variation for iron and zinc content in the polished rice grains. Therefore, IRRI shifted its entire screening work to polished rice starting 2004. Scientists at IRRI
evaluated 15 promising genotypes in replicated trials at two locations IRRI, PhilRice Maligaya and in 3 seasons 2004WS, 2005DS, 2005WS. The range of
average iron content in the polished rice was from 2.97 to 7.4 ppm with an average of 4.75 ppm, while for zinc content the range is from 14.33 to 23.26 ppm with an
average of 20.02 ppm. These results suggested that ample genetic variation exists in the germplasm and it would be worthwhile to breed for enhancing the iron and zinc
content in the rice grains Virk et al. 2006; 2007.
Selection of Donor Genotypes
A total of twelve rice genotypes were used in this study Table 1. Four promising elite breeding lines namely IR75862-206-2-8-3-B-B-B, IR69428-6-1-1-
3-3, IR68144-2B-2-2-3-1-166 and IR68144-2B-2-2-3-1-127 were identified from a multi-environmental evaluation study by Virk et al. 2006; 2007. Two popular
indica varieties namely IR64-released in 1985 and PSBRc82 IR64683-87-2-2-3-3-
released in the Philippines in 2000 were also included as potential recipient genotypes. In addition 2 temperate japonica varieties namely Joryeongbyeo and
Areumbyeo with high zinc content Virk et al. 2006 and 3 nutritionally known Korean rice varieties namely Heugjinjubyeo, Baegjinjubyeo and Goami2 Lee et al.
2006; 2007. Lastly, a promising entry namely Zuchein from China possessing high iron and zinc content in the brown rice was identified from previous screening
conducted at IRRI Gregorio, Personal communication was also included in this study.
3.3. Materials
3.3.1. Production of F
1
Hybrids
A set of 39 F
1
crosses including reciprocals were produced Table 2. The parents of these F
1
s included micronutrient rich donor lines and high yielding popular varieties as explained in the previous section.
17
Table 1. A brief description of selected genotypes No Genotype
Group Country of
Origin GID Iron
Content Zinc
content
1 IR75862-206-2-8-3-B-B-B Tropical
Japonica Philippines 1876352
High High
2 IR69428-6-1-1-3-3 Tropical
Japonica Philippines 777182
High High
3 IR64 Indica
Philippines Several Low
Low 4 IR68144-2B-2-2-3-1-166 Indica
Philippines 1183120 High
High 5 PSBRc82
Indica Philippines 94801
Low Low
6 JORYEONGBYEO Temperate
Japonica Korea 575600
High High
7 HEUGJINJUBYEO Temperate
Japonica Korea 1994065
High -
8 BAEKJINJUBYEO Temperate
Japonica Korea 2174341
Low -
9 AREUMBYEO Temperate
Japonica Korea 923791
High -
10 GOAMI2 Temperate
Japonica Korea 2174338
Low -
11 ZUCHEIN Tropical
japonica China 85719
High High
12 IR68144-2B-2-2-3-1-127 Indica Philippines 1183121
High High
High = 6-8 ppm; Low 2-4 ppm; High = 20 ppm; Low= 10-16 ppm; - not available
3.3.2. Production of F
2
and Backcross Populations
F
1
plants were planted in F
1
nursery and were sekefed to produce F
2
seeds. In addition F
1
plants were crossed to their respective recurrent parents to produce seeds of backcross populations as explained below.
Preparation of the land, seeds, and establishment of plants was as per IRRI’s standard practices Appendix 3.
1. One row of each of the parents as check were planted next to the F
1
populations. Recurrent parents were planted in three staggerred sowings to synchronize flowering, i.e. two weeks before, together, and two weeks after the
F
1
were planted. 2.
When the seedling growth was around 2 cm or two weeks old, one young leaf was collected from each seedling for DNA extraction using “quick and dirty”
method Collard et al. 2007; Appendix 4. Deoxyribo Nucleic Acid was extracted from 15 plant of each F
1
population. 3.
The hybridity of F
1
plants was tested using polymorphic SSR markers Appendix 5. SSR markers are co-dominant in nature Powell et al. 1996 and
therefore were used to distinguish between homozygote parents and
18
heterozygous F
1
s. Accidental selfed plants are expected to show only the maternal allele. Accidental selfed plants were discarded.
Table 2. List of F
1
successful crosses in Dry Season of 2006
No Code
IR Number Cross Combination
Number of seeds
1 US6WSC1 IR
84749 IR75862-206-2-8-3-B-B-B IR64
627 2
US6WSC7 IR 84833
IR64 IR75862-206-2-8-3-B-B-B
some 3 US6WSC2
IR 84842 IR75862-206-2-8-3-B-B-B
IR69428-6-1-1-3-3 253
4 US6WSC5 IR
84723 IR69428-6-1-1-3-3 IR75862-206-2-8-3-B-B-B
259 5 US6WSC3
IR 84750 IR75862-206-2-8-3-B-B-B
PSBRc82 347
6 US6WSC4 IR
84841 IR75862-206-2-8-3-B-B-B IR68144-2B-2-2-3-1-166
112 7 US6WSC12 IR
84838 IR68144-2B-2-2-3-1-166 IR75862-206-2-8-3-B-B-B
26 8 US6WSC6
IR 84840 IR69428-6-1-1-3-3
IR68144-2B-2-2-3-1-166 41
9 US6WSC8 IR
84831 IR64 IR68144-2B-2-2-3-1-127
some 10 US6WSC9
IR 84832 IR64
IR68144-2B-2-2-3-1-166 1165
11 US6WSC13 IR 84837 IR68144-2B-2-2-3-1-166
IR64 635
12 US6WSC10 IR 84834 IR64
Zuchein 56
13 US6WSC33 IR84849 Zuchein
IR64 9
14 US6WSC11 IR 84830 IR64
Baekjinjubyeo some
15 US6WSC26 IR 84820 Baekjinjubyeo
IR64 17
16 US6WSC14 IR 84836 IR68144-2B-2-2-3-1-166
Conde 35
17 US6WSC15 IR 84839 IR68144-2B-2-2-3-1-166
PSBRc82 some
18 US6WSC18 IR 84848 PSBRc82
IR68144-2B-2-2-3-1-166 100
19 US6WSC16 IR84835 IR68144-2B-2-2-3-1-166
Baekjinjubyeo some
20 US6WSC17 IR 84847 PSBRc82
IR68144-2B-2-2-3-1-127 25
21 US6WSC19 IR 84822 Conde
Heugjinjubyeo some
22 US6WSC25 IR 84827 Heugjinjubyeo
Conde 27
23 US6WSC20 IR 84823 Conde
Joryeongbyeo some
24 US6WSC21 IR 84845 Joryeongbyeo
IR69428-6-1-1-3-3 15
25 US6WSC22 IR 84846 Joryeongbyeo
IR75862-206-2-8-3-B-B-B 41
26 US6WSC23 IR 84828 Heugjinjubyeo
IR64 514
27 US6WSC24 IR84829 Heugjinjubyeo
PSBRc82 55
28 US6WSC27 IR84821 Baekjinjubyeo
PSBRc82 some
29 US6WSC28 IR 84819 Baekjinjubyeo
Conde some
30 US6WSC29 IR 83287 Areumbyeo
IR64 132
31 US6WSC30 IR 84825 Goamy2
IR64 46
32 US6WSC31 IR 84826 Goamy2
PSBRc82 13
33 US6WSC32 IR 84824 Goamy2
Conde 18
34 US1 IR
85601 IR64 Joryeongbyeo
68 35 US2
IR 85602 Joryeongbyeo
IR64 511
36 US3 IR
85604 PSBRc82 Joryeongbyeo
some 37 US4
IR 85603 Joryeongbyeo
PSBRc82 50
38 US5 IR
83317 IR69428-6-1-1-33 IR64
443 39 US6
IR 84725 IR69428-6-1-1-33
PSBRc82 111
19
4. True F
1
plants were crossed to its resppective recurrent parents to produce BC
1
F
1
seeds. The crossing technique is described in Appendix 2. Table 3. List of F
2
populations developed
No IR Number
Cross Combination 1 IR
84749 IR75862-206-2-8-3-B-B-B IR64
2 IR 84833 IR64
IR75862-206-2-8-3-B-B-B 3 IR
84842 IR75862-206-2-8-3-B-B-B IR69428-6-1-1-3-3
4 IR 84723 IR69428-6-1-1-3-3
IR75862-206-2-8-3-B-B-B 5 IR
84750 IR75862-206-2-8-3-B-B-B PSBRc82
6 IR 84841 IR75862-206-2-8-3-B-B-B
IR68144-2B-2-2-3-1-166 7 IR
84838 IR68144-2B-2-2-3-1-166 IR75862-206-2-8-3-B-B-B
8 IR 84840 IR69428-6-1-1-3-3
IR68144-2B-2-2-3-1-166 9 IR
84831 IR64 IR68144-2B-2-2-3-1-127
10 IR 84832 IR64
IR68144-2B-2-2-3-1-166 11 IR
84837 IR68144-2B-2-2-3-1-166 IR64
12 IR 84834 IR64
Zuchein 13 IR
84830 IR64 Baekjinjubyeo
14 IR 84820 Baekjinjubyeo
IR64 15 IR
84836 IR68144-2B-2-2-3-1-166 Conde
16 IR 84839 IR68144-2B-2-2-3-1-166
PSBRc82 17 IR
84848 PSBRc82 IR68144-2B-2-2-3-1-166
18 IR 84847 PSBRc82
IR68144-2B-2-2-3-1-127 19 IR
84822 Conde Heugjinjubyeo
20 IR 84827 Heugjinjubyeo
Conde 21 IR
84823 Conde Joryeongbyeo
22 IR 84845 Joryeongbyeo
IR69428-6-1-1-3-3 23 IR
84846 Joryeongbyeo IR75862-206-2-8-3-B-B-B
24 IR 84828 Heugjinjubyeo
IR64 25 IR84829
Heugjinjubyeo PSBRc82
26 IR84821 Baekjinjubyeo
PSBRc82 27 IR
84819 Baekjinjubyeo Conde
28 IR 83287 Areumbyeo
IR64 29 IR
84825 Goami2 IR64
30 IR 84826 Goami2
PSBRc82 31 IR
84824 Goami2 Conde
32 IR 85601 IR64
Joryeongbyeo 33 IR
85602 Joryeongbyeo IR64
34 IR 85604 PSBRc82
Joryeongbyeo 35 IR
85603 Joryeongbyeo PSBRc82
36 IR 83317 IR69428-6-1-1-33
IR64 37 IR
84725 IR69428-6-1-1-33 PSBRc82
20
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
