免费文献传递   相关文献

Genome Wide Analysis of NBS Encoding Disease Resistance Genesin Populus trichocarpa

毛果杨全基因组 NBS 类型抗病基因分析


Abstract: A full set of disease resistance ( R ) candidate genes encoding nucleotide-binding sites (NBS) in a complete genome of Populus trichocarpa was identified and characterized by structural diversities, physical positions, phylogenetic relationships. Based on structures of N-terminal motif and leucine-rich repeat domains motif, we found 381 NBS-coding sequences with 122 non-regular NBS genes and 259 regular NBS genes that were further classified into 13 types such as TNL, CNL, NL, XNL, TN and other minor types. Meanwhile 81.9% of the NBS genes were distributed in cluster, and 81.8% of the cluster genes had duplicates. The results showed that there were many duplicate phenomenon occurred in the evolution of disease resistance genes of P. trichocarpa. After analysis of NBS standard phylogenetic tree in the genome of P. trichocarpa, the structure of tree exhibited a star topology, and the regular NBS genes were classified into 68 groups by less than 30% amino acid sequence diversity in each domain.


全 文 :! !"" ! ##
# $ $ %$ # %
& ’ ( )
&’()*+(, &(-.,) &(*(’,)
./01!"**/1#
2345*
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
# $ $ %
4JpWXY/ *6&*mhCXY)V"
#&L 9 ’ ( ] ) +
$%&* u’>.iE45).F?;O.*6&.>%#32
’()*+#9:;" ,-./0#, ,12+#<$$< = :!;;3#$$%4$# = $<"# = $8
89:#*#$$: = <$ = 7$&
;<=>*xWU(Ae=hgF[(A_=<$;$<$7$#$:7MbxWUIEJFG=><>?#$$;,<#;M&
"=pOPQRS&lJxWq/[)i2())@qI1HIG+J%rsb#32/KdK*7Dy&
!"#$%"@&’(" )#*+,-’- $. /01@2#3$(’#4 5’-"*-" 6"-’-7*#3" !"#"-
’# !"#$%$& ’()*+"*,(#,
AB CDEFDGH ’DF ’I3GH A3F JB/ KFDGH LDG
)8-79*37* , MB00 N3O /M PFN3DN3 Q3NFNODGR3<9MRDGPFPDO3 H3G3N 3GR/PFGH GBR03/OFP3@4FGPFGH NFO3N<*6&MFG D R/ST03O3 H3G/S3
/M :$03%3* +(2"#$",(0, UDN FP3GOFMF3P DGP RIDQDRO3QFV3P 4W NOQBROBQD0 PFX3QNFOF3N*TIWNFRD0 T/NFOF/GN*TIW0/H3G3OFR Q30DOF/GNIFTN 5
6DN3P /G NOQBROBQ3N /M *@O3QSFGD0 S/OFM DGP 03BRFG3@QFRI Q3T3DO P/SDFGN S/OFM*U3 M/BGP 7;< *6&@R/PFGH N3EB3GR3N UFOI <##
G/G@Q3HB0DQ *6& H3G3N DGP #"% Q3HB0DQ *6& H3G3N OIDO U3Q3 MBQOI3Q R0DNNFMF3P FGO/ <7 OWT3N NBRI DN +*-*’*-**-*K*-*+*
DGP /OI3Q SFG/Q OWT3N 5 Y3DGUIF03 ;< 5%Z /M OI3 *6& H3G3N U3Q3 PFNOQF4BO3P FG R0BNO3Q*DGP ;< 5 ;Z /M OI3 R0BNO3Q H3G3N IDP
PBT0FRDO3N 5 +I3 Q3NB0ON NI/U3P OIDO OI3Q3 U3Q3 SDGW PBT0FRDO3 TI3G/S3G/G /RRBQQ3P FG OI3 3X/0BOF/G /M PFN3DN3 Q3NFNODGR3 H3G3N
/M : 5 +(2"#$",(0, 5 ,MO3Q DGD0WNFN /M *6& NODGPDQP TIW0/H3G3OFR OQ33 FG OI3 H3G/S3 /M : 5 +(2"#$",(0,*OI3 NOQBROBQ3 /M OQ33
3[IF4FO3P D NODQ O/T/0/HW*DGP OI3 Q3HB0DQ *6& H3G3N U3Q3 R0DNNFMF3P FGO/ 8; HQ/BTN 4W 03NN OIDG 7$Z DSFG/ DRFP N3EB3GR3
PFX3QNFOW FG 3DRI P/SDFG5
:", ;$9(-* :$03%3* +(2"#$",(0,.4F/FGM/QSDOFRN.PFN3DN3 Q3NFNODGR3 H3G3.GBR03/OFP3 4FGPFGH NFO3.TIW0/H3G3OFR OQ33
?EKrN6E7i9Uk*6Eg?EK:
-9+"h#3=><\/0B4*#$$EK7%tz>@A5&QX’b?EKey*5
1?EKey7%d>%7FBF#:mXT(X
vE8]G3|??EK17uIo;O
:-77]R2=>*&.6EF?7=;O7.
/ 4^8bR2@^*FzT6E?9)f6EF?
E~>?17IGqK<6D^3Q )+ ,% 5*<%%:M&UZ
7|?F?;OvO *6&F?;O*%-E~]G
F3;O*5bc.i@FFBr\[.nSG
<*6&MbW0[7oks<-]]NM&*6&su/F7
72-6EF?;O7|?b+)*}BsuSm
@F’Fy()xd7;d&e *6&@-]]./b
c7}F);O1+T #[)+bc.i *c@FFB +(]<+/00DGP FGO3Q03B^FG@<
Q3R3TO/Q*Tg’u +/00.ifv8lEiE+L-
= -]])k;Orzv +*-)k;O&56)k7
*6&@-]];OSJbc +(]./suO( +(]@*6&@-]])k;OrSw G/G@+*-)k;
OM*;}F);O44+f7 * =c’’FB"p
mn./<’’*’/F03P@R/F0M*Ae}y;O%v$ ’’@
*6&@-]])k;OrSzv ’*-)k;O&
>2T8ZziE*;O‘%\bij/a%
!<&O3MDG )+ ,% 5*#$$:M*O;ROF~&6VwiE
>?Zs=o*5afa7+)\]C$pibE
9piM[/KgT<&O3Q^W )+ ,% 5*#$$!M&;>2
7F#V(2~u’><:$03%3* +(2"#$",(0,M7^;
O‘;d[XgT<+BN^DG )+ ,% 5*#$$8M&>2F?
;ORO>2;O17FB[BK*T9>Y[’
%KmX+p-qyF3SG<-3NR/O )+ ,% 5*#$$!.
LFG )+ ,% 5*#$$!M*+BN^DG l<#$$8M%[nu’>F
?;O+WR#d#$.|S*}T^;O‘ *6&
)F?;O7#Fh+)b>%#3=>l:m7
>?$G-VwiEWlho6<."/0# )#*%1#M’((.M&<%PmiE45):;>?u’>^;
O‘1 /!0)k7F?;O’H*Sn5#$+)
b>%Ki)+0*e#OB!uE>27F?;
O >^?F?@^b62F?;OB!&6F?E
~]|;Z&
1 TUiYZ
121 pq;w <>?eu’>O>?n]*u’
>^;O‘Zgb 34/5ZgY]-u’>/*Z
gn< 121&;O‘N_ +667*889%:,;%& <9=>7?@ & ,A98
B,76A1 C 18B,76A1 C 1 & +,;%& +6;"&
12’ pqP& 1M/!0)k;O7|,g+)
<>?+ D%:%!#:$12F?3=jx?A%?=?6#:3%D^
?E=?%#?%Db?/!0DZ,F?;O&@Z,‘7;O
Pm B@#; Zgg+2P2 4/5FGGH0<=#$ 6!H50F:<5"6?I+-" 2* #, &*
1JJ(M<3 K#"-% L (2((1M*+[K:7u’>;O‘
34/5Zgg1eF?;O7 /!0sudfOr‘
df#$ !H50F ~!O#df&!H50F .’1O
#r\[dfPm B@#;<.iBKZgg*+667*88
7@#;& M-?6" & %E-8+;; ?%#A3+& ?+6;"M@I /!0 ./7
;OdfRp*.’Pm NHO0F5H P#, &*1JJ.M:;#$Udf]n&Pm B@#; b
NGQH07 /c./b HRR./*/!0)F?;O#$+)&
’M/!0)k;OslzE9|S /!0)k;
OPm /N!QZgg?\7slz|S+2#$u
’>slzE9|S&cM62 /N!Q17u’>
;O‘ !H50F +2#$ !H50F ]n*+P2
?D%:,;% S=%MD+2*|, /!0)k;O7slzS
U&A’7 /!0 )k;OP2}~:;|,E9
SUG*P2 D%:,;%B=I%"=T%A< +667*88MMM& :=U"AA? &
-3E#K=?& %E-8D%:,;%B=I%"=T%A8D%:,;%B=I>%"=T%AC P%"3,;%&
+6;"M<=@A’F’g] /!0./7\{F?;O
+u’>slzd7E9SU{^Tp&
)M/!0)k;O]^+0 A’Ty /!0)k
;OK:<3Zgg*+@eFByd7;O62
4/5FGGH0<=#$ !H50F:]n*_-eF;Og
56;O773a*e||T!9+;O]^U]&
.M/!0)k;O>%Ki)+0 _-F?;
O7 /!0./70;[df2’xaxd3*qd . ’*6 2 VWD5 )21 < =< +667*88MMM&
;%9#?,@6M#A%& :%68MnA’u’> /!0 F?;O /!0
./su0;[dfP2op;X,=:=:9
;%6+,EM%#32&
’ MJi)V
’21 /!0hHkl‘#/ + /N!QZgg17Z
,*\04);6EO&7 ’’~6E7h 1YZBF
/!0su7r\[df2- !H50F ]n&Xm<
3 !H50F*@eBr‘df7]n.’7Fndf
Rp*/H_‘ )JY BF’ /!0 ./7-N;O&
9GPmn )JYB 34/5df#$ B@#;+0bUd
fqd*RpI /!0 ./su7-N;O*/H_
)[1BF /!0./7;O&
’2’ /!0hHkl‘Wh Pm B@#;Zggh|
|_‘ ’\JB2’\{./7 /!0)k;O*56
1’’B;OT9 B@#;.’9+ /!0 ./}Qg{ /!0 9+)IFG*&#Tja%P*AeQ-
(UR /!0;O&>G*\{./ /!0)k;OX
m NGQH0b B@#;+0*h J.B;OdfF’ NN;
d*’[B;OdfF’ FQR;d&
Q ! 4Jp "#$hCXY)*uJW!
%&’(! )*&++,-,.&/,01 &12 134’56+ 0- "#$ 2,+5&+5
65+,+/&1.5 7515+ ,1 !"#$%$& ’()*+"*,(#,
;O)?
0,A6 ,@ 9%:%
zv
H%66%A 3,E%
;OZ>
/-;U%A
,@ 9%:%?
UR /!0;O
R%9-"#A /!0 9%:%?
NN>/!0>HRR N/H [)
NN>/!0 N/ 11
FQR>/!0>HRR F/H ’’
FQR>/!0>HRR>FQR F/HF .
FQR>/!0>HRR>/!0>HRR F/H/H ’
FQR>/!0 F/ ).
/!0>HRR>FQR /HF ]>/!0>HRR ]/H \[
/!0>HRR>] /H] ’
]>/!0 ]/ )
/!0>] /] 1
/!0 / 11
/!0>HRR /H ’)
UR /!0;O’h
F,6#" A%9-"#A /!0 9%:%? ’\J
(UR /!0;O
/,:>A%9-"#A /!0 9%:%?
/!0 Z 111
/!0>HRR Z 11
(UR /!0;O’h
/,:>A%9-"#A /!0 9%:%? 1’’
/!0;O’h
F,6#" /!0 9%:%? )[1
!]*W0;d O:$:,M: 9%:% ?%^-%:3%?&
+Wlh1*/!0 )k;O\e)\1! ’# B[$l*u’>^;O‘ /!0)kF?;O+0
!"#suB~7g+T $ [)*}27sug++
!c9+ %&’./rSJ9+&;+u’>19+
[H7 !"#./J\{7 !"#)kF?;O&Y
QWlhbo67F?;O+):;<"()*+ !" #$ ,*
$--../012 !" #$ ,*$--3M*d1 !cb 4’’su;d
7s?n}y !"#F?;O#$.+)*h+O 56
);O<( 5M&
p 5 u’> !"#;O+slzdE9SU
789, 5 :0;<8=)( (1=)>81?< 1@ >0+ !"# 9+?+< 8? %&’($() "*+,-&,#*’#
slz0+ =0F1G1<1G+<
<=0FG.9F); B)F+ >0+ )HHF1A8G)>+ (1=)>81?< 1@ +)=0 !"# 9+?+,
$I. !"#hHkl‘JMGI p 5 O\{ !"#
)k;O+u’> 5Jxslzd7+E*\H-}
y;O+slzd+ETJMe7*O 5K8slz
I\{ !"#)k;O; 5J8slz’ L3x*WK
UC)k7;O+slzd7+E’)IU’3&
;O1h 5LJB;O<6 L6I.NM+E+ $$ B;O
y1*xMeB;OyF K , 6B;O&_p\0!
5J8slz;Oy/UX K B*F?;Ofax
a&p.;Oy;*K’ 55‘##:-+Ex+7
;O\yb K ‘ . B;O:-+Ex+7;O365 & ’ ( ) 36"
p ! u’> "#$;O "#$;d0;[df>%Ki)+0
%&’( ! )*+,-’./.0&1 2/2,+3.3 -4 0*. "#$ ’./.3 &/ !"#$%$& ’()*+"*,(#, 523.6 -/ 0*. 27&/- 21&6 3.89./1.3 &/ 0*. "#$ 6-72&/
^C>%Ki=>_ :;<= >(?<=7op;+0 "#$./u70;[df_-&A’7 !@ABdf@A B> B C"D*??B C"*!!B E"D*
F B E"DE*! B E"D"D*>F B E"*@ B "DE*@B B G"D*! B "DG*> B G"*?B "G*?? B "*!> B "D&H/&I.J32, K*+,-’./.0&1 J.,20&-/3*&K L23
2/2,+M.6 93&/’ 0*. /.&’*5-JNO-&/&/’ 7.0*-6 523.6 -/ 27&/- 21&6 3.89./1.3 &/ 0*. "#$ 6-72&/ 5+ :;<= >P? ( E-02, !@A 3.89./1.3 1-/02&/ B> C"D*?? C"*
!! E"D*F E"DE*! E"D"D*>F E"*@ "DE*@B G"D*! "DG*> G"*? "G*?? "*!> "D(
@@?! !# B[$l*u’>^;O‘ "#$)kF?;O+0
y*h !"! B;O9+-;Oyr\‘1*O-
#"$%&7 ’();O+E+F|7ya&F’ *+ B
’())k;Oh.+E-u’>slzd*6\{
’())k;O7 "#$"&*;Wlh1});O6
!, -#&<(./01 !" #$ -*!223M^o66 !* - "&<4567 !"
# . -*!22*M&O-*u’>;O‘1h;O6 ’())
k;O’Z7]k]o6bWlh1l\*b)u
’>7 ’())k;OF]rF‘^=7Ki*+
Sa&
!$* ’()hHkl‘JK O)H>2;O‘1
[H ’())k;OT_-;O]^**{|,;O
]^PmeY !B\{<87 !" #$ - !22!M*FT4G
7;Oja#[- #2&*8T4Gd7su#[-
+9&7df733&’h "# B9+]^;O7y
7|,*6 !!B;Oy7 #"$#&*O! "#;Oy1
7 :;9%,332 g! !2 ;Oy7 :;,+*,+, 9+;O
]^&}b)u’>F?;OTy+ES_[b+
T_-;O]^U]ST*)HF?;O]^TS
TF?;O+u’>;O‘1TW7EO&
!$9 ’()hHkl‘QRcOWa +0 ’()s
u*u’>h’ 3#"B ’())k;O*F’ !9%B;
O#$. ’();d7>%Ki+0*}TOOb+
;O7 ’();dmP<\-")7 !<3 jM*AeV
’i}Fb+;Ofw+0n]&
Wlh7 ’() ./u>%#32’ ! B)I
7+;<=>?5.@ !" #$ -*!22!M*;o67 ’() suV
’)Iy7+)*h’ "%!B\7+‘*dUe/7
Ky0>%+E<4567 !" #$ -*!22*M*u’>7 ’()
./u7#32

d1 32&7.ildfFGa*>21h’ ,#B+
‘*xMe‘ 3$#"B;O&}e‘7;OZFGx
[*51 3! B‘F " B;O*# B‘F ! B;O*9
B‘F 3B;O*!B‘F *B;O*!"B‘F 9B
ed7;O*/[7‘F’ !,B;O&
3 "6
A.6B<"%+"M?-F?;O#37?;On;OD
|?‘?EKIo;O<#%&M7vE:G*6Ek8
vi’t7F?\u&}F#3Vk729.’X
T*?EK+#37Fm:Y/TU23 FGB.i*
qh5g6E17uF?.i77]R2;I_6
E7|?*}}~U23 FGB.i238^]5r
H48;S#FhT4gT5Rii-#$ -*!22,M&7\*&9NORz\#3b^]6E
F?lS;OU23X;T[5n?EK7]G3
|?+$"nF?;O7./ 4^8b!#,|’
.<<37Q3*;OF]TST;OZ>Tc7
/&#EO<87 !" #$ -*!22!M*Xp "\0 ’();O
+u’>JOslzd7+E9+@[FG*"+8
slzI+E*; "%8slz+EZX‘ ,**O-*
u’> ’() ;O9+JMe7]G&#Fh+0
() ’();O+slz+Ed9+IG+Esl
z*O! "%8b! "8slz*;}yslzd7
’();OO#3%a*Pm;O]^^=+0\0*
+u’>7[b+F?;Oy1*[b+T_-;
O]^ST7*}go6 W^lh7F:*}%#F
hb);O]^TSTF?;O+;O‘1Tc7
&#EO&OP #" - %&C~)k ’() ;O+E+
;Oy1*u’> ’();O+slz+EdUU5
6k1+E7]G*}go6<4567 !" #$ -*!22*Mb
Wlh<=>?5.@ !" #$ -*!22!M’();O+E7]^%
())3*_ww[-o6 9"&F?;OS-y
S*]o6bWlh’Sfk7U]}~U]vi
7EO\8T}yF?;OPm;OF‘ F^;O
]^l:w+EslzJ+SU#$Tc*e>F
UQ7?EK*S/H/TFyslzE9+EI
Gsu&
Pm^;O‘ ’() ;O>%#32+0\e
H‘*u’>7#32go6<4567 !" #$ -*!22*M^W
lh<=>?5.@ !" #$ -*!22!MFG%[*Wlh’ !B)
I7+;;o6+‘UX "%!B*u’>+‘’ ,#
B*TL-#S:-7Vw*b)>27F?;O#
32’.]7]G*>">2F?;O7uEb4
8I)k8_‘#Fh.e&
d e , -
F.IJK57. ) A*8>J5 L*M>..1B L*!" #$ - "%%2 - (/J>?.6?/. /.>CDN1DI J1/B?5
I66. - O M6. (>6.*!"9**23 P *"2-
(/01B (*4/NQB@J0> :*)I/J0/R>?S (*!" #$ - "%%+ - )>CD/.>DC >D T./DIUN>?B6Q1
>DI1B/?I>6DJ- )?>1D?1*!+,*+!, P +33-
(./01 V*M1@1BJ ( V*F.1K/DW1B X6S>0*!" #$ - !223 - 81D6N1UR>W1 /D/.@J>J
6E ’()UY==UZD?6W>DC C1D1J >D FB/Q>W6TJ>J- :./DI V1..*"9*#2% P #3*-
H/DC. Y O*M?H6R1.. O M- !22, - [R6 N6W1J 6E T/I56C1D B1?6CD>I>6D Q@
T./DIJ- :B6? ’/I. F?/W )?> \)F*"23*#9+9 P #9+,-
A.6B ]- "%+" - V7BB1DI JI/I7J 6E I51 C1D1UE6BUC1D1 ?6D?1TI - FDD7 =1G
:5@I6T/I56.*%*!+9 P !%,
87 4*V/G/.?/DI> F*V51D A V*!" #$ - !22! - ZKI1DI 6E C1D1 W7T.>?/I>6D >D I51
C1D6N1J 6E ’&()(*+,$#*D1N/I6W1*/DW @1/JI - M6. (>6. ZG6.*"%*!9, P
!,!-
]6.7Q Z- !22" - FBNJ B/?1 >J /D /D?>1DI 5>JI6B@ >D -&#.,/(*),)*I51
,9" & ’ ( ) *9"
!"#$%#&!’( ) *+, -’. /’0’,*1*234 5 216)
789+: ;*<+0 =>?’ @*A,&B? C) 3DD3 ) E(’$"B,"0F B&#’$ G&"#: %(&H 9(&,’"0
:’I8’0B’:) AB"’0B’*121*3341 5 334J)
7’:B&, @*-&HK+8,: A*LM+0F C*!" #$ ) 1NNJ ) ;00&,+,"&0 &% + D2 5 ?K
%&’($() *!$"&+*!) F’0&H"B :’I8’0B’ (’.’+#: + $":’+:’ (’:":,+0B’ F’0’
B#8:,’( +0$ 0&.’# B#+:: ! +0$ B#+:: " ,(+0:9&:+K#’ ’#’H’0,:) OM’&(
;99# /’0’,*3ND*3N 5 11)
@’>’(: P G*="B?’(H+0 ; Q*@"BM’#H&(’ - Q*!" #$ ) 3DDD ) E#+0, $":’+:’
(’:":,+0B’ F’0’: ’0B&$’ H’HK’(: &% +0 +0B"’0, +0$ $".’(:’ 9(&,’"0 %+H"#>
!",M"0 ,M’ 08B#’&,"$’RK"0$"0F :89’(%+H"#>) E#+0, C*1N*S36 5 SS1)
-"BM#> T*U8(,M C*7’":,’( =) 1NN1 ) @&$’ &% +H9#"%"B+,"&0 +0$ (’&(F+0"V+,"&0
&% (’:":,+0B’ F’0’: $8("0F (’B’0, ,-#.+*&’)+) "/#$+#0# ’.,"&0) @&#
P"&# T.&#*3D*64 5 WJ)
A,’%+0 C*G+(# C =&8F#+:) 1NN6 ) %&’($()*; @&$’# A>:,’H %&( E#+0, P"&#&F>)
;008 -’. E#+0, P"&#*2W*JS2 5 J2W)
A,’(?> X*PM+#’(+& - -*Y00’K’(F E*!" #$ ) 1NNJ ) ; E&98#8: TAO (’:&8(B’
%&( 9#+0, %80B,"&0+# F’0&H"B:) E(&B *+,# ;B+$ AB" YA;*3N3*3SD23 5
3SD24)
OM&H9:&0 C =*Z"FF"0: = /*/"K:&0 O C) 3DDJ ) G7YAO;7 Q*[H9(&."0F ,M’
:’0:",".",> &% 9(&F(’::".’ H8#,"9#’ :’I8’0B’ +#"F0H’0, ,M(&8FM :’I8’0B’
!’"FM,"0F*9&:","&0R:9’B"%"B F+9 9’0+#,"’: +0$ !’"FM, H+,("\ BM&"B’)
*8B#’"B ;B"$: -’:*11*J46S 5 J4WN)
O8:?+0 / ;*="X+V"& A*C+0::&0 A* !" #$ ) 1NN4 ) OM’ /’0&H’ &% P#+B?
G&,,&0!&&$*%&’($() "-+1/&1#-’#M) AB"’0B’*S3S*32D4 5
34NJ)
"^0 O @*="X+V"& A E*/80,’( 7 T*!" #$ ) 1NNJ ) /’0’,"B +0$ 9M>:"B+#
H+99"0F &% 2!$#3’)&-# (8:, (’:":,+0B’ F’0’: "0 %&’($() +0$
BM+(+B,’("V+,"&0 &% #"0?+F’ $":’I8"#"K("8H +0$ %#+0?"0F F’0&H"B
:’I8’0B’) *’! EM>,&#&F":,*34J*D2 5 3N2)
LM&8 O*Q+0F ^*GM’0 C _*!" #$ ) 1NNJ ) /’0&H’R!"$’ "$’0,"%"B+,"&0 &% *PA
F’0’: "0 C+9&0"B+ ("B’ (’.’+#: :"F0"%"B+0, ’\9+0:"&0 &% $".’(F’0, 0&0RO[-
*PAR7-- F’0’:) @&# /’0’, /’0&H"B:*3N*31JS 5 312J)
01234 3458
623! 1# B[$l*u’>^;O‘ *PA)kF?;O+0