simple sequence repeats (ssr) polymorphisms and the

Federal University Ndufu Alike Ikwo, Nigeria

Simple sequence repeats (SSR) polymorphisms and the relationship with phytochemical composition in

Nigerian sesame (Sesamum indicum L) cultivars

Friday, Nwalo Nweke

Our Study

Products and Nutrients of sesame seeds

Phytochemicals in Sesame seeds

Summary and policy implication

1

2

3

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Introduction

�Sesame (Sesamum indicum L.) is very important in Nigerian food.

�One of the oldest crops known to humans.

� It originated from India and later introduced to Africa and Asia.

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Fig. 1 Sesame production

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�Sesame seed is an importantsource of edible oil and protein

�Rich in some minerals, vitaminsand some essential amino acids.

�Sesame seed oil is high in MUFA,PUFA.

Health Benefits

Proportion of Fatty Acids in Sesame Oil

14.89%

35.71%

46.19%Saturated

Monounsaturated

Polyunsaturated

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Nutrients in Sesame Seeds

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Fig: 5 Nigeria Sesame Producing Regions

Simple sequence repeats (SSRs)

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� Detects high level of polymorphisms, btw closely related individuals and heterozygotes

�Simple, quick to analyze, reliable and

�Genetic fingerprinting, population genetic studies and molecular breeding.

Phytochemical screening

�Titrimetric method of Harbone -tannins and glycosides.

� Spectrophotometric method of the Association of Analytical Chemists- -saponins, alkaloids and flavonoids.

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Plant Materials

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�Thirty accessions from the National Cereals Research Institute(NCRI) Badeggi, Nigeria.

�Seeds were grown in pots at the FABI, University of Pretoria using growth media and Hoagland’s nutrient solution.

DNA extraction

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�DNA was extracted from leaves plant/seed DNA KITTM D6020.

�DNA concentration and purity was determined using nanometer.

� Integrity of the DNA was confirmed by 2% Agarose gel electrophoresis.

SSR(s) Analysis

� Two primer pairs were used.

� PCR (20μL) included 20 ng genomic DNA, 2 ul of 10×PCR buffer, 2 ul of 25mM MgCl2, 0.5 ul of 10mM dNTP, 0.5ul of 25uM of each primer and 0.1ul of 5unit/ul Taq polymerase.

� PCR profile: 94°C for 3 mins. (pre-heating),35 cycles at 94°C for30 secs (denaturing), 55–60 °C for 45 secs (annealing), 72°C for 30 secs. with a final extension at 72°C for 10 mins. and stored at - 4 °C.

� Amplified products were resolved on 2% agarose gel, visualized under UV light and photographed

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PCR (product/ fragment) purification

� PCR products were purified using QIAquick purification kit.

�PCR sequencing reaction: (10 ul): 2ul BigDye

Terminator, 3ul DNA sample,1ul forward primer,1ul of dilution buffer and 3ul dist. water

� PCR program: 35 cycles of 96oC for 10 sec, 57oC for 5

sec, 60oC for 4min, 72oC for 10min, store at 4oC

� Fragment purification: Sephadex S-500 spin columns, sample dried in a vacuum drier at 60oC for 40min and sequenced

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Locu

s

Gen

Ban

kA

cces

sio

n n

o.

Pri

mer

Seq

uen

ce

Rep

eat

mo

tif

Ta

(�C

)

Na

Siz

e ra

ng

eo

f alle

les

(bp

)

GBssr-sa-123 AY838916 F: 5-GCAAACACATGCATCCCT-3

R: 5-GCCCTGATGATAAAGCCA-3

(TC)21,

(TC)15

61 4 272–282

GBssr-sa-184 AY838922 F: 5`-TCTTGCAATGGGGATCAG-3`R: 5`-CGAACTATAGATAATCACTTGGAA-3`

(TC)20 55 4 179–193

Ta, annealing temperature; Na, number of alleles; HO

, observed heterozygosity; HE

, expected heterozygosity; PIC, polymorphism information content.

Table 1: SSR Marker

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A A AC T T C T C A C G G A A AC T C AT C T C T C T C T C10

T C T C T C T C T C20

T C T C T C T C T C30

T C T C T C T C T C40

T C T C C T C T T T50

T T A T A T A T AT60

C T T C A

A G T T160

G A A AC C T C T C170

A C A A A C GT C T180

C T C T C T C T C T190

C T C T C T C T C T200

C T C T C T C T T C210

TGT C TG C T C T220

C A T CT CT CT C230

G C AG AA AGG

2nd Region TC 15

SSR - V

1st RegionTC22

Fig. 6 Microsatellite sequences generated from sesame genotype Adaukiari using primer p7-GBssr-sa-123; Source: Nweke et al. 2011.

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I

II

IV

V

VI

VII

VIII

III

Fig: 7 Phylogenetic Tree

Result and Discussion

Ciano” in Group I, with a TC12,

.Yobe machina”,in Group VIII (TC15

Genotypes Repeats Tannins Flavonoids Saponins Cyanogens Alkaloids

Group I

Ciano 27 TC12 1.68 L 0.28 L 1.26 L 0.59 H 4.75 L

Group VIII

Yobe

machina

TC15 1.24 L 0.83 L 3.8 H 0.72 H 6.86 H

Results and Discussion

Group II

Kwander TC20 1.69 L 1.91 H 1.93 H 0.21 L 4.24 L

Incriben0.2

m

TC20 1.30 L 2.01 H 2.83 H 0.28 L 6.13 H

Pachequeno TC20 1.81 L 0.81 L 3.76 H 0.39 L 5.84 H

43-9-1 TC20 1.29 L 1.30 H 2.79 H 0.24 L 5.60 L

Y GW TC20 1.25 L 0.81 L 3.77 H 0.44 L 7.09 H

Group III

Ciano 16 TC19 2.32 H 0.79 L 2.78 H 0.30 L 4.18 L

69-1-1 TC19 1.61 L 1.20 L 2.78 H 0.80 H 5.37 L

Group II of five accessions (TC20

sequence repeat Low in T&G, High in S Group III (TC

19) has 2 Genotypes low in F&A; high in S

Results and Discussion

Group IV

Adaukiari TC22 1.68 L 2.02 H 2.38 H 0.32 L 5.86 H

Chimkwale TC22 2.20 H 1.62 H 1.57 L 0.18 L 6.87 H

Chimkwale

yellow

TC22 1.10 L 1.76 H 2.06 L 0.21 L 6.14 H

E.8 TC22 1.27 L 0.91 L 1.19 L 0.21 L 6.19 H

Eva TC22 1.27 L 0.97 L 2.38 H 0.78 H 4.11 H

Zuru TC22 2.51 H 0.77 L 2.21 L 0.31 L 6.27 H

34-4-1 TC22 2.07 H 1.12 H 3.04 H 0.60 H 5.71 H

Group IV consisted of seven accessions, with a TC22

high in A

Result and Discussion

Group V

Alaide TC21 2.42 H 1.24 L 2.04 L 0.76 H 6.06 H

Cau w TC21 2.50 H 1.77 H 2.83 H 0.46 L 5.18 L

Domu TC21 1.39 L 0.84 L 2.70 H 0.44 L 6.74 H

Otobi TC21 1.62 L 0.76 L 3.68 H 0.28 L 6.47 H

Yorri TC21 2.20 H 0.78 L 3.00 H 0.46 L 6.87 H

Group VI

ABBS TC25 2.20 H 0.75 L 3.79 H 0.60 H 5.18 L

Incri0.3L TC25 1.35 L 0.81 L 2.04 L 0.46 L 6.44 H

Cross 95 TC25 2.59 H 0.82 L 2.21 L 0.31 L 4.82 L

Groups V has 5 Genotype TC21with sub cluster low G&F high S&AGroup VI has 3 Genotype TC25 Low F sub cluster Low S&G

Results and Discussion

Group VII

Jigawa TC18 1.26 L 2.16 H 0.90 L 0.60 H 6.68 H

Kachia TC18 1.10 L 1.90 H 1.24 L 0.32 L 5.84 H

Incriben 0.1m TC18 2.99 H 0.78 L 1.24 L 0.17 L 6.16 H

NCRI (Iwo) TC18 1.82 L 0.79 L 2.08 L 0.72 H 5.57 L

Yobe GB TC17 1.61 L 0.58 L 3.68 H 0.24 L 6.05 H

69-882 TC17 1.69 L 0.91 L 3.12 H 0.31 L 6.33 H

Group VII cultivars, consisting of six cultivars with TC18and TC

17sequence repeats with identical sub group

Descriptive statistics of phytochemicals (mg/100 g seed fresh weight).

Phyto N Mean Std Dev Sum Minimum Maximum

Tannins 60 1.72 0.35 106.84 1.10 2.99

Flavonoids 60 1.14 0.37 70.69 0.28 2.16

Saponins 60 2.40 0.66 148.99 0.90 3.80

Glycoside

cyanogens

60 0.41 0.14 25.97 0.17 0.80

Alkaloids 60 5.88 0.58 365.00 4.11 7.09

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D N- Number of variables; Alkaloids showed the highest value of 7.09 mg/100 g, followed by saponins 3.8 mg/100 g, while glycoside cyanogens had the lowest value of 0.17 mg/100 g followed by flavonoids 0.28 mg/100 g.

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Summary

� Sesame provides excellent food, nutrition, health care, edible oil and phytochemicals.

� SSRs distinguished the tested accessions into 6 main clusters and 2 other cultivars.

� Accessions formed clusters and sub-clusters in relation to phytochemicals.

� Tannins; flavonoids; saponins; alkaloids and ; cynogenic glycoside were determined.

� Significant variability was found in these phytochemicals among the tested cultivars (p < 0.001)

Policy Implications

� This study investigated, first time any possible association btw phytochemical and genetic diversity sesame accessions to identify accessions for selection and exploitation in plant breeding programs and health care.

� Presently, petroleum production brought easy and windfall earnings, which diverted Nigeria’s attention and encouraged the neglect and underutilization of sesame.

� Nigerian sesame has high oil content for bakery and confectionery and Nigeria plays a strategic role in the stability of sub-Saharan Africa

�Policy and capacity building should appraise and evaluate the research, trials and extension services available to the sesame sector

�This will encourage self-sufficiency in food and raw materials for industries and improve the socio-economic welfare of rural people and diversification of the sources of foreign exchange and create jobs.

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