6.2 Materials and methods
6.2.5 Field and screenhouse evaluation of C 1 seed and recombination of nematode resistant
Field and screenhouse evaluation of the 20 C1 lines for resistance to P. zeae and Meloidogyne spp. was conducted at IITA-Namulonge farm between March 2009 and June 2009 (Table 6.2; Fig. 6.1). Four cultivars used as checks were the C0 line (for each population), Gandajika 8022 (susceptible to P. zeae), Western Yellow (both P. zeae and Meloidogyne resistant) and Meloidogyne spp. susceptible lines from the S1 selection (named S1-5, S1-201 and S1-91 for Longe 1, ZM521 and Longe 4, respectively). The field and screenhouse experimental designs were split plot with two replicates. In the field, nematode treatment (nematode infested vs nematicide treatment) was the whole plot factor while the C1 lines were the subplot factor arranged in a 6 x 4 α-lattice design (Appendex 6.2). Double rows were planted per genotype, each composed of 16 plants spaced at 75 cm between rows and 30 cm between plants in a row. A total of 32 plants were thus maintained in each genotype in the field, of which 12 were used for root nematode assessment and the rest (20 plants) used for yield assessment. The screenhouse type used for evaluation was a detached Quonset greenhouse with curved metallic rafters, and roofing made of twin polycarbonate material. The sides were fitted with transparent, rain-proof netting. The average temperature and relative humidity estimates in the screenhouse were 25.5oC and 65%, respectively. Sterilized loam and sand soils were used in the screenhouse. These were mixed in equal proportions to form about 2 500 ml of soil sufficient to fill the plastic pots. For each line in the screenhouse trial, 12 pots were prepared and each pot sown with two seeds, later thinned to one seedling one week after planting (Appendix 6.3). Separate trials were
121 conducted for Meloidogyne spp. and Pratylenchus spp. evaluation in the screenhouse. Each pot was inoculated with 5 000 adults and juveniles of Meloidogyne spp. and Pratylenchus zeae. The inocula were administered using 10 ml pipette to ensure uniformity of application.
Data collected from the field and screenhouse trials was the basis upon which the best 10 nematode resistant C1R lines were selected.
Remnant C1R seed from 10 of the 20 S1 nematode resistant lines with superior C1R progeny tests (field and screenhouse trials) were planted (23.8% of the 42 C0 plants originally selected; approximately 1.295 selection intensity). Planting was done in single rows of 30 hills per line to form a total of 300 plants per population spaced at 80 x 40 cm.
Recombination of these 10 lines was done at flowering per population by bulking the pollen and randomly mating the plants in all possible combinations between November 2009 and February 2010. Cycle 2-nematode resistant (C2R) seed was generated from this recombination. According to Weyhrich et al. (1998), genetic drift becomes a stronger force in altering allele frequencies than selection when fewer than 10 lines are recombined.
6.2.6 Multi-locational field evaluation of the original and advanced S1 progeny
Evaluation of C0, C1R, C1S and C2R populations for nematode resistance was conducted in three-replicate trials at Namulonge (1 200 m above sea level (a.s.l); 0°32’N, 32°34’E; 1 300 mm bimodal rainfall), Kabanyolo (1 150 m a.s.l; 0º 28’N, 32º 37’E; 1300 mm bimodal rainfall) and Bufulubi (1 130 m a.s.l; 00º 49’ N, 033º 42’ E; 1 345 mm bimodal rainfall) between February 2010 to July 2010 (Table 6.2; Fig. 6.1). The design was a split-plot at all sites with whole plots being the levels of nematode treatment (nematode infested vs nematicide treated plots); and the subplot levels were the original and advanced seed cycles (C0, C1R, C1S and C2R) (Appendix 6.4). The nematode infested plots comprised an average Pi of 500 P. zeae and 100 Meloidogyne spp. per 100 g of soil per plot and lesser populations of other nematode species. Each subplot (4.2 x 2.8 m2) was made up of five rows of 15 hills, providing a plant population of 75 plants per sub-plot spaced at 0.75 m between rows and 0.3 m within rows. Of the 75 plants in each sub-plot, 15 were used for nematode assessment through destructive sampling, whereas the remaining 60 plants were used for assessment of agronomical traits and grain yield. Screenhouse evaluation was not conducted for the multilocation trial to compare performance of original and advanced populations.
122 Table 6.2: Time line for the recurrent selection activities
Season Activity
November 2007 – March 2008 500 plants per original (source) population of Longe 1, Longe 4 and ZM521 planted at IITA-Namulonge for selfing.
April 2008 – August 2008 Field evaluation for nematode resistance and susceptibility of the selected 42 S1 progeny per population. Selection of 20 nematode resistant and 10 nematode susceptible progeny for advancement using their remnant seed.
November 2008 – February 2009 Remnant seed of both resistant and susceptible S1 lines sown ear-to-row. Random mating of S1 lines in nematode resistant and susceptible populations by bulking pollen and making crosses in all possible combinations. Some of the harvested seed from each of the 20 lines was kept separately for later use in initiating C2 seed; the remaining seed was bulked to form C1R and C1S seed.
March 2009 – June 2009 Remnant seed of the individual 20 lines comprising the nematode resistant C1R families subjected to field and screenhouse evaluation for P. zeae and Meloidogyne spp. resistance at IITA- Namulonge for each of the 3 populations. 10 progeny with the highest weighted selection index picked for advancement to C2R using their remnant seed.
November 2009 – February 2010 Planting of remnant seed of the 10 selected progeny, pollen bulked and plants randomly mated but reserving some seed from each of the 10 lines. C2R seed generated from this recombination.
February 2010 – July 2010 Field evaluation of C0, C1R, C1S and C2R done in 3 replicate trials at Kabanyolo, Bufulubi and Namulonge for each of the three maize populations.
123 Figure 6.1: Flow diagram for the selection scheme applied to a single source population