Читать книгу Principles of Plant Genetics and Breeding - George Acquaah - Страница 238

Near‐isogenic lines (NILs) that carry small introgressed chromosome fragments from related wild species in a cultivated tomato background are most useful pre‐bred in a breeding program. To develop NILs that only differ in Ol genes for resistance to O. neolycopersici, resistant donor accessions were crossed with susceptible S. lycopersicum cv. Moneymaker (MM). BC crosses were made starting from crossing F1 plants back to MM (Figure B5.3). During the backcrossing, selection of resistant BC plants can be performed in two ways. One is by testing BC plants with powdery mildew and the other is to genotype them with markers linked to individual resistant loci. Since we have markers linked to each Ol‐genes, we could apply MAS. We did a disease test since (i) it is a relatively easy disease assay which can be carried out at seedling stage; and (ii) the resistance phenotype is clear to be scored. In case that the disease assay is not easy to perform, for example due to (i) quarantine pathogens or (ii) a disease test that has be done at the late developmental stage, MAS would be a convenient way to select resistant plants (Figure B5.4).

Figure B5.3 Cross‐pollinating scheme of generation of near‐isogenic lines (NIL) harboring dominant resistance genes to tomato powdery mildew. Via backcrosses, new traits are introduced from wild tomato relatives. During the backcrosses, selection of resistant plants can be performed via (1) disease test and/or (2) marker‐assisted selection (MAS, Figure B5.4). In addition, select plants should have similar morphology to the recurrent parent. * and **: Usually it takes many generations to remove the deleterious genes that go along with the introduced genes due to linkage drag. Therefore, it is useful to start with advance breeding lines having introgression from the wild species in order to shorten the backcrossing procedure. In our practice, we used advance breeding lines derived from S. habrochaites G1.1560 (donor of the Ol‐1 gene) to produce the F₁. We checked BC₃S₁ plants for the uniformity of their genetic background by genotyping these plants with 12 AFLP primer combinations that produce genome‐wide markers. Of the 30 AFLP marker alleles specific for S. habrochaites G1.1560, they were only present in the BC₃S₁ plants with two fully cosegregating with the Ol‐1 gene, suggesting that the genetic background of these BC₃S₁ plants are genetically similar to MM. For the Ol‐4 genes derived from S. peruvianum LA2172, we started with the wild accession. With 12 AFLP primer combinations, 48 AFLP marker alleles were identified from S. peruvianum LA2172 and 11 of these alleles still segregated in the tested BC₃S₁ plants. Thus, when the backcross is started from wild accessions, more backcross generations are needed to make NILs.

Figure B5.4 Illustration of marker‐assisted selection (MAS). On the left, a genetic linkage map of tomato chromosome 6 showing that the Ol‐1 and Ol‐3 genes, conferring resistance to tomato powdery mildew, are located at the same locus and are flanked by Markers 3 and 4. On the right, electrophoretic patterns of PCR markers showing marker genotypes of 6 plants; the upper panel for Marker 3 and the lower panel for Marker 4. Plant 1–4 are either BC3 plants (for Marker 3) and BC3S1 plants (for Marker 4). Plant 5 and 6 are parental plants that are susceptible and resistant to tomato powdery mildew, respectively. M indicates DNA size marker of 1 kb ladder. For MAS, marker flanking the target gene is often used. For Marker 3, BC3 plants no. 1 and 3 are selected and expected to be resistant to powdery mildew since they have the marker allele of the resistant parent (plant no. 6). For Marker 4, plant 1 to 3 are selected and expected to be resistant since they have the resistant marker allele as the resistant parental plant no. 6 (homozygous plant no. 1 and heterozygous plant no. 2 and 3).

After several backcrossing generations, homozygous BC_nS₁ resistant plants of these crosses were selected (Figure B5.3). Since we have facilities for genome‐wide analysis, we genotyped all selected plants with AFLP markers to compare their genetic background with the recurrent parent MM. BC_nS₁ resistant plants that were genetically most similar to MM were maintained as NILs.