Genetic integrity of the International Transit Centre (ITC) collection: field verification and reference collection

(1)

Genetic integrity

of the ITC collection :

DArT genotyping

(2)

Rationale and objectives

Objective: reducing and managing the loss of genetic integrity of

conserved germplasm.

• genetic integrity

: identity of the genetic composition of the sample

conserved at ITC to that of the original collected, bred or improved.

• To detect

loss of genetic integrity

:

– compare an (ITC) accession to its most original sample (MOS),

– or be able to determine that the accession doesn’t behave as it

should.

Bioversity has adopted a workplan to identify accessions that have

eventually undergone a genetic change.

(3)

ITC

in vitro collection

712 accessions

CIRAD

Guadeloupe

QDPI

Australia

. . .

MOS

Most Original sample

sent to ITC

Introductions from ITC

Field collections:

Field verification

morphological characters

site 1

site 2

site 3

TTT true to type

OT off type

ML mislabelling

Genotyping

ITC accessions

DArT

498 markers

712 accessions

ITC/CIRAD

DArT

836 markers

156 accessions

241 markers shared

113 accessions:

70 MOS + 43 intro

Classification

C well classified

MC misclassified

D dubious

diversity analysis

Cytology

Ploidy level

workplan of the

verification of the

genetic integrity of

ITC Musa accession

ITC accessions compared to

MOS or REF by genotyping

ITC/MOS-REF

DArT

1088 markers

+220 accessions

1088 markers

110 accessions pairs:

63 MOS + 47 REF

Flow cytometry

Periodical verification

of trueness-to-type

(accessions

maintained in vitro for

(4)

Diversity Arrays Technology (DArT)

GCP: 168 accessions from IITA and CIRAD analysed with 836 DArTs

markers :

• «DArTs can be used for genome wide analyses»,

• Despite the dominant nature of DArT markers, they can be used to

«compare different genomes at a large number of loci in a single

assay»,

• «The analysis cluster genotypes consistently with the accepted

classification knowledge».

(5)

ITC

in vitro collection

712 accessions

CIRAD

Guadeloupe

QDPI

Australia

. . .

MOS

Most Original sample

sent to ITC

Introductions from ITC

Field collections:

Field verification

morphological characters

site 1

site 2

site 3

TTT true to type

OT off type

ML mislabelling

Genotyping

ITC accessions

DArT

498 markers

712 accessions

ITC/CIRAD

DArT

836 markers

156 accessions

241 markers shared

113 accessions:

70 MOS + 43 intro

Classification

C well classified

MC misclassified

D dubious

diversity analysis

Cytology

Ploidy level

workplan of the

verification of the

genetic integrity of

ITC Musa accession

ITC accessions compared to

MOS or REF by genotyping

ITC/MOS-REF

DArT

1088 markers

+220 accessions

1088 markers

110 accessions pairs:

63 MOS + 47 REF

Flew cytometry

(6)

Analysis of 712 ITC accessions with DArTs

• 498 DArT markers.

• The phylogenetic tree produced by analyzing the DArT markers show

the separation of accessions in species / groups and eventually

subspecies/ subgroups, confirming the separation from morphological

observations and previous molecular markers (RFLP, SSR)

.

• DArT markers are able to spot accessions which are not grouping with

what was expected. These are clearly misclassified accessions.

• In many cases DArT analysis allowed to complement a classification

(eg. the subgroup of a poorly identified accession can be identified).

(7)

AAw malaccensis/Malaccensis

AAw burmannica/Long Tavoy AAw burmannicoides/Calcutta 4

AAw banksii/banksii0341 AAw banksii/Higa

AAw malaccensis/Selangor 2 x AAw malaccensis/Kluai Pal

AAw zebrina/zebrina1177 AAw banksii/banksii1216

AAw malaccensis/malaccensis0250

AAw banksii/Madang

AAw burmannica/Long Tavoy

AAw banksii/banksii UPLB

AAw truncata/truncata AAw malaccensis/malaccensis0399 AAw banksii/banksii0453 AAw banksii/Hawain 3 AAw malaccensis/Pahang AAw banksii/Hawain 2 AAw banksii/Hawain 3 AAw banksii/banksii0619 AAw banksii/banksii0620 AAw banksii/banksii0623 AAw banksii/banksii0624

AAw siamea/Khae (Phrae) x

AAw malaccensis/Pisang Cici (Brasil) AAw zebrina/Maia Oa AAw banksii/Paliama AAw banksii/banksii0805 AAw banksii/banksii0806 AAw banksii/banksii0852 AAw banksii/banksii0853 AAw banksii/banksii0867 AAw banksii/banksii0879 AAw banksii/banksii0885 AAw banksii/banksii0897 AAw banksii/banksii0955

AAw malaccensis/Pisang Kra

AAw errans/Agutay

AAw zebrina/Monyet AAw banksii/Hawain No.2 AAw banksii/banksii1219

AAw malaccensis/Pisang Serun 404

AAw malaccensis/Pisang Serun 400

banksii

malaccensis

siamea/burma

zebrina

NJ tree analysis wild M. acuminata ssp.

44 accessions 468 markers

banksii, malaccensis, zebrina,

siamea/burma ssp form distinct clusters

wrong accession

held at ITC

accession

misclassified in the

field genebank

(8)

ITC0004_AAA/Pisang Nangka

ITC0836_AAB ind./Kunaimp ITC0871_AAB ind./Kimbem

ITC1062_AAB ind./Pisang Nangka

AAB

ABB

AAB

AAA 1

AAA 2

ITC0343 Pisang Raja Bulu

consistent clustering at the

group and subgroup level

NJ tree analysis triploides

(9)

AAB ABB AAB AAA1 AAA2

accessions

classified “AAB”

to reclassify

“AAA”

consistent clustering at the

group and subgroup level

Cavendish

Gros Michel

Ibota

accessions

classified “AAA

undetermined”

that should be

reclassified as

“Cavendish”

NJ tree analysis triploides

(10)

Analysis of 712 ITC accessions with DArTs

Combined with ploidy checking, the analysis of 712

ITC accessions resulted in :

• 582 are well classified (81%)

• classification of 67 accessions has been

specified

• 42 (less than 6%) are truly misclassified (e.g.

an accession classified AAB while it is a AAA)

Include accessions that were introduced

under a false identification and errors at ITC.

• 29 (4%) accessions to be eliminated

(redundancy)

well classified

classification specified

misclassified

to eliminate

(11)

ITC

in vitro collection

712 accessions

CIRAD

Guadeloupe

QDPI

Australia

. . .

MOS

Most Original sample

sent to ITC

Introductions from ITC

Field collections:

Field verification

morphological characters

site 1

site 2

site 3

TTT true to type

OT off type

ML mislabelling

Genotyping

ITC accessions

DArT

498 markers

712 accessions

ITC/CIRAD

DArT

836 markers

156 accessions

241 markers shared

113 accessions:

70 MOS + 43 intro

Classification

C well classified

MC misclassified

D dubious

diversity analysis

Cytology

Ploidy level

workplan of the

verification of the

genetic integrity of

ITC Musa accession

ITC accessions compared to

MOS or REF by genotyping

ITC/MOS-REF

DArT

1088 markers

+220 accessions

1088 markers

110 accessions pairs:

63 MOS + 47 REF

Flew cytometry

(12)

Comparison of ITC and CIRAD common accessions

Methods

• Joint analysis of 241 DArTs markers in common on 113 genotypes in

common in ITC collection and CIRAD Guadeloupe field genebank.

• Dissimilarity index calculated between each pair of accessions of the

same genotype

Results

• Definition of a statistical threshold by permutation test

• Estimation of a dissimilarity between ITC and Guadeloupe accessions

• Comparison with field verification results

(13)

Dissimilarit y index >12% = > genet ic diff erence 113 pairs of accessions analysed

•

92 pairs are considered as ident ical

•

21 pairs have dissimilarities exceeding 12% (inc lude miss ing data and outgroups ) The errors may have occured at I TC collect ion or at CI RAD genebank

Comparison of ITC and CIRAD common accessions

0 10 20 30 40 50 60 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80

mismatches %

frequencies

0 10 20 30 40 50 60 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80

mismatches %

frequencies

Dissimilarity %

Frequency

dissimilarty > 12%

(14)

Comparison of ITC and CIRAD common accessions

Genotyping

(DArTs)

D>12%

D<12%

Phenotyping

(fied

v

e

r

i

f

i

c

a

t

i

o

n

)

Mislabeled

6/10

4/56

Offtypes

0/10

4/56

True to type

4/10

48/56

Comparision with field verification

results

•4 accessions out of 10 considered as

mislabelled in the field are not

detected by DArTS

•Offtypes are NOT detected by DArTs

• 4 accessions out of 66 considered as

true to type in the field are considered

different with DArTs

(15)

Factorial analysis: Axes 1 / 2

-.45 -.4 -.35 -.3 -.25 -.2 -.15 -.1 -.05 .05 .1 .15 .2 .25 .45 .4 .35 .3 .25 .2 .15 .1 .05 -.05 -.1 -.15 -.2 ITC0020_ICE_CREAM ITC0082_INTOKATOKE ITC0084_MBWAZIRUME ITC0093_LONG_TAVOY ITC0109_OBINO_l_EWAI ITC0121_IHITISM ITC0123_SIMILI_RADJAH ITC0243_PISANG_RAJAH ITC0245_SAFET_VELCHI ITC0247_HONDURAS ITC0249_CALCUTTA4 ITC0253_BORNEO ITC0277_LEITE ITC0283_LONG_TAVOY ITC0298_BERAM ITC0312_PISANG_JARI_BUAYA ITC0314_PISANG_SIPULU ITC0334_NZIZI ITC0335_POPOULU ITC0345_POYO ITC0373_UWATI ITC0394_CARDABA ITC0409_PA_PATTHALONG ITC0415_PISANG_CICI_ALAS ITC0420_PISANG_KAYU ITC0424_PISANG_USTRALI ITC0428_HIGA ITC0431_KAWIJAM ITC0432_PAMOTION ITC0438_DIPLOIDE_BASILAN ITC0439_SAING_HIL ITC0440_MORONG_PRINCESA ITC0443_INABACA ITC0445_MORONG_PRINCIPE ITC0449_PISANG_LAWADIN ITC0450_PISANG_PALEMBANG ITC0472_PELIPITA ITC0473_BALONKAWE ITC0475_PISANG_ANGLENG_HITAM_{ITC0477_INARNIBAL} ITC0517_ORISHELE ITC0575_RED_DACCA ITC0582_LADY_FINGER ITC0587_PISANG_RAJAH ITC0609_PAHANG ITC0623_BANKSII ITC0637_ORNATA ITC0649_FOCONAH ITC0652_KLUAI_TIPAROT ITC0653_PISANG_MAS ITC0654_PETITE_NAINE ITC0659_NAMWA_KHOM ITC0660_KHAE_PHRAE ITC0662_KHAI_THONG_RUANG_{ITC0683_PISANG_GLINTONG} ITC0688_ID077 ITC0698_PISANG_KEPOK_BUNG ITC0735_DIBY1 ITC0737_KINGALA_NO1 ITC0766_PALIAMA ITC0767_DOLE ITC0769_FIGUE_POMME_GEANTE ITC0824_KUMUNAMBA ITC0843_PISANG_RAJA_BULU ITC0855_BENGANI ITC0856_SCHIZOCARPA ITC0898_SAR ITC0910_PALANG ITC0962_PRATA_ANA ITC0975_PISANG_GUNINCHIO ITC0983_AUKO ITC0987_AUKO ITC0990_VUNAPOPE ITC0993_YAWA_DWARF ITC0996_MANAMEG_RED ITC1034_KUNAAN ITC1062_PISANG_NANGKA ITC1064_PISANG_BAKAR ITC1072_TE9TILIS ITC1077_PISANG_KLUTUK ITC1120_TANI ITC1122_GROS_MICHEL ITC1123_YANGAMBI_KM5 ITC1138_SABA ITC1139_ZEBRINA ITC1140_RED_YADE ITC1152_WOMPA ITC1156_PISANG_BATU ITC1166_NGENGE ITC1177_ZEBRINA ITC1187_TOMOLO ITC1238_YAWA2 ITC1253_MJENGA_GROS_MICHEL ITC1275_YANGAMBI_NO2 ITC1287_PISANG_BERANGAN ITC1325_ORISHELE ITC1365_TAY_TIA ITC1373_CHUOI_VA_HUONG ITC1374_CHUOI_TIEU_LIEN ITC1382_CHUOI_NAM ITC1423_CHUOI_TIEU_VUA ITC1425_CHUOI_9IEM_MAT ITC1432_CHUOI_RUNG_HOA_9OAN ITC1438_CHUOI_BOM ITC1441_PISANG_CEYLAN ITC1482_POYO ITC1483_MONTHAN ITC1586_GRANDE_NAINE ITC1587_PISANG_KLUTUK_WULUNGITC1588_LAL_VELCHI ITC0020_ICE_CREAM ITC0082_INTOKATOKE ITC0084_MBWAZIRUME ITC0093_LONG_TAVOY ITC0109_OBINO_l_EWAI ITC0121_IHITISM ITC0123_SIMILI_RADJAH ITC0243_PISANG_RAJAH ITC0245_SAFET_VELCHI ITC0247_HONDURAS ITC0249_CALCUTTA4 ITC0253_BORNEO ITC0277_LEITE ITC0283_LONG_TAVOY ITC0298_BERAM ITC0312_PISANG_JARI_BUAYA ITC0314_PISANG_SIPULU ITC0334_NZIZI ITC0335_POPOULU ITC0345_POYO ITC0373_UWATI ITC0394_CARDABA ITC0409_PA_PATTHALONG ITC0415_PISANG_CICI_ALAS ITC0420_PISANG_KAYU ITC0424_PISANG_USTRALI ITC0428_HIGA ITC0431_KAWIJAM ITC0432_PAMOTION ITC0438_DIPLOIDE_BASILAN_{ITC0439_SAING_HIL} ITC0440_MORONG_PRINCESA ITC0443_INABACA ITC0445_MORONG_PRINCIPE ITC0449_PISANG_LAWADIN ITC0450_PISANG_PALEMBANG ITC0472_PELIPITA ITC0473_BALONKAWE ITC0475_PISANG_ANGLENG_HITAM ITC0477_INARNIBAL ITC0517_ORISHELE ITC0575_RED_DACCA ITC0582_LADY_FINGER ITC0587_PISANG_RAJAH ITC0609_PAHANG ITC0623_BANKSII ITC0637_ORNATA ITC0649_FOCONAH ITC0652_KLUAI_TIPAROT ITC0653_PISANG_MAS ITC0654_PETITE_NAINE ITC0659_NAMWA_KHOM ITC0660_KHAE_PHRAE ITC0662_KHAI_THONG_RUANG ITC0683_PISANG_GLINTONG ITC0688_ID077 ITC0698_PISANG_KEPOK_BUNG ITC0735_DIBY1 ITC0737_KINGALA_NO1 ITC0766_PALIAMA ITC0767_DOLE ITC0769_FIGUE_POMME_GEANTE ITC0824_KUMUNAMBA ITC0843_PISANG_RAJA_BULU ITC0855_BENGANI ITC0856_SCHIZOCARPA ITC0898_SAR ITC0910_PALANG_PNG ITC0962_PRATA_ANA ITC0975_PISANG_GUNINCHIO ITC0983_AUKO ITC0987_AUKO ITC0990_VUNAPOPE ITC0993_YAWA_DWARF ITC0996_MANAMEG_RED ITC1034_KUNAAN ITC1062_PISANG_NANGKA ITC1064_PISANG_BAKAR ITC1072_TE9TILIS ITC1077_PISANG_KLUTUKITC1120_TANI ITC1122_GROS_MICHEL ITC1123_YANGAMBI_KM5 ITC1138_SABA ITC1139_ZEBRINA ITC1140_RED_YADE ITC1152_WOMPA ITC1156_PISANG_BATU ITC1166_NGENGE ITC1177_ZEBRINA ITC1187_TOMOLO ITC1238_YAWA2 ITC1253_MJENGA_GROS_MICHEL ITC1275_YANGAMBI_NO2 ITC1287_PISANG_BERANGAN ITC1325_ORISHELE ITC1365_TAY_TIA ITC1373_CHUOI_VA_HUONGITC1374_CHUOI_TIEU_LIEN ITC1382_CHUOI_NAM ITC1423_CHUOI_TIEU_VUA ITC1425_CHUOI_9IEM_MAT ITC1432_CHUOI_RUNG_HOA_9OAN ITC1438_CHUOI_BOM ITC1441_PISANG_CEYLAN ITC1482_POYO ITC1483_MONTHAN _GRANDE_NAINE _PISANG_KLUTUK_WULUNG _LAL_VELCHI

AFTD

110 accession pairs

1088 DArTs

ITC (red)

63 MOS and 47 REF (green)

P. sipu

lu

**Comparison of ITC and MOS/REF accessions* by DArTs**

(16)

Conclusions

Morphological and molecular characterization are complementary tools :



_{DArT markers are able to detect ‘Mislabelled accessions’ if the exchange has}

happened between genetically distant accessions but if mislabelling occurs

between two accessions from the same subgroup, our observations suggest

that DArT markers would not be powerful enough to detect the error.



_{DArT markers do not detect ‘Off-types’ that are due to somaclonal variations.}