研修テキスト[PDF:11.3MG] - 農業・食品産業技術総合研究機構
Contents
1. 192. jus Y lt DNA
3. 029 838 6474 0543 69 7108 200611 PE f 305 8605 2 1 P hHttp fruit naro affrC gO D
4. TGTGTGTGAAGAACCA NARO DNA SSR A 17 DNA 9 NARO H15 15 07 9 2 ji 58 SSR O O NO IR nn BCT23b 214 226 196 226 196 226 196 206 196 226 196 196 196 214 196 214 196 206 196 206 BGA35 136 136 128 136 136 136 136 136 136 136 136 136 136 136 136 136 128 136 136 136 KA14 184 184 184 184 184 184 184 184 184 184 184 184 184 184 184 184 184
5. DNA MixAssort 2 DNA DNA DNA DNA
6. DNA DNA 2 9 DNA DNA Snes os me me
7. gt r 5 Nas UV Print UV freeze UV 96 18
8. PCR 53 4 DNA Spm EO sr OTAR
9. PCR 20 NARO SS5R 13 100 DNA 21 2
10. SSR PCR 10 X PCR buffer MgCI2 gt 30 C 4C dNTP 30 4 30 20 MgCI2 50mM 30 4 Taq 20 C DNA 4 C DNA 1 5ml dNTP 2mM 10pmol ul each MgCl 50mM Taq DNA 2 5ug ul milliQ total PCR 9ul PCR PCR
11. 25 30 lt 0 2 2323 21478 SD BE 82
12. 2 7 700 s IERD 4 1 10 DNA 4 1 35mm 1 1 03 6744 2118 1 2
13. 1 10kb 2 A A oss DNA ADNA A 1 x TAE ZAIT 8 8 50 MOOR 1 x TAE 1 100V
14. SPE S 5 28 pk B JJ AA I EU NS BEI 1 s 82 20 10 4 3 53
15. Distinctness 25 Uniformity off type Stability N J J propagation off type 26 1 4 6
16. 2009 7 10 1 10 1000 3
17. DNA DNA DNA Sa DNA DNA DNA M m cc pO S 517 DNA SSH DNA ERR 20 DNA w
18. 1 DNA 12 _ SSR oo SSR 25 7 SSR 46 5 SSR marker k Ho KTOO1b 0 894 KTOO4a 0 848 5 0 818 k 1 0 439 HO 0 788 0 758 HE CsCAT18 0 712 CsCAT24 0 455 Cervus 2 0 SSR Average 0 681 66 66 6 60 0 24 0 16 0 08 coefficient 13 6enomic Tip DNA 17777 400 mg E 7 Qiagen FEE 3 7 DNA ng 10 30 100 300
19. v J MinimalMarker v 3 v PM O
20. 100V 2 29 15 30
21. EK Tk SSR A bp 14 Z8 V V V VI V V ITV V 8I V V ITV V ITV V ITV V V V ITV V V V V V V V V V V LI V V Lc V 8 V V V Lc V 8 V Lc V V LI V V Lc V V Lc V Lc V LI V V Lc V LI V Lc V Lc V Lc V V LI V V et LOHN 98 V SS V V 8 8 CtV 3 V V 8 V 8 8 qTIOHN c9 V 8 V V V 8I V V V V V V V q600HN yST V V V V VI V V 8Z V 1 8 01 V 8Z V V 8Z V V 8Z V 0I V 8C V V V V 8Z V V 8Z V V V qLOOHN V 9 V 91 91 V V b V V V 9I V V 9I V V V V 9I V V 9I V TV V V 9I V V 9I V b V V qsOOHN SS 18 Le V cct V V Y LEtV V OctV CCtV LEtV LEtV Le V cc V Le V cc V Le V cc V Le V cc V LEtV V FOOHN 8
22. 3 29 II 30 2 10 1 000 3 Warning Civil Relief Import Prohibition nu PE EXE 4 17 4 1 31
23. DNA 13767 2006 2 27 4 3 3N N28 H16 18 ARO JA EU TA 55 3 SSR SSR SSR Total 88 43 rr 52 28 59 27 1 1 rh 31 16 SSR SSR SSR 4N 1 CHO03g06 178 178 180 178 178 178 178 176 180 4 94 102 102 102 102 102 102 94 94
24. x 36 ul 52 10XPCRbuffer MgCI2 32 96 I 36 0 3960 0 1 36 0 3960 0 0 5 18 0 1980 0 0 4 14 4 1584 0 0 05 1 8 198 0 6 05 217 8 23958 0 10 324 35640 DNA1hI 8 PCR Program gt user yama or yam accept run 52c35 start Volume 10Hl 34 DNA 2 3 30 30 C PCR X10 1 8 1 10TE 27ul 2 PCR 3ul
25. A1 Plate Buffer MilliQ 4 Plate Record Plate Position Indicator Linked Plate Records Plate Record Plate Position Indicator Run View Run 1 D States View Status Run 1 Run 16 40 GeneScan36_POP4_short Run D NAppliedbio 3100 DataExtractor Extracted Runs 41 1 Windows NT Ctr Alt Del User Name 3100user Password 2 OrbixWeb Daemon Start menu Applied Biosystems OrbixWeb Daemon 3 3100 4 3100 Data Collection 5 Tools Change Polymer Wizard 5 3100 1 Data CR So
26. 28 10ul 10 30 100 300 ng ul 4 DNA 10ul lt F f 4 4 2 DNA 1 lt 2 30 Dye 10uIl
27. 2007 7 3 1 2 3 4 DNA 9034 DNA 16 12 25 17 3 1 100 1 6 4 18 2 1 41
28. 2 Clip Art FacTory 1 2 3 SSR DNA 50 DbNA SSR 1717 NARO DNA EE gt DNA N e gt ug O 2 amp 0 Bn 850 HH 5 m w we eg DNA 100 F SSR RARE 8 GERALA LTAT CAPS SSR
29. 10 DNA DNA DNA A 7G T 4 MEDIR 13215 LUCHO WIRIO ERAI PCR
30. DNA DNA DNA Genomic tip 20 G G2 DNA 1 50 ml 10 ml G2buffer 200 ul 100 mg Polyclar AT 2 hl O RNase 2 1 g s 3 2 50C2 2 3 4 50A TV 50 12 6 000 rpm 30 4C 50m 5 2
31. 1 A B C D E 1 2 B1 B2 C1 C2 D1 D2 E1 E2 A1 A4 B2 B3 C1 C3 02 04 1 4 B3 B4 C3 C4 03 04 E3 E4 0 2 8 17 4 SSR 1 DNA Sawamura Y et al 2004 Identification of of Japanese pear Housui J Soc Hort Sci 73 511 518 85 JP 953 1955 SSR DNA x 33 2 _33x 1 DNA 10 DN4 17 15
32. DNA 42 E
33. 40 CAPS 3 Epe CAPS Cleaved Amplified Polymorphic Sequence CAPS DNA PCR PCR 2 gt 0 gt PCR RFLP DNA PCR SNP markers 9 PIP potential intron polymorphism markers 81 SNP markers established out of 288 esteq a n n as F MdomPIP1 MdomPIP288 http ibi zju edu cn pgl pip download html EST derived SNPs 24 SNP markers established out of 94 tested A mr L k G G P uc _ DNA sequencer ddGTP J SNaPshot ABI SSR SSR Simple Sequence Repeat
34. 80C DNA 80C 1 3 20C 84901 LDLLODLV LLOODOLVODLLVDD VVDVDVDVIDDVDVDVDVVVDVDVDHo215 II 8 691 SG PCCIHN 8 ODVVVOVOOOVVVVLVVVDVY O LLV LLODOO LO IO ILO LIO LD L19115 55 cI 091 OLV LIDLV LDVVVDD VVD00 O10115 DVDLVOODDVDLLDVLVTLLLV 55 P O0CHN SI LET DDL LVVLVVVVVO VVVO VLVMMS VODVDLVVOLLLDLLVVVLVVVOVL 55 eyOCHN 01 DDLDVDLVOVVOVLDVVODVY VLDLDVVVDVDOODTLLDDL 55 6COHN 6 SS VLLVLVOOVOOOLOLD OL SS 6COHN SI 66 DOV VVVV LOVVVDV VOV DDDVOVVOLLVOVVNOVOVODD LO PcCOHN Lcl L L L L LOVLV LL LD VOODLVDLVVVVDDDVLDLL 0 COLHN 8 98 1 119111518 CS eyIOHN 181 DDVDDDVDDLLDDDDILILI35 15 DVOVOVDVDVDV LVOVO LLDD SG 8 661 LOLLO OOVLLL LOLODDMMS VOLLVOOV LDVNODNVDOO 0 d600HN 91 8051 DIDVLLVVDDLLVDVILDVILVV loD 15 O2
35. 96 13 SSR DNA SSR Tm 50CC60C gtttctt
36. 1 5 ml 3 6S5C 450 ul Buffer 8 ul 1 5 ml 65 10 2 3 RNase 4 ul RNase A 100 mg ml 4 150 ul Buffer AP2 3 5 TOMY MX 160 TMA 24 14 000 rpm 10 15 6 5 7 EYF spin column 14 000 rpm 2 2718 15 4 9 2 7 QIAshredder spin column 800 ul Buffer AP3 E 8 7 650 ul DNeasy Mini spin column 9 12 000 rpm 15C 10 7 D
37. 9 No 5 gt TEKEN A K 072 14 4 1 14 EEX 17 10 1 6 DNA DNA DNA 9 9 DNA
38. SM 800 mM 100 ml Tris HCI pH 8 0 30 mM 30 ml 0 5 EDTA pH 8 0 30 mM 60 ml Tween 20 5 50 ml Triton 100 0 50 5 ml jk kal 755 ml 22 1000ml QBT buffer Jee he 5M NaCl 750 mM 30 ml 1 pH 7 0 50 mM 10 ml Isopropanol 15 30 ml Triton X 100 0 15 0 3 ml J 017 129 7 ml Yi 200ml buffer RE x SM NaCl IM 80 ml IM pH 7 0 50 mM 20 ml Isopropanol 15 60 ml 017 240 ml 400ml buffer SM 1 1 25 M 50 ml Tris HCI 8 5 50 mM 10 ml Isopropanol 15 30 ml 017 110 ml 200ml 4 5 DNA 2 DNeasy Plant Mini Kit DNeasy Plant Mini Kit DNA 1 100 mg 2 1 5
39. SSR 96 SSR 8 DNA 8 M04 MOS VO VO2 VO4 VO5 AB BB AA HH AD AD AA AC VOG 07 VO8 BC VO3 V04 VO6 V0 VO9 AD BB AA MinimalMarker 8
40. 1 6 1 59 6 2 Agalose 3 00 1 200ml 300ml 2 9 7 8 30 gt DNA 21 6 96 F wasa 8 96 oa tasa eee aa 9 ar 9 TALDE TE DNA 2ul DNA2Lul 2 x 2 dye 2 xdye 8
41. 4 C 20 C 3100 1 96 Well Reaction Plate ABI 384 Well Reaction Plate 5 Hi Di Formamide 400HD ROX 2 PCR 1 5ul 3 4 95 C 5 94c PCR 5 5 6 7 Hi Di Formamide 400HD ROX X1 X2 X3 X4 HiDi 960 1920 2880 3840 400HD ROX 20 40 60 80 total u 980 1960 3920 7840 35 E lgM 96 1 2 3 4 p 2 Formamide ROX 2
42. 1 2 3 5 6 7 8 9 10 71 00 gt gt gt 2 gt g T T gt T DD m G G m O O O O O gt rm rm gt O U gt p gt O DU gt gt gt gt gt gt gt gt D gt gt m mD gt m gt gt gt gt gt gt gt gt gt gt gt gt O gt gt gt G gt gt gt gt gt gt gt gt gt gt MixAssort 1 A A A 06 11 1 2 3 4 5 6 7 8 9 10 gt gt gt gt 07 08 09 10 11 gt gt gt
43. 1 2 SSR PCR DNA 17 SSR AG TC AC TG 2 US lt GH 2 DNA 80 DNA
44. DNA 1 SNP 1 DNA C U r 04 MRK 7 5 amp ES 05 SSR X X AIEA AA
45. 22 45 DNA 7 DNA HERBARIUM DF NATIONAL CENTER FOR SEEDS AND SEEDLINGS Materials examined for Dlstinciness Uniformity and Stability lrrmamlhemami pacifi Qhaymma Prel City ehou Staten Mu isnal for Seeds FH S TS FE EL Dal Guit Yoshihiro Data 2008 11 12 21029 Bamagnusmaess mamas i 47 DNA 2009 6 26 895 715 G 5 DNA Bisa 0 fz 9 BH 321 6 zl Sa T LLT ANK 1 5 ENEE UOI1BZIUB
46. 19 DNeasy Mini spin column 500 ul Buffer AW 12 000 rpm 1 157C 20 8 12 000 rpm 1 15 C 21 DNeasy Mini spin column 1 5 ml 22 65C 100 p191 10 TE 5 10 000 rpm 1 15 C DNA 23 65 C 100 401 10 TE DNeasy Mini spin column 5 min 10 000 rpm 1 15 DNA ww K Buffer imitant R36 38 5813 26 36 46 Buffer R22 30r 38 513 26 38 46 RNase A solution F Finrmf en Se 24 26 36 37 j intant 942 43 X QIAGEN Sciences Maryland 20874 USA Tel 1 240 686 7700 n DNeasy Mini spin column QIAshredder spin column DNA 1 50 x TAE Tris acetate EDTA Tris 0 5M EDTA RT DNA 2M 2M 0 05M 2 1 XTAE Tris acetate
47. 32 TEL 029 838 6589 FAX 029 838 6583 Email hinsyu_gmen ncss go jp 17 132 gt gt 30 70 17 4 1 21 3 31 33 35 Y33 390 emas GOCO sam 36 2893 37 2003 6 A 1999 B 1 B
48. H FHA 21 DNA 21 10 19 Ness G N A 22 AK gt 23 20 F 3 30 2306 03 07 24
49. DNA DNA P Rs gt 62 QO X gt 2 MixAssort DNA 3 MixAssort SSR 74 1 A 01 02 03 04 05 12806 1307 14808 1409 44810 11 12 50 50 80 50 A 01 02 03 04 05 06 12 10 DNA
50. G 2005 7 K nn FE ODIE ER 2 2005 11 38 O E g 2005 12 DNA 39
51. 1990 18 5 19 DNA 43 DNA DNA 20 21 S L gt z gt Pumap 4 P
52. 1 0 1g 1g 2 80C 20 C 1000 200 10 1 5ml 1 1 5ml 450 Buffer AP1 8 ul 65C 4 100 mg ml 2 1 3 1 3 0 1g
53. 17 SSR 1 PCR 2 PCR BV pip SS 13 0
54. 250 500 1 062 3 Microsofte Visual Basic 6 0 Microsoft Excel 2003 Windowse XP 6 3 MarkerToolKit CD R 5 MixAssort MixAssort DNA 1 1 MixAssort
55. 5 6 10 pmolul FER 1 10 TE 384 e Genetic Analyzer Buffer with EDTA Hi Di 3100 POP 4 3100 3130 1 36cm Capillary Array 47 cm x 50 um GeneScan 400HD ROX 0 0056 mS m 25 C 121C 13 JIS C p TA
56. STR A66 A 464646464646 B AGAGAGAGAGAGAGAGAGA6G A610 DNA SSR p v J NARO SSR B6T23b 9 100 SSR AB219800 55 1 CACATTCAAAGATTAAGAT 2 gtttcttACTCAGCCTTTTTTTCCCAC gtttctt TC 18 5 DDp 203 8
57. DNA DNA 3 DNA NARO DNA gt 320 00 1 1 e i PAPA 5 a 1 PI DNA DNA DNA mim nami mu m E 2 ta DNA 3
58. NARO DNA NARO bDNA co DNA STR SSR DNA DNA DNA DNA DNA DN4
59. DNase 1 PCR DNA Polymerase 0 1 ul ZN 2 0 ul dNTPs 2 0 ul SSR 1 0 ul 13 9 ul DNA 5 10 1 0 ul 20 0 ul ju 2 PCR GeneAmp PCR System 9700 PCR 1 94 S 94 1 50 55 1 72 2 35 72 10C eo 3 20C SSR PCR 1 DNA DNA 5 10 2 4 4 DNA 1 Genomic tip 20 G G2 DNA PCR 3 PCR
60. B GA C SNP Single Nucleotide Polymorphism 1 SNP SNP CAPS SSR DNA VN 9 02 72 29 3 4 03 PELOR O gt CUYS 9 5 22 J SSR SSR SSR CAPS SNP ODDS SO 7K DEDERE 100 28 m TRUT 100
61. nm 95 5 500 4 000 z 11 1 i pei F j 1 Wa 1 4 ri P 1 E e SE Fm ri r _ z Ta 1 F F F Baz 7 gt a n r mi LO CET WA 1 s T i i TOS H 3 4 A i AN i gt ar vi 4 1 p WE 1 una Q 19 z k 4 5 4 F pa F 1 15658 2007 10 22
62. 40 2 3 a cm 50 C 30 E 58 17 2 273071 189 2 1 x TAE 1 xTAE 1 x TAE 4 C 1 DNA 6 x dye
63. 1 1 1 DNA 2 DDNA 3 DNA 4 10 ng 5 30 ng ADNA 6 100 ng ADNA 7 300 ng ADNA 10 DNA DNA 2 DNA 1 2 1 2 1 2 2 3 3 DNA 4 1 DNA 5 10 ng ADNA 6 30 ng ADNA 7 100 ng ADNA 8 300 ng 4 7 PCR PCR PCR DNA DNA
64. 10 1 2 1 10 gl 1 x TAE 1cm TAE DNA 2 lt 3 Q 10cm mw 6 6 x Dye 2 DNA DNA 201 DNA DNA 7 Dye DNA
65. 5 6 7 8 polyclar AT 30mg 2 9 10 65C 10 77 89 2 3 21 11 150 ul Buffer AP2 5 12 T0MY MX 160 TMA 24 14 000 rpm 10 15 09 S 13 12 QIAshredder spin column 14 000 rpm 2 15C 14 QIAshredder spin colum
66. 3 GeneScan L lt TET 4 50 ml 5 1 5 2 mm 6 Mupid 2 8 9 10 12 4 4 1 DNA 7 4 2 DNA DNA 4 3 50 ml 1 5 ml 2 ml Polyclar AT kE u Y LARE 180C2
67. ina 508 MN gH PP gog E 77 LENAO 58 03817 8 PEOS 88 SALAA G e MEHO m lt 0 1 EZ 4 1 GY L CaA 54 P A 28 52258 FEN 56469 078 O EE S0 QEP FEGH 457 9 8015 2 EJ 1 CC 7 Ho FEY 4 1 5200811 auy fl ENEDES FEA FEN gt WA Wg ALELY FE j m HEUL gt 5588 w Y 63 B gt s ET Z 2 2 2 37 0 Q Ya HA Bul 2 222 2 5 J ik H uy Hii 1961 007
68. DNA 70 3 MarkerToolKit MixAssort MarkerToolKit MixAssort http fruit naro affrc go jp q 69 HE of Fruit Tree Sci B isis papers ps em 22 L LE TATA E Li q l L SAri h 2 Vergi 1 D LES CELTI TECE
69. 2 227 6 H 6 2 VNG HERO OO 3229 6 226 52002 2 C H YM ZO wu 0 2922 62 SW HZ e cq 4 0 es gt 1 68220002 ES HUO 26 6 21212 6 JL MEIN 2 VNA Amlueuelduoo AA WMO CU K o r LT 226 6 297 6 HREJ ANOLA pa 03 9 199 2126200 O gt MC SLSR D O VO 2981
70. ADNA10 30 100 300 ng ul i milliQ 100 ul 1L 6 x dye W 1 xTAE T 10 30 100 300 1 1 xTAE 500ml 2 OO 4C 200ul PP 3 1 TAE 50 HI 5 PP imi m
71. RU SDS ERr RGE j oT gt Weton MixAssort 1 0 Windows 4 IHAT RR hu L ERRAT j TVR ndows Vista L T uE T ROP iF 2007 Framework Version 20 hr 1 SE Z IR ia L rE LE tA z T CN f DORRERA PITERA TARNE Th ART SN ER ZL DL U ET L ITARA A RIKI TT 2 SITE SEE 1 bT EB KARATE it Ta 4 NERTH ENAUT 5 54241 p L SU UFAZ 21513
72. 80C 1 3 DNA SSR 20 80 DNA DNA DNA DNA DNA
73. 17 96 101 FREDY 1 00 E L T 78 ra U TA F E MU mm 7741 RB Minis ibit t a bti 707S DEML m 4 MarkerToolKit MarkerToo1Kit DNA DNA 9 2 1 9 1 SSR MinimalMarker 2 2 1 3 4
74. 36cm POP 4 Genetic Analyzer Buffer with EDTA 5 GenaScan SSR A bp 2 12 BGT23b 196 196 10 A A 30 226 21 A A 30 A 307A 30 A A 30 A A 30 RE i sj Teis o qo Sp 13 17 SSR 2 17 SSR 4
75. 6 DNA MarkerToolKit MixAssort MinimalMarker EST MAEZATO 7 DNA DNA DNA SSR
76. PCR GeneAmp PCR System 9700 SSR PCR 2 5C 2 52C 4 8 SSR SSR PCR DNA 1 PCR TE 20 100 PCR 2 1 5 ul PCR 0 17 ul 400HD ROX 8 33 Hi Di 96 X PCR 384 3 PCR PCR System 9700 72 2 2 HOT 9505 3 4 ABI PRISM 3100 Genetic Analyzer
77. 3 CAPS Cleaved Amplified Polymorphic Sequence 64 gt CAPS PCR PCH RHFLP SSR Simple Sequence Repeat o G D 010100 10001 Antw itil 24 44 DNA SSR PCR DNA A GAGAGAGAGAGAGA SSR
78. DNA DNA DNA DNA 9 MarkerToolKit DNA DNA
79. DNA DNA DNA DNA 19 bNA DNA 1 2 DNA PCR RE 2 3 3 DNA gt gt gt 3 lt 1 2 3 A CH A B C DNA
80. 180 C 2 G2 buffer buffer QC buffer buffer RNase 100 mg7ml Genomic tip 20 6 1710 TE EDTA 0 1 mM TE Buffer Buffer AP2 Buffer AP3 E Buffer AW DNeasy Plant Mini Kit QIAshredder spin column DNeasy Mini spin columnm DNeasy Plant Mini Kit 30 0 25 0 25 10 10 ng 30 ng 100 300 ng 0 8 1 5 Ay TE 0 3 1 0 ug ml 96 PCR j AmpliTaq DNA Polymerase PERAS Z T T ZARRA A VAT ARI 2 mM dNTPs dATP dTTP dCTP dGTP Z TE ESSR
81. 94C1 S0 S 1 3594 7 72C2 72C7 10C PCR HiDi DNA GeneScan 17 1 Yamamoto T T Kimura Y Sawamura Manabe K Kotobuki T Hayashi Y Ban and N Matsuta 2002 Simple sequence repeats for genetic analysis in pear Euphytica 124 129 137 2 Yamamoto T T Kimura M Shoda Y Ban T Hayashi and N Matsuta 2002 Development of microsatellite markers Japanese pear Pyrus pyrifolia Nakai Molecular Ecology Notes 2 14 16 3 Yamamoto T T Kimura Shoda T Imai T Saito Y Sawamura K Kotobuki T Hayashi and N Matsuta 2002 Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears Theoretical and Applied Genetics 106 9 18 4 Sawamura Y T Saito N Takada T Yamamoto T Kimura T Hayashi and K Kotobuki 2004 Identification of parentage of Japanese pear Housul Journal of the Japanese Society for Horticultural Science 73 511 518 5 Kimura T Y Z Shi M Shoda K Kotobuki N Matsuta T Hayashi Y Ban and T Yamamoto 2002 Identification of Asian pear varieties by SSR analysis Breeding Science 52 115 121 6 Kimura T Y Sawamura K Kotobuki N Matsuta T Hayashi Y Ban
82. DNA 1 2004 1 DNA DNA CO 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 DNA FEDER DNA 1 2 DNA PCR SSR 4 10 1 2 PCR
83. L lt SE AZAL 75020 V AT 2 VNO ZE0 Z Kl BI O C C k 21 MEJA 1 s s 2 4 gt SU HIS SS 0 ni COo 6 MEB 2 Ez 02 L SZ E 14 222 6 215 fas 9 5 Hy amp DAPA 0 0 2 266 Hy Q 2 rH f VNG H i lt 624559 28 HOP 50015894 UIPU 39SeI9UIAIod a N s se H li lt 69 27 026927 ju
84. 21 DNA 10 19 1 DNA 10 00 11 00 1 2 11 00 12 00 SET H F 3 22 DNA 13 00 16 00 1 4 DNA 16 15 17 15 2 10 20 5 DNA 9 00 10 00 55 6 DNA 10 00 11 45 pp 70 7 11 45 12 00 8 1 DNA 2 DNA 1 DNA National Agriculture and Food Research Organization
85. DNA DNA 1 DNA 1 Genomic tip 20 G G2 DNA 2 DNA 2 DNeasy Plant Mini Kit DNA 2 DNA RZ 65 1 10 TE 1 5 ul 2 DNA 10 uu 10 ng 30 ng 100 ng 300 ng ADNA 0 8 1 5 10u 3 100V30 4 15 45 HELT 9 2 5 254 nm 366 nm 2 6 SSR 3 10 ng ul DNA W x ERT 7 20C
86. 5 04 185 185 187 185 185 185 185 185 187 NH007b 129 141 147 129 129 147 147 129 141 14 164 182 188 164 164 182 182 135 188 NB114a 133 137 137 137 137 137 137 133 133 PCR bp 4N 1 4N 1 2 1 Watanabe al 2008 J cE Soc Hort Sci 77 388 394 17 NARO DNA DNA DNA ji DNA NARO DNA DNA DNA 18 DNA DNA DNA o DNA
87. 76 SSR 2 80 84 2 2 2 1 DNA
88. V V V V V V V V V V V V V V V V V V V V V V V V V V 961 5 05 8 Oc V OI V OI V V V V 5 OI V V Oc V V Oc V V Oc V 0c V Oc V V dcCLDd gt WE 24 7 NAE c x Y Mz 2 5 15 1 6ZT V E91 V 8ZT V 6TT V 08 V Z8 V 28 V V V V V V OI V V II V V V V I V SI V gt 7164 V V V V V V V V V V V V 9I V CI TV V V V V V V OI V V II V V 9 V V V 9 V 9I V V V V ZtV V V C V V V V C V 9I V cI V V V V V ZtV V 9 V V V V V V 9T V 9 V V V V V ZtV V V C V OI V V V V V V I V 0 V V C V V V V V V C V OI V V 9 V V 9 V V V V V V V V V V V V V V 9T V ST V 1 5 V V V V ZtV V 9 V V 0T V C V OI V V 9 V V V 9 V V V V V V V V V V C V V V V V 9 V 0 V x C x V V V V V V V V 0I V Z V V V V V 9T V ST V V V V V C V V V V V C V V V V V 9T V ST V V V V V ZtV V V V OI V V II V V V V 9I V V 41714 eyTISN eLOZHN gbOZHN 6cOHN e6ZOHN eSZOHN amp amp 55 RW 5 5 1 16 4 10 PCR 94 5
89. 193 197 193 197 195 197 193 197 193 197 193 197 193 197 193 197 4 195 177 195 195 195 177 195 177 195 177 195 177 195 177 195 177 5 136 136 136 136 132 136 136 136 136 136 116 136 136 136 136 136 Yamamoto et g 2003 2 1 M1 M2 V1 0 4 0 16 V2 0 4 0 16 V3 4 4 0 10 V4 0 4 2 16 V5 0 0 2 16 V6 0 0 4 16 V7 0 4 2 16 V8 0 4 0 16 0 4 0 4 2 4 0 4 0 4 0 4 0 4 0 4 4 0 18 18 18 0 18 0 18 0 18 0 18 0 18 0 18 M5 0 0 0 0 4 0 0 0 0 0 20 0 0 0 0 0 Tds 34431233 1 1 2 3 1 V1 0 6853662506 V2 0 1438471797 V3 0 0017077071 V4 0 6853662506 V5 0 3576934570 V6 0 0236768299 V7 0 6853662506 V8 0 6853662506 2 1 2
90. 5 6 SSR 7 SSR 2 2 8 9 2004 3 0 1 SS 2 1 SSR V1 V2 V3 V4 V5 V6 V7 V8 M1 84 80 84 80 84 84 84 80 80 80 80 80 84 80 84 80 Microsoft Exce12003 Nore 2 94 78 94 78 88 78 94 80 94 80 94 74 94 80 94 78
91. 2820 60 4 3760 Hl 80 20C ey 3100 Data Collection 1 Windows NT Ctrl Alt Del 95 User Name 3100user Password 2 OrbixWeb Daemon Start menu Applied Biosystems OrbixWeb Daemon 3 3100 4 3100 Data Collection 38 Plate Record Plate Editor 3100 Data Collection Software Version 1 1 File Instrument Tools Sevice Plate via Status view Array view view Penrlina Recorde Piste weis Place a rito Place a plate mta piale pate postion Linked Plate Recorda Plale Application atus Processed Plate Records Plate Name Application Vells 98 EE 55 384 processed 0 384 processed 95 384 processed 56 processed 55 Import 2 Plate
92. RAHAAN IALA Fy 2 2 1 5 ml 2 0 ml 4 4 DNA 1 Genomic tip 20 G G2 Genomic tip 20 6 02 DNA 1 DNeasy Plant Mini Kit DNA 2 DNA 1 DNA DNA DNA 2
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97. EDTA _ 50 x 200ml 10L 242g ma a 57 1ml 500ml Tris 100ml EDTA 500ml 1000ml 1000ml buffer 3 agarose S 4 2 Mupid 2 200ml Mupid 2 24 196 17 2 0 6g 1 x 60ml 200ml TAE 40
98. a 06 73 88 6 4412 2 O 25 NAHIZ LO MEFR OHAL 598 HARTY 61 EE 0 2 SE 1 G 2 B L 1 15 202 2 1 E yxcC 8 HH P AN REE LA HR 4 0 7 30 f AENEAM J H eg 7 B a aaa m QERA Og 4 TTE gt AMORE LH u A 1 2121 OLERE lt LEE 62 wy ee Ww CO 20896 zk TA E 16 m EN lt L 50 lJ R E HARALI TE FEE 845 2 2 E s 09 aug
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100. 0 G 2 ml QBT buffer 6 5 2 ml 7 buffer 50 9 2 ml O QC buffer 9 2 ml buffer 10 0 2ml 50C 1ml 0 QF buffer DNA 11 2 ml 10 DNA 12 10 11 0 7 ml x2 ml 1 f 20 30 13 20 30 TOMY MX 160 TMA 24 14 000 rpm 20 4 C 14 S00 ul 70 14 000 rpm 10 4 15 16200 ul 1 10 17 4C 3 DNA DNA G2 buffer
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107. NSPIII 3 C Documents and SettingsNNSP NMy Documents NSP7000II 5 7000 A 1 96 _96 2_ 96PCRnp3 2455 Rovervok _36MTP amp 96PGR 96MTP amp Reservoir 96MTP np3 _ 4 amp 96 4_384 3 274 t 3250 Reservoir sa4wTFsgeTP4 384MTPmp9 gt 2740 5 7000 7 5294 9 9 Q 4 Start NSF Executation gt Set 2 ns Pi h ume a Ti Cx Da RMTP QA MTF Ap 5 Program END 36 96 Well Reaction Plate ABI L lt 14 384 Well Reaction Plat ABI 1 5 ul Hi Di Formamide 400HD ROX 8 5 HI 95 C 5 PCR 5 Hi Di Formamide 400HD ROX 96 Hi Di Formamide 400HD ROX 1 940 20 ul 2 1880 Ll 40 Hl 3
108. Neasy Mini spin column 12 000 rpm 15C 11 DNeasy Mini spin column 300 ul Buffer AW 12 000 rpm 1 15 C 12 DNeasy Mini spin column 500 Buffer AW 2 JUL MERA 12 000 rpm 1 15C 13 12 000 rpm 1 15C 14 DNeasy Mini spin column 1 5 ml 15 6 5C 100 ul 1 10 TE 3 7 46 10 000 rpm 1 15C DNA 16 65C 100 ul 1 10 DNeasy Mini spin column 5 min 10 000 rpm 15C DNA 4 6 DNA DNA DNA DNA
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111. and T Yamamoto 2003 Parentage analysis in pear cultivars characterized by SSR markers Journal of the Japanese Society for Horticultural Science 72 182 189 7 Yamamoto T T Kimura T Hayashi and Y Ban 2006 DNA profiling of fresh and processed fruits in pear Breeding Science 56 165 171 Yamamoto T T Kimura S Terakami C Nishitani Sawamura T Saito K Kotobuki and T Hayashi 2007 Integrated reference genetic linkage maps of pear based on SSR and AFLP markers Breeding Science 57 321 329 18 0 10 DNeasy Plant Mini Kit 19 Genomic tip 207G G2 DNA R DNA DNA DNA 1
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113. n 800 Buffer AP3 E 15 14 650 ul DNeasy Mini spin column 16 12 000 rpm 1 15C gt L j Plant Mini 50 gt Cat Nt Buffer AP3 E jl Buffer AP1 Bufe 183898 5 IR22 38 58 5132 20 18 48 52224 26 36 27 Sciences Maryian Buffer AP Buffer Binding P is buffer 30 ml conc 40 ml Buter Sefora Fes obtain top room tem Vy 977 gt Buffer AW Buffer AP2 17 14 DNeasy Mini spin column 12 000 rpm 1 157C 18 DNeasy Mini spin column 500 ul Buffer AW 12 000 rpm 1 15C
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