African Journal of Biotechnology

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Afr. J. Biotechnol.


Vol. 2 No. 10



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Agrama HA

Tuinstra MR


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African Journal of Biotechnology Vol. 2 (10), pp. 334-340, October 2003

ISSN 1684–5315  © 2003 Academic Journals

 


Full Length Research Paper

 

Phylogenetic diversity and relationships among sorghum accessions using SSRs and RAPDs

 

Agrama H.A.* and Tuinstra M.R.

 

1Department of Agronomy, Kansas State University, 2004 Throckmorton, Manhattan, KS 66506-5501, USA.

 

*Corresponding author; Present address: Plant Pathology Department, North Dakota State University and NCSL, USDA, Fargo, ND 58105-5677, USA. Tel.: 701-239-1345. Fax: 701-239-1369, E-mail: agramah@fargo.ars.usda.gov.

 

Accepted 31 August 2003

 
   

Abstract


Abstract
Introduction
Materials and Methods
Results
Discussion
References
 

 

 

Two DNA-based fingerprinting techniques, simple sequence repeats (SSR) and random amplified polymorphic DNA (RAPD) analyses, were applied in sorghum germplasm analysis to compare suitability for quantifying genetic diversity. Twenty-two sorghum genotypes, representing an array of germplasm sources with important agronomic traits, were assayed for polymorphism using 32 RAPD primers and 28 sets of sorghum SSR primers. The results indicated that SSR markers were highly polymorphic with an average of 4.5 alleles per primer. The RAPD primers were less polymorphic with nearly 40% of the fragments being monomorphic. An analysis of genetic diversity among sorghum lines indicated that the genetic distances calculated from SSR data were highly correlated with the distances based on the geographic origin and race classifications. Based on the results of these studies, SSR markers appear to be particularly useful for the estimation of genetic similarity among diverse genotypes of sorghum.

 

Key words: cluster, diversity, polymorphism, RAPD, Sorghum, SSR.

 

 
   

Introduction

 
Abstract
Introduction
Materials and Methods
Results
Discussion
References

 

 

Sorghum [Sorghum bicolor (L.) Moench] is ranked the fifth most important cereal crop in the world. The United States, India, Nigeria, Mexico, Sudan, and China currently produce the most grain sorghum. More than half the world's sorghum is grown in the semi-arid tropics, where it is a staple food for millions of people in India and Africa; however, livestock feeding accounts for most of the U.S. sorghum usage.

 

Many studies have been devoted to assessing patterns of sorghum genetic variation based on morphology (Appa-Rao et al., 1996; Djč et al., 1998) or pedigree (Jordan et al., 1998). More recently, DNA-based techniques have been used successfully in DNA fingerprinting of plant genomes (Hongtrakul et al., 1997; Cervera et al., 1998) and in genetic diversity studies (Paul et al., 1997; Sonnate et al., 1997; Barrett and Kidwell, 1998; Chowdari et al., 1998b; Zhu et al., 1998; De-Bustos et al., 1999). 

 

Among them, random amplified polymorphic DNA (RAPD) analysis is quick (Colombo et al., 1998; Fahima et al., 1999) and well adapted for nonradioactive DNA fingerprinting of genotypes (Cao et al., 1999). However, problems with the reproducibility in amplification of RAPD markers and with data scoring have been reported (Jones et al., 1998). Although major bands from RAPD reactions are highly reproducible, minor bands can be difficulty to repeat due to the random priming nature of this PCR reaction and potential confounding effects associated with co-migration with other markers (Tessier et al., 1999).

 

SSR markers are attractive for DNA fingerprinting studies for several reasons. They are codominant and highly informative. They generally display high levels of polymorphism (Beckmann and Soller, 1990; Brown et al., 1996; Senior et al., 1998) and are amenable to automated genotyping strategies. They also can be amplified by PCR and efficiently detect DNA polymorphism (Pejic et al., 1998). Finally, radioisotopes are not required in the detection of SSR markers, because sequence polymorphism usually can be detected by separation in agarose gels (Burr, 1994).

 

Although SSRs are well established for human and mammalian genetics, these markers have only recently become available in plant species. They have been identified in many plant genomes including those of maize (Senior and Heun, 1993; Shatuck-Eidens et al., 1990; Taramino and Tingey, 1996); soybean (Akkaya et al., 1992; Morgante and Olivieri, 1993); Brassica spp. (Poulsen et al., 1993); rice (Wu and Tanksley 1993); barley (Saghai-Maroof et al., 1994); pearl millet (Chowdari et al., 1998a); Arabidopsis (Depeige et al., 1995); tomato (Broun and Tanksley, 1996); conifers (Tsumura et al., 1997); and sorghum (Brown et al., 1996; Taramino et al., 1997; Dean et al., 1999). The results of studies using SSR markers in these species suggest that they may provide an outstanding new tool for genetic analysis of plant species.

 

Harlan and DeWet (1972) classified cultivated sorghum based on agronomic and morphological characteristics. The utilities of isozymes (Morden et al., 1989; Aldrich et al., 1992), RFLP (Aldrich and Doebley, 1992), and RAPD (de Oliveira et al., 1996; Menkir et al., 1997; Ayana et al., 2000) markers have been used to study genetic diversity in sorghum germplasm. Several efforts have been made to utilize SSR markers in plants to study genetic diversity, characterize germplasm, and evaluate population dynamics (Zhang et al., 1997; Liu and Wu 1998; Senior et al., 1998; Struss and Plieske, 1998). A comparison of RAPD and SSR marker techniques in sorghum is timely, even though the utility of different molecular markers for corn (Smith and Helentjaris, 1996), soybeans (Powell et al., 1996) and barley (Russell et al., 1997) germplasm already has been reported. The objectives of the present study were to: (1) compare the application and utility of RAPD and SSR marker techniques for analysis of genetic diversity among sorghum genotypes, (2) compare genetic similarity quantified by molecular markers with regional and race information.

 

 
   

Materials and Methods

 

 
Abstract
Introduction
Materials and Methods
Results
Discussion
References

 

 

Plant Materials

 

Twenty-two sorghum accessions including landraces, improved lines, and wild accessions were evaluated in this study (Table 1). Most of these accessions represent landrace varieties and are described in detail in the USDA-ARS Germplasm Resources Information Network available at http://www.ars-grin.gov/npgs. Less information is available on wild accessions and improved varieties that were obtained from plant breeders from different parts of the world.

   

 

Table 1. Country of origin, race classification, and other distinguishing characteristics for sorghum accessions used in this genetic diversity studies.

 

No

Germplasm Accession

GRIN Designation

Origin

Region

Race

Distinguishing Characteristics

1

P954035 (SC 33)

PI 534132

Ethiopia

1

D

Drought tolerance

2

B35 (SC 35)

PI 534133

Ethiopia

1

D

Drought tolerance

3

SC 1158

PI 597957

Ethiopia

1

D

Disease resistance

4

SC 326

IS 3758C

Ethiopia

1

C

Disease resistance

5

SC 414

PI 533831

Sudan

2

C

Disease resistance

6

SC599

PI 534163

Sudan

2

C

Drought tolerance

7

ShanQuiRed

-

China

7

B

Cold tolerance

8

SanChiSan

GRIF 620

China

7

B

Cold tolerance

9

PI 550590

PI 550590

Russia

8

B

Cold tolerance

10

12-26

-

Egypt

5

W

Wild sorghum

11

47-121

-

Kenya

1

W

Wild sorghum

12

PI 465483

PI 465483

Yemen

1

D

Large seed size

13

PI 559761

PI 559761

Yemen

1

D

Large seed size

14

Dorado

-

El-Salvador

6

M

Elite tropical variety

15

Malisor

-

Mali

3

M

Elite tropical variety

16

Sureno

-

Honduras

6

M

Elite tropical variety

17

Macia

-

Mozambique

4

M

Elite tropical variety

18

M91051

-

USA

6

M

Elite tropical variety

19

TX430

PL-140

USA

6

M

Elite U.S. pollinator

20

TX2737

GP-82

USA

6

M

Elite U.S. pollinator

21

TX2741

GP-86

USA

6

M

Elite U.S. pollinator

22

PL1

-

USA

6

M

Large seed size

 

Region: 1 = East Africa, 2 = Central Africa, 3 = West Africa, 4 = South Africa, 5 = Northern Africa, 6 = North America, 7 = Asia, 8 = Europe. Race: D = Durra, C = Caudatum, B = Bicolor, W = Wild, M = Breeding.

 

 

DNA extraction and SSR and RAPD markers

 

Genomic DNA was extracted from etiolated hypocotyls 5- to 7-day-old plants of each genotype according to the method of Djč et al. (2000). Initially the five individual plants of each accession were assayed for RAPDs or SSRs using 5 primers. No polymorphisms were detected between individuals within a genotype.

 

Seventeen SSR markers described by Brown et al. (1996) and 11 described by Taramino et al. (1997) were used for genotyping assays (Table 2). Eighty different RAPD primers obtained from OPERON Technologies (Kits A-D) were used to generate markers as described by Tao et al. (1993). Thirty-two primers that generated clear and reproducible fragments were used to fingerprint the 22 sorghum genotypes. The SSR and RAPD reaction products were evaluated for polymorphisms on 3% Metaphor agarose gels (FMC Products, Rockland, ME, USA) and 1.6% agarose gels, respectively. Gels were stained with 1 mg mL-1 ethidium bromide for 30 to 60 min.

 

   

                        Table 2. SSR marker used to diverse the 22 sorghum accessions.

 

No.

SSR Markers1

No of alleles

Size range i