Bulked Segregant Analysis Using the GoldenGate Assay to Locate the \u3ci\u3eRpp3\u3c/i\u3e Locus that Confers Resistance to Soybean Rust in Soybean

Few resistance loci to soybean rust (SBR), caused by Phakopsora pachyrhizi Syd., have been genetically mapped and linked to molecular markers that can be used for marker assisted selection. New technologies are available for single nucleotide polymorphism (SNP) genotyping that can be used to rapidly map traits controlled by single loci such as resistance to SBR. Our objective was to demonstrate that the highthroughput SNP genotyping method known as the GoldenGate assay can be used to perform bulked segregant analysis (BSA) to fi nd candidate regions to facilitate effi cient mapping of a dominant resistant locus to SBR designated Rpp3. We used a 1536 SNP GoldenGate assay to perform BSA followed by simple sequence repeat (SSR) mapping in an F2 population segregating for SBR resistance conditioned by Rpp3. A 13-cM region on linkage group C2 was the only candidate region identifi ed with BSA. Subsequent F2 mapping placed Rpp3 between SSR markers BARC_Satt460 and BARC_Sat_263 on linkage group C2 which is the same region identifi ed by BSA. These results suggest that the GoldenGate assay was successful at implementing BSA, making it a powerful tool to quickly map qualitative traits since the Golden- Gate assay is capable of screening 1536 SNPs on 192 DNA samples in three days

Side-population of HBM.

<p>(a) Sorting profile of side-population (SP) and non side-population (NSP) from breast milk. (b) Scatter plots showing no relationship between the percentage of SP and duration of breastfeeding and (c) age of mother. (d) Immunocytochemistry illustrating expression of nestin exclusively only on SP (top left) and expression of CK 18 exclusively on NSP (bottom right).</p

Antigens expressed on CD133+ and CD133- cells in HBM.

<p>Antigens expressed on CD133+ and CD133- cells in HBM.</p

Mesenchymal Culture of Cells.

<p>(a, b) Adherent colonies of fetal MSC emerged after low density seeding at 4 cells per cm². Osteogenic induction of fetal MSC resulted in the deposition of extracellular calcium crystals staining positive with Alizarin red (c) and Von Kossa (d).CFU-F assays of SP and NSP as well as Stro1 positive and negative fractions of HBM did not establish any colonies as shown by the absence of colonies (e, h). WCP (f) and Stro-1 positive cells (g) cultured in D10 remained as non-adherent cells (red arrows) which did not undergo any proliferation in culture.</p

Antigens expressed on the cells in HBM.

<p>Antigens expressed on the cells in HBM.</p

RT-PCR on Messenger RNA (mRNA) of milk samples from three individuals.

<p>(a) mRNA of CD133 and CD34 were present in WCP of HBM (Lane 1–3). (b) Osteonectin (ON), alkaline phosphatase (ALP) and osteopontin (OP) (Lane 1–3) as well as (c) musashi-1 (Msi), nestin (NES) and neurofilament-M (NFM) in observed in WCP of HBM (Lane 1–3). (d) Messenger RNA of CK5, 14 and 18 were present in WCP of HBM (Lane 1–3). The negative controls in Lane 4, were MCF-7 for hematopoietic, mesenchymal and neural markers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014421#pone.0014421-Calvanese1" target="_blank">[49]</a> and mononuclear cells in peripheral blood for epithelial cell markers of CK5 and CK14. Positive controls (Lane 5) are cells isolated from umbilical cord blood (a), fetal MSC (b), cells from snap-frozen fetal brain (c) and MCF-7 (d) respectively.</p

Cellular concentration in human breast milk did not vary in relation to the duration of breastfeeding.

<p>Cellular concentration in human breast milk did not vary in relation to the duration of breastfeeding.</p

CD133 Sorting.

<p>(a) Flow cytometry of CD133 staining on cellular component of HBM (i), as compared to the isotype control (ii). (b, c) Three was no relationship between the frequency of CD133 cells and the duration of breastfeeding, nor the age of the mother. (d) CD133+ cells from cord blood formed multi-lineage colonies on CFC assays.</p

Identification of nestin-positive putative mammary stem cells in human breastmilk

10.1007/s00441-007-0390-xCell and Tissue Research3291129-13