F
2
seeds from all the 39 F
1
populations except two crosses namely US6 WSC16 and US6WSC33.were harvested Table 3. One F
2
population developed from a cross between Joryeongbyeo and IR64 was selected as a mapping
population. A set of 12 backcross populations were developed Table 4. Table 4. List of Backcross Populations
No Cross combination Seeds
1 IR64 Joryeongbyeo
IR64 123
2 Joryeongbyeo IR64
IR64 98
3 PSBRc82 Joryeongbyeo
PSBRc82 45
4 Joryeongbyeo PSBRc82
PSBRc82 13
5 IR69428-6-1-1-3-3 IR64
IR64 184
6 IR69428-6-1-1-3-3 PSBRc82
PSBRc82 202
7 IR75862-206-2-8-3-B-B-B IR64 IR64
463 8 IR75862-206-2-8-3-B-B-B PSBRc82
PSBRc82 345
9 IR64 IR68144-2B-2-2-3-1-166 IR64
376 10 IR68144-2B-2-2-3-1-166 PSBRc82
PSBRc82 407
11 PSBRc82 IR68144-2B-2-2-3-1-127 PSBRc82
411 12 Areumbyeo
IR64 IR64
248
One backcross population, IR75862-206-2-8-3-B-B-BIR64IR64 BC
1
F
1
seeds was selected to be used as a mapping population. The remaining 11 backcross populations are being used by the breeding team at IRRI for the
development of anther culture derived doubled haploids or single seed recombinant inbred lines.
3.3.3. Development of RIL Populations
RILs are being developed by the breeding team at IRRI, for mapping and breeding purposes. The target was to develop 200 RILs for each of the population.
RILs from 23 populations are at F
5
stage Table 5. The procedure of developing RILs population is explained in Appendix 2.
21
Table 5. List of crosses from which RILs production is in progress WS 2008 as F
5
No. IR Number
Cross Combination No. of
individual 1 IR
85601 IR64 Joryeongbyeo
280 2 IR
85602 Joryeongbyeo IR64
280 3 IR
85604 PSBRc82 Joryeongbyeo
46 4 IR
85603 Joryeongbyeo PSBRc82
22 5 IR
83317 IR69428-6-1-1-3-3 IR64
196 6 IR
84725 IR69428-6-1-1-3-3 PSBRc82
280 7 IR
84749 IR75862-206-2-8-3-B-B-B IR64
280 8 IR
84842 IR75862-206-2-8-3-B-B-B IR69428-6-1-1-3-3
280 9 IR
84750 IR75862-206-2-8-3-B-B-B PSBRc82
280 10 IR
84841 IR75862-206-2-8-3-B-B-B IR68144-2B-2-2-3-1-166 246
11 IR 84723 IR69428-6-1-1-3-3
IR75862-206-2-8-3-B-B-B 260
12 IR 84840 IR69428-6-1-1-3-3
IR68144-2B-2-2-3-1-166 260
13 IR 84833 IR64
IR75862-206-2-8-3-B-B-B 260
14 IR 84832 IR64
IR68144-2B-2-2-3-1-166 280
15 IR 84839 IR68144-2B-2-2-3-1-166
PSBRc82 264
16 IR 84847 PSBRc82
IR68144-2B-2-2-3-1-127 280
17 IR 84845 Joryeongbyeo
IR69428-6-1-1-3-3 265
18 IR 84846 Joryeongbyeo
IR75862-206-2-8-3-B-B-B 240
19 IR 84828 Heugjinjubyeo
IR64 244
20 IR84829 Heugjinjubyeo PSBRc82
220 21 IR
83287 Areumbyeo IR64
215 22 IR
84825 Goami2 IR64
147 23 IR
84826 Goami2 PSBRc82
200
3.3.4. Mapping Populations
Following two populations from the above materials were selected as mapping populations.
1. IR75862-206-2-8-3-B-B-BIR64IR64 backcross mapping population, BC
1
F
1
. A total of 463 seedlings were raised for this population. However, because of
poor germination and seedlings death 120 plants could be transplanted. 2.
JoryeongbyeoIR64 F
2
mapping population. A total of 300 seeds were seeded. However because of poor seedling viability
178 plants could be transplanted. Both populations were grown along with 15 plants of parents and F
1
as control. Planting of the materials was executed as follows:
1. Seed preparation, seedling, transplanting and maintaing the plants used the
technique as described in Appendix 3. Single plant per hill was strictly
22
implemented. Leaf collection for DNA extraction was done around two weeks after transplanting.
2. At the maximum tillering stage, the plants were splitted into three equal plants
and transplanted into field following Completely Randomized Design with three replications clone from splitted plant as replication and with a planting
space of 20 cm x 20 cm. 3.
Sticks were used as needed to support the plants from lodging caused by strong wind. Bird net was applied to keep the grains from birds.
4. Each mature plant was harvested separately by cutting from the base of the
stem to measure the plant height. Sun drying for five sunny days was sufficient to get the grain moisture content of around 14.
5. The grains were kept free from contaminants such as dust, water, and other
material that might be affecting the accuracy of measurements. 6.
The grains were threshed manually and cleaned removing unfilled grains, then kept in unfumigated seed storage until ready for iron and zinc measurements.
3.4. Molecular Methods
