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 Rp1-D21 and the Identification of Hrml1

As mentioned earlier, Rp1-D21 is an aberrant allele at the Rp1 locus which confers resistance to specific races of the common rust pathogen, Puccinia sorghi [14]. Rp1 is a complex locus consisting of a family of tandemly-repeated, tightly linked and closely related resistance genes or paralogs whose number/organization (haplotype) differs from gene to gene [15]. For instance, the Rp1-D haplotype is composed of Rp1-D plus eight paralogs that exhibit 91-97% DNA identity with Rp1-D. The Rp1-D21 allele was derived from a crossover event between paralog 2 and paralog 9 (the Rp1-D gene itself), such that it acquired the NBS domain and the first 13 of the 15 repeats of the LRR region from paralog 2 and the remainder of the LRR region from paralog 9 [68]. It still contains intact paralog 1, but paralogs 3 through 8 are missing from Rp1-D21.

Fig 2. A 3-week-old Rp1-D21(H95) heterozygote in the H95 background

Plants carrying Rp1-D21 exhibit HR constitutively under both greenhouse and field conditions, albeit in a developmentally-programmed fashion (Fig. 2). Developing Rp1-D21 lesions accumulate both superoxide and hydrogen peroxide, two hallmarks of the HR response (Fig. 3). They also induce a number of defense markers (Fig. 4), suggesting the HR lesions induced by Rp1-D21 are identical to the bona fide HR lesions triggered at the site of infections. It has been reported that Rp1-D21 requires some biotic stimulus to form HR lesions [14]. This conclusion was based on data from Rp1-D21 seedlings that were grown axenically in 2L bottles. However, the higher humidity and lower incident light of these growing conditions, both of which are known to dampen the HR response [18,33,69], could also explain these results and we have not detected any requirement for a biotic stimulus.


Fig 3. In situ staining of developing Rp1-D21 lesions with nitroblue tetrazolium (A) and di-amino benzadine known to dampen the HR response (B), showing the production of superoxide and H2O2.



We identified the background-dependent nature of Rp1- D21 because of Slm1, which we had found earlier in the Mo20W inbred background as a QTL able to suppress cell death associated with les23, as well as a number of other les mutants. However, Slm1 did not suppress the HR associated with Rp1-D21, if anything, it appeared to enhance it (Fig. 1). Control crosses were also made between Rp1-D21 and B73 and Mo17. Interestingly, in the F1, B73 suppressed Rp1-D21, while Mo17 enhanced the Rp1-D21 phenotype (Fig. 1). The HR phenotype of Rp1-D21 was so severe in the H95/Mo17 background that we were unable to propagate the plants further. This raised the question of how to look into the genetic basis of suppression in B73 and of enhancement in Mo17. Weil’s suggestion that the IBM population could be used worked well and led to the identification of Hrml1.


Fig. 5. A QTL scan of chromosome 10 showint the size and location of Hrml1. The Rp1 locus is also located on ch 10, but many cM away.


Hrml1 was mapped as follows. Rp1-D21(H95) was crossed to 174 of the 302 IBM lines. Since the Rp1-D21(H95) plants were heterozygous for the Rp1- D21 gene, each of the 174 resulting F1 families segregated 1:1 for Rp1-D21 but were otherwise isogenic. This population of 174 families was assessed in three different environments: in the greenhouse (scored at six times between 17 and 44 days after planting) in the field in West Lafayette, IN (one replication) and in the field at Clayton, NC (two randomized replications). In each case at least 8 plants from each F1 family were scored for 1) average lesion severity of the Rp1-D21 segregants, scored on a 1-10 scale, and 2) average height of the Rp1-D21 plants divided by average height of the wild type segregants from the same F1 family. The Windows QTL cartographer version 2.5 software package was used to detect the QTL using standard methodology [22]. A moderate size QTL was found to map on chromosome 10, bin 3, somewhere between 144-192 map units on the IBM2 map (Fig. 5). (http://www.maizegdb.org) (Fig. 5). Since it was identified in all three experiments (replications), we considered it to be genuine and have named it Hrml1.

In summer 2007, we generated an 100 additional Rp1-D21(H95) x IBM F1 families. We will therefore run field trials using at least 270 F1 families in 2008. This should give us some extra precision and power for QTL mapping. We are also in the process of introgressing Rp1-D21 into B73. Four backcrosses have been made and the 5th will be finished this winter in the greenhouse. Plans have been made to cross the entire MDL (maize diversity lines) population with both Rp1-D21 in both H95 and B73 backgrounds.