dna fingerprinting of popular indian varieties using ssr...

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DNA fingerprinting of popular Indian varieties using SSR and ISSR

markers

M.S.SARASWATHI et al.Senior Scientist (Hort.)

National Research Centre for BananaTiruchirappalli, Tamil Nadu, INDIA,

By saraswathimse.com


• Protection of biodiversity

• Identification of marker for specific traits

• Assessment of genetic diversity in crop germplasm

• Assist in the management and conservation of germplasm accessions

INTRODUCTION


MATERIALS S.No. Name of

the varietyGenomic

groupSub group Special characteristics

1. Grand Naine

AAA Cavendish High yield

2. Robusta AAA Cavendish3. Dwarf

CavendishAAA Cavendish High yield, ability to withstand

strong winds, short crop cycle and good response to micro-irrigation and high economic returns per unit area

4. Red banana

AAA Unique –Red Dacca

Pleasant flavoured banana with high carotenoids

5. Poovan AAB Mysore Wilt resistant6. Rasthali AAB Silk Mealy pulp with apple flavour7. Nendran AAB Plantain Starchy fruits with high

carotenoids, suitable for chips making


Contd…….8. Pachanadan AAB Pome Coolant9. Monthan ABB Monthan Vegetable10. Kothia ABB Bluggoe Dual purpose variety

11. Karpuravalli ABB Pisang Awak

Suitable for the preparation of value added products like fig, juice, wine etc.

12. Udhayam ABB Pisang Awak

High yield and suitable for the preparation of value added products.

13. Neypoovan AB Neypoovan Soft and tasty pulp, suitable for the preparation of baby weaning foods.

14. Peyan ABB Peyan Pulp is juicy, sweet witharomatic flavours, orangecoloured pulp. Resistant toYellow Sigatoka.


FOURTEEN POPULAR AND RULING COMMERCIAL VARIETIES

Grand Naine Robusta Dwarf Cavendish


Contd…..Contd…..

RasthaliRed banana Poovan


Contd…..

Nendran Pachanadan Monthan


Contd….Contd….

KarpuravalliKothia Udhayam


Neypoovan Peyan

Contd….Contd….


Methods• ISSR (10) and SSR (20) markers

RESULTS AND DISCUSSION

ISSR• Nine primers (90%) amplified products resulting indiscrete, repeatable amplicons.

• Total alleles amplified - 105

• Mean no. of alleles amplified per primer - 11.7

• Maximum alleles amplified - 20 (UBC 811)

• Minimum alleles amplified - 7 (UBC 836)


Unique bands produced by different ISSR markers

Accession Name Primer pair Allele size in bp

Red banana (AAA) UBC 834 867,1258, 1366

UBC 842 801Poovan (AAB) UBC 836 967

UBC 840 529Rasthali (AAB) UBC 841 1497

Nendran (AAB) UBC 840 2118

UBC 841 1649Neypoovan (AB) UBC 808 2017, 2117

UBC 840 608, 1637


876 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

500

1000

1500

1.Grand Naine 2.Robusta 3.Dwarf Cavendish4.Red banana 5.Poovan 6.Rasthali7.Nendran 8.Pachanadan 9.Monthan10.Kothia 11.Karpuravalli 12.Udhayam13.Neypoovan 14.Peyan



1258 bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

867 bp 1366 bp



M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

2118 bp

529 bp608 bp



Common bands produced by varieties of the same genomic group in different ISSR markers

Common bands produced by varieties of the same genomic group in different ISSR markers

Genome Varieties Primer Allele size in bp

AAA1.Grand Naine2.Robusta3.DwarfCavendish4.Red banana

UBC 841 527

AAB1.Poovan2.Rasthali3.Nendran4.Pachanadan

UBC 811 876

ABB1.Monthan2.Kothia3.Karpuravalli4.Udhayam5.Peyan

UBC 811 414


Microsatellite markers•Twenty primer pairs (100%) amplified products resulting indiscrete, repeatable amplicons.

• Total alleles amplified - 178

• Mean no. of alleles amplified per primer – 8.9

• Maximum alleles amplified - 14 (AGMI 113/114)

• Minimum alleles amplified - 4 (Ma SSR 2-10 and Mb SSR 1-149)

• Unique alleles - 16 primer pairs (different varieties of the same or different genomic group)

• Common alleles (different varieties of the same genomic group) -14 primer pairs


Unique bands produced by different microsatellite markers

Unique bands produced by different microsatellite markers

Accession Name Primer pair Allele size in bpGrand Naine (AAA) AGMI 67/68 127, 146

AGMI 95/96 272, 278AGMI 103/104 207AGMI 113/114 214AGMI 123/124 322AGMI 197/198 94Ma 8a/8b 241Ma 24a/24b 111

Robusta (AAA) AGMI 103/104 199AGMI 105/106 307AGMI 133/134 408

Red banana (AAA) Ma SSR 24a/24b 115Poovan (AAB) AGMI 24/25 223

Ma SSR 2-10 153Rasthali (AAB) AGMI 35/36 110

AGMI 123/124 315


Pachanadan (AAB) AGMI 67/68 131AGMI 105/106 245

Nendran (AAB) AGMI 113/114 231Ma SSR 1-19 218

Monthan (ABB) AGMI 103/104 193Kothia (ABB) AGMI 67/68 108

AGMI 95/96 164AGMI 103/104 191AGMI 105/106 242AGMI 123/124 292AGMI 129/130 240

Karpuravalli (ABB) AGMI 197/198 111Mb SSR 1-149 196

Udhayam (ABB) AGMI 24/25 238Neypoovan (AB) AGMI 197,198 142Peyan (ABB) AGMI 197/198 120

Accession Name Primer pair Allele size in bp


Common bands produced by varieties of the same genomic group in different microsatellite markersCommon bands produced by varieties of the same genomic group in different microsatellite markers

Genome Varieties Primer pair Allele size in bp

AAA 1.Grand Naine2.Robusta3.Dwarf Cavendish4.Red banana

AGMI 33/34 129, 161AGMI 35/36 110AGMI 93/94 125, 126AGMI 129/130 215Ma SSR 24a/24b 167Mb SSR 1-100 157, 208

AAB 1.Poovan2.Rasthali3.Nendran4.Pachanadan

AGMI 67/68 121, 126AGMI 95/96 255AGMI 103/104 194, 199AGMI 113/114 252AGMI 129/130 211, 217AGMI 197/198 137Ma SSR 24a/24b 167, 172, 176Ma SSR 2-10 154, 159Ma SSR 3-132 177Mb SSR 1-100 157

ABB1.Monthan2.Kothia3.Karpuravalli4.Udhayam5.Peyan

AGMI 129/130 242AGMI 103/104 194Ma SSR 1-19 200


DNA fingerprints produced by AGMI 113/114, Mb SSR 1-149 and AGMI 95/96

DNA fingerprints produced by AGMI 113/114, Mb SSR 1-149 and AGMI 95/96

196 bp

231 bp

M - Marker1, 15, 29 – Peyan 8, 22, 36 - Nendran2, 16, 30 - Ney poovan 9, 23, 37 - Rasthali3, 17, 31 – Udhayam 10, 24, 38 - Poovan4, 18, 32 – Karpuravalli 11, 25, 39 - Red banana5, 19, 33 – Kothia 12, 26, 40 - Dwarf Cavendish6, 20, 34 - Monthan 13, 27, 41 - Robusta7, 21, 35 – Pachanadan 14, 28, 42 - Grand Naine

M - Marker1, 15, 29 – Peyan 8, 22, 36 - Nendran2, 16, 30 - Ney poovan 9, 23, 37 - Rasthali3, 17, 31 – Udhayam 10, 24, 38 - Poovan4, 18, 32 – Karpuravalli 11, 25, 39 - Red banana5, 19, 33 – Kothia 12, 26, 40 - Dwarf Cavendish6, 20, 34 - Monthan 13, 27, 41 - Robusta7, 21, 35 – Pachanadan 14, 28, 42 - Grand Naine


Primer Label Product range (bp) Number of alleles

Expected Observed Expected observed

AGMI 24 /25 240-300 227-266 12 13AGMI 33/34 151 150-179 6 10AGMI 35/36 106 108-126 7 9AGMI 67/68 100-160 104-117 7 12AGMI 93/94 114-156 131-159 12 7AGMI 95/96 240 249-280 4 7AGMI 103/104 181 162-196 4 7

AGMI 105/106 267 239-306 9 9

AGMI 113/114 210 230-270 - 14

AGMI 123/124 250-344 230-345 3 6


AGMI 129/130 221 167-196 9 6AGMI 133/134 200-250 281-459 3 13AGMI 197/198 173 100-175 - 11Ma SSR 08a/08b 200-250 230-261 5 8

Ma SSR 24a/24b 150-200 167-189 6 11

Ma SSR 1-19 200-250 177-195 - 7

Ma SSR 2-10 100-200 131-162 - 4

Ma SSR 3-132 200-250 150-180 - 10

Mb SSR 1-100 190-235 184-223 6 8

Mb SSR 1-149 180-200 181-235 3 4


Primer Label PolymorphicInformation content

Effective Multiplex ratio

Marker Index

AGMI 24 /25 0.34 13 4.42AGMI 33/34 0.42 10 4.20AGMI 35/36 0.35 9 3.15AGMI 67/68 0.31 12 3.72AGMI 93/94 0.42 7 2.94AGMI 95/96 0.23 7 1.61AGMI 103/104 0.21 7 1.47AGMI 105/106 0.31 9 2.79AGMI 113/114 0.30 14 4.20AGMI 123/124 0.28 6 1.38AGMI 129/130 0.38 8 3.04AGMI 133/134 0.38 13 4.94AGMI 197/198 0.23 11 3.08Ma SSR 08a/08b 0.38 8 3.04Ma SSR 24a/24b 0.35 11 3.85Ma SSR 1-19 0.39 7 2.73Ma SSR 2-10 0.32 4 1.28Ma SSR 3-132 0.37 10 3.70Mb SSR 1-100 0.39 8 3.12Mb SSR 1-149 0.39 4 1.56

6.75 178 60.210.34 8.9 3.01


PrimerLabel

PolymorphicInformation

content

Effective Multiplex

ratio

Marker Index

UBC 841 0.24 9.09 2.18UBC 868 0.40 8.10 3.24UBC 842 0.32 7.11 2.27UBC 811 0.38 20.0 7.78UBC 834 0.28 10.08 2.91UBC 836 0.32 5.14 1.65UBC 840 0.30 14.0 4.20UBC 807 0.41 9.0 3.70UBC 808 0.22 9.30 2.04

2.87 91.82 29.970.31 10.20 3.33


ISSR SSR


Conclusion

• Genetic identity in terms of DNA fingerprintsdeveloped in the present study would have greaterapplications in aiding variety registration, DUScharacterization and in detecting infringement ofbreeders’ rights and biopiracy.

• Specific bands could also be converted into STSmarkers of great value to detect any mix upbetween the cultivars and as DNA fingerprints.

dna fingerprinting of popular indian varieties using ssr...

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