Genetic integrity of the ITC collection : DArT genotyping. J.P. Horry, X. Perrier, N. Roux, S. Channeliere

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1 Genetic integrity of the ITC collection : genotyping J.P. Horry, X. Perrier, N. Roux, S. Channeliere

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 Field collections: MOS Most Original sample sent to ITC Introductions from ITC ITC in vitro collection 712 accessions CIRAD Guadeloupe QDPI... Australia workplan of the verification of the genetic integrity of ITC Musa accession Field verification morphological characters site 1 site 2 site 3 TTT true to type OT off type ML mislabelling Cytology Flow cytometry Ploidy level Genotyping ITC accessions 498 markers 712 accessions diversity analysis ITC accessions compared to MOS or REF by genotyping ITC/CIRAD 836 markers 156 accessions ITC/MOS-REF 1088 markers +220 accessions Periodical verification of trueness-to-type (accessions maintained in vitro for >10 years) Classification C well classified MC misclassified D dubious 241 markers shared 113 accessions: 70 MOS + 43 intro 1088 markers 110 accessions pairs: 63 MOS + 47 REF

4 Diversity Arrays Technology () GCP: 168 accessions from IITA and CIRAD analysed with 836 s markers : «s can be used for genome wide analyses», Despite the dominant nature of 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 Field collections: MOS Most Original sample sent to ITC Introductions from ITC ITC in vitro collection 712 accessions CIRAD Guadeloupe QDPI... Australia workplan of the verification of the genetic integrity of ITC Musa accession Field verification morphological characters site 1 site 2 site 3 TTT true to type OT off type ML mislabelling Cytology Flew cytometry Ploidy level Genotyping ITC accessions 498 markers 712 accessions diversity analysis ITC accessions compared to MOS or REF by genotyping ITC/CIRAD 836 markers 156 accessions ITC/MOS-REF 1088 markers +220 accessions Classification C well classified MC misclassified D dubious 241 markers shared 113 accessions: 70 MOS + 43 intro 1088 markers 110 accessions pairs: 63 MOS + 47 REF

6 Analysis of 712 ITC accessions with s 498 markers. The phylogenetic tree produced by analyzing the 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). markers are able to spot accessions which are not grouping with what was expected. These are clearly misclassified accessions. In many cases analysis allowed to complement a classification (eg. the subgroup of a poorly identified accession can be identified).

7 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 AAw banksii/banksii0620 AAw banksii/banksii0341 AAw banksii/banksii0453 AAw banksii/higa AAw banksii/banksii0623 AAw banksii/banksii0619 AAw banksii/banksii0806 AAw banksii/banksii0805 AAw banksii/banksii0885 AAw banksii/banksii0879 AAw banksii/banksii0624 AAw banksii/hawain 3 AAw banksii/hawain 3 AAw banksii/banksii0867 AAw banksii/banksii0853 AAw banksii/banksii0852 AAw banksii/banksii1219 AAw banksii/banksii0955 AAw banksii/paliama AAw banksii/hawain No.2 AAw banksii/hawain 2 AAw banksii/banksii0897 AAw banksii/banksii1216 AAw errans/agutay AAw banksii/banksii UPLB AAw malaccensis/pisang Serun 400 AAw malaccensis/kluai Pal AAw malaccensis/pisang Kra AAw malaccensis/pahang AAw banksii/madang AAw malaccensis/pisang Serun 404 AAw malaccensis/selangor 2 x AAw malaccensis/malaccensis AAw malaccensis/malaccensis0399 AAw malaccensis/malaccensis0250 AAw zebrina/maia Oa AAw zebrina/zebrina1177 AAw zebrina/monyet AAw malaccensis/pisang Cici (Brasil) AAw siamea/khae (Phrae) x AAw burmannica/long Tavoy AAw burmannicoides/calcutta 4 AAw burmannica/long Tavoy AAw truncata/truncata banksii malaccensis zebrina siamea/burma

8 NJ tree analysis triploides 292 accessions, 498 markers consistent clustering at the group and subgroup level AAB ABB AAB ITC0343 Pisang Raja Bulu ITC0836_AAB ind./kunaimp ITC1062_AAB ind./pisang Nangka ITC0004_AAA/Pisang Nangka ITC0871_AAB ind./kimbem AAA 2 AAA 1

9 NJ tree analysis triploides 292 accessions, 498 markers consistent clustering at the group and subgroup level AAB ABB AAB Cavendish accessions classified AAA undetermined that should be reclassified as Cavendish AAA2 AAA1 Gros Michel Ibota accessions classified AAB to reclassify AAA

10 Analysis of 712 ITC accessions with s 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 Field collections: MOS Most Original sample sent to ITC Introductions from ITC ITC in vitro collection 712 accessions CIRAD Guadeloupe QDPI... Australia workplan of the verification of the genetic integrity of ITC Musa accession Field verification morphological characters site 1 site 2 site 3 TTT true to type OT off type ML mislabelling Cytology Flew cytometry Ploidy level Genotyping ITC accessions 498 markers 712 accessions diversity analysis ITC accessions compared to MOS or REF by genotyping ITC/CIRAD 836 markers 156 accessions ITC/MOS-REF 1088 markers +220 accessions Classification C well classified MC misclassified D dubious 241 markers shared 113 accessions: 70 MOS + 43 intro 1088 markers 110 accessions pairs: 63 MOS + 47 REF

12 Comparison of ITC and CIRAD common accessions Methods Joint analysis of 241 s 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 92 pairs are considered as ident ical 21 pairs have dissimilarities exceeding 12% (inc lude miss ing data and outgroups ) Dissimilarit y index >12% = > genet ic diff erence 113 pairs of accessions analysed The errors may have occured at ITC collection or at CIRAD genebank Comparison of ITC and CIRAD common accessions Frequency 50 dissimilarty > 12% Dissimilarity mismatches %

14 Comparison of ITC and CIRAD common accessions Phenotyping (fied Genotyping (s) D>12% D<12% Mislabeled 6/10 4/56 Offtypes 0/10 4/56 True to type 4/10 v e r i f i c 48/56 a t i o n ) Comparision with field verification results 4 accessions out of 10 considered as mislabelled in the field are not detected by S Offtypes are NOT detected by s 4 accessions out of 66 considered as true to type in the field are considered different with s

15 Comparison of ITC and MOS/REF Factorial analysis: Axes 1 / 2 accessions* by s.45 ITC1072_TE9TILIS ITC1072_TE9TILIS ITC0898_SAR.4 AFTD 110 accession pairs 1088 s ITC (red) 63 MOS and 47 REF (green) ITC1432_CHUOI_RUNG_HOA_9OAN ITC1432_CHUOI_RUNG_HOA_9OAN ITC0637_ORNATA ITC0660_KHAE_PHRAE ITC0993_YAWA_DWARF ITC0856_SCHIZOCARPA ITC0283_LONG_TAVOY ITC0283_LONG_TAVOY.2 ITC0249_CALCUTTA4 ITC0637_ORNATA ITC0093_LONG_TAVOY ITC0247_HONDURAS ITC1588_LAL_VELCHI ITC1587_PISANG_KLUTUK_WULUNG ITC1077_PISANG_KLUTUK ITC1120_TANI ITC1156_PISANG_BATU ITC1120_TANI ITC0652_KLUAI_TIPAROT ITC0473_BALONKAWE _PISANG_KLUTUK_WULUNG ITC0312_PISANG_JARI_BUAYA ITC0314_PISANG_SIPULU ITC0312_PISANG_JARI_BUAYA ITC0440_MORONG_PRINCESA ITC1156_PISANG_BATU ITC1139_ZEBRINA ITC0996_MANAMEG_RED ITC0623_BANKSII.05 ITC0084_MBWAZIRUME ITC1238_YAWA2 ITC1187_TOMOLO ITC0898_SAR ITC0415_PISANG_CICI_ALAS ITC0623_BANKSII ITC0084_MBWAZIRUME ITC0082_INTOKATOKE ITC0253_BORNEO ITC0428_HIGA ITC1152_WOMPA ITC1253_MJENGA_GROS_MICHEL ITC0996_MANAMEG_RED ITC0334_NZIZI ITC1152_WOMPA ITC1166_NGENGE ITC0439_SAING_HIL ITC0766_PALIAMA ITC0020_ICE_CREAM ITC0334_NZIZI ITC1238_YAWA2 ITC0432_PAMOTION ITC0440_MORONG_PRINCESA ITC0432_PAMOTION ITC0653_PISANG_MAS ITC0767_DOLE ITC0975_PISANG_GUNINCHIO ITC0093_LONG_TAVOY ITC0735_DIBY1 ITC0659_NAMWA_KHOM ITC0409_PA_PATTHALONG ITC1138_SABA ITC0659_NAMWA_KHOM -.45 ITC0993_YAWA_DWARF ITC0394_CARDABA ITC0443_INABACA.25 ITC0987_AUKO ITC1483_MONTHAN ITC0123_SIMILI_RADJAH ITC1138_SABA ITC0472_PELIPITA ITC0424_PISANG_USTRALI ITC1034_KUNAAN ITC1423_CHUOI_TIEU_VUA ITC1123_YANGAMBI_KM5 ITC1438_CHUOI_BOM ITC0472_PELIPITA ITC0767_DOLE ITC0983_AUKO ITC0910_PALANG_PNG ITC0662_KHAI_THONG_RUANG ITC1139_ZEBRINA ITC0975_PISANG_GUNINCHIO ITC0990_VUNAPOPE ITC0855_BENGANI ITC0475_PISANG_ANGLENG_HITAM ITC0082_INTOKATOKE ITC0662_KHAI_THONG_RUANG ITC0688_ID077 ITC1077_PISANG_KLUTUK ITC0688_ID077 ITC0438_DIPLOIDE_BASILAN ITC0445_MORONG_PRINCIPE ITC1483_MONTHAN ITC1187_TOMOLO ITC0373_UWATI ITC0431_KAWIJAM ITC0373_UWATI ITC0855_BENGANI ITC0314_PISANG_SIPULU ITC0683_PISANG_GLINTONG ITC0298_BERAM ITC0575_RED_DACCA ITC0439_SAING_HIL ITC0298_BERAM ITC1287_PISANG_BERANGAN ITC0987_AUKO ITC0431_KAWIJAM ITC0245_SAFET_VELCHI ITC1275_YANGAMBI_NO2 ITC0654_PETITE_NAINE ITC0475_PISANG_ANGLENG_HITAM ITC0020_ICE_CREAM ITC0438_DIPLOIDE_BASILAN ITC1166_NGENGE ITC0698_PISANG_KEPOK_BUNG ITC0450_PISANG_PALEMBANG ITC1382_CHUOI_NAM ITC0245_SAFET_VELCHI ITC1275_YANGAMBI_NO2 ITC0737_KINGALA_NO1 ITC0277_LEITE ITC0477_INARNIBAL ITC1253_MJENGA_GROS_MICHEL ITC0420_PISANG_KAYU ITC0683_PISANG_GLINTONG ITC0394_CARDABA -.05 ITC0769_FIGUE_POMME_GEANTE ITC1365_TAY_TIA ITC1365_TAY_TIA ITC1122_GROS_MICHEL ITC1373_CHUOI_VA_HUONG ITC1123_YANGAMBI_KM5 ITC0698_PISANG_KEPOK_BUNG ITC0121_IHITISM ITC0345_POYO ITC1586_GRANDE_NAINE ITC0654_PETITE_NAINE ITC0445_MORONG_PRINCIPE ITC1034_KUNAAN ITC1482_POYO ITC1064_PISANG_BAKAR ITC1382_CHUOI_NAM ITC1423_CHUOI_TIEU_VUA ITC1482_POYO ITC1373_CHUOI_VA_HUONG ITC0449_PISANG_LAWADIN ITC0345_POYO ITC1374_CHUOI_TIEU_LIEN ITC1438_CHUOI_BOM ITC1122_GROS_MICHEL ITC0443_INABACA ITC0450_PISANG_PALEMBANG ITC0109_OBINO_l_EWAI ITC0517_ORISHELE ITC0109_OBINO_l_EWAI ITC0121_IHITISM -.1 ITC1062_PISANG_NANGKA ITC0335_POPOULU ITC1325_ORISHELE ITC1140_RED_YADE ITC1062_PISANG_NANGKA ITC0335_POPOULU ITC0582_LADY_FINGER ITC1325_ORISHELE ITC1140_RED_YADE ITC0517_ORISHELE ITC0735_DIBY1 ITC0824_KUMUNAMBA ITC1425_CHUOI_9IEM_MAT ITC0769_FIGUE_POMME_GEANTE ITC1425_CHUOI_9IEM_MAT ITC0449_PISANG_LAWADIN ITC0587_PISANG_RAJAH ITC1441_PISANG_CEYLAN ITC0587_PISANG_RAJAH ITC0843_PISANG_RAJA_BULU -.15 ITC0649_FOCONAH ITC0424_PISANG_USTRALI ITC0243_PISANG_RAJAH ITC0843_PISANG_RAJA_BULU ITC0582_LADY_FINGER ITC1441_PISANG_CEYLAN ITC0962_PRATA_ANA ITC0962_PRATA_ANA ITC0649_FOCONAH ITC0243_PISANG_RAJAH P. sipulu ITC0609_PAHANG ITC1177_ZEBRINA ITC0609_PAHANG ITC0415_PISANG_CICI_ALAS ITC0428_HIGA *partner collections : CIRAD (France), FAVRI (Vietnam), FHIA (Honduras), IITA (Nigeria), NARI (Papua New Guinea)

16 Conclusions Morphological and molecular characterization are complementary tools : 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 markers would not be powerful enough to detect the error. markers do not detect Off-types that are due to somaclonal variations. Morphological observation stays the most precise way to detect any loss of genetic integrity, provided that the modification / mutation affects a visible character. Recommendations Misclassification: use molecular markers and ploidy to check the classification of the accessions before being introduced in the ITC. Mislabelling: to regularly analyse accessions by batches, using molecular markers (SSR or ), which will allow to detect around half of the Mislabelled accessions. Off-types: so far, only the morphological observations can detect somaclonal variations.

Genetic Heritage of Wild Lake Trout in Lake Michigan

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