Marker assisted foreground & background selection in soybean
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This document describes the development of near-isogenic lines (NILs) in soybean containing a recessive bacterial leaf pustule resistance gene (rxp) introgressed from donor parent SS2-2 into two recurrent parents, Hwangkeumkong and Taekwangkong. The researchers performed three rounds of backcrossing followed by two generations of selfing. They used marker-assisted selection with over 100 SSR markers to select plants with the highest percentage of recurrent parent genome. The resulting NILs will be used to map and characterize the rxp gene through transcript analysis when infected with the bacterial pathogen.
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Thymidine kinase (TK), dihydrofolate reductase (dhfr), and chloramphenicol acetyl transferase (CAT) are examples of selectable marker genes used for animal cells. Marker genes help monitor transfection by allowing detection of whether the transgene was successfully transferred. Selectable marker genes enable transformed cells to survive selection conditions that kill non-transformed cells.
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The document discusses mutation breeding in plants. It describes selecting parents and handling the M1 to M3 generations for mutant selection. The objectives of mutation breeding are outlined, such as improving specific traits or inducing male sterility. The process involves mutagenic treatment of parent seeds, then selection of mutants in segregating M2 and subsequent generations up to M5. Guidelines are provided for planning M1 generation sowing and handling the M1 seeds. Molecular mechanisms of induced mutations like DNA damage and repair systems including mismatch repair and double strand break repair are also summarized.
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- 1. MARKER-ASSISTED FOREGROUND & BACKGROUND SELECTION BACTERIAL LEAF PUSTULE RESISTANT GENE IN SOYBEAN 3/15/2020 1
- 2. LEAF PUSTULE DISEASE Bacterial pustule is caused by the bacterium Xanthomonas axonopodis pv. glycines. Like bacterial blight, bacterial pustule resides in crop residue during winters and is carried by wind or water droplets from the ground to the plant. In addition, the disease can be spread during cultivation while the foliage is wet. The bacterium will enter the plant through natural openings and wounds. 3/15/2020 2
- 3. SYMPTOMS This disease is typically present on the upper leaves. Initial infection results in the development of tiny pale green spots on the new leaves. As the disease progresses, small light colored pustules will form in the center of the spots. These spots may merge together to form irregular areas that appear as lesions. As spots mature the lesion and pustule turns brown. 3/15/2020 3
- 4. 3/15/2020 4 A resistance gene to BLP, supposedly recessive and designated as rxp was identified in CNS (ancestral cultivar). The rxp locus has been found to be located on linkage group (LG) D2 using the phenotypic variation of the BLP reaction in segregation mapping populations including an F2 or F2-derived recombinant inbred line (RIL) populations from a cross of distinct genotypes of the BLP resistance trait.
- 5. The simple sequence repeat (SSR) markers, Satt372 and Satt014 were observed to flank the rxp locus, which was 3.9 cM away from Satt372 . Furthermore, comparisons between phenotypic values for the BLP resistance and the SSR marker genotypes in the RIL populations suggested that Satt372 showed the most significant association with the trait variation. In the recent soybean consensus map Satt486 was mapped between those flanked markers 3/15/2020 5
- 6. 3/15/2020 6 Resolutions of the markers surrounding rxp in the RIL populations are not high enough to isolate rxp by Mapbased cloning. In the present study, the NILs with respect to the BLP resistance gene, rxp were developed by introducing the rxp gene into two Korean BLP-susceptible elite cultivars, Hwangkeumkong and Taekwangkong through consecutive backcrosses. Korean genotype SS2-2 showing BLP-resistant response to Xag was used as a donor parent. Hwangkeumkong and Taekwangkong were selected as a recurrent parent because they are Korean elite cultivars but BLP-susceptible
- 7. 3/15/2020 7 Two sets of NILs were made from the crosses Hwangkeumkong x SS2-2 (HS) & Taekwangkong x SS2-2 (TS) by backcrossing three times in succession. Five BC3F3 and three BC3F2 lines were obtained from the HS and TS crosses, respectively . For molecular marker genotyping and BLP phenotyping five or eight seeds per BC lines were sown for growth of BC plants.
- 8. 3/15/2020 8
- 9. BACTERIA CULTURE & INOCULATION The parents and four-week-old BC plants were inoculated by spraying the bacterial suspension of pathogen strain 8ra of Xag to both sides of leaves using an automizer. The infected plants were incubated in growth chambers under the conditions of 28 ℃, 100% Relative humidity and 12 h illumination 3/15/2020 9
- 10. DNA EXTRACTION & SSR MARKER ANALYSIS Total DNA was isolated from young leaves of Hwangkeumkong, Taekwangkong, SS2-2, and all BC plants using the modified CTAB procedure. Background selection was performed using the SSR markers on all chromosomes.In the first background selection, a total of 26 and 21 non-carrier markers were only used in HS and TS lines respectively. For the second background selection, an additional 92 and 69 markers in HS and TS lines were used to obtain the BC plants with the highest RPG content. 3/15/2020 10
- 11. RESULTS & INTERPREATION To develop the NILs for BLP resistance gene rxp, they’ve carried out three cycles of backcrossing and advanced two more selfing generations. Since the desirable allele of the BLP resistance gene is recessive, the recessive homozygote is needed. In general BC breeding programs, additional selfing process should be included to introgress a recessive gene, particularly in odd generations such as BC1F2, BC3F2 etc 3/15/2020 11
- 12. The four-stage selection strategy is one of the selection strategies for marker-assisted backcrossing proposed which consists of four selection steps: i. select individuals having the target allele ii. select homozygous individuals for recurrent parent allele at the flanked markers iii. select homozygous individuals for recurrent parent allele at additional markers on the carrier chromosome iv. select one homozygous individual at the maximum number of markers across all chromosomes. 3/15/2020 12
- 13. In the future, whole transcript analysis in the resistant and susceptible NILs infected with Xag using GS-FLX or Solexa should allow the rxp gene or candidate genes for BLP resistance to be characterized. 3/15/2020Footer Text 13