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S   6, `P   Z*    6 `   \*  H  0޽h ? ̙3380___PPT10.- H 0(    0`v P    X*   0Lw     Z*   6P `P   X*   6D `   Z* H  0޽h ? ̙3380___PPT10.- Pn @ g_P(   c $ C"?`E  A  0n  ]Bill Finch-Savage Warwick HRI, University of Warwick,Wellesbourne, Warwick, CV35 9EF, UK. 6] K*6   f  c >AHRI Logo new Un  s 8ADefra1" HB  s *@޽h ? 3f3y___PPT10Y+D=' = @B +  ,(  ,J , <ߠh  Traits measured in B. oleracea: Germination rate with and without a range of stresses (water, heat, oxygen) Pre-emergence root growth rate Pre-emergence shoot growth rate Ability for shoot to grow upwards in strong soil Post emergence growth rate (ground cover) |!.% f  f ff @`H , 0@޽h ? ff3̙f___PPT10i.4~`+D=' = @B + -K0 23D(  Dd D <1?0`Kd D <1?  5 D <5E 3Brassica oleracea QTL associated with seed vigourB4 fff0" L k P $D#  lB %D <A)?  lB &D <A)?<<prB 'D BA)?k   (D <  |P X Conductivity    8 % 3D%l D <1? D  ` xaxa1? g01 4    D Zxaxa1?H g02 4    D Zxaxa1?Q g03 4    D  `#xaxa1?%  [04 (    D  `@#xaxa1? '  g05 4    D  `#xaxa1? i  h06 4    D  `,#xaxa1?5g g07 4     D  `#xaxa1?J| g08 4     D  `#xaxa1?` g09 4   T )eX  D# S#`B  D 0)?er `B  D 0)?)X`B D 0)?  `B D 0)? J ` `B D 0)?  `B D 0)? `B DB 0)?qxS `B D 0)?de `B D 0)? \T P1  D# H`B D 0̙)?  `B DB 0̙)?UU `B D 0̙)?+ P+ 8`B D 0̙)?X fB D 6̙)?k J K  D <## 1  vGermination rate - no stress*   'T `  D# `B !D 0)?p fB "D 6)?k  #D <<)# ` yGermination rate - water stress*   6T k S )D#  H %}`B *D 0f)?SS@ fB +D 6f)?k   ,D N.# f? Sj v Germination rate - heat stress:! f T Hr -D# 1B .D ZDf>?HLH| B /D TDf>?P B 0D TDf)?s 0 0 1D Nx4# f? r u Germination rate - low oxygen: fB D s *@޽h ? a(y___PPT10Y+D=' = @B + "t C(  t t 0<#$  ]Linkage Group 12 2 ff" t N`@# f?fI b   Substitution lines used to fine map QTL. SL101 has a c. 1cM introgressed region (ROG1) in an otherwise uniform genetic background which accounts for much of the difference in germination rate between the parents. 7? B t ZDf?4ttd 8 cx "tPw;HlB t <D>  h lB t <DԔ ( (lB t <DԔ  lB t <DԔ  lB t <DԔ  lB t <DԔ lB t <DԔ lB  t <DԔ h h   t 6M#   G108cM& 2  t 6R#  8  F98cM& 2  t 6xV# X`x L88cM, 2  t 6[# ` F78cM& 2` t 0 h lB t <DԔH  H  rB t BD8c h h rB t BD8c   xB t HD8c|   rB t BDԔT X X rB t BDԔ(h rB t BDԔ8 x t 6t`#( H KSL101* 2 t 6e# c  OQTL0 2B t  `Df>?#x ;  t Tb# f?   KROG 1* B t s *@޽h ? ̙33___PPT10e.+D=' = @B +$ $ $7G0 #(  5 : H(  /Brassica oleracea: introgressed region in SL1014006  A 6"`#   k = 120 Kbp$ 0 2    fCgN/ŀAa H>repeat 6"`g    B"`9   centromere T0     l  6"`* "    H "`*   L"0    Nȣ"`*   L"0    B"` q  AA12"0XB @ 0D -    B"`* $  k telomere F 0      B"` N?  G At3g01040" 0 RB  @ s *D XB   0De@f   0"`e|  63"`*   F 0   B"`   BGD33"0  T??"6?@`NNN?N@+[f  0"`f  0"`d  B"`  n  B.nigra tandem repeat40    BÙ"`' x H"0    Bř"` 1  G At3g01200" 0 f  0"`i ? f  0"`U,~  0S"`?6R QRB  s *DT    B|ʙ"` C ?  G At3g03380" 0 RB ! s *D % % :  " BPϙ"`  G At3g02420" 0 f $ 0"`  ' Bhԙ"`L3 c H"0  ( Nיf"`* Jg  L"0  RB ) s *D* :  * Bڙ"`M   G At3g07130" 0 RB - s *D RB . s *D  / Hߙ"` c  B. oleracea Sfr140   0 H"`   B. rapa S-receptor kinase40RB 1 s *D RB 2 s *D  3 Bp"` }  AA12"0 4 B"` }  BGD33"0 5 B,"` *}  CSL101"0RB 6 s *D  7 B("`O ?  G At3g01190" 0  8 B$"`! ]  G At3g03450" 0 RB 9 s *D R R 1 XB < 0D w RB = s *Dxx RB > s *D M  ? B"` m  {+ Markers GD33 in SL101 Markers A12 in SL1014,0   @ B"`~   H"0  l B 6"`b   G <@ d.In collaboration with Guy Barker (Warwick HRI)//fB  s *@޽h ? 33___PPT10i.P^,,+D=' = @B + 0 HE(  H H  T1 ?`   q H S ~xaxa1 ? @,  # OIn this slide I will show the principle QTL we have identified No stress Water stress Where there was strong correlation water stress and no stress we identified the same QTL so there is clear evidence for a genetic basis to this correlation Where the correlation was weaker, eg low oxygen and heat shock the principle QTL do not coincide although minor ones may. For example, there is a non significant local maxima in lod score for germination rate after heat shock that coincides with the principle oxygen QTL on linkage group 8 However, it is clear that each of these measures of germination performance the principle genetic influence is different, although there are a number of non-significant local maxima in LOD score that coincide. In a different population and with more lines to screen these maxima may prove to be significant QTL.@     H H 0޽h ? a($ 0 x(  xd x c $X   y x s *' @   9Line SL101 hasa single small introgresssed region of the GD 33 parent on linkage group O1 that enhances germination rate in an otherwise A12 parent background and is therefore the equivalent of a near isogenic line (NIL) In line SL101 we have the equivalent of a near isogenic line line that differs from the A12 parent by only the alleles in this small genomic region of about 1 Cm. We have therefore reduced the complexity of this polygenic trait by identifying most phenotypic variation for the trait to be governed by this single QTL that we have called Rate Of Germination 1 (ROG1). With this so called mendelised QTL we can begin to get away from considering this as a quantitative trait Usefully this introgresed region is collinear with the region on BO3 and more importantly with the top of chromosome 3 in Arabidopsis, I will come back to this. We are Currently backcrossing this line into the recurrent A12 parent and using a range of markers to further refine this QTL, will come back to this. In the mean time we have begun to characterise the phenotype of this QTL and therefore the impact of natural allelic variation in this refined QTL. We can see that this introgressed region carries much of the improvement in emergence seen in the GD33 parent over the A12 parent in field sowings as well as in the laboratory. $:E  ;L [  H x 0޽h ? ̙33f 0 v(  X  C X     S 䔠 @   xdI aim to present a tremendous amount of work in a short period of time, and cover four projects which I will present in chronological order. The relevant project will appear at the bottom of the slide. Because of the limited time available I will concentrate on the more recent work and Project HH3711SFV which is less than half way through ending in 2007.H  0޽h ? ̙3380___PPT10.`9bd- 0 t(  X  C X     S A @   v`In the final two results slides I will briefly mention what we are doing to further refine the introgressed region in SL101 and also to identify candidate genes We identified a number of markers from the collinear region of arabidopsis that were polymorphic between the two parents. Illustrated by the banding patterns in the insert. We used these to challenge a BAC (section of bacterial articficial chromosome) library and to select those that were specific to this introgressed region and coupled with BAC end sequencing and further maker development we constructed the tiling path shown. This indicates a minimum tiling path of 4-5 BACs each of approximately 120 kilobases, say max 50 genes, probably many less as they contain a lot of tandom repeats etc and the BACs significantly overlap. Red bits colinear with Arabidopsis, clear is deletion in BO, red markers in GD33 introgression and Blue in A12 parent background We are currently nebulising these selected BACs& This randomly shears the BACs. We will then sequence 48 clones to idicate the genes present on the BAC and to provide Brassica specific markers to identify and characterise recombination in this region we have produced by backcrossing the SL101 line onto the background A12 parent. WE should be down to very few genes some of which we could likely eliminate as house keeping genes by analogy with Arabidopsis. (to get genes can Blast the Arabidopsis sequence or we have 22,000 Ests in house and many more are currently being produced and will be put in the public domain. We think this represents the furthest anybody within the public domain has got with Brassica oleracea. In the next slide I hope to summarise the backcrossing we have carried out. (For info overlapping BACs are a tiling path. A BAC contig is a stack over the same area and must have minimum overlapo of 40Kb. 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