Nature Biotechnology: doi:10.1038/nbt · 2016-06-16 · Supplementary Figure 2 Southern blot analyses to verify BAC clone integrity and NB-LRR copy number (a) Southern blot of BglII-

Supplementary Figure 1

Fine-mapping of the CcRpp1 locus in the Cajanus cajan accession G119-99

(a) Physical and genetic map interval for the CcRpp1 locus based on the C. cajan reference genome scaffold (variety “Asha”). (b) Gain and loss of functions key recombinants closing the CcRpp1 genetic interval.

Nature Biotechnology: doi:10.1038/nbt.3554


Southern blot analyses to verify BAC clone integrity and NB-LRR copy number

(a) Southern blot of BglII- and HindIII-digested DNA of BAC 6G on the identified NB-LRR genes (NB 1-4) and, as a control, digested genomic DNA from the resistant parent G119-99. (b) Southern blot of HindIII- digested DNA of BACs 3F, 6G and the BAC clone obtained from DNA isolated from the resistant parent G59-95. The biotin labelled probe was designed on the region coding for the so-called “P-loop”, a highly variable structural feature present in NB-LRR proteins. The gene coding for CcRpp1 is indicated in bold.



Visualization of RNAseq reads aligned to the four NB-LRR candidate genes NB-1, -2, -3 and -4.

(a) RNAseq reads from the resistant parent G119-99 aligned against the CcRpp1 locus sequence of 205,344 bp. (b) Close-up of the NB-LRR genes -1 to -4 paired-end read coverage.



CcRpp1 expressing plants are not more resistant against another fungal plant pathogen

Disease score on CcRpp1 expressing plants homozygous for the transgene compared with segregating null plants when challenged with Fusarium virguliforme. Each dot represents a technical replicate consisting of 4-5 plants; different colours indicate different biological replicates; different symbols indicate technical replicates. Blue error bars indicate non-parametric (bootstrapped) limited 95% confidence interval of the mean. (see Supplementary Files 8-10 for details on the statistic analyses and figure generation).



Expression of CcRpp1 does not impact basic agronomic traits

(a) Null segregants compared to plants expressing CcRpp1 at 8 weeks after germination. (b) Canopy area*of null segregants and plants

homozygous for the CcRpp1 transgene (event CcRpp1 7.1) (c) Germination rate plotted against the expected germination rate. Numbers below zero indicate a lower than expected germination rate. Blue error bars indicate non-parametric (bootstrapped) limited 95% confidence interval of the mean. (see Supplementary Files 11-16 for details on the statistic analyses and figure generation). *Surface area determined with ImageJ 1.48a.


Supplementary, Table, 1:! Chi%Square!analysis! (!!)!of! the!phenotypic! segregation! in! crosses!between!different!C.#cajan!accessions!challenged!with!the#P.#pachyrhizi!isolate!PPUFV02.!!

crossesa,plants, hypothesisa,

R,:,S,,

phenotype, observed, expected, χ2!! ! ! ! ! !G119%99♀!x!G48%95♂! resistant! 195! 201! ! !or! susceptible! 73! 67! 3:1! 0.71!

G48%95♀!x!G119%99♂! total! 268! 268! ! !! ! ! ! ! !! resistant! 112! 111,75! ! !

G48%95♀!x!G59%95♂! susceptible! 37! 37,25! 3:1! 0.002!! total! 149! 149! ! !! ! ! ! ! !! resistant!! 88! 93,75! ! !

G146%97♀!x!G48%95♂! susceptible! 37! 31,25! 3:1! 1.41!! total! 125! 125! ! !

a! expected! segregation! based! on! the! hypothesis! that! one! dominant! gene! confers! resistance! (R! =!resistant;!S!=!susceptible).!!!,,, ,


Supplementary,Table,2:,Geographical!location!of!the!P.#pachyrhizi!isolates!that!were!used!to!challenge!the!C.#cajan!accessions!described!in!this!study.!Double!lines!separate!countries.!Coordinates!of!samplings!locations!are!indicated!when!known.!!,sample,, Country,and,State, isolate, coordinates,

name, latitude, longitude,

1! Brazil,#Minas#Gerais# PPUFV02! %20.766808! %42.869733!2! MG!02_14! %19.671190! %47.620300!3! MG!04_14! %19.569900! %47.010150!4! MG!07_14! %20.012680! %48.165560!5! MG!08_14! %19.761230! %47.808320!6! MG!09_14! %19.733610! %47.633980!7! MG!14_14! %19.613030! %47.198300!8! MG!15_14! %19.583910! %46.446180!9! MG!17_14! %19.133300! %48.173870!17! Brazil,#Paraná# PR!01_14! %25.254500! %50.016140!18! PR!02_14! %25.345670! %49.844600!19! PR!03_14! %25.449560! %49.739970!20! PR!04_14! %25.474600! %49.660370!25! PR!10_14! %23.765364! %51.145450!26! PR!11_14! %24.879330! %53.555550!28! PR!13_14! %22.846625! %51.031419!29! PR!14_14! %22.849756! %51.017389!30! Brazil,#Rio#Grande#do#Sul# RS!02_14! %29.251060! %52.161890!31! RS!03_14! %28.373330! %54.133710!32! RS!05_14! %28.404110! %54.542640!33! RS!08_14! %28.506710! %54.552490!34! RS!09_14! %28.593400! %53.679190!35! RS!10_14! %28.594000! %53.679190!36! RS!13_14! %28.635280! %53.471830!37! RS!16_14! %28.682150! %52.594770!38! RS!15_14! %28.647340! %53.178210!39! RS!17_14! %28.706080! %52.575300!40! RS!19_14! %28.954550! %52.373040!41! RS!20_14! %28.604820! %52.687290!42! RS!21_14! %28.642060! %52.877400!44! Brazil,#Goiás# GO!13_14! %18.570810! %52.628890!45! GO!14_14! %18.675160! %52.622190!46! GO!16_14! %17.386261! %51.366951!47! GO!17_14! %17.550200! %51.099290!48! GO!18_14! %17.794220! %50.963960!49! GO!20_14! %18.238820! %49.713500!50! GO!21_14! %17.401056! %52.682344!52! GO!23_14! %17.896389! %49.238056!54! Brazil,#Mato#Grosso#do#Sul# MS!08_14! %18.771750! %52.648310!55! MS!09_14! %18.771750! %52.648310!56! MS!11_14! %19.232170! %53.126290!57! MS!12_14! %19.242940! %53.169800!


58! MS!16_14! %18.212770! %53.121260!59! Brazil,#Mato#Grosso# MT!01_14! %15.797230! %55.397290!60! MT!03_14! %16.019750! %54.903870!61! MT!04_14! %16.061370! %54.881480!62! MT!05_14! %16.828310! %54.044580!63! MT!06_14! %16.939110! %53.567000!65! MT!17_14! %18.009580! %53.176780!66! MT!18_14! %17.689650! %53.328350!67! MT!20_14! %16.867260! %53.891320!68! MT!21_14! %15.577950! %54.383570!69! MT!22_14! %14.471190! %56.242100!70! MT!23_14! %14.510670! %56.225800!71! MT!24_14! %13.002710! %55.369350!72! MT!25_14! %13.002710! %55.369350!81! MT!42_14! %13.731806! %57.634056!82! MT!43_14! %13.676750! %57.795639!83! MT!44_14! %13.676750! %57.795639!84! MT!45_14! %16.169494! %54.793606!85! MT!46_14! %15.951261! %54.986500!86! MT!47_14! %13.743936! %57.564367!87! MT!48_14! %13.526233! %58.951864!88! MT!49_14! %15.534153! %55.186139!89! MT!50_14! %14.395078! %56.408857!90! MT!51_14! %14.377638! %56.377646!91! MT!52_14! %12.441361! %55.657544!92! MT!53_14! %16.703700! %54.662108!93! MT!54_14! %11.855883! %55.543892!94! MT!55_14! %13.853475! %56.085303!95! MT!56_14! %17.288606! %53.245183!96! MT!60_14! %15.470656! %55.590767!97! MT!64_14! %15.529878! %54.293441!98! MT!93_14! %17.817641! %53.286883!99! MT!98_14! %17.210253! %54.759944!100! MT!105_14! %16.487011! %54.647336!101! MT!106_14! %16.449317! %54.666011!102! U.S.A.,#Georgia! G05! %! %!103! U.S.A.,#Mississippi## M08! %! %!104! Japan,#Tsukuba# T1%4! %! %!

!!, ,


Supplementary,Table,3:,Primers!and!probes!used!in!this!study.,,

name, sequence,(5'C3'), description,dCAPS52491%F! ATGCGATCGACGCTGGCAT

CGGGGTAGAGGCA!Oligonucleotide!for!the!marker!dCAPS52491!for!the!CcRpp1!fine!mapping.!Restriction!enzyme!SphI.!Fragment!size!of!121!bp.!!

dCAPS52491%R! GGGATTTAGCGTACATAGTGTATT!

Oligonucleotide!for!the!marker!dCAPS52491!for!the!CcRpp1!fine!mapping.!Restriction!enzyme!SphI.!Fragment!size!of!121!bp.!!

SSR12872%F! GGTAGTAGTAGTATAGTATTCATGATTGAT!

Oligonucleotide!for!the!marker!SSR12872!for!the!CcRpp1!fine!mapping.!Fragment!size!of!129!bp.!!

SSR12872%R! TGAAACATTTACAGGAAGTGAA!

Oligonucleotide!for!the!marker!SSR12872!for!the#CcRpp1!fine!mapping.!Fragment!of!129!bp.!!

dCAPS140555%F! TTTAGCACACATTCTAGAGCTGAGGAGGCATA!

Oligonucleotide!for!the!marker!dCAPS140555!for!the!CcRpp1!fine!mapping.!Restriction!enzyme!NdeI.!Fragment!size!of!289!bp.!Marker!used!to!screen!the!BAC!library.!!

dCAPS140555%R! AATAGCTATCAGAGTTCATGTCCACATTC!

Oligonucleotide!for!the!marker!dCAPS140555!for!the!CcRpp1!fine!mapping.!Restriction!enzyme!NdeI.!Fragment!size!of!289!bp.!Marker!used!to!screen!the!BAC!library.!!

CAPS20006%F! AAGACTACTTGTACGAGCTCATTG!

Oligonucleotide!for!the!marker!CAPS20006!for!the!CcRpp1!fine!mapping.!This!marker!has!homology!to!the!CCFNBFLRR!genes!1!to!4.!Restriction!enzyme!BbsI.!Fragment!size!of!205!bp.!!

CAPS20006%R! TTACAATGAATGGACAGTCTG!

Oligonucleotide!for!the!marker!CAPS20006!for!the!CcRpp1!fine!mapping.!This!marker!has!homology!to!the!CCFNBFLRR!genes!1!to!4.!Restriction!enzyme!BbsI.!Fragment!of!205!bp.!!

SSR2152%F! ATTAGCTTTCCGTTTCCCTTCT!


SSR2152%R! AAGTCCTGTCATTAGGGCTGAA!


CC%NBLRR%1%nested%F!!

TTGTTCATGGTTGACGGAGA!

CCFNBLRRF1#sequence.#Fragment!size!of!3478!bp.!!#

CC%NBLRR%1%nested%R!!

TTTGAGATCAAAAACACAATTAATCC!

CCFNBLRRF1#sequence#


CAAACACAGAGGAGATTATATCACAGTG!

CCFNBLRRF2#sequence.#Fragment!size!of!3229!bp.!#


CAATTTTGTTGTCCCGTTCTG!



CACTCTCCCTTGCATGAACA! CCFNBLRRF3#sequence.#Fragment!size!of!3950!bp.#


CAATTACAATACTTGAGATCAAAAACA!


CC%NBLRR%4%nested%F! ACAACAGGCAAGGGATCG CCFNBLRRF4#sequence.#Fragment!size!of!4382!bp.!!


! GGAGCC! #CC%NBLRR%4%nested%R!

!TATATATCATTTGATACATTAACG!


CC%NBLRR%1%codingregion%F!

!

ATGGCGGACAGTGTGGTTTCCTTT!

CCFNBLRRF1#coding!region!size:!2862!bp.#

CC%NBLRR%1%codingregion%R!

!

CTAACAAATACCACGTGAATTAAATGC!

CCFNBLRRF1#coding!region!size:!2862!bp.!


!

ATGGCAGACAGTGTGGTTTCCTTT!

CCFNBLRRF2!coding!region!size:!2778!bp.#


!

CTAGTTGTCATGATCATCGACTATG!

CCFNBLRRF2!coding!region!size:!2778!bp.!


!


CCFNBLRRF3!coding!region!size:!2793!bp.#


!

CTACGGTTCAACTATAAGTTCAACC!

CCFNBLRRF3!coding!region!size:!2793!bp.!


!


CCFNBLRRF4#coding!region!size:!2847!bp.#


!

TCATCCATAGATAATCACAGTAGAATC!

CCFNBLRRF4#coding!region!size:!2847!bp.!

Ra%NBLRR!9F! GCTATTACAAAGCCTAGGGT!

Forward!primer!for!RT%PCR!analysis!of!CcRpp1!expression!in!T0!plants;!fragment!size:!543!bp.!!

UBQ%R! CCACCAAAGTTTTACATGAAACG!

Reverse!primer!for!RT%PCR!analysis!of!CcRpp1!expression!in!T0!plants;!fragment!size:!543!bp.!

Ra6F4! TGGGCAAGGACCTGTGAAG!

Forward!primer!for!T1!CcRpp1!qRT%PCR!expression!analysis;!fragment!size:!66!bp.!!

Ra6R4! CGGAGATTCCACATAACCTCATT!

Reverse!primer!for!T1!CcRpp1!qRT%PCR!expression!analysis;!fragment!size:!66!bp.!!

Cons7%4342F! GAGCAGCTGGGGTACATTACTGT!

Forward!primer!for!T1!internal!control!qRT%PCR!expression!analysis;!fragment!size:!118!bp.!!

Cons7%4344R! CCTCGACCCTTTGCTCAATG! Reverse!primer!for!T1!internal!control!qRT%PCR!expression!analysis;!fragment!size:!118!bp.!!

Ra6Probe4! TTCAAGGCCACTATTCAGGAT!(FAM%MGB)!

Probe!for!detection!of!CcRpp1!amplification!products!of!Ra6F4!and!Ra6R4.!!

Cons7%QP139! TGGCATACGTGGACTAC!(VIC%MGB)!

Probe!for!detection!of!internal!control!(Zinc%metallopeptidase)!amplification!products!of!Cons7%4342F!and!Cons7%4344R.!

,, ,


Supplementary,Table,4:!Average!lesion!count!and!transgene!copy!number!on!leaves!harvested!from!individual!T1!plants!challenged!with!P.#pachyrhizi.!

sample,number, copy,number, scored,(DAI), R,geneCevent,average,lesion,count,

per,cm2,leaf,

1! 2! 29! CcRpp1.5.1! 3.33189!2! 4! 29! CcRpp1.5.1! 3.86E%02!3! 2! 29! CcRpp1.5.1! 2.136475!4! 2! 15! CcRpp1.5.1! 11.47195!5! 2! 29! CcRpp1.5.1! 1.272032!6! 2! 29! CcRpp1.5.1! 9.483924!7! 0! 15! CcRpp1.5.1! 6.121527!8! 0! 15! CcRpp1.5.1! 14.19253!9! 0! 15! CcRpp1.5.1! 25.92788!10! 2! 29! CcRpp1.5.1! 12.88536!11! 2! 29! CcRpp1.5.1! 1.390825!12! 0! 15! CcRpp1.5.1! 5.786687!13! 2! 29! CcRpp1.5.1! 0.8108795!14! 0! 15! CcRpp1.5.1! 4.717413!15! 0! 15! CcRpp1.5.1! 3.858117!16! 0! 15! CcRpp1.5.1! 9.783814!17! 0! 15! CcRpp1.5.1! 7.657857!18! 2! 29! CcRpp1.5.1! 4.026186!19! 4! 29! CcRpp1.5.1! 0!20! 2! 29! CcRpp1.5.1! 1.396809!21! 4! 29! CcRpp1.5.1! 0!22! 2! 29! CcRpp1.5.1! 3.35737!23! 2! 29! CcRpp1.5.1! 1.746732!24! 4! 29! CcRpp1.5.1! 0!25! 0! 15! CcRpp1.5.1! 8.030923!26! 2! 29! CcRpp1.5.1! 5.197651!27! 0! 15! CcRpp1.5.1! 21.99426!28! 0! 29! CcRpp1.5.1! 3.638567!29! 2! 29! CcRpp1.5.1! 5.89774!30! 2! 15! CcRpp1.5.1! 10.46497!31! 0! 15! CcRpp1.5.1! 3.888193!32! 2! 29! CcRpp1.5.1! 1.223785!33! 2! 29! CcRpp1.5.1! 3.985063!34! 4! 29! CcRpp1.5.1! 0!35! 0! 15! CcRpp1.5.1! 6.479619!36! 0! 15! CcRpp1.5.1! 6.038405!37! 4! 29! CcRpp1.5.1! 3.97E%02!38! 4! 29! CcRpp1.5.1! 3.42E%02!39! 2! 29! CcRpp1.5.1! 0.294127!40! 4! 29! CcRpp1.5.1! 0!41! 2! 29! CcRpp1.5.1! 3.607623!42! 2! 29! CcRpp1.5.1! 1.622122!


43! 0! 15! CcRpp1.5.1! 4.705379!44! 2! 29! CcRpp1.5.1! 1.711984!45! 4! 29! CcRpp1.5.1! 0!46! 2! 29! CcRpp1.5.1! 2.608662!47! 0! 15! CcRpp1.5.1! 10.96335!48! 2! 29! CcRpp1.5.1! 2.488083!49! 4! 29! CcRpp1.5.1! 0!50! 2! 29! CcRpp1.5.1! 2.694437!51! 2! 29! CcRpp1.5.1! 1.400268!52! 2! 29! CcRpp1.5.1! 1.860417!53! 0! 15! CcRpp1.7.1! 7.354984!54! 1! 29! CcRpp1.7.1! 4.286582!55! 2! 29! CcRpp1.7.1! 0!56! 1! 29! CcRpp1.7.1! 4.120177!57! 1! 29! CcRpp1.7.1! 2.654222!58! 0! 15! CcRpp1.7.1! 20.87732!59! 0! 15! CcRpp1.7.1! 7.43335!60! 0! 15! CcRpp1.7.1! 15.41043!61! 1! 29! CcRpp1.7.1! 3.6223!62! 0! 15! CcRpp1.7.1! 10.05334!63! 1! 29! CcRpp1.7.1! 0.1612305!64! 1! 29! CcRpp1.7.1! 2.812133!65! 1! 29! CcRpp1.7.1! 2.42304!66! 0! 15! CcRpp1.7.1! 8.00108!67! 1! 29! CcRpp1.7.1! 5.819004!68! 1! 29! CcRpp1.7.1! 4.355444!69! 0! 15! CcRpp1.7.1! 3.939862!70! 2! 29! CcRpp1.7.1! 0!71! 1! 29! CcRpp1.7.1! 3.541141!72! 1! 15! CcRpp1.7.1! 3.941022!73! 1! 29! CcRpp1.7.1! 4.059883!74! 1! 29! CcRpp1.7.1! 3.710176!75! 2! 29! CcRpp1.7.1! 0!76! 1! 29! CcRpp1.7.1! 3.638881!77! 2! 29! CcRpp1.7.1! 0!78! 0! 29! CcRpp1.7.1! 4.383701!79! 0! 15! CcRpp1.7.1! 24.99284!80! 1! 29! CcRpp1.7.1! 5.179039!81! 1! 29! CcRpp1.7.1! 2.368011!82! 1! 29! CcRpp1.7.1! 1.47432!83! 2! 29! CcRpp1.7.1! 0!84! 1! 29! CcRpp1.7.1! 2.109487!85! 0! 29! CcRpp1.7.1! 5.914096!86! 2! 29! CcRpp1.7.1! 0!87! 2! 29! CcRpp1.7.1! 0!88! 1! 29! CcRpp1.7.1! 1.392124!


89! 1! 29! CcRpp1.7.1! 1.668585!90! 1! 29! CcRpp1.7.1! 5.910942!91! 0! 15! CcRpp1.7.1! 3.045878!92! 2! 29! CcRpp1.7.1! 0!93! 0! 15! CcRpp1.7.1! 10.67294!94! 1! 29! CcRpp1.7.1! 2.746685!95! 2! 29! CcRpp1.7.1! 4.12E%02!96! 1! 29! CcRpp1.7.1! 2.792706!97! 2! 29! CcRpp1.7.1! 0!98! 0! 15! CcRpp1.7.1! 14.55001!99! 2! 29! CcRpp1.7.1! 0!100! 1! 29! CcRpp1.7.1! 2.52511!101! 2! 29! CcRpp1.7.1! 2.52E%02!102! 0! 15! CcRpp1.7.1! 3.483124!103! 2! 29! CcRpp1.7.1! 0!104! 1! 29! CcRpp1.7.1! 2.865822!105! 1! 29! CcRpp1.7.1! 1.505028!106! 2! 29! CcRpp1.7.1! 0!107! 0! 15! CcRpp1.7.1! 28.74361!108! 1! 29! CcRpp1.7.1! 0.6321178!109! 2! 29! CcRpp1.7.1! 0!110! 0! 15! CcRpp1.7.1! 5.196421!111! 1! 29! CcRpp1.7.1! 1.980603!112! 1! 29! CcRpp1.7.1! 2.271328!113! 2! 29! CcRpp1.7.1! 0!114! 0! 15! CcRpp1.7.1! 5.045407!115! 1! 29! CcRpp1.7.1! 9.17E%02!116! 1! 29! CcRpp1.7.1! 3.11872!117! 2! 29! CcRpp1.7.1! 0!118! 1! 29! CcRpp1.7.1! 10.57436!119! 2! 29! CcRpp1.7.1! 0!120! 2! 29! CcRpp1.7.1! 0!121! 1! 29! CcRpp1.7.1! 0.539324!122! 1! 29! CcRpp1.7.1! 11.19678!123! 0! 15! CcRpp1.7.1! 4.785155!124! 0! 15! CcRpp1.7.1! 12.98839!125! 0! 15! CcRpp1.7.1! 5.380365!126! 2! 29! CcRpp1.7.1! 0!127! 0! 29! CcRpp1.7.1! 8.696402!128! 1! 29! CcRpp1.7.1! 0.5453197!129! 2! 29! CcRpp1.7.1! 0!130! 0! 15! CcRpp1.7.1! 4.549024!131! 0! 15! CcRpp1.7.1! 8.379582!132! 1! 29! CcRpp1.7.1! 0.6510592!133! 1! 29! CcRpp1.7.1! 0.1505407!134! 0! 29! CcRpp1.7.1! 4.804328!


135! 1! 29! CcRpp1.7.1! 1.236849!136! 0! 15! CcRpp1.7.1! 5.471978!137! 0! 15! CcRpp1.7.1! 15.80409!138! 1! 29! CcRpp1.7.1! 0.8199618!139! 1! 29! CcRpp1.7.1! 1.457871!140! 0! 15! CcRpp1.7.1! 13.28372!141! 2! 29! CcRpp1.7.1! 0!142! 1! 29! CcRpp1.7.1! 0.4952864!143! 2! 29! CcRpp1.7.1! 0!144! 1! 29! CcRpp1.7.1! 5.617968!145! 1! 29! CcRpp1.7.1! 0.1235167!146! 2! 29! CcRpp1.7.1! 4.00E%02!147! 0! 29! CcRpp1.7.1! 7.049789!148! 1! 29! CcRpp1.7.1! 0.9767177!149! 1! 29! CcRpp1.7.1! 3.65398!150! 2! 29! CcRpp1.7.1! 0!151! 2! 29! CcRpp1.7.1! 0!152! 1! 29! CcRpp1.7.1! 2.191817!153! 2! 29! CcRpp1.7.1! 8.70E%02!154! 1! 29! CcRpp1.7.1! 1.539256!155! 0! 15! CcRpp1.7.1! 5.054699!156! 1! 29! CcRpp1.7.1! 4.475985!157! 0! 15! CcRpp1.7.1! 9.403294!

,, ,


Supplementary,Table,5:!Average!transgene!expression!level!and!transgene!copy!number!in!individual!T1!plants!from!the!events!CcRpp1.5.1!and!CcRpp1.7.1!using!TaqMan!analyses.!!

event,and,plant,nr.,, zygosity, quantity,mean,,,,,,,

Ra6,probe4,quantity,mean,,,,,,,,,,,cons7,probe,

relative,expression,(Ra6/cons7),

5.1%plant!1! null! 183.753! 15,537.563! 0.012!5.1%plant!2! null! 71.714! 18,094.055! 0.004!5.1%plant!3! null! 109.927! 22,266.697! 0.005!5.1%plant!4! null! 59.701! 20,025.637! 0.003!5.1%plant!5! null! 52.510! 16,395.643! 0.003!5.1%plant!6! hemiz! 28,760.875! 21,317.842! 1.349!5.1%plant!7! hemiz! 27,209.100! 24,129.838! 1.128!5.1%plant!8! hemiz! 30,247.977! 25,839.969! 1.171!5.1%plant!9! hemiz! 31,895.838! 20,722.572! 1.539!5.1%plant!10! hemiz! 26,904.416! 20,187.881! 1.333!5.1%plant!11! homoz! 65,722.727! 24,584.119! 2.673!5.1%plant!12! homoz! 64,346.531! 25,010.396! 2.573!5.1%plant!13! homoz! 57,664.473! 25,038.955! 2.303!5.1%plant!14! homoz! 78,134.063! 28,913.422! 2.702!7.1%plant!1! null! 353.186! 17,125.797! 0.021!7.1%plant!2! null! 207.388! 21,275.822! 0.010!7.1%plant!3! null! 321.715! 19,357.057! 0.017!7.1%plant!4! null! 102.394! 18,372.391! 0.006!7.1%plant!5! null! 142.047! 21,292.803! 0.007!7.1%plant!6! hemiz! 41,172.703! 26,593.268! 1.548!7.1%plant!7! hemiz! 35,885.332! 22,117.625! 1.622!7.1%plant!8! hemiz! 41,297.172! 25,181.729! 1.640!7.1%plant!9! hemiz! 42,479.230! 24,137.807! 1.760!7.1%plant!10! hemiz! 33,602.324! 24,390.070! 1.378!7.1%plant!11! homoz! 56,353.895! 20,796.107! 2.710!7.1%plant!12! homoz! 72,690.680! 30,512.900! 2.382!7.1%plant!13! homoz! 101,600.195! 34,955.363! 2.907!7.1%plant!14! homoz! 71,352.375! 31,408.506! 2.272!7.1%plant!15! homoz! 51,255.434! 26,683.084! 1.921!

!! !


Supplementary,Table,6:!Chi%Square!analysis!(!!)!of!observed!versus!expected!germination!rates!in!selfed!plants!that!are!hemizygous!for!the!CcRpp1!transgene!(event!7.1).!!!copy,number, observed, expected, deviation, (deviation)2, χ2,

! ! ! ! ! !null! 116! 122.75! %6.75! 45.5625! 0.371!hemizygous! 245! 245.5! %0.5! 0.25! 0.001!homozygous! 109! 122.75! %13.75! 189.0625! 1.54!

a!expected!germination!based!on!the!hypothesis!that!hemizygous!CcRpp1!plants!segregate!1:2:1.!!!,!


Documents

Nature Biotechnology: doi:10.1038/nbt · 2016-06-16 · Supplementary Figure 2 Southern blot analyses to verify BAC clone integrity and NB-LRR copy number (a) Southern blot of BglII-