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Screening of different AFLP fragments between near-isogenic lines of male sterile and fertile Salvia miltiorrhiza and their comparison analysis

丹参雄性不育与可育近等基因系差异片段的发掘



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ScreeningofdiferentAFLPfragmentsbetweennearisogeniclinesofmale
sterileandfertileSalviamiltiorrhizaandtheircomparisonanalysis
LIANGWenjie1,LUCongyu1,SHUZhiming1,ZHOUZili1,YEJiayu2,
ZHANGYuejin1,LIANGZongsuo1,GUOHongbo1
(1.StateKeyLaboratoryofStresBiologyinAridAreas,StateandLocalJointResearchCenterofTraditonal
ChineseMedicineFingerprintandNaturalProducts,ColegeofLifeSciences,NorthwestA&FUniversity,Yangling712100,China;
2.DepartmentofBiology,TheUniversityofBritishColumbia,KelownaV1V1V7,Canada)
[Abstract] Thereisdistinctiveadvantageofusingmalesterilelinestobreednewcultivarandproducehybrids,whencomparedwith
generalbreedingmethodonyieldandqualityInourpreviouswork,nearisogeniclines(NILs)ofmalesterileandfertileSalviamilti
orhizahavebeenobtainedthroughcontinuoushybridizationinmanyyearsInthisinvestigation,378primercombinationwerescreened
byusingAFLPandBSAtechnique,inwhich26markersamplifiedfromsevenprimerswerefoundtotightlylinktomalesterilegene
Basedonthesemarkers,twolinkagegeneticmapswereconstructedA2027,2028bpfragmentwasamplifedfromNILsoffertileand
sterileSmiltiorhiza,respectively,usinggenomewalkingtechniqueandpreviousE11/M4208markerastemplateFourbasemuta
·5141·
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Vol41,No.8 April,2016
tionswerefoundinintronwhencomparingbothfragmentsAmongaldiferentmarkersbetweenNILsofmalesterileandfertileSmilt
iorhiza,fourwasfoundtohave100% identitiestochromosome1,3and5ofArabidopsis,namely,E01/M09418,E05/M13308,
E05/M04750andE01/M01204TheE01/M09418markerwasveryclosetomalesterilegeneofSmiltiorhizawithdistanceof21
cM,whichalsohad100% identitiestomalesterilegeneMS2inArabidopsisBothweredistributedinchromosome3ofArabidopsis
The2028bpfragmentalsohad100% identitiestoMS2geneAnotherE05/M04750markerthathad100% identitiestochromosome
5ofArabidopsiswasfoundtohavehighidentitiestoPOP085M05geneofpoplarsandlowafinitycalciumantiporterCAX2ofArabi
dopsiswithverylowEvalueTheconstructedgeneticmapanddiferentialfragmentswithpotentialfunctionsfoundinthisstudyprovide
asolidfoundationtolockmalesterilegenesinSmiltiorhizagenomeandtodiscovertheirfunctions
[Keywords] Salviamiltiorhiza;malesterility;nearisogeniclines;fertilelines;genomewalking;diferentfragments;genetic
map;AFLP
doi:10.4268/cjcmm20160808
  
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Table1 TheEcoRIandMseIprimersdesignedtoamplifydiferentfragmentsbetweenNILsofmalesterileandfertileSalviamiltior
rhiza
No
ó¯
(5′3′) No
ó¯
(5′3′)
E00 GACTGCGTACCAATTC M00 GATGAGTCCTGAGTAA
E01 GACTGCGTACCAATTCACC M01 GATGAGTCCTGAGTAACAG
E02 GACTGCGTACCAATTCAAT M02 GATGAGTCCTGAGTAACAA
E03 GACTGCGTACCAATTCAGC M03 GATGAGTCCTGAGTAACTC
E04 GACTGCGTACCAATTCACA M04 GATGAGTCCTGAGTAACTG
E05 GACTGCGTACCAATTCAGG M05 GATGAGTCCTGAGTAACTA
E06 GACTGCGTACCAATTCAAG M06 GATGAGTCCTGAGTAATGC
E07 GACTGCGTACCAATTCAGT M07 GATGAGTCCTGAGTAACAC
E08 GACTGCGTACCAATTCAAC M08 GATGAGTCCTGAGTAACAT
E09 GACTGCGTACCAATTCCTA M09 GATGAGTCCTGAGTAACCC
E10 GACTGCGTACCAATTCCTC M10 GATGAGTCCTGAGTAACTT
E11 GACTGCGTACCAATTCCAG M11 GATGAGTCCTGAGTAAGAT
E12 GACTGCGTACCAATTCCAA M12 GATGAGTCCTGAGTAAGAG
E13 GACTGCGTACCAATTCCCT M13 GATGAGTCCTGAGTAACCG
E14 GACTGCGTACCAATTCACT M14 GATGAGTCCTGAGTAAGAC
E15 GACTGCGTACCAATTCAAA M15 GATGAGTCCTGAGTAAGTA
E16 GACTGCGTACCAATTCATA M16 GATGAGTCCTGAGTAATAA
E17 GACTGCGTACCAATTCATC M17 GATGAGTCCTGAGTAATAC
E18 GACTGCGTACCAATTCAGA M18 GATGAGTCCTGAGTAAGCA
E19 GACTGCGTACCAATTCCTG
E20 GACTGCGTACCAATTCGAA
E21 GACTGCGTACCAATTCGTA
  
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Table2 PrimersusedtoamplifydiferentfragmentsbetweennearisogeniclinesofmalesterileandfertileSalviamiltiorhizaingenome
walking
=É ó¯
(5′3′)
=É ó¯
(5′3′)
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F1131 AGAATGGCTTGTGAGGTTAGAG
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r/
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F7273 TTTTGCTTCGCAATAGCCTGTG
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Vol41,No.8 April,2016
  
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Table3 SpecificfragmentsbetweennearisogeniclinesofmalesterileandfertileSalviamiltiorhizaamplifedbysevenAFLPprimers
=É´H
ÕÓÙ ÒÓÙ
+ -
ÆÛϕ˜
/% + -
ÆÛϕ˜
/%
ÆÛÏu•˜
/%
E+ACC/M+CAG 9 55 140 40 88 313 255
E+ACC/M+CCC 1 15 60 10 22 313 229
E+AGC/M+CTC 1 7 125 5 11 313 250
E+AGC/M+CTT 3 13 188 8 24 250 250
E+AGG/M+CTG 9 23 281 12 52 231 219
E+AGG/M+CTT 3 29 93 22 42 344 260
E+AGG/M+CCG 12 28 30 33 47 413 375
q
1 
=ÉÄc=€ÏÐѹÒÓÔÕÓ¡Ö×ØÙ
AFLP
ÚۀÜÄUN¨©
Fig1 ElectrophoresisdetectionofdiferentAFLPfragements
betweennearisogeniclinesofmalesterileandfertileSalvia
miltiorhiza
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q
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2 E01/M01(E+ACC/M+CAG)
=ÉÄcÏÐѹÒÓ
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Fig2 ElectrophoresisdetectionofE01/M01amplificationbe
tweennearisogeniclinesofmalesterileandfertileSalviamilti
orhiza
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Table4 Statisticsof26AFLPmarkersconstructedingeneticmapsthattightlylinkedwithmalesterilityofSalviamiltiorhiza
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Fig3 Twocontructedlinkagegeneticmapsbasedon26AFLP
markersthattightlylinkedwithmalesterilegeneofSalviamilti
orhiza
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Fig4 Amplificationoffragmentsinnearisogeniclinesofmale
sterileandfertileSalviamiltiorhiza
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Ù$@•nRm¸ê()âÄ
2028bp
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MS2
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100%
QÐr

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Vol41,No.8 April,2016
+
5 4
YÔR9Ð×Ø´B
100%
QÐrÄ
AFLP
õ
Table5 FourAFLPmarkersthathave100% identitieswithArabidopsisgenome
=ɶ
GenBank
Wã< R9Ð×Ø´ó¯

QÐr
/%
E01/M09418 NC0030748
R9ÐÓ

093 100
E05/M13308 NC0030768
R9ÐÓ

066 100
NC0030709
R9ÐÓ

066 100
E05/M04750 NC0030768
R9ÐÓ

046 100
E01/M01204 NC0030748
R9ÐÓ

51 100
E01/M01230 NC0030748
R9ÐÓ

88 100
  
-VB
AFLP
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308,E01/M01204,E01/M01230
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E05/M04750
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f

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+
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Table6 ComparisonoffiveAFLPmarkersobtainedfromnearisogeniclinesofmalesterileandfertileSalviamiltiorhizawithother
species
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GenBank
Wã<

•¶ó¯ •¶É,

QÐr
/%
E01/M09418 AC239879.3

BAC
^VI[
RP23429N16
:·Ó
13
41 100
AC1543891

BAC
^VI[
RP232H9
:·Ó
13
41 100
E05/M13308 APGK010439061
w×Ø´\]ó¯
Seq01043916
$”yào:^
Dendroctonusponderosae 29 100
E05/M04750 AC2103411 POP085M05
w(ó¯ §×
Populustrichocarpa 6E-33   85
E01/M01204 HG9754471
Ó

Solanumpenneli 63 100
E01/M01230 LL9109141  
×Ø´´§ó¯
Ssolidus_NST_G2,cafold,
SSLNcontig0000479
Schistocephalussolidus 32 100
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7 
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QÐr
/% GenBank
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E01/M09418 3epi6deoxocathasterone23monooxygenase 060 42 NP5664621
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E05/M04750
±A»çB—îâj¾0
CAX2
lowafinitycalciumantiporterCAX2
8E-25  75 NP5664521
E01/M01204 ubiquitinspecificprotease26 650 48 NP5669221
E01/M01230 uncharacterizedprotein 270 69 NP1886852
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bifunctionalaspartokinase/homoserinedehydrogenase1 760 64 NP1744081
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·1241·