the non-relatedness of landraces from the sameregion could be due to selection of unconscious favorablealleles by farmers with better adaptation to local agro-climatic conditions and exchange of seeds by farmers fromdistant regions. Migration of landraces among regions,followed by mixing and introgression with preexistinggermplasm, could be another reason.

This plentiful diversity at both the genotypic andphenotypic levels may flow largely from the fact that maizeis an open pollinating species. However, in addition, thelandraces were sampled from a very variable set ofenvironmental and geographical locations (Fig. 1), whichalso tends to increase the level of diversity (Angelo et al.2008). Maize is cultivated in Turkey under a variety ofconditions that differ for temperature, soil, precipitation,intercropping condition, rotational practices and localagricultural practices. Conscious and unconscious selectionby farmers is one of the main factors affecting the diversity.Various authors reported that multiple factors such asbiological, socio-cultural, economic as well as price policyfactors influence a farmer’s decision to select, replace ormaintain a particular variety at any given time (Jarvis et al.2000). In the process of planting, managing, harvesting andselecting seeds, farmers make crucial decisions that affectgenetic diversity of crop populations over time.

Conclusion

In summary, we have provided some detail regarding thegenetic diversity of Turkish maize landraces, which havehad not been much exploited to date by local breedingprograms. The data provide an objective means ofidentifying and preserving the diversity of this germplasm.According to an unpublished field survey carried out in theBlack Sea region during 2007, a few farmers still growmaize landraces in certain restricted mountainous areas,thanks mainly to their better adaptation to the localenvironment than is achieved by commercial hybrids.Maintaining locally well adapted landraces would be anasset for our future and may contribute to Turkish maizebreeding programs as well as other growers worldwideinterested in Turkish maize genetic resources.

Acknowledgement We thank the Menemen gene bank (AegeanAgricultural Research Institute, Izmir, Turkey) for the kind provisionof landrace seed stocks. The authors express their gratitude toTÜBİTAK (The Scientific and Technological Research Council ofTurkey, TOVAG-104O186) and University of Cukurova, ScientificResearch Projects Unit (ZF2004BAP17) for their financial support.

References

Angelo M, Pinheiro de Carvalho A, Ganança JFT, Abreu I, Sousa NF,Marques dos Santos TM, Vieira MRC, Motto M (2008)Evaluation of maize (Zea mays L.) diversity on the Archipelagoof Madeira. Genet Resour Crop Evol 55:221–223

Angioi SA, Rau D, Nanni L, Bellucci E, Papa R, Attene G (2011) Thegenetic make of the European landraces of the common bean.Plant Genetic Resources: Characterization and Utilization 1–5.doi:10.1017/S1479262111000190

Azar C, Mather DE, Hamilton RI (1997) Maize landraces of the St.Lawrance-Great lakes region of North America. Euphytica98:141–148

Baloch FS, Kurt C, Arıoğlu H, Özkan H (2010) Assaying of diversityamong soybean (Glycin max (L.) merr.) and peanut (Arachishypogaea L.) genotypes at DNA level. Turk J Agric For 34:285–301

Bang TC, Raji AA, Ingelbrecht IL (2011) A multiplex microsattelitemarker kit for diversity assessment for large cassava (Manihotesculenta Crantz) germplasm collection. Plant Mol Biol Rep.doi:10.1007/s11105-010-0273-2

Baraket G, Chatti K, Saddoud O, Abdelkarim AB, Mars M, Trifi M,Hannachi AS (2011) Comparative SSR and AFLP markers forevaluation of genetic diversity and conservation of fig. Ficuscarica. Genetic resources in Tunisia. Plant Mol Biol Rep 29:171–184

Beyene Y, Botha AM, Myburg AA (2005) A comparative study ofmolecular and morphological methods of describing geneticrelationships in traditional Ethiopian highland maize. Afr JBiotechnol 4(7):586–595

Beyene Y, Botha AM, Myburg AA (2006) Genetic diversity amongtraditional Ethiopian highland maize accessions assessed bysimple sequence repeat (SSR) markers. Genet Resour Crop Evol00:1–10

Bogyo BT, Proceddu E, Perrino P (1990) Analysis of samplingstrategies for collecting genetic materials. Econ Bot 34:11–86

272 Plant Mol Biol Rep (2012) 30:261–274

Botstein D, White RL, Skolnick M, Davis RW (1980) Construction ofgenetic linkage map in man using restricted fragment lengthpolymorphism. Am J Human Genet 23:314–331

Bourguiba H, Krichen L, Audergon JM, Khadari B, Trifi-Farah N(2010) Impact of mapped SSR markers on the genetic diversityof apricot (Prunus armeniaca L.) in Tunisia. Plant Mol Biol Rep28:578–587

Bracco M, Lia VV, Gottlieb AM, Hernandez JC, Poggio L (2009)Genetic diversity in maize landraces from indigenous settlementsof Northeastern Argentina. Genetica 135:39–49

Brandolini A, Brandolini A (2001) Classification of Italian maize (Zeamays L.) germplasm. Plant Genet Resour Newslett 126:1–11

Brown AHD (1978) Isozymes, plant population genetics structure andgenetic conservation. Theor Appl Genet 52:145–157

CIMMYT/IBPGRI (1991) Descriptor of maize, RomeChoukan R, Hossainzadeh A, Ghannadha MR, Talei AR, Mohammadi

SA, Warburton ML (2006) Use of SSR data to determinerelationships and potential heterotic groupings within mediumto late maturing Iranian maize inbred lines. Field Crops Res95:212–222

Dubreuil P, Rebourg C, Merlino M, Charcosset A (1999) The DNApooled sampling strategy for estimating RFLP diversity of maizepopulation. Plant Mol Biol Rep 17:123–138

Dubreuil P, Warburton M, Chastanet M, Hoisington D, Charcosset A(2006) More on the introduction of temperate maize into Europe:large scale bulk SSR genotyping and new historical elements.Maydica 51:281–291

Dice LR (1945) Measures of the amount of ecologic associationbetween species. Ecology 26:297–302

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh leaftissue. Focus 12:13–15

Ege H, Karahocağil P (2001) Yemlık Tahıllar Arpa, Mısır durum vetahmin 2001/2002 TEAE Yayını No 82, Ankara

Eschholz TW, Peter R, Stamp P, Hund A (2008) Genetic diversity ofSwiss maize (Zea mays L) assessed with individual and bulks onagarose gel. Genet Resour Crop Evol 55:971–983

Enoki H, Sato H, Koinuma K (2002) SSR analysis of genetic diversityamong maize inbred lines adapted to cold regions of Japan.Theor Appl Genet 104:1270–1277

FAOSTAT (2009) http://faostat.fao.org/Gouesnard B, Dallard J, Panouille A, Boyat A (1997) Classification of

French maize populations based on morphological traits. Agron-omie 17:491–498

Harlan JR (1975) Our vanishing genetic resources. Science 188:618–621

Hartings H, Berardo N, Mazzinelli GF, Valoti P, Verderio A, Motto M(2008) Assessment of genetic diversity and relationship amongmaize (Zea mays L.) Italian landraces by morphological traits andAFLP profiling. Theor Appl Genet 117:831–842

Huson DH, Bryant D (2006) Application of phylogenetic networks inevolutionary studies. Mol Biol Evol 23:254–267

Huh MK, Moon SG (2001) Chlorophyll deficient gene and morpho-logical variations in Korean populations of maize (Zea mays). JPlant Biol 44(3):141–147

Ilarslan R, Kaya Z, Tolun AA, Bretting PK (2001) Genetic variabilityamong Turkish Pop, Flint and Dent corn (Zea mays L. spp. mays)races: enzyme polymorphism. Euphytica 122:171–179

Ilarslan R, Kaya Z, Kandemir I, Bretting PK (2002) Geneticvariability among Turkish flint, pop and dent corn (Zea maysL) races. Morphological and agronomic traits. Euphytica128:173–182

Jambrovic A, Simic D, Ledencan T, Zdunic Z, Brkic I (2008) Geneticdiversity among maize (Zea mays L.) inbred lines in EasternCroatia. Period Biol 110(3):251–255

Janick J, Caneva G (2005) The first images of maize in Europe.Maydica 50:71–80

Jarvis DI, Meyer L, Klemick H, Guarino L, Smale M, Brown AHD,Sadiki M, Sthapit B, Hodgkin T (2000) Training guide for in situconservation on-farm. Biodiversity International. InternationalPlant Genetic Resources Institute, p 161

Kırtok Y (1998) Mısır Üretimi ve Kullanımı. Kocaoluk Basımı veYayınevi (In Turkish)

Kün E (1985) Tahıllar II (Sıcak iklim tahıllar). Ankara ÜniversitesiZiraat Fakultesi Yayınları 953. Ders Kitabı 275. Ankara Üniv.Basımevi, Ankara (In Turkish)

Kün E (1994) Tahıllar II (Sıcak iklim tahıllar). Ankara ÜniversitesiZiraat Fakultesi Yayınları 1360:141–206 (In Turkish)

Laborda PR, Oliveira KM, Garcia AAF, Paterniani MEAGZ, de SouzaAP (2005) Tropical maize germplasm: what can we say about itsgenetic diversity in the light of molecular markers? Theor ApplGenet 111:1288–1299

Legesse BW, Myburg AA, Pixley KV, Botha AM (2007) Geneticdiversity of African maize inbred lines revealed by SSR markers.Hereditas 144:10–17

Leng ER, Tavcar A, Trifunovic V (1962) Maize of SoutheasternEurope and its potential value in breeding programs elsewhere.Euphytica 11:263–272

Liu YJ, Huang YB, Rong TZ, Tian ML, Yang JP (2005) Comparativeanalysis of genetic diversity in land-races of waxy maize fromYunnan and Guizhou using SSR markers. Sci Agric Sin 4:648–653

Matsuoka Y, Mitchell SE, Kresovich S, Goodman M, Doebley J(2002) Microsatellites in Zea—variability, patterns of muta-tions, and use for evolutionary studies. Theor Appl Genet104:436–450

Magorokosho C (2006). Genetic diversity and field performance ofmaize varieties from Zimbave, Zambia and Malawi. Dissertation,Texas A&M University, South Africa

Okumus A (2007) Genetic variation and relationship between Turkishflint maize landraces by RAPD markers. Am J Agri Biol Sci 2(2):49–53

Ögel B (2000) Türk Kültür tarihine Giriş Cilt-II. ‘Türk köy ve Şehirhayatı GökTürklerden Osmanlılara’. T.C. Kültür BakanlığıYayınları/638 Yayınlar Başkalığı Kültür Eserleri Dizisi/46 (inTurkish)

Özkan H, Kafkas S, Ozer MS, Brandolini A (2005) Geneticrelationship among South-East Turkey wild barley populationand sampling strategies of Hordeum spontaneum. Theor ApplGenet 112:12–20

Pressoir G, Berhaud J (2004) Patterns of population structure in maizelandraces from the Central Valleys of Oaxaca in Mexico.Heredity 92:88–94

Qi-Lun Y, Ping F, Shu-Xian Z (2008) Constructing a core collectionfor maize (Zea mays L.) landrace from Wuling mountain regionin China. Agr Sci China 7(12):1423–1432

Rao NK (2004) Plant genetic resources: advancing conservation anduse through biotechnology. Afr J Biotechnol 3:136–145

Rebourg C, Gousnard B, Charcosset A (2001) Large scale molecularanalysis of European maize populations. Relationship withmorphological variation. Heredity 86:576–584

Reif JC, Hamrit S, Heckenberger M, Schipprack W, Peter MaurerH, Bohn M, Melchinger AE (2005) Genetic structure anddiversity of European flint maize populations determined withSSR analysis of individual and bulks. Theor Appl Genet111:906–913

Rohlf FJ (2004) NTSYS-pc ver 2.11 T. Exter Software. Satauket, NYRuiz de Galarreta JI, Alvarez A (2001) Morphological classification of

maize landraces from Northern Spain. Genet Resour Crop Evol48:391–400

Saeed A, Hovsepyan H, Darvishzadeh R, Imtiaz M, Panguluri SK,Nazaryan R (2011) Genetic diversity of Iranian accession,improved lines of chickpea (Cicer arietinum L.) and their wild

Plant Mol Biol Rep (2012) 30:261–274 273

relatives using simple sequence repeats. Plant Mol Biol Rep.doi:10.1007/s11105-011-0294-5

SAS Institute (2002) The SAS system for windows, release 9.0. SASInstitute Inc, Cary

Senior ML, Murphy JP, Goodman MM, Stuber CW (1998) Utility ofSSRs for determining genetic similarities and relationships inmaize using agarose gel system. Crop Sci 38:1088–1098

Sharma L, Prasanna BM, Ramesh B (2010) Analysis of phenotypicand microsattelite-based diversity of maize landraces in India,especially from the North East Himalayan region. Genetica.doi:10.1007/s10709-010-9436-1

Sharma SS, Negi MS, Sinha P, Kumar K, Tripathi SB (2011)Assessment of genetic diversity of biodiesel species pongamiapinnata accessions using AFLP and three endonuclease-AFLP.Plant Mol Biol Rep 29:12–18

Schuelke M (2000) An economic method for the fluorescentlabelingof PCR fragments. Nat Biotechnol 18:233–234

Smith JSC, Smith OS (1992) Fingerprinting crop varieties. Adv Agron47:85–140

Taşdan K (2005) Maize market in turkey. PhD thesis, Institute ofNatural and Applied Sciences University of Çukurova, Adana,Turkey

Uzun A, Yesiloglu T, Polat I, Kacar YA, Gulsen O, Yildirim B, TuzcuO, Tepe S, Canan I, Anil S (2011) Evaluation of genetic diversity

in lemon and some of their relatives based on SRAP and SSRmarkers. Plant Mol Biol Rep. doi:10.1007/s11105-010-0277-y

Warburton ML, Xia XC, Crossa J, Franco J, Melchinger AE, FrischM, Bohn M, Hoisintong D (2002) Genetic characterization ofCIMMYT maize inbred lines and open pollinated populationsusing large scale fingerprinting methods. Crop Sci 42:1832–1840

Wu YS, Zheng YL, Sun R, Wu SY, Gu HB, Bi YH (2004) Geneticdiversity of waxy corn and popcorn landraces in Yunan by SSRmarkers. Acta Agron Sin 30:36–42

Xia X, Reif J, Hoisington D, Melchinger A, Frisch M, Warburton M(2004) Genetic diversity among CIMMYT maize inbred linesinvestigated with SSR markers: I. Lowland tropical maize. CropSci 44:2230–2237

Xie C,WarburtonM, LiM, Li X, XiaoM, Hao Z, Zhao Q, Zhang S (2008)An analysis of population structure and linkage disequilibrium usingmultilocus data in 187 maize inbred lines. Mol Breed 21:407–418

Yao Q, Yang K, Pan G, Rong T (2007) Genetic diversity of maize (Zeamays L.) landraces from Southwest China based on SSR data. JGenet Genom 34(9):851–860

Yücel C, Baloch FS, Özkan H (2009) Genetic analysis of somephysical properties of bread wheat grain (Triticum aestivum L.em Thell). Turk J Agric For 33:525–535

Zeven AC (1998) Landraces: a review of definition and classification.Euphytica 104:127–139

274 Plant Mol Biol Rep (2012) 30:261–274