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The scheme for development of Ciherang-Sub1-AG1 is summarized in the following chart (Fig. 1) including the markers used in each stage of selection. Initially, all foreground markers for a specific population were used to narrow down the plants prior to background selection. However, results showed that markers for a certain locus showed the same genotype for a particular plant such as ART5 and RM8300 for SUB1. Thus, in the consecutive generations, less foreground markers were initially used. Plants which were selected after background selection were genotyped again using DNA from re-collected leaf samples, and then the rest of the foreground markers were added.
Foreground selection of the F1 progeny revealed the approximate expected ratio of the homozygotes and heterozygotes for the tolerant allele which was 50:50 for B and H in the SUB1 locus (Appendix Fig. 1), and A and H genotypes in the AG1 locus (Appendix Fig. 2). Actual results approximated the expected, namely 55:45 ratio for SUB1 and 54:46 for AG1. The slight deviation might be due to sampling of seeds during planting. From the 542 plants, 72 plants which were homozygous for the SUB1 tolerant allele, as well as heterozygous in the AG1 locus, were selected. However to increase the chances that plants with favorable background may be selected, some plants which were heterozygous for both AG1 and SUB1 loci were randomly selected to increase the total to 92 for background selection.
Figure 1. Development of Ciherang-Sub1-AG1 including markers used for selection.
Though it was expected that all plants being F1 would be heterozygous for all loci, random background genotyping with SSR markers was still done. Of the 26 polymorphic SSR markers, 14 were used for a rough background. Genotyping revealed a product of selfing and that approximately 66% of the selected plants had a foreign allele not belonging to IR64, Ciherang-Sub1 or KHO at the tip of chromosome 2 (RM279) (Appendix Fig. 3). Finally, seven plants which were heterozygous for the IR64 and Ciherang alleles for all genotyped loci except the tip of chromosome 9 (1Mb) which was fixed for the SUB1 tolerant allele were selected for backcrossing to Ciherang-Sub1. These were homozygous for the tolerant SUB1 allele and heterozygous for the tolerant AG1 allele, except for one which was heterozygous for both loci.
Since the ratooned plants were used as the pollen parent, a significantly lower number of seeds were produced. Thirty-two BC1F1 plants were subjected to foreground selection using the same markers used for genotyping of the F1 plants. From these, 12 plants which were heterozygous at the AG1 locus and were homozygous or heterozygous for the tolerant allele at the SUB1 locus were selected for background genotyping with the 26 SSR markers (Appendix Fig. 4.). Plants which had more than 50% of these loci fixed as similar to Ciherang and without foreign alleles were chosen for backcrossing to Ciherang-Sub1. Of the five plants selected, all were fixed at the SUB1 locus (23-1-25-1, 23-1-25-2, 23-1-25-3, 30-2-27-1) except for one (42-11-22-4). Selfed seeds from three of the plants (23-1-25-1, 30-2-27-1, 42-11-22-4) were also collected for use in the AG tolerance phenotypic confirmation.
Initially, the goal of this study was to develop the AG and submergence tolerance improved Ciherang cultivar with one generation of backcrossing followed by one generation of selfing. A study by Sanchez et al. (2009) proved that a cultivar may be improved using this approach provided that the source of tolerance is a related line. Ciherang-Sub1 was developed using this approach starting with selection from a Ciherang/IR64-Sub1 population with 520 BC1F1 plants. This was narrowed down to 216 plants heterozygous for SUB1 and finally two BC1F1 plants with the best background were selected for selfing. Then, 1,395 BC1F2 plants were subjected to the same selection wherein 303 plants fixed for SUB1 were screened with the rest of the heterozygous markers. One plant which was fixed for the Ciherang genotype except for the SUB1 locus was selected and seed increase was done before release. Their results showed that having a large BC1F2 population (about 1,000 plants) is necessary to do successful transfer in one backcross.
Although, their study involved only one trait, this approach may also be extended to a study such as this with two traits provided that the BC1F2 population is large enough. If there had been successful selection of only F1 plants homozygous for the tolerant SUB1 allele, the resulting BC1F1 population will be similar to the population in the previous study with foreground selection being concentrated on heterozygotes for the AG1 locus only. The background selection will also be similar since the same background markers used in their study was adopted for this research. However, in this study, the BC1F1 population was very small and the parents were not the best plants in the F1 generation, which were already fixed for SUB1. Thus, another backcross generation was added.
Foreground selection was done on 446 BC2F1 plants using the same process for the BC1F1 and 228 were chosen for background genotyping using 9-12 markers representing the previously heterozygous loci in the respective BC1F1 parents. Background genotyping revealed that eight plants were fixed as similar to Ciherang in all but 2-3 heterozygous background loci. After confirmation of foreground genotyping, four of these (23-1-25-3-52, 30-2-27-1-85, 30-2-27-1-103 and 30-2-27-1-109) were selfed to fix the remaining heterozygous background loci, as well as the AG1 locus.
A total of 570 BC2F2 plants were grown and these were initially genotyped using SC3 and Dreb4bp to select for plants which were fixed at both loci with the tolerant alleles. A total of 140 plants were selected and genotyping was done using the remaining previously heterozygous background markers. Plants which were fixed as similar to Ciherang in all loci except for the presence of the tolerant allele for both AG1 and SUB1 were identified. DNA was then re-extracted from these plants and genotyping was repeated for the foreground, as well as all 26 background loci. Confirmation revealed that the best four plants (1-109-116, 1-109-136, 1-103-10, 1-85-174) were homozygous for the tolerant allele at the AG1 and SUB1 loci. These were similar to Ciherang-Sub1 at all background loci except one which was fixed as similar to IR64 instead.
This locus represented by marker RM524 was located on 12.9Mb on chromosome 9. However, the next marker which is less than .5Mb from RM524 shows that the adjacent region is already conserved between the two cultivars. Since this region is small and IR64 is also an elite cultivar, the study recommends that either of these four plants are sufficiently similar to Ciherang and may be used for seed increase and in further studies as the improved Ciherang with tolerance to submergence and anaerobic germination. After these lines have been sufficiently evaluated in the field for tolerance, as well as desirable agronomic characteristics, these NILs may be very useful to farmers.
Phenotypic Confirmation for AG Tolerance
A total of 202 BC1F2 plants from selected BC1F1 plants (23-1-25-1, 30-2-27-1, 42-11-22-4) were genotyped for SUB1 and AG1. All plants were homozygous for the tolerant SUB1 allele, but were segregating for the AG1 locus (data not shown). From these, 20 plants which were homozygous for the AG1 tolerant allele and 10 of those homozygous for the intolerant allele were randomly selected. Scoring for AG tolerance was done at 16 and 21 days after seeding (DAS), and percent germination was computed. To increase accuracy, plants which had a germination rate lower than 80% in the control setup (unsubmerged) were removed. This reduced the number of expected tolerant plants to 16 and the susceptible ones to six.
The following figure (Fig. 8) shows the average germination rates at 21 DAS. The mean percent germination rate of the intolerant and tolerant BC1F3 plants were combined into the –AG1 and +AG1 groups, respectively. Checks used were the AG intolerant IR42, slightly tolerant Ciherang and highly tolerant KHO. As expected, the intolerant check IR42 had no germination, whereas the highest germination rate among the checks was 45% for the tolerant donor, KHO. The samples without AG1 showed an average rate of 9.44%, which is comparable to the original cultivar (8.33%), with a standard error of 3.12. On the other hand, those with AG1 had a value of 47.29% (standard error = 2.85). This was slightly higher than the value for KHO, although these are comparable. The variation among individual plants was reflected in the standard error which was higher than those exhibited by the checks.
Based on the phenotypic data, generally plants homozygous for the AG1 tolerant allele had greater tolerance than those without. But, since the background was not yet fixed as similar to Ciherang, variations in the background loci might also have affected the effect of the alleles resulting to varying degrees of tolerance. It is possible to have variations in the germination rate since the plants tested were not fixed in all loci as similar to Ciherang. The variations in these loci may interact to result to higher or lower tolerance as compared to the original Ciherang cultivar. Although without the tolerant AG1 allele, these plants are not expected to show similar germination rates to KHO.
Figure 8. Average germination rate of IR64-Sub1/IR64-AG1//Ciherang-Sub1 BC1F3 plants with and without AG1 tolerance alleles after 21 days of submergence. The vertical bars represent + S.E.
It is likewise possible that some of the BC1F2 plants might really have greater anaerobic germination tolerance compared to KHO. Similarly, this advantage might be due to variations in the background. It is possible that interaction among the background loci or interaction of the loci with the AG tolerance locus from KHO might have further increased tolerance. Interactions may also prevent the full effect of the AG allele from KHO. As a result, although all lines had the tolerance allele, not all exhibited the same degree of tolerance as KHO. Also, there may be other minor QTLs in KHO contributing to its tolerance which were not transferred to the improved Ciherang.
Phenotypic Confirmation for AG Tolerance