图1. 筛到的分布在水稻12条染色体上的多态性分子标记的连锁图
4.3 TF13突变位点的初定位
采取群体分离分析方法，从F2代分离群体中随机取60个样品，取等量叶片分别提取DNA，再取1-20,21-40,41-60号样品的等量DNA构建三个突变池，以突变池为模板，用筛到的多态性分子标记进行扩增反应，同时扩增两亲本及其混合DNA模板作为对照。在突变池DNA中与突变基因连锁的座位的遗传背景偏向突变体来源的亲本，通过凝胶分析可以看出，突变体来源的亲本条带和对照相同，而用于杂交的另一亲本条带没有或弱于对照。
图2. 初定位时的PAGE电泳条带
电泳结果显示，八号染色体上的Os801与Os804处出现重组子。进一步用这两个引物对120个突变株进行分析，以初步定位。后又用Os802（Os803扩增产物无条带）与HY804将此120个突变株进一步分析，以确定初定位无误。
TF13初定位的引物统计信息如下： 染色体 无多态性 有连锁反应 编号 的分子标记 的分子标记 1 Os105，Os110,Os117
2 Os209-212,Os214,Os215 Os204,Os208
5
3 Os307,Os309-312
4 Os401-403 Os407,CH407 5 Os505,Os507,Os509 Os512,Os513 6 Os601,Os603-605,Os607,Os608 Os610,Os611 7 Os701-706,Os708 Os707,Os709 8 Os801-807 Os809,Os810 9 Os905,Os906 Os901,Os902 10 Os1004,Os1006,Os1007 11 Os1101-1102,Os1104-1106 12 Os1201,Os1202,Os1205
4.4 TF13突变位点的精细定位
为了分离TF13的突变基因，必须在HY804和Os804之间设计新的分子标记，进行基因的精细定位。将粳稻与籼稻在HY804和Os804之间的BAC克隆序列进行比较，然后在差异区段的两侧利用REDB网页设计缺失标记，定位群体扩大到284株，进行定位。
新设计的引物在八号染色体上的信息如下：
13.9 13.9 ~13.9 16.4 16.4 16.7 20.2 20.2 ~20.2 21.6 21.6 ~21.6 ~21.6 ~21.6 25.2 ~25.2 26.3 27.1 28.4 29.4 29.4 31.6 P0582D05 OSJNBb0009H02 P0509D04 P0680F05 OJ1613_G04 OJ1119_D01 OJ1368_G08 P0473D02 P0025F03 P0665F09 P0571B09 P0470H09 P0015A04 P0443G08 OJ1349_D05 OJ1066_B03 OJ1177_E11 P0438H08 P0498E12 P0672D01 P0577B11 OJ9990_A01 AP004591 AP005250 AP005618 AP005620 AP003896 AP003876 AP003911 AP005542 AP004381 AP005506 AP005526 AP005741 AP005740 AP004461 AP005467 AP003940 AP003905 AP004460 AP004698 AP004635 AP005504 AP005847 OS802 HY804 TF13-1 TF13-2 TF13-3 TF13-4 TF13-5 OS803 TF13-6 119944 + 155388 - 140736 - 185181 + 97862 - 107132 + 145287 - 138995 - 124745 + 49086 + 131617 + 66700 - 162139 + 55180 + 115339 + 69828 - 148626 + 79506 + 146954 + 111844 + 77500 + SO802-9311 136186 - 6
32.7 32.7 34.6 34.6 ~34.6 35.7
OJ1449_C01 P0462E11 P0458D06 OJ1590_E05 OSJNBa0054L03 OJ1134_B10 AP005605 AP004560 AP005501 AP005606 AP005164 AP003882 RM5432 RM4085 OS804 150950 + 167313 - 166879 + 144450 - 185650 - 117713 + TF13-1
Query 53883 GATACTTCTCCTATAGTCAAATCCTGGTGAAAATCAGCTCTTCCCAGCACTTTTACTACA 53942 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15330 GATACTTCTCCTATAGTCAAATCCTGGTGAAAATCAGCTCTTCCCAGCACTTTTACTACA 15271
Query 53943 GTCCTTAATCCTGGTTAATTCAAACAAAATTGGCTTGACCTTTAACAACAttttttttta 54002 ||||||||||| | ||||||||||||||||||||||||||||| | Sbjct 15270 GTCCTTAATCC--------T---------TTGGCTTGACCTTTAACAACATTTTTTTTAA 15228
Query 54003 aaaaaaaaaCTATCATCTGACTCCATTTTTAGGTAAGAAATGAGTCCTCCAAGGGATTCA 54062 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15227 AAAAAAAAACTATCATCTGACTCCATTTTTAGGTAAGAAATGAGTCCTCCAAGGGATTCA 15168
Query 54063 ATTTCTAGTAAACATTCTTTTTAATCTCTTAACGCAACACAAGTTCTCTGTTTGATGCTT 54122 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 15167 ATTTCTAGTAAACATTCTTTTTAATCTCTTAACGCAACACAAGTTCTCTGTTTGATGCTT
OLIGO start len tm gc% any 3' seq LEFT PRIMER 28 23 59.14 43.48 4.00 2.00 TF13-1F: CTTAACGCAACACAAGTTCTCTG
RIGHT PRIMER 220 22 59.60 40.91 4.00 0.00 TF13-1R: GAGCTGATTTTCACCAGGATTT
TF13-2
Query 126470 GTTGTGTTGCATATCTCTGAAAATATTTAATGTCTGTAATGCTTATTGCAAATACAGGTG 126529 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 4263 GTTGTGTTGCATATCTCTGAAAATATTTAATGTCTGTAATGCTTATTGCAAATACAGGTG 4322
Query 126530 CAAACATCCCaaaaaaaaaaaGAATCTCAAGTGAGAAGCAATGCGTTTATATTACGAAAC 126589 |||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||| Sbjct 4323 CAAACATCCCAAAAAAAAAA-GAATCTCAAGTGAGAAGCAATGCGTTTATATTACGAAAC 4381
7
Query 126590 GCCAACCAGCTTAACTGTGGTATCTAACAAAGGAAATAGGATGGTTCCAGGGAGAAAACA 126649 |||||| | | | || | || |||||||||||||||||||||||| Sbjct 4382 GCCAAC-A----A---G-GG-A---AA---------TAGGATGGTTCCAGGGAGAAAACA 4419
Query 126650 AAGAATAATGGGGGCAAAAGGCAACCAAACTCAATTTAATGAAGCCAAACAAACTGGTCC 126709 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 4420 AAGAATAATGGGGGCAAAAGGCAACCAAACTCAATTTAATGAAGCCAAACAAACTGGTCC 4479
Query 126710 AGAAGTGAGCTTATCCACCATTTCAGCAACACATAAACTGCCAGATACCAATATAGGAAC 126769 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 4480 AGAAGTGAGCTTATCCACCATTTCAGCAACACATAAACTGCCAGATACCAATATAGGAAC
OLIGO start len tm gc% any 3' seq LEFT PRIMER 88 22 60.43 40.91 5.00 3.00 TF13-2F: CAAGTGAGAAGCAATGCGTTTA
RIGHT PRIMER 282 22 60.17 40.91 3.00 3.00 TF13-2R: GGCAGTTTATGTGTTGCTGAAA
TF13-3
Query 58512 ATAGGGGTTTGGGAAACAAGCAAAATCTTCTCTCATTATTTGTAGGTCTCGCATAGTAGC 58571 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 19015 ATAGGGGTTTGGGAAACAAGCAAAATCTTCTCTCATTATTTGTAGGTCTCGCATAGTAGC 18956
Query 58572 TGAGTGGTGATGAACTGATCACTAGCTGTGGTAGATTCACCGCTACCCATACCATCTTTT 58631 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 18955 TGAGTGGTGATGAACTGATCACTAGCTGTGGTAGATTCACCGCTACCCATACCATCTTTT 18896
Query 58632 AATTACTGATTCAGATTATAAGACGTTTTGATTTTAGTCAAAGTTAAACTGCTTATAACT 58691 |||||||||||||| || ||||||||||||||||||||||||||| |||||||| Sbjct 18895 AATTACTGATTCAG-------GA-GTTTTGATTTTAGTCAAAGTTAAACTGTTTATAACT 18844
Query 58692 TTGATCAAGTTTGTAGAGAAAAACAGTAACatttttaacccaaaacaaatttattatgaa 58751 |||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 18843 TTGACCAAGTTTGTAGAGAAAAACAGTAACATTTTTAACCCAAAACAAATTTATTATGAA 18784
Query 58752 aatatattcaattattgattttataaaattaatttagttttataaatattactatatttt 58811 ||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||| Sbjct 18783 AATATATTCAATTATTGATTTCATAAAATTAATTTAGTTTTATAAATATTACTATATTTT
OLIGO start len tm gc% any 3' seq LEFT PRIMER 92 22 60.22 50.00 3.00 0.00 TF13-3F: TAGATTCACCGCTACCCATACC
RIGHT PRIMER 324 23 60.31 43.48 6.00 2.00
8