2012 Annual Report
1a.Objectives (from AD-416):
Develop SNP markers for soybean aphid resistance genes for marker assisted breeding for Rag3 and Rag4 genes.
1b.Approach (from AD-416):
SNP (single nucleotide polymorphism) markers will be identified in the genomic regions of the aphid resistance genes using appropriate RIL mapping populations and near isogenic lines. The Solexa sequencing and Illumina and other genotyping platforms will be used for mapping the SNPs.
Over 200 recombinants between markers flanking the rag3 gene were tested with 18 newly developed single nucleotide polymorphism (SNP) markers located between the flanking markers used to identify the recombinants. The genomic regions containing rag3 gene has been narrowed down to a 216 kb region, which contains eight candidate genes. Three F8:9 plants that are still segregating for aphid resistance were indentified from over 1,000 plants. The progenies from these three plants were tested for aphid resistance in the greenhouse. Over 1,000 F7:8 or F8:9 plants were tested with SNP markers linked to the rag3 gene to identify individuals that are still heterozygous for the genomic region containing the rag3 gene. The heterozygous plants are needed to develop near isogenic lines that are almost identical except for the rag3 genes. The near isogenic lines are necessary to identify the candidate gene responsible for aphid resistance. Six plants were found heterozygous based on the markers. Over 1,000 progeny from these six plants are currently being evaluated for aphid resistance in the greenhouse.
Life time summary: In 2007, Rag1 was mapped between Satt299 and Sat_244 (about 10 cM) and in 2009, Rag4 was mapped between Satt648 and Satt348 (about 15 cM). To fine map these two genes, we created two large populations, one (050016) has individuals of 386 and another (070070) has individuals of 927. These two populations were genotyped with the closest flanking markers (Satt435 and Satt245 for rag1, and Satt569 and Satt348 for rag4). A total of 77 and 68 lines were selected from population 050016 and 070070. All those lines recombined at one of these two intervals. The phenotype data for those lines were collected in two years (one in the field cage in 2009 and one in the greenhouse in 2010). To further reduce the intervals where rag1 and rag4 located, we added three more simple sequence repeat (SSR) markers (SSR-13-417, SSR-13-392, SSR-13-320) in the rag4 interval and three more markers (SSR-07-282, SSR-07-328, SSR-07-411) in the rag1 interval using 170 lines in population 070070. These rag1 and rag4 intervals were further narrowed down to 7.2 cM and 2.7 cM. The rag3 gene was previously mapped between Sat339 and Satt414 (about 20 cM) using 249 lines from a large population (050017), which has a total of 797 lines. All these 797 lines have been phenotyped in the greenhouse. From that experiment, we also selected 19 pairs of isogenic lines, each pair had opposite phenotypes and derived from a same line. To narrow down the interval, we added seven more SSR markers (SSR-16-418, SSR-16-444, SSR-16-467, SSR-16-486, SSR-16-538, SSR-16-507, and SSR-16-549) in that interval using those 249 lines. The interval was narrowed down to about 13 cM. The parents and subsets of the mapping populations were genotyped with over 52,000 SNP markers on the Illumina iSelectHD BeadChips. Polymorphic SNP markers in the candidate regions containing the three aphid resistant genes were identified. These polymorphic SNP markers were used to genotype more than four thousand lines segregating for the three resistant genes to further narrow down the genomic region containing the resistant genes. Additional mapping populations segregating for the three aphid resistant genes were tested in the greenhouse for aphid resistance.
This project enhances/expands on objective 2B (Characterize and map aphid resistance genes in three soybean plant introductions) of the parent project.