Genetic Characterization of Maize Landraces from Central ... · Diplomarbeit von Santiago J....

Universität Hohenheim

Institut für Physiologie und Biotechnologie der Pflanzen

Prof. Dr. Andreas Schaller

Genetic Characterization of Maize Landraces

from Central and South America

Diplomarbeit von Santiago J. Ramírez Aguilar

Betreut von Dr. Susanne Dreisigacker und Dr. Marilyn Warburton

(CIMMYT)

Stuttgart, November 2006

- 1 -

Table of Contents

1. Introduction 3

1.1 Importance of maize in the world 3

1.2 The genus Zea: the relatives of maize and its origin 4

1.3 Conservation of the diversity of maize of the Americas 8

1.4 Genetic Characterization of maize using molecular markers 9

2. Materials and Methods 12

2.1 Plant Materials 12

2.1.1 Germination and Harvest 13

2.1.2 Lyophilization 17

2.2 DNA Fingerprinting 17

2.2.1 DNA Extraction 17

2.2.2 DNA Quantification 19

2.2.3 DNA Quality control 19

2.2.4 Polymerase Chain Reaction (PCR) 20

2.2.5 DNA Electrophoresis 23

2.3 Statistical Methods 24

3. Results 27

3.1 DNA Quality Control 27

3.2 Allelic frequencies and genetic distances 27

3.3 Neighbor Joining Clustering 28

3.4 Mean genetic distance within groups of populations 35

3.5 Principal Components Analysis 36

3.6 Binary analysis of frequent and rare alleles 39

4. Discussion 40

- 2 -

4.1 Characterization of the groups and the landraces 40

4.1.1 Genetic relationships within and among groups of

populations 40

4.1.2 Genetic diversity within and among populations 53

4.1.3 Conclusion 54

4.2 Critical evaluation 55

5. Appendices 60

5.1 References 60

5.2 Tables 64

5.3 Figures 67

5.4 Abbreviations 70

6. Acknowledgements 73

7. Eidesstattliche Erklärung 74

- 3 -

1. Introduction

1.1 Importance of maize in the world

As many important cultivated crops, maize belongs to the family of Poaceae.

This family has a large agricultural importance, because it includes the three

most important sources for staple foods in the world, which are rice, wheat and

maize. According to the current statistics from the Food and Agriculture

Organization (FAO) of the United Nations (table 1.1), these three crops deliver

almost half of the calories (45%) consumed worldwide through direct human

consumption. Maize alone delivers 6% of the total calories in the world, and in

some regions of the world it is the principal source of energy in food. In Latin

America, and especially in Mexico, northern Central America and northern

South America maize is the principal carbohydrate source. Among indigenous

populations of Latin America, maize has an additional cultural and

socioeconomic significance. Outside America, maize is especially important as

a human food source in many Sub-Saharan African Countries, in some of which

(Malawi, Zambia and Lesotho) it delivers half of the total ingested calories

(48%).

Moreover, according to the FAO, maize is the crop with the highest average

yield per hectare and with the highest total production worldwide. However

maize production does not reach its highest yield productivity in most

developing countries, since poor farmers usually cultivate maize for self

consumption under difficult environmental conditions, such as low rainfall or

emergence of different diseases. Additionally, they cannot afford the technology

and do not have the infrastructure to reach the optimum productivity. It is

therefore important to improve new maize varieties that are especially

developed for specific environments in those regions in which they will be

cultivated.

- 4 -

Table 1.1: Calorie consumption in the sixteen countries with the highest percentage of maize consumption per person per day (data from the FAO, November 2006)

Country

Calory consumption per day per capita 2002-

2004

Percentage calories delivered by maize

Country

Calory consumption per day per capita 2002-

2004

Percentage calories delivered by maize

Malawi 2120 49.34 Venezuela 2340 19.96

Zambia 1950 48.41 Ethiopia 1850 19.51

Lesotho 2580 48.10 Cameroon 2260 17.61

Guatemala 2230 38.97 Paraguay 2530 17.39

Zimbabwe 1980 36.36 Angola 2120 16.75

Kenya 2150 36.05 Benin 2590 16.22

South Africa 2980 35.64 Seychelles 2460 15.33

El Salvador 2560 34.96 Burkina Faso 2500 15.12

Mexico 3170 34.10 Swaziland 2300 13.43

Bosnia and Herzegovina

2730 33.88 Ghana 2690 13.05

Tanzania, United Rep of 1960 32.96 Burundi 1660 12.65

Moldova, Republic of 2720 32.02 Uganda 2370 12.19

Honduras 2340 25.26 Colombia 2580 12.09

Togo 2350 25.19 Central African

Republic 1960 11.99

Jordan 2730 24.14 Democratic Republic

of Congo 1590 11.70

Mozambique 2080 24.04 Botswana 2150 10.79

Nicaragua 2290 21.57 World 2731 6.53

1.2 The genus Zea: the relatives of maize and its origin

Maize shares the genus Zea together with four other species that are native to

Mexico and northern Central America (Buckler, 2006). These species are

named teosinte and include Zea diploperennis, Zea perennis (both grass

species found in the highlands of western Mexico), Zea luxurians (found in

Guatemala and Honduras), and Zea nicaraguensis. The species Zea mays

contains four annual subspecies including domesticated maize and three

teosinte wild taxa:

• Zea mays ssp. huehuetenangensis (Iltis and Doebley) Doebley, limited to

a few highlands of northwestern Guatemala.

• Zea mays ssp. mexicana (Schrader) Iltis, restricted to the highlands of

central and northern Mexico.

- 5 -

• Zea mays ssp. parviglumis (Doebley), is a common teosinte originated in

the middle and low elevations of the southwest of Mexico.

• Zea mays ssp. Mays (Doebley), which is what is generally referred to as

maize.

It is widely accepted that the teosintes are the wild relatives of domesticated

maize; Zea mays ssp. parviglumis being the most closely related subspecies

(Kato 1976; Doebly et al., 1984, Smith et al., 1985) (Figure 1.1). This teosinte

grows in the same regions of central and southern Mexico where maize is

cultivated and produces intergenetic hybridizations with maize resulting in a

fertile offspring that can be backcrossed again to maize or to teosinte. In

regions where teosinte is still part of the ecosystem today, such intergenetic

hybridizations can even lead to a loss or diminish positive traits in the offspring.

Therefore, teosinte has been considered as a weed by maize producers in

Mexico (Buckler, 2006). However, these hybridizations can also introduce

greater genetic diversity into maize and in some cases even introgress new

desirable traits.

The theory that maize originated from teosinte has also been considered in

detail by McClintock et al. (1981) and Kato (1984). Both studies have shown a

very similar chromosome knob configuration in teosinte and maize, and

supported the idea that gene flow from teosinte to maize occurred since the

beginning of maize cultivation. This gene flow is considered to be responsible

for the large genetic and phenotypic variability in maize today.

According to Kato (1984), maize was the product of several independent

domestication events that occurred 10 000 to 5 000 years ago in Mexico and

northern Central America. The author declared that the remarkable diversity in

current maize would not have been gained from a single domestication only.

Other important crops, such as rice (Second, 1982) and bean (Sonnante, 1994)

also evolved from multiple origins and contain large variation.

- 6 -

Figure 1.1: Zea mays ssp. parviglumis (teosinte) at CIMMYT, Mexico

However, later studies (Matsuoka, 2002b) showed that maize evolved from just

a single domestication event between 8000 and 10000 years ago. According to

Matsuoka, the diversification of maize races was the product of continuous

hybridizations among diverse populations of maize with teosinte in many

including novel environments. This resulted in the introgression of a large

diversity into maize and might have established the maize races known today.

The diversification is the expected result of hybridizations between maize and

teosinte, as teosinte contains higher genetic diversity than maize (Matsuoka et

al., 2002a). Teosinte has a much longer developing history than maize, thus,

teosinte has had more time to adapt to different environments. This allowed the

introgression of new traits into the different types of maize and selection within

them, creating many different morphological and physiological phenotypes in

maize (figures 1.2, 1.3, 1.4 and 1.5).

- 7 -

Figure 1.2: Diversity in maize varieties at CIMMYT

Figure 1.3: Diversity in maize varieties at CIMMYT

Figure 1.4: Ears of two different varieties of maize

- 8 -

1.3 Conservation of the diversity of maize of the Americas

Demands for increasing amounts of maize, in particular during the last 1000

years, lead to a continuous replacement of established maize races and

populations with improved varieties. Yields increased dramatically as breeders

moved away from open pollinated cultivars and began developing hybrids at the

beginning of the 20th century. Due to the fact that a limited number of maize

varieties have been included in that new hybrid concept, the genetic base in

most areas of intense cultivation dropped (Becker, 1993). If this trend would

have continued, many of the original landraces would have become extinct

today with the result of loosing a very important genetic potential. To conserve

and maintain wild relatives and early developed maize races and populations,g

germplasm banks were established in various places, including CIMMYT in

Mexico and the National Center for Genetic Resources Preservation (NCGRP)

in Colorado, USA (Taba, 2005). Networks for landrace conservation were

created together with new national germplasm banks in several Latin American

countries such as Argentina, Colombia and Peru, just to mention some of them.

These germplasm banks deposit duplicates of the materials at NCGRP and

CIMMYT. CIMMYT alone stores 24,463 accessions (Taba, 2005) and new

accessions are added continuously.

Having a given percentage of the genetic basis of domesticated maize stored

and maintained in ex-situ collections of germplasm banks, is only an initial step.

For the exploitation and further use of germplasm bank accessions in plant

breeding programs, their detailed characterization is necessary. Scientist and

gene bank managers alike are interested particularly in accessions collected in

the center of origin of any crop. In maize, many of the Mexican and South

American landraces have already been characterized using morphological

characteristics (Wellhausen, 1952, Goodman and Bird, 1977), chromosome

knobs (McClintock et al., 1981) and isozymes (Sánchez et al., 2000, Sánchez et

al., 2004). By characterizing the landraces of maize it is be possible to identify

the genetically most distinct varieties and use these in breeding programs in a

more effective way.

- 9 -

Figure 1.5: Morphological diversity of maize ears

1.4 Genetic characterization of maize using molecular markers

One way to investigate the relationship among diverse genetic materials, such

as maize accessions is the detection of their similarity or dissimilarity. These

measures are based and can be calculated using agronomic or morphological

characteristics of each accession and more recently using molecular markers.

Many marker technologies of are available to date. Examples are Amplified

Fragment Length Polymorphism (AFLP) marker, Restriction Fragment Length

Polymorphism (RFLP) markers, Random Amplified Polymorphic DNA (RAPDs),

and Simple Sequence Repeats (SSRs).

In addition to their genetic cause and their application, molecular markers can

be distinguished with respect to the allelic information they provide (Reif et al.,

- 10 -

2005). Non-informative markers or so called dominant markers show only the

presence or the absence of a specific allele, e.g. AFLPs. Allelic informative or

co-dominant markers are markers that can distinguish among alleles at a given

locus and allele frequencies can be calculated, such as with SSRs.

SSRs, also called microsatellites were chosen for this study They are a special

class of tandem repeat loci which contain motifs of one to eight base pairs

repeated up to 100 or 200 times. SSRs are highly polymorphic due to their high

minimal mutation rate of 10-3 or 10-4 (Goldstein et al., 1995). Normal mutation

rates in most organisms account on average for 10-5 or 10-6 (Falconer, 1989).

The high mutation rate of SSRs appears to be due to a slippage effect of the

template during DNA replication (Voet and Voet, 2004). The high degree of

polymorphism makes SSRs a very useful tool for diversity studies as shown in

many crops (Dreisigacker et al., 2004 for wheat; He et al., 2003 for tomato;

Diwan et al., 1997 for soybean, to mention some examples) and previous

studies in maize (Matsuoka et al, 2002a; Reif et al., 2005; Warburton et a.,

2002; Sharopova et al., 2002).

The quantification of similarity or dissimilarity between different plant accessions

is based on the principle of genetic distance. Reif et al., (2005) summarized the

genetic and mathematical properties of a set of distances that are suitable to

characterize plant materials. Generally it applies:

A dissimilarity d is called distance when it assumes the following properties:

( ) 0, ≥jid , and ( ) 0, =jid only if ji =

( ) ( )ijdjid ,, =

( ) ( ) ( )kjdjidkid ,,, +≤ , and ( ) ( ) ( )kjdjidkid ,,, += only if j is the offspring of i

and k.

for which d is the dissimilarity between the operational taxonomic units (OTUs,

e.g. a landrace accession) i and j, j and k or i and k.

One frequently used example is the Euclidian distance, defined by the equation:

- 11 -

( )2

1 1

∑∑=

= =

−=lL

l

A

a

j

al

i

alE

l

ppd ,

for which ialp and j

alp represent the frequency of allele a at locus l for the OTUs

i and j, Al the total amount of alleles at locus l, and L the total number of loci.

This distance represents a straight line distance between two OTUs which is

important for an unbiased demonstration of observed relationships.

The detection of the genetic diversity with SSR markers among Central and

South American maize landraces was the main objective within this study. The

knowledge of the relationships among maize landraces can help to further

understand the evolution of maize, optimize germplasm bank management e.g.

building core collections or identify duplications, and give indications of which

materials to consider for the identification of new traits that can be transferred

into maize.

- 12 -

2. Materials and Methods

2.1. Plant Materials

Hundred and one maize landraces from ten South American countries and

Guatemala were chosen for this study (Table 2.1. See supplementary table 3 at

http://www.cimmyt.org/english/webp/support/publications/support_materials/wor

d/table3-GermplasmInfo.xls for further information). The landraces are assumed

to represent common and typical maize races in the region or country where

they were collected and are supposed to have been cultivated by native groups

over a long period of time. The chosen landraces were concentrated more on

the western and northern parts of South America, and less in southern and

eastern South America. Northwest and Western South America include regions

where maize is know to be cultivated for more than 3000 years (Eubanks Dunn,

1979, McKelvy et al., 1990). There is even evidence that maize could have

been cultivated in these specific areas of the subcontinent for about 7000 years

(Staller & Thompson, 2001), although such an early date is very controversial.

The collection of landraces was therefore expected to have a good

representation of South American maize diversity. Guatemalan landraces were

added to allow for a comparison to Mesoamerican races. This region comprises

the descendants of the oldest landraces of maize. The collection site of each

landrace is shown on two maps in figures 2.1 and 2.2.

Figure 2.1: Map of Guatemala. Collection sites of eight maize landraces are marked with dots.

- 13 -

Figure 2.2: South America map. Collection sites of maize landraces are marked with dots.

2.1.1. Germination and harvest

The seeds for the landraces were provided by CIMMYT’s germplasm bank.

Fifteen seeds per landrace population were planted in pots in the greenhouse at

CIMMYT.1

Table 2.1: List of 101 maize landraces their country of origin, dedicated primary race, elevation, and grain type.

Country ID Name PrimaryRace Elevation Grain Type

1 Altiplano Altiplano 2500 dent 2 Chileno Manfredi Camelia 62 floury 3 Amarillo de ocho Capia Amarilla de Ocho n/a dent 4 Capie White Capia Blanco 2500 flint dent 5 Rosarini Cateto 1006 flint

Argentina

6 Culli Culli 2500 flint

1 Cultivation and harvest of plant tissue, as well as DNA extraction were done with help of the ABC staff from CIMMYT

- 14 -

Table 2.1: continued

7 Bolivia 90 Acre Interlocked 500 floury 8 Osco Aysuma 1947 flint floury 9 Blando Amazónico Cuzco cristal amarillo n/a floury 10 Gris Checchi 1976 dent 11 Chullpi Chuspillu 1947 dent floury 12 Morado Coroico 156 floury 13 Duro Amazónico Duro Amazónico n/a floury 14 Chejura Haulco 2900 floury 15 Hualtaco Hualtaco n/a dent flint 16 Huillcaparu Huillcaparu 2500 floury 17 Kellu Kellu 2700 floury 18 Kulli Kulli 2150 dent 19 Puca Sara Paru 3000 20 Pisankalla Pisankalla 1790 21 Amarillo Blando o Bayo Pojoso 1050

Bolivia

22 Uchuquilla Uchuquilla 2600 flint dent 23 Vermelho Duro Cateto Assis 274 flint 24 Brazil 60 Cateto Grande 500 dent 25 Amarelo Mole Dente Riograndense Rugoso 274 flint dent 26 Brazil 2305 Dente Paulista n/a dent 27 Brazil 2441 Cateto Pag. n/a flint 28 Cristal BP n/a dent 29 Brazil PE012 Dentado n/a flint

Brazil

30 Cateto Nortista Cateto Nortista n/a sweet floury

31 Ocho Corridas Ocho Corridas 250 dent 32 CHZM 09 030 Araucano 100 flint 33 Camelia Camelia 625 flint 34 Choclero Choclero 220 flint 35 Chulpe Chulpi 2300 flint dent

Chile

36 Curagua Pisankalla 450 flint 37 Maíz de Ano Amagaceño 1600 flint

38 Caquet. 321 Andaqui n/a flint

39 Chaparrito Cabuya 1920 flint

40 Amapolo Cacao 1252 flint

41 Cariaco Cariaco 229 flint

42 Blanco Común Chococeño 183 floury

43 Maíz Amarillo Común 896 flint

44 Blanco Costeño 7 floury

Colombia

45 Magdal. 443 Guirua 1860 flint

- 15 -


46 Nariño 534 Montaña n/a flint dent 47 Colombia 613 Negrito 540 flint dent 48 Calilla Pira n/a floury 49 Maicena Pira Naranja 1554 flint 50 Cundin 465 Pollo n/a dent 51 Puyita Puya 229 dent 52 Amarillo Puya Grande 978 flint 53 Blanco de Harina Sabanero 1960 flint 54 Tolima 389 Yucatán n/a 55 Boyaca 462 Pira n/a flint 56 Cundin 480 Pira n/a

Colombia

57 Porva Pira 2377 58 Canguil Canguil 2213 flint 59 Morocho Amarillo Chillo 2560 sweet floury 60 De Pollo Chococeño 27 61 Chulpi Chulpi 2579 flint floury 62 Morocho o Tusilla Kcello 1800 flint 63 Mishca Mishca 2286 flint 64 Morocho Huandango Montaña 2259 flint 65 Morocho Morochón 2195 dent 66 Renegrido Racimo de Uva 2423 floury flint 67 Ecuador X14237 Shima n/a flint

Ecuador

68 Uchima Uchima 1737 dent flint 69 Negro Negro Alt. 2316 flint dent floury

70 Llanero Negro Chimaltenango 1219 71 Amarillo NalTel Amarillo tierra baja 914 dent floury 72 Zapalote Chico Nal Tel blanco tierra alta 2774 dent flint 73 Oaxaqueño Nal Tel blanco tierra baja 1006 flint dent 74 Guatem. SH22 Olotón n/a flint 75 Pinto Quicheño 610 dent 76 Blanco Salpor 688 dent

Guatemala

77 Chimbo Blanco San Marceño 206 floury 78 Avati Caigua Avati Ti 240 flint pop 79 Avati Moroti (3 Meses) Avati Mita 210 flint 80 Avati-Tupi Avati Moroti Guapy 400 flint dent 81 Avati-Mita Avati Moroti Mita 140 floury 82 Avati-Moroti Avati-Moroti 400 floury 83 Avati-TI Avati Moroti Ti 230 84 Avati Pichinga Avati Pichinga 300

Paraguay

85 Avati Pichinga Avati Pichinga Ihu 200 floury 86 Maíz Común Ancashino 2900 flint 87 Chullpi Chullpi 2800 floury sweet 88 Llamellán Cuzco 3100 floury 89 Cuzco 56 Cuzco cristal amarillo 3268 floury 90 Cuzco 363 Cuzco cristal 3200 floury 91 Cuzco 77 Cuzco Gigante 3200 92 Poccho Cruzado Kculli 2300 93 Blanco Criollo Mochero 23 94 Amarillo L.M.(S.M.) Perla 253

Peru

95 Pun. 14 Uchuquilla 2000

- 16 -


96 Cuarentón Cateto Sulino Escuro 183 flint 97 Uruguay 1131A Cateto Sulino n/a dent 98 Colorado Cateto Sulino 183 flint

Uruguay

99 Uruguay 1187A Dentado Brasil. n/a dent 100 Cariaco Cariaco 101 floury

Venezuela 101 Cariaco Cariaco 365 floury

Four to six weeks after planting, when the plants were between 20 and 25 cm

tall, the second and the third leaf was harvested (Figure 2.3). Thick and tough

midribs were removed. The leaves of all fifteen individuals of each population

were cut to the same length (~10–15 cm) and put together in plastic screen

mesh bags in an ice container to keep the tissue cool and ventilated.

Figure 2.3: Maize plants before tissue harvest.

After harvest the samples were quick-frozen with liquid nitrogen and stored at

-80 ºC before lyophilization.

- 17 -

2.1.2. Lyophilization2

The samples containing the tissue from equal amounts of 15 plants were

transferred from a -80 ºC freezer to a lyophilizer (Luph-Look, Labconco). The

temperature of the vacuum chamber was always kept lower than -50 ºC and the

initial pressure was 10 µm Hg. During lyophilization, the pressure was always

kept under 100 µm Hg. After 72 hours, the dried samples were removed into

sealed plastic bags and stored at -20 ºC. The dried and frozen samples were

then ground to fine powder with a mechanical mill (Tecator Cyclotec Sample

Mill, Model 1093) in plastic scintillation vials. The vials were stored at -20 ºC

until DNA extraction.

2.2. DNA Fingerprinting

2.2.1. DNA Extraction2

Reagents for DNA Extraction:

• CTAB extraction buffer: 10 ml

amount final concentration

dH2O 6.5 ml

1 M Tris-HCl pH 7.5 1.0 ml 100 mM

5 M NaCl 1.4 ml 700 mM

0.5 M EDTA pH 8.0 1.0 ml 50 mM

CTAB 0.1 g 1% w/v

14 M β-mercaptoethanol 0.1 ml 140 mM

A stock solution was prepared containing tris-buffer, NaCl and EDTA and water.

Shortly before DNA extraction, β-mercaptoethanol and cethyltrimethyl-

ammonium bromide (CTAB, Sigma M-7635) were given to the stock solution to

result in freshly prepared CTAB extraction buffer.

2 Cultivation and harvest of plant tissue, as well as DNA extraction were done with help of the ABC staff from CIMMYT

- 18 -

• WASH 1: 100ml

volume final concentration

dH2O 16 ml

100% EtOH 76 ml 76%

2.5 M Sodium Acetate 8 ml 0.2 M

• WASH 2: 100 ml


dH2O 23 ml

100% EtOH 76 ml 76%

1 M Ammonium Acetate 1 ml 10 mM

• Chloroform:Octanol (24:1): 100 ml


Chloroform 96 ml 96%

Octanol 4 ml 4%

• 10X TE buffer: 10 ml


dH2O 8.8 ml

1 M Tris-HCl pH 7.5 1 ml 100 mM

0.5 M EDTA pH 8.0 200 µl 10 mM

Was diluted 1:10 with dH2O to get 1X TE buffer

The genomic DNA isolation was based on the method of Saghai-Maroof et al.

(1984). Dry tissue powder (300 to 400 mg) was gently mixed by inversion with 9

ml of warm (65 ºC) freshly made CTAB extraction buffer (prepared as described

above). The homogenized mixture was then incubated for 90 min at 65 ºC

under continuous agitation. After 90 min the tubes were removed from the oven,

cooled down, and 4.5 ml of chloroform/octanol (24:1) were added and mixed

with the buffer containing the tissue. The mixture was gently rocked for 5 to 10

min at room temperature and centrifuged for 10 min at 1500 g. At this step, it

was important to keep the extract above 15 ºC to avoid precipitation of the

- 19 -

DNA-CTAB complex. The top aqueous layer was poured into new 15 ml tubes,

4.5 ml of chloroform/octanol were added, and tubes were rocked gently for 5 to

10 min and centrifuged for 10 min at 1500 g at room temperature. The top

aqueous layer was transferred with a pipette to a new 15 ml tube containing 30

µl of 10mg/ml RNase A, mixed a few times by inversion, and incubated at room

temperature for 30 min. Six ml of isopropanol were added and gently mixed by

inversion for DNA precipitation. The precipitated DNA was then removed with a

glass hook and placed in a 5 ml plastic tube containing 1 ml of TE buffer. To

dissolve the DNA it was shaken gently overnight at room temperature. DNA was

again precipitated by adding 50 µl of 5 M NaCl and 2.5 ml 100% EtOH.

Precipitated DNA was then removed with a glass hook and placed in a new 5 ml

plastic tube with 3.5 ml of WASH 1 for 20 min. The hook with DNA was rinsed

with 2 ml of WASH 2 and transferred to a 2 ml centrifuge tube. 0.5ml TE buffer

was added and DNA was dissolved at room temperature overnight. The DNA

solution was stored at 4 ºC.

2.2.2. DNA quantification:

Fifteen µl of each DNA sample were diluted in 735 µl of TE buffer and mixed.

UV absorption at 260 nm and 280 nm was read with a Beckman DU-65

spectrophotometer and each DNA sample was subsequently diluted to a

working dilution with a concentration of 200 ng/µl. The equation to determine

the DNA concentration of a DNA sample is:

where OD260 is the, optical density of the DNA dilution measured at a

wavelength λ of 260nm.

2.2.3. DNA Quality control

Reagents for DNA qualification:

• 10 X TAE gel buffer pH 8: 1 l

amount final concentration

- 20 -

Tris Base 48.4 g 400 mM

Sodium Acetate 4.1 g 50 mM

Na4EDTA 2.92 g 7.7 mM

Glacial acetic acid to adjust to pH 8.0

Fill up with dH2O to 1 l

• Ethidium bromide solution 1 l


dH2O 1 l

Ethidium bromide 10 mg/ml 100 µl 1 mg/l

The quality of DNA was estimated based on the method of T. Helentjaris et al.

(1985) using agarose gels. For each agarose gel, 300 ml 1 X TAE gel buffer

was prepared by diluting 30 ml of the 10 X TAE stock with 270 ml dH2O. To

make a 0.7% agarose gel 2.10 g agarose were added to the TAE gel buffer and

heated under continuous stirring to dissolve the agarose. The solution was then

poured into a 20 × 25 cm gel tray. A thirty-well comb was inserted and after 20

to 30 min the gel was solidified. The gel was put into a rig and 1 X TAE gel

buffer was poured into the gel rig until the gel was covered by at least 0.5 cm.

DNA solutions from 20 populations were randomly chosen to check the DNA

quality. For each of these samples, 10 ng/µl dilutions were made and 10 µl (100

ng DNA) were loaded onto the gel. As a positive control, 10 µl of a 10 ng/µl

uncut λ-DNA were loaded. The samples were then run at 50 mA for 90 min.

After the run, the gel was removed from rig and stained in the ethidium bromide

solution for 20 minutes under gentle shaking. The stained gel was rinsed 20 min

in dH2O, exposed to UV light, and photographed.

2.2.4. Polymerase Chain Reaction (PCR)

For each SSR a reaction mix was prepared according to table 2.2, using the

following stock solutions:

Stock reagents for PCR master mix

ddH2O Sigma cell culture water, catalog # W-3500

10 X Taq buffer, Mg-free

MgCl solution 50 mM

- 21 -

dNTP Mix, 2.5 mM each

Taq Enzyme, 5 U/µl

• 10X Taq buffer Protocol (Mg2+-free) for 40 ml

Volume Concentration

ddH2O Sigma cell culture water 15.6 ml

1M KCl 20 ml 500 mM

1M Tris-HCl pH 8.5 4 ml 100 mM

Triton-X 0.4 ml 1%

Each SSR primer was fluorescent labeled. Three phosphoramidite fluorescent

dyes 6-carboxyflouresein (6-Fam), tetrachloro-6-carboxyflouresein (Tet) or

hexachloro-6- carboxyflouresein (Hex) were used as SSR labels.

Table 2.2: Format for the PCR Master mix of 34 SSRs used for genetic characterization

# PCR React'ns

SSR

Primer (ul/

react’n)

Buffer (ul)

NTPs (ul)

MgCl (ul)

Taq (ul)

Primer (ul)

Water (ul)

Total (ul/

react’n)

PCR program

3

120 nc133 1.5 120 144 48 18 180 510 8.5 SSR54

120 phi014 0.8 120 144 48 18 96 594 8.5 SSR64

120 phi029 1 120 144 48 18 120 570 8.5 SSR56

120 phi031 1 120 144 48 18 120 570 8.5 SSR60

120 phi034 1 120 144 48 18 120 570 8.5 Q62

120 phi046 1 120 144 48 18 120 570 8.5 Q60

120 phi059 1 120 144 48 18 120 570 8.5 Q58

120 phi062 1 120 144 48 18 120 570 8.5 SSR56

120 phi063 1 120 144 48 18 120 570 8.5 Q64

120 phi069 1 120 144 48 18 120 570 8.5 Q58

120 phi072 0.8 120 144 48 18 96 594 8.5 SSR56

120 phi075 2 120 144 48 18 240 450 8.5 Q54

120 phi076 2 120 144 48 18 240 450 8.5 Q60

120 phi079 1 120 144 48 18 120 570 8.5 SSR62

120 phi083 1 120 144 48 18 120 570 8.5 SSR54

120 phi084 0.8 120 144 48 18 96 594 8.5 SSR54

120 phi085 1 120 144 48 18 120 570 8.5 Q60

3 The number of every PCR program corresponds to the annealing temperature.

- 22 -


120 phi090 1.8 120 144 48 18 216 474 8.5 Q62

120 phi102228 1 120 144 48 18 120 570 8.5 SSR54

120 phi108411 1 120 144 48 18 120 570 8.5 Q60

120 phi109188 1 120 144 48 18 120 570 8.5 Q58

120 phi109275 1.5 120 144 48 18 180 510 8.5 SSR56

120 phi112 1 120 144 48 18 120 570 8.5 Q62

120 phi114 2 120 144 48 18 240 450 8.5 SSR62

120 phi115 1 120 144 48 18 120 570 8.5 SSR54

120 phi127 1 120 144 48 18 120 570 8.5 Q62

120 phi227562 2.5 120 144 48 18 300 390 8.5 SSR56

120 phi299852 1 120 144 48 18 120 570 8.5 Q60

120 phi308707 0.8 120 144 48 18 96 594 8.5 SSR54

120 phi331888 1 120 144 48 18 120 570 8.5 Q56

120 umc1196 0.6 120 144 48 18 72 618 8.5 SSR58

120 umc1266 1 120 144 48 18 120 570 8.5 Q52

120 umc1332 1.5 120 144 48 18 180 510 8.5 SSR60

120 umc2047 1.5 120 144 48 18 180 510 8.5 Q58

The sequences and their corresponding SSR repeat unit of all primers used in

this DNA fingerprinting study can be found on the web:

http://www.cimmyt.org/ambionet/85%20coremarkersfordiversitystudy.pdf.

A master mix was prepared in a 1.5 ml plastic tube containing all reagents

except the taq-polymerase. The taq-polymerase was added shortly before

adding the master mix to the DNA samples. All reagents and solutions were

kept on ice. For each PCR with a different primer set, 1.5 µl of a 20 ng/µl DNA

solution from every population bulk was mixed with 8.5 µl of the corresponding

master mix to get a 10 µl reaction mix. Ten microliters of mineral oil were added

to each tube containing the PCR reaction mix to avoid evaporation during the

PCR reaction. Amplification was carried out in MJ Research DNA Engine

Tetrad™ System Thermocyclers according to the PCR program of the SSR that

was amplified:

PCR Programs

SSR

- 23 -

1) Denature at 94 ºC for 2 min

⇒ 1 cycle of step 1

2a) Denature at 94 ºC for 1 min

2b) Annealing temperature for 2 min (54 ºC, 56 ºC, 58 ºC, 60 ºC, 62 ºC or

64ºC)

2c) Incubate at 72 ºC for 2 min

⇒ 30 cycles of steps 2a), 2b) and 2c)

3) Terminate at 72 ºC, 5 min

4) Store at 10 ºC

Quick SSR (Q)

1) Denature at 94 ºC for 2 min

⇒ 1 cycle of step 1

2a) Denature at 94 ºC for 30 sec

2b) Annealing temperature for 1 min (54 ºC, 56 ºC, 58 ºC, 60 ºC, 62 ºC or

64ºC, depending on the program chosen, e.g. 60 ºC for Q60)

2c) Incubate at 72 ºC for 1 min

⇒ 30 cycles of steps 2a), 2b) and 2c)

3) Terminate at 72 ºC, 5 min

4) Store at 10 ºC

2.2.5. DNA Electrophoresis

PCR products were separated based on the principle of electrophoresis using a

ABI PRISM 3100 capillary sequencer (Applied Biotechnology). Therefore,

Hi-Di™ formamide was mixed with 30 µl GS 500 ROX size standard, for every 1

ml formamide. To 1.2 µl of each PCR product nine micro liters formamide/size

standard mix were added. All samples were then denatured for seven min at

95 ºC and immediately put in ice. Some PCR products were run together by

multiloading the samples. This means that PCR products with different

expected fragment sizes and dyes were sampled and run together in the

- 24 -

sequencer to improve the efficiency of fingerprinting and reduce costs. Up to

five different PCR products were run at the same time. PCR products labeled

with the same dye were loaded together when the minimum size difference was

at least 10 base pairs to avoid errors in the subsequent detection and analyses

of fragments

2.3 Statistical Methods

Fragment sizes were calculated automatically with the computer software

GeneScan® 3.1 (Applied Biotechnology Systems) by comparing to fragments of

the internal size standard GS 500 ROX. GeneScan fragments were assigned to

alleles using the category function of the software Genotyper® 2.1 (Applied

Biotechnology system). Fragments were labeled with their size in base pairs,

peak height, category’s name (bin number), and a modulation score value

(MSV). The MSV is a value that characterizes the quality of a band, taking into

account the peak height and form in comparison to the gel background. The

lower the quality of a peak, the lower the MSV. The data labels were then

extracted and saved in form of tables.

A script written by CIMMYT for the software package R v. 2.2.1 was used to

calculate the allelic frequencies for each fragment based on its peak heights as

described in Dubreuil et al. (2006). This script first filters the fragments for low

peak heights and low MSV. Fragments with a peak height lower than 100

and/or MSV below 10 were not considered. Allelic frequencies p for each of the

residual fragments are then calculated by dividing the peak height of each

fragment by the sum of peak heights of all peaks observed within one

population and SSR marker.

∑=

=

=ni

i

i

l

l

a

ap

1

1

,

where 1

la is the allelic height of peak 1 in locus l. As fifteen individuals per

population were bulked, the maximum number of peaks expected was 30.

For some SSRs, peaks had to be corrected due to stuttering. The stutter ratios

of those SSRs were already determined by Dubreuil et al., 2006. The stutter

- 25 -

ratio r “is defined as the ratio of peak heights between the α-stutter band (the

stutter band one repeat unit smaller than the allele) and the allele itself. Each

locus-specific r value was estimated as the average of individual-allele

estimates (Dubreuil et al., 2006). It was assumed that r was constant for a

given locus among alleles and among experiments. In case of stuttering, for

each lane, observed peak heights were corrected sequentially starting from the

band with the highest molecular weight (MW), by using the following formulas:

nn QQˆ* ====

nnn QrQQˆˆ* −−−−==== −−−−−−−− 11

122 −−−−−−−−−−−− −−−−==== nnn QrQQˆˆ*

…

1++++−−−−−−−−−−−− −−−−==== mnmnmn QrQQˆˆ* ,

where n is the length in repeat units of the band with the highest MW, m is the

number of repeat units between the two extreme bands, Q̂ and *Q are the

observed and corrected peak heights, respectively, and r is the stutter ratio

estimated at this locus. Corrected peak heights were set to 0 if negative.

The genetic distances between the populations were determined using the

Modified Rodgers’ Distance (MRD) equation (Wright 1978):

( )21 1

2

2

11 jal

i

al

Ll

l

Aa

a

W ppL

dl

−= ∑ ∑=

=

=

=

where ialp and j

alp are the allelic frequencies of allele a at locus l in

populations i and j , respectively. lA is the total number of alleles at locus l

and L is the total number of loci. A distance matrix was built in the software

package R by calculating the distances between all pair wise comparisons of

populations. This matrix was used as the base for neighbor joining cluster

- 26 -

analyses and principle coordinate analyses carried out with the DARwin

5.0.130© software (Perrier et al. 2003).

For each population group, the number of rare alleles was determined. Rare

alleles are alleles with a frequency equal or lower than 5%.

The arithmetic mean of the distances between the populations of each group

was calculated to analyze the diversity within groups, as well as the mean

distance among all populations and the mean of the distance mean of all

groups.

- 27 -

3. Results

3.1 DNA Quality Control

To asses the quality of genomic DNA used for SSR analysis, DNA was

extracted from 20 populations, randomly selected and analyzed by agarose gel

electrophoresis (figure 3.1). It can be seen from the figure that no significant

DNA degradation has occurred during extraction, and the DNA will thus be

suitable for PCR amplification.

Figure 3.1: DNA quality gel

3.2 Allelic frequencies and genetic distances

Allelic frequencies were determined for the 34 SSR markers in all 101

populations and are given in supplemental table 1

(http://www.cimmyt.org/english/webp/support/publications/support_materials/wo

rd/table1-allelicfrequencies.xls). For the determination of genetic distance

Modified Rodgers’ Distance (MRD) was employed. The matrix of Modified

Roger’s Distances between all pairs of populations can be found in

supplemental table 2 at:

- 28 -


d/table2-GeneticDistances.xls

3.3 Neighbor joining clustering

A neighbor joining clustering tree was calculated with the MRDs between all

pairs of populations using the DARwin 5.0 software package (Figure 3.2). The

populations from each country can be differentiated by the color of the line.

The neighbor joining clustering tree (Figure 3.2) shows eight principal clusters

of maize landraces which are described here and discussed in the Discussion

section.

- 29 -

Figure 3.2: Neighbor joining cluster with all 101 populations. The operational taxonomic units (OTUs) are labeled with the ID numbers of each population (see table 2.1 for information about each landrace). The color coding for the countries of origin is indicated.

- 30 -

Group I contains 17 populations, and is mainly represented by northeastern

Colombian populations. Only one Venezuelan, one Brazilian and one

Argentinean population belong also to this group. The races are mostly flints,

but include one dent and one popcorn. This group contains landraces of maize

from different elevations in more or less equal proportions (3 landraces from the

lowlands, 5 landraces from the highlands and 4 landraces from mid-elevations;

Table 3.1).

Table 3.1: Landraces in group I

ID CommonName PrimaryRace Country Elevation Grain Type

Grain Color

4 CAPIE WHITE Capia Blanco Argentina 2500 flint dent yellow

26 BRAZIL 2305 Dente Paulista

Brazil n/a dent yellow, white, sun red

37 MAIZ DE ANO Amagaceño Colombia 1600 flint white 38 CAQUET 321 Andaqui Colombia n/a flint white 39 CHAPARRITO Cabuya Colombia 1920 flint white 40 AMAPOLO Cacao Colombia 1252 flint yellow

41 CARIACO Cariaco Colombia 229 flint blue, white, yellow

43 MAIZ AMARILLO Común Colombia 896 flint white

45 MAGDAL 443 Guira Colombia 1860 flint yellow, white,

brown 47 COLOMB 613 Negrito Colombia 540 flint dent brown, sun red 49 MAICENA Pira Naranja Colombia 1554 flint white, yellow 53 BLANCO DE HARINA Sabanero Colombia 1960 flint blue 54 TOLIMA 389 Yucatán Colombia n/a 55 BOYACA 462 Pira Colombia n/a flint yellow 56 CUNDIN 480 Pira Colombia n/a white

57 PORVA Pira Colombia 2377 dark blue, sun

red 100 CARIACO Cariaco Venezuela 101 floury white, sun red

Group II contains 16 populations, and is a mix of flints, dents and one floury

maize. It is characterized by populations from three defined regions: (i) lowlands

from Chile, Argentina, and Uruguay (ii) tropical lowlands from Brazil and Peru,

and (iii) mid-elevation Guatemala. This group, together with group III, includes a

high proportion of lowland landraces and fewer highland landraces. It is also

evident that the regions of origin of all the landraces contained in this group

exclude to some extent the regions of western South America and the central

Andes. Only one landrace (Amarillo ID #94) from Peru and one landrace from

Ecuador (Canguil ID #58) are originally from western South America.

- 31 -

Table 3.2: Landraces in group II

ID Common Name Primary Race Country Elevation Grain Type

Grain Color

3 AMARILLO DE

OCHO Capia Amarilla de

Ocho Argentina n/a dent

yellow, varigate, dark blue

24 BRAZIL 60 Cateto Grande Brazil 500 dent yellow, sun red

25 AMARELO MOLE Dente

Riograndense Rugoso

Brazil 274 flint dent

yellow

28 CRISTAL BP Brazil n/a dent yellow

31 OCHO

CORRIDAS Ocho Corridas Chile 250 dent yellow

32 CHZM 09 030 Araucano Chile 100 flint yellow 34 CHOCLERO Choclero Chile 220 flint yellow

36 CURAGUA Pisankalla Chile 450 flint sun red, yellow,

blue 58 CANGUIL Canguil Ecuador 2213 flint white

70 LLANERO Negro

Chimaltenango Guatemala 1219

white, blue, sun red

73 OAXAQUENO Nal Tel Blanco de

Tierra Baja Guatemala 1006

flint dent

white, blue, yellow

74 GUATEM SH22 Olotón Guatemala n/a flint red, blue,

75 PINTO Quicheño Guatemala 610 dent white, sun red,

yellow

77 CHIMBO BLANCO San Marceño Guatemala 206 floury orange yellow,

yellow

94 AMARILLO L.M.(S.M.)

Perla Peru 253

97 URUGUA 1131A Cateto Sulino Uruguay n/a dent white

Group III is a heterogeneous group of thirteen races similar to Group II.

Although it contains also some highland races it is mostly a lowland group with

representatives from the central part of South America, from Peru and Ecuador

to Brazil. It displays a noteworthy exclusion of Venezuelan, Colombian and

Chilean landraces in this group. It contains also different grain types with no

special type to emphasize.

Table 3.3: Landraces in group III


Grain Color

13 DURO

AMAZONICO Duro Amazónico Bolivia n/a floury dark blue, red,

20 PISANKALLA Pisankalla Bolivia 1790 29 BRAZIL PE012 Dentado Brazil n/a flint yellow

30 CATETO NORTISTA

Cateto Nortista Brazil n/a sweet floury

yellow

60 DE POLLO Chococeño Ecuador 27 yellow, red, sun

red

69 NEGRO Negro de Altura Guatemala 2316 flint dent floury

yellow, sun red, white

71 AMARILLO Nal Tel Amarillo de Tierra Baja

Guatemala 914 dent floury

yellow, sun red

72 ZAPALOTE CHICO

Nal Tel Blanco de Tierra Alta

Guatemala 2774 dent flint white

76 BLANCO Salpor Guatemala 688 dent yellow, white,

- 32 -

sun red

79 AVATI MOROTI (3 MESES)

Avati Mita Paraguay 210 flint yellow, white

85 AVATI-

PICHINGA Avati Pichinga

Ihu Paraguay 200 floury white

96 CUARENTON Cateto Sulino

Escuro Uruguay 183 flint orange yellow

99 URUGUA 1187A Dente Branco Uruguay n/a dent yellow

Group IV is a small group (four populations) containing only lowland populations

from north eastern Colombia and Venezuela.

Table 3.4: Landraces in group IV

ID CommonName Primary Race

Country Elevation Grain Type

Grain Color

44 BLANCO Costeño Colombia 7 floury yellow, sun red, 48 CALILLA Pira Colombia n/a floury yellow, white 51 PUYITA Puya Colombia 229 dent yellow 101 CARIACO Cariaco Venezuela 365 floury white

Group V contains 26 populations divided into three subgroups of closely related

Andean populations. Geographical origins of this group encompass southern

Bolivia and northern Chile, and the southern and central Andes to the border of

Ecuador. Most populations in subgroup Va were previously classified into a well

defined complex of central Andean races (Goodman & Bird 1977, Goodman &

Brown 1988); including the races Huilcaparu, Kulli, and Chulpi. Flint, floury, and

dent grain types are present in equal proportions.

Subgroups Vb and Vc are closely related to subgroup Va. Included are

populations from Peru, Ecuador, and Colombia previously classified into the

complexes of central but also northern Andean races (Goodman & Brown,

1988, Sánchez et al., 2006).

Table 3.5: Landraces in group V

ID Common Name

Primary Race Country Elevation Grain Type

Grain Color Group

8 OSCO Aysuma Bolivia 1947 flint floury

red, yellow 5a

9 BLANDO

AMAZONICO Cuzco cristal amarillo

Bolivia n/a floury blue, white, yellow

5a

10 GRIS Checchi Bolivia 1976 dent yellow 5a 14 CHEJURA Hualco Bolivia 2900 floury sun red 5a 15 HUALTACO Hualtaco Bolivia n/a dent flint white 5a

16 HUILLCAPARU Huillcaparu Bolivia 2500 floury blue, brown, yellow

5a

18 KULLI Kulli Bolivia 2150 dent white 5a

22 UCHUQUILLA Uchuquilla Bolivia 2600 flint dent white, sun

red 5a

35 CHULPE Chulpi Chile 2300 flint dent yellow 5a 86 MAIZ COMUN Ancashino Peru 2900 flint yellow 5a

- 33 -

87 CHULLPI 9 Chulpi Peru 2800 floury sweet

yellow, white 5a

89 CUZ. 56 Cuzco cristal amarillo

Peru 3268 floury yellow, blue 5a

91 CUZ. 77 Cuzco Gigante Peru 3200 5a

61 CHULPI Chulpi Ecuador 2579 flint floury

yellow, white 5b

63 MISHCA Mishca Ecuador 2286 flint yellow, white 5b 65 MOROCHO Morochón Ecuador 2195 dent yellow, white 5b

66 RENEGRIDO Racimo de Uva Ecuador 2423 floury flint

white, yellow 5b

67 ECUADO X14237

Shima Ecuador n/a flint yellow 5b

88 LLAMELLAN Cuzco Peru 3100 floury brown, white 5b

93 BLANCO CRIOLLO

Mochero Peru 23 5b

95 PUN. 14 Uchuquilla Peru 2000 5b

42 BLANCO COMUN

Chococeño Colombi

a 183 floury

yellow, varigate, red

5c

46 NARINO 534 Montaña Colombi

a n/a flint dent

yellow, sun red, white

5c

59 MOROCHO AMARILLO

Chillo Ecuador 2560 sweet floury

yellow, white 5c

68 UCHIMA Uchima Ecuador 1737 dent flint white 5c

92 POCCHO CRUZADO

Kculli Peru 2300 5c

Group VI covers a broad area of the central Andes. This area goes from

Ecuador over Peru to Bolivia and northwest of Argentina, all over the highlands

of the region, being also present in regions where groups II and V are dominant.

This group of eight populations contains only one that comes originally from the

lowlands of the west Amazonian Basin in Bolivia, the Coroico Morado (ID # 12).

Flint, floury and dent representatives are included, which are mostly yellow or

white, with also some red, red-orange and one blue population.

Table 3.6: Landraces in group VI

ID CommonName PrimaryRace Country Elevation Grain Type

Grain Color

6 CULLI Culli Argentina 2500 flint orange yellow

11 CHULLPI Chuspillu Bolivia 1947 dent floury

dark blue, red, yellow

12 MORADO Coroico Bolivia 156 floury yellow 17 KELLU Kellu Bolivia 2700 floury sun red, white 19 PUCA SARA Paru Bolivia 3000

62 MOROCHO O TUSILLA

Kcello Ecuador 1800 flint white, yellow

64 MOROCHO HUANDANGO

Montaña Ecuador 2259 flint white, yellow

90 CUZCO 363 Cuzco cristal Peru 3200 floury yellow, red, white

- 34 -

Group VII is represented mainly by flint populations from southeastern South

America. Catetos from Argentina, Uruguay and Brazil are the most evident. The

four populations of this group originated in lowland elevations, except one

Argentinean population, which is native from a region near the Andes from the

mid-elevations.

Table 3.7: Landraces in group VII


Grain Color

2 CHILENO MANFREDI Camelia Argentina 62 floury white

5 ROSARINI Culli Argentina 1006 flint yellow 27 BRAZIL 2441 Cateto Pag. Brazil n/a flint yellow 98 COLORADO Cateto Sulino Uruguay 183 flint yellow

Group VIII includes seven populations only from the lowlands of central

southern South America, with and important portion of Paraguayan races. The

Paraguayan race Avati is dominant, but this group contains all grain types. The

Avati landraces are also very homogeneous, and cluster in the same branch of

the group.

Table 3.8: Landraces in group VIII


Grain Color

7 BOLIVI 90 Acre

Interlocked Bolivia 500 floury

yellow, white, varigate

21 AMARILLO

BLANDO O BAYO Pojoso Bolivia 1050

23 VERMELHO DURO

Cateto Assis Brazil 274 flint yellow

33 CAMELIA Camelia Chile 625 flint white

50 CUNDIN 465 Pollo Colombia n/a dent white, yellow, sun red

78 AVATI CAYGUA Avati Ti Paraguay 240 flint pop white

80 AVATI-TUPI Avati Moroti Guapi

Paraguay 400 flint dent

white

81 AVATI-MITA Avati Moroti

Mita Paraguay 140 floury yellow, blue

82 AVATI-MOROTI Avati Moroti Paraguay 400 floury yellow, blue,

white 83 AVATI-TI Avati Ti Paraguay 230 84 AVATI-PICHINGA Avati Pichinga Paraguay 300

The elevation from where the landraces were collected was also used as a

category to describe them. Three different elevation categories were arbitrarily

defined as Lowland (0 m to 600 m over the sea level), Mid-elevation (600 m to

1800 m above sea level), and Highland (more than 1800 m above sea level).

- 35 -

Figure 3.3: Neighbor joining tree with 101 landraces. Categories are described by color of the OTU and correspond to elevation group

Using geographic and elevational origin to establish equivalent populations

(those that should have more in common) and make comparisons among

‘equals’, a neighbor joining tree was done discerning between the elevation

categories (Figure 3.3), This tree clusters into separate groups with populations

from the Southern Andes and the Northern Andes grouping together. The

graphical analysis of the highland landraces confirms the division of two main

branches of highland landraces from South America. The first main branch

(right half in figure 3.3) is formed by populations from the Central and Southern

Andes (Ecuador, Peru, Bolivia, Chile and Argentina). The second main branch

is formed by populations from the northern Andes and Guatemala. Argentinean

landraces are also present in this branch.

3.4 Mean genetic distance within groups of populations

The arithmetic mean of genetic distances between populations of the same

group was calculated for all eight groups and for all populations, as well as its

standard deviation. This shows the similarity that the populations have to each

other and can be used as a measure of diversity within families of landraces.

The mean genetic distances and their standard deviations are shown in figure

3.4 and table 3.9.

- 36 -

Table 3.9: Mean genetic distance and its corresponding standard deviation between landraces within every group of populations.

Group Mean Distance within group Standard deviation I 0.1503 0.0470 II 0.2036 0.0627 III 0.1235 0.0554 IV 0.1062 0.0314 V 0.0987 0.0319 VI 0.1097 0.0338 VII 0.1539 0.0610 VIII 0.1520 0.0535 Total 0.1710 0.0613

Mean of Means 0.1321 0.0438

Mean genetic distance within group and standard deviation

0.00

0.05

0.10

0.15

0.20

0.25

I II III IV V VI VII VIII Total Mean ofmeans

Groups

Figure 3.4: Mean of genetic distances between landraces of each group and their corresponding standard deviations

The group with the largest mean genetic distance was group II, with a mean

distance higher than the mean distance between all populations. The smallest

mean genetic distance was in group V, followed by group IV and VI.

3.5 Principal Components Analysis

The Principal Components analysis was done using the genetic distance matrix

(http://www.cimmyt.org/english/webp/support/publications/support_materials/wo

- 37 -

rd/table2-GeneticDistances.xls) created from the allelic frequencies (chapter

2.3).

The Principal Components Analysis, projecting axes 1 and 2 (figure 3.5),

suggests a grouping of the landraces from each country in a common area. For

example, all Colombian landraces are clearly grouped together. Likewise,

Brazilian, Paraguayan and Guatemalan landraces occupy more or less a

specific area of the plot.

Figure 3.5: Principal components analysis between axes 1 and 2. Each color stands for a country. Dots are labeled with corresponding country of origin.

- 38 -

Landraces from the Central Andean Complex (Ecuador, Bolivia, Peru and North

Argentina and Chile) occupy a very specific area in the graph, which is a strong

indication for its close relationship and its identity as a group.

However, for some countries, the populations do not group together in the same

area but spread all over the plot indicating diverse genetic origin. This is the

case for the Argentinean, Chilean and Uruguayan landraces.

The PCA projection of axes 1 and 3 (figure 3.6) shows a slight separation

between highland landraces (blue dots) from the southern and central Andes,

and the other landraces from mid-elevations (purple dots), and lowlands (red

dots). The clear separation between the groups as defined in the neighbor

joining cluster cannot be confirmed in this scatter plot.

Figure 3.6: Principal components projected onto axes 1 and 3. Numbers refer to populations as described in Table 2.1. Elevation categories are characterized by color. Red: Lowlands Blue: Highlands Purple: Mid-elevations Black: N/A

- 39 -

3.6 Binary analysis of frequent and rare alleles

A binary table of all alleles in all populations was created. All allele frequencies

in every population were translated to 1 if the allele was present or 0 if absent.

Binary frequencies were added. When the alleles were present in 5 or fewer

populations, they were considered rare (5% or less). Alleles that were present in

more than 95 landraces (95%) were considered common alleles. This was

useful to get an impression of the frequency of presence of each non-common

allelic component among the groups.

The list of populations containing rare alleles and lacking very common alleles

within the 101 populations of this study are shown in table 3.10 and 3.11

(appendix 5.2).

- 40 -

4. Discussion

4.1 Characterization of the groups and the landraces

4.1.1 Genetic relationships within and among groups of populations

South America has a very broad variety of landraces, since maize has been

cultivated in this continent for thousands of years. The huge range of diversity

encompassed by the continent makes it a very important object for research. It

has been of special interest to identify the different population groups and the

specific characteristics of each group, and use this information to improve

productivity and quality of maize. Several earlier investigations have already

worked to characterize and describe South American landraces. Using the data

of this study, a new characterization can be done, and relationships established

by earlier studies can be analyzed, compared and confirmed or rejected.

The data obtained in this study allows the grouping of landraces with respect to

genetic distance. The first and most important grouping that can be derived

from the data is the separation between highland and lowland landraces, where

highlands were defined as elevations greater than 1800 meters above sea level,

and lowlands were defined as elevations 600 meters above sea level or lower.

This separation was evident in the neighbor joining cluster tree (figure 3.3).

The separation of clusters based on elevation is not complete, however, since

some clusters in the tree contained both highland and lowland landraces in

more or less equal proportions. Taking these characteristics into account the

subcontinent was tentatively separated in three general regions:

• Northwestern South American Region, including Colombia,

Venezuela and parts of Ecuador. This region is the only where maize

is cultivated in both highlands and lower altitudes in geographic

proximity.

• Central and South Andean Region, with populations from Ecuador,

Peru, Bolivia, and northern Chile and Argentina. This region is

characterized by mostly higher altitudes (above 1500 m above sea

level).

- 41 -

• Northeast, East and Southern South America, where low

elevations are common. This region would include the lowlands of

Chile, Argentina, Paraguay, Uruguay, Brazil and eastern Venezuela.

Obviously there are some parts of these regions which intercalate with the

neighboring regions. Such is the case for Venezuela, where groups or

populations from the eastern region are present in immediate proximity to

populations that belong to the Northwestern South American Region. A similar

case are the regions of Bolivia and Paraguay, where the highlands of the Andes

and the lowlands from the Amazonian Basin or the tropical dry forests from

Paraguay and Brazil interfere closely (Strasburger, 1999).

In addition to the geographical separation of South America in three principal

regions, eight groups were defined by demonstrating the genotypic data of

landraces via neighbor joining clustering (Figure 3.2). The following discussion

highlights probable relationships within and among landrace groups in Central

and South America to the rest of the world as well as possible origins.

Group I is characterized by its mostly flinty grain type from the Northwest of

South America (Colombia and Venezuela) for 15 of its 17 landraces. The

genetic distance between groups I and II is relatively small (Table 3.9). This is

an indication of the similarity of Colombian and Venezuelan landraces to some

of the landraces from Guatemala and Mexico which characterize group II. This

relationship has also been documented by McClintock et al. (1981). They

described that maize landraces from the “Northern Territory” of South America,

which included the region that is composed of half of Central America from

Nicaragua to Panama, together with Colombia and Venezuela and parts of the

Roraima Territory of Brazil, are directly related with south Mexico, Guatemala

and in general with the “North American Sphere of Influence”. From the analysis

of Northern Territory maize McClintock et al. (1981) concluded that the maize

landraces Zapalote Chico from Oaxaca (Mexico) and Nal Tel from Chiapas

(Mexico) and Guatemala must have been close relatives to the maize types that

were introduced into Colombia and Venezuela in a very early period. The

landraces Capia Blanco (ID #4) from Argentina and Dente Paulista (ID #26)

from Brazil are an exception to this mostly exclusively northern South American

- 42 -

group. The presence of these exceptions in group I can be explained by the

several maize introductions that Southern South America has had in the last

decades from Mexico and the USA (Goodman and Bird, 1977), which are more

related to maize from the “Northern Sphere of Influence”.

Figure 4.1: Area of dispersion of landraces of group I. Places of origin are marked by dots.

Group II is genetically the most diverse of the eight groups. The genetic

distances among landraces of this group are on average the highest (figure 3.4

and table 3.9) and its populations appear most often in the lists of landraces

with uncommon allelic components (tables 3.10 and 3.11, appendix 5.2). The

group mainly includes races from Southern South America (lowlands from

Chile, Argentina, Uruguay and southern Brazil) and mid elevation Guatemalan

races. It is possible that the South American landraces of this group descend

from Mesoamerican Landraces that were brought to South America relatively

late, since it includes many Guatemalan races.

Chile, Uruguay and Argentina have introduced landraces from other regions of

the world for their production. This results in the grouping of Chilean,

Uruguayan and Argentinean landraces with Central American landraces. It has

been documented that germplasm from the USA, Mexico and Europe has been

intensively imported in the last decades of the 20th century (Goodman & Bird

1977) into these countries of the “Cono Sur” (Southern Cone: Argentina, Chile,

- 43 -

Uruguay, southern Brazil and Paraguay). Therefore these landraces could

actually be a mixture between indigenous and/or imported varieties, and for this

reason show a strong relationship with Mesoamerican and North American

races.

Two Chilean landraces of this group are mentioned by McClintock et al. (1981):

Choclero (ID #34) and Araucano (ID #32). According to her study, these

landraces belong to the southern half of the East Coast of South America, but

are closely related to the landraces from the highlands of the southern and

central Andes. These two landraces cluster in our study in the same branch,

and thus show no special relationship to the groups of landraces that are

commonly found in the highlands of the Andes (group V and VI). If they

contained any influence from these Andean landraces, gene flow with

populations that do not contain the “Andean” genetic characteristics must have

occurred. On the other hand, it is possible that this influence from the Andean

region in the landraces Araucano and Choclero was strongly pronounced at the

time, when McClintock et al. collected those landraces in the late 50’s. The

collections in the present study were performed four decades later, in 1999 and

1994 respectively; probably enough time for the populations to have developed

and changed genetically.

The Brazilian landraces included in this group come from more tropical regions

of the subcontinent (figure 4.2). Nevertheless these regions are relatively close

to the southern border of Brazil to Argentina and resemble the environmental

characteristics of the other regions, where the Uruguayan and Argentinean

maize of this group comes from.

The single Ecuadorian landrace Canguil (ID #58) from the highlands does not

really fit to any other of the regions that were mentioned. Although its origin is

near to the lowland Peruvian landrace Perla (ID #94), the altitude is very

different. Maybe these landraces are related to the group through the influence

of the Guatemalan landraces.

- 44 -

Figure 4.2: Area of dispersion of landraces of group II in South America. Places of origin are marked by dots.

Group III is less described by a geographic pattern than most of the other

groups, and includes populations from Guatemala, Bolivia, Brazil, Ecuador,

Paraguay and Uruguay. It contains a mixture of races from all elevations and

grain types.

McClintock et al. (1981) describes a very important area of influence of the

Andean Complex into the lowlands east of the Andes. This group could be

related to the descendents of crosses between Andean Complex’s races with

other races from the lowlands of central and eastern South America (figure 4.3).

The Guatemalan landraces from the highlands present in this group could give

a clue about the origin of the Andean Complex, supposing that this group

descends from crosses between Andean and lowland races from the east of the

Andes. McClintock et al. affirmed that some Guatemalan landraces from the

Highlands were extremely similar in their chromosomal configuration to the

highland landraces from the south and central Andes that have the “Andean

Complex” (Almost no chromosome knobs in total; one large chromosome knob

at the long arm of the chromosome 6). Surprisingly the Guatemalan landraces

present in this group all come from locations higher than 600 meters above sea

level, and the highest two are from above 2300 meters above sea level, which

could support the theory of a relationship with Andean highland races.

- 45 -

The Guatemalan populations represent four out of five landraces in this group

that are not strict lowland landraces. The inclusion of the lowland landraces in

this group is curious. The possibility that these landraces were recently

imported is not very probable. The highland landraces from Guatemala (or other

parts of North America), if brought to the lower altitudes of South America,

would not have had time to adapt to the new growing conditions, a process that

typically takes many generations.

Nevertheless, the origin and relationships of this group are quite unclear. If this

group is to some extent the product of crosses of two or more different

population groups, it would probably have a heterogeneous identity. This means

that the genetic distances between populations within this group would be

relatively wide. This is not the case, compared to other groups, as will be

discussed later in this chapter. It is also remarkable that McClintock et al.

included one of her landraces, Pisankalla (ID #20) from Bolivia, in the “North

American Sphere of Influence”, which could also be an explanation for the

presence of Guatemalan landraces in this group. Pisankalla is also the fifth

landrace from higher altitudes in this group, together with the other four

Guatemalan landraces.

Three of the other eight landraces that belong to this group have been classified

in earlier studies. Goodman & Brown (1988) classify Chococeño from Ecuador

as a lowland northern South American popcorn that groups together with

landraces that belong in our study to groups I, II, IV and V. There is even a

Chococeño from Colombia that clustered in our study together with the highland

landraces in group V. Morphologically the Chococeño race appear to be very

specific popcorn landrace with short conical ears, high row numbers (Goodman

& Bird, 1977), tall and heavily tillered plants and late flowering (Goodman &

Brown, 1988). Sánchez et al. (2006) classify in their isozyme and morphological

characterization the Chococeño landrace also as a specific popcorn landrace.

Despite its clear identity as a specific landrace, the two Chococeño landraces in

this study appear to be genetically far from each other. The genetic distance

between the two landraces is 0.1333 (supplemental table 2:


d/table2-GeneticDistances.xls), which is higher than the mean genetic distance

within group III (0.1235) and group V (0.0987, supplemental table 2:

- 46 -


d/table2-GeneticDistances.xls), the two groups that contain Chococeño

landraces. Consequently, these two landraces do not seem to be significantly

related to each other, and their common name may be the result of the

morphological likeness without a near evolutive past in common.

The landrace Chococeño, which seems to have an unclear past, could also

provide some explanation with respect to the presence of Guatemalan

landraces in this group. According to Goodman and Bird (1977) the

Chococeños from Ecuador and Colombia are part of the “Northwestern South

American Landraces”, the same group which contains the Montaña complex.

This complex has presumably a very wide geographic range. Representatives

of this complex can be found from northern Peru to southern and central

Mexico, including Guatemala. This complex is supposed to be related to many

common landraces from Guatemala, like Olotón, Serrano and Salpor (Goodman

and Bird). In this way, the results of Goodman and Bird were confirmed by our

results: the latter landrace (Salpor ID #76) from Guatemala was present in our

study and clustered also in group III.

The landrace Cateto Sulino (ID #97 & #98) from Uruguay was classified by

Goodman & Brown (1988) to the Southern South American landraces. Two

other Catetos Sulinos from Uruguay were part of this study and grouped with

the populations of groups II and VII respectively. Cateto Nortista from Brazil (ID

#30) also clustered in this group. Like the Chococeño race, the populations with

the name ‘Cateto’ did not have a specific genetic identity that related them

closely with each other. Catetos were present in our study in groups II, III, VII

and VIII. The designation ‘Cateto’ appears to be only a name for maize that has

the phenotypic characteristics of coastal tropical flints from the Caribbean,

which in many cases derive from these regions and sometimes from other

regions of the world (McClintock et al., 1981).

- 47 -

Figure 4.3: Area of dispersion of landraces of group III in South America. Places of origin are marked by dots.

Group IV is a small group of lowland populations from the north of South

America (Colombia, Venezuela). It appears to be less related to group I, II, and

III, probably because of a stronger influence of Caribbean tropical flints, as

described in McClintock et al. (1981). This group could be related to the

northern East Coast of South America and to some extent to landraces from the

south and southeast of the subcontinent, as all the East Coast build a

continuum of very alike and homogeneous landraces (McClintock et al. 1981).

Three out of the four landrace representatives of this group were catalogued by

Goodman and Bird (1977) in the group of Northwestern South American Races.

Although it is obvious that these landraces are from the North of South America,

Goodman and Bird catalogued also other landraces into this group, which in our

study resulted to be in other groups. Landraces mainly from groups I and V, but

also from groups II, III, IV, VI and VIII were catalogued in the Northwestern

South American Races.

- 48 -

Figure 4.4: Area of dispersion of landraces of group IV. Places of origin are marked by dots.

Group V is divided in three subgroups of closely related Andean highland

populations. The area of cultivation of this group encompasses southern Bolivia,

northern Chile, all over the southern and central Andes to Peru, Ecuador, and

equatorial Colombia. Group V represents a subset of ancient landraces present

in pre-Columbian South America (McClintock et al., 1981). The landraces in this

group are supposed to be derived form a single introduction into the central and

southern Andes followed by an expansion over all highland regions. The

landraces were highly isolated before new introductions came from the northern

regions. Strong adaptation leads to the development of strong characteristics,

which influenced almost one third of the South American subcontinent. This

influence is still clearly visible as this investigation shows.

Subgroup Va includes Bolivian populations, some populations from Peru and

one population from the northern part of Chile near to Bolivia. Most populations

were previously classified into a well defined complex of Central Andean races

(Goodman & Bird 1977, Goodman & Brown 1988); including e.g. the races

Huilcaparu (ID #16), Kulli (ID #18), Chulpi (ID #61), Ankashino (ID #86). Flint,

floury, and dent grain types were present in equal proportions. All Bolivian

landraces clustering in group V are present in this first subgroup Va.

Subgroup Vb is very closely related to subgroup Va. Some populations from

Ecuador and Peru are included, the corresponding races of which were

classified into the complexes of Central and Northern Andeans (Goodman &

Brown, 1988, Sánchez et al., 2006). Subgroup Vc contains Colombian and

Ecuadorian highland landraces. These landraces are not as closely related as

the first two subgroups, but rather derive from several branches in the neighbor

clustering tree (Fig. 3.2). This subgroup was most likely influenced by the

- 49 -

complex of Northern Andean Landraces described in group I, and consequently

clustered more separately (Goodman & Brown. 1988).

There is no clear agreement in the classification of landraces and the

establishment of relationships when comparing group V with the classifications

done by Goodman & Bird (1977) and Goodman & Brown (1988). Especially the

study of Goodman & Bird (1977) classifies many of the landraces that belong to

Vb in our study into a Northwestern South American complex, which is more

related to landraces from group I (Colombia, Venezuela and north of Ecuador).

Additionally, representatives from group Vc are classified together with

populations from Northern areas by the same authors. Thus it seems that the

genetic influence of Central Andean highland races in Northern South American

regions was more important than originally thought.

Figure 4.5: Area of dispersion of landraces of group V. Places of origin are marked by dots.

Group VI covers a very broad area over the borders of the Andes; Bolivia as a

major part of it. Possibly this group represents populations from crosses

between typical Andean races with races from the lowlands near to the Andean

areas. The group contains flint, floury and dent representatives and no racial

group is particularly numerous. Populations were collected in the highlands with

one exception from Bolivia.

This group shares most of its characteristics with group V. Its representatives

are in its majority from high altitudes in the Andes with a similar spread over

countries the (Bolivia, Peru, Ecuador, Argentina). The populations have been

considered to belong to the complexes of northern highland and south and

- 50 -

central Andean landraces. Similar to group V, the diversity within group VI is low

with a low mean genetic distance. Due to the results presented here, this group

has a higher influence from other landraces, most probably from landraces in

groups I, II and III. This is demonstrated in a neighbor joining figure 3.7

(appendix 5.3) clustering only highland landraces, where the landraces Culli

from Argentina (ID #6), Kellu (ID #17) and Paru (ID #19) from Bolivia, Kcello (ID

#64) from Ecuador and Cuzco Cristal (ID #90) from Peru cluster separately from

landraces in group V and near to landraces from groups I, II and III. One

exception in group VI is the landrace Coroico (ID #12) from Bolivia. This

landrace has clearly been classified into a complex of Amazonian landraces

(Goodman & Bird, 1977; Goodman & Brown, 1988; Sánchez et al. 2006). It is

the only landrace in group VI that was collected in a lowland tropical region, but

clusters with southern Andean landraces. The two most related landraces to

Coroico are Hualco (ID #14) and Paru (ID #19) from Bolivia, with genetic

distances of 0.087 and 0.094 respectively. Both landraces belong to the Andean

highland groups V and VI. The landrace Coroico probably has genetic

compartments form highland landrace but was adapted or selected for to grow

in the lowlands

Figure 4.6: Area of dispersion of landraces of group VI. Places of origin are marked by dots.

Group VII represents mainly populations from the south eastern part of South

America. Most of the populations belong to the Cateto race from Argentina,

Uruguay and Brazil, with a flinty grain type. Only one population was collected

in intermediate elevations (1006 meters) in western Argentina. All other

- 51 -

landraces were collected in the lowlands. This group, as well as group VIII, has

no clear relationship with any other group of this study. The branch from which

all landraces of this group descend in the clustering tree deviates from all other

branches. The Catetos race is designated in the eastern coast of South

America and has common phenotypic characteristics with Coastal tropical flints

from the Caribbean. These landraces may have been brought in from the

Caribbean or southern Central America (McClintock et al., 1981) during the last

few centuries.

The Culli race from Argentina (ID #5), which is the landrace with the highest

collection site (1006 m) among the landraces of this group, has the peculiarity of

having been classified by Sánchez et al. (2006) into the group of “Central

Andean Flours and Fonts” and by Goodman and Brown (1988) into the group of

“Cuban flints”. According to our data, the race Culli is probably more related to

the Caribbean landraces, since it clusters together with the Catetos, a landrace

that is in part descendant from the Caribbean races (Goodman & Brown, 1988).

Figure 4.7: Area of dispersion of landraces of group VII. Places of origin are marked by dots.

Group VIII includes mainly populations from the lowlands of central southern

South America, especially from Paraguay. The Paraguayan race Avati is

dominant although no specific grain type (flints, dents, flours, or popcorns) is

particularly present. The area were this group is most prominent was originally

populated by Caingang and Guarani tribes (McClintock et al., 1981). These

cultures probably had an influence on the spread of some of these landraces

that are now common in this area, such as Avati Moroti.

Our study confirms that the Avati races have a close relationship with each

other. The landrace Pollo (ID #50) included in this group originates from

Colombia, which is not consistent with the geographical location of the

- 52 -

remaining landraces. How this relationship was caused is not clear. An

introduction of this landrace or of one of its ancestors into Colombia is possible.

However, it should then have a large genetic distance to other Colombian

landraces. The results of this study demonstrate that Pollo’s closest races are

Costeño (ID #44) from Colombia, Chococeño (ID #60) from Ecuador and Puya

Grande (ID #52) from Colombia; the latter race could not be catalogued into any

group. This gives evidence of relations to northern landraces. Pollo was

catalogued by Sánchez (2006) into a single complex, named likewise. He

described this group as an independent group from Colombia and Venezuela

with associations to Araucano (ID #32) from Chile (in the present study in group

II) and Nal Tel Amarillo de Tierra Baja (ID #71) from Guatemala (group III).

Avati races were not taken into account for Sánchez’s investigation.

On the other hand, an influence from the central Andean landraces can be

expected due to their geographic proximity to the central and southern Andes,

taking into account that the Andean races mixed with other landraces in most of

the areas surrounding the Central Andean “core” of the groups V and VI, like

the Amazonian basin and the Chilean and central Argentinean lowlands

(McClintock et al., 1981). Especially the Bolivian and the only Chilean landraces

are in the possible area of influence of the Andean groups. Despite this, no

clear genetic evidence of a special relationship to the Andean landraces can be

found, based on the results of this study. It is evident from tables 3.10 and 3.11

(appendix 5.2) that landraces that belong to group VIII have more allelic

components typical to the first three groups and frequently lack the same

common alleles that are also missing in the first three groups.

- 53 -

Figure 4.8: Area of dispersion of landraces of group VIII. Places of origin are marked by dots.

4.1.2 Genetic diversity within and among populations

When allele frequency was assessed in the different groups (table 3.10 and

3.11, appendix 5.2) it could be observed that the landraces in groups V and VI

did not have as many rare alleles as groups I, II and III. Within populations from

the first three groups much more rare alleles were found to be typical only for

these groups, whereas only six alleles were found to be typical for the Andean

groups.

The data demonstrate that (i) all populations are divided into two big blocks.

The first block is formed by the first three groups and, to a less important

degree, by group VIII. The second block is formed by groups V and VI; and (ii)

the groups V and VI are genetically more uniform and do not present as much

diversity from other areas of America or the world as groups I, II, III and VIII.

The mean genetic distance within groups also indicates a higher uniformity and

lower diversity within the central Andean groups V and VI (table 3.9 and figure

3.4), in comparison with the groups I, II, III and VIII.

The highest genetic diversity was observed within group II. This group is

geographically most widely distributed containing populations from all over the

subcontinent plus Guatemala. This makes it possible to find new clusters of

landraces that have more in common. It is also possible that this group is too

diverse to be one group. Its arithmetical mean genetic distance is even higher

- 54 -

than the arithmetical mean of the genetic distances between all populations,

which would take also distances between groups into account.

The lowest diversity was observed within group V. This supports the theory that

Andean landraces are descendants of a single introduction followed by

cultivation with low degrees of migration and new introductions.

All other groups evolved from several independent introductions. These

introductions took place during the pre-Columbian period, and were later

enriched by new introductions from other parts of the continent after the

Spanish conquest. Probably many of the Caribbean and northeastern landraces

were brought from the Atlantic Coast from Mexico to Panama, Colombia and

Venezuela. Newly introduced landraces spread then to other regions; for

example, from the Venezuelan coast to the Caribbean, and from there back

aga

Genetic Characterization of Maize Landraces from Central ... · Diplomarbeit von Santiago J....

Documents

Transcript of Genetic Characterization of Maize Landraces from Central ... · Diplomarbeit von Santiago J....