Map position

 

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Cloning by map position:

The Rhg4 locus on Linkage Group A2 confers resistance to race 3 of the soybean cyst nematode.  We are attempting to clone the Rhg4 by map-position-based cloning techniques. The Rhg4 locus is very tightly linked (0.35 cM) with the I locus conferring black seed coat.  Chalcone synthase is encoded at the I locus and a restriction fragment length polymorhism (RFLP) identified by probe pBLT65 also is closely linked to the I locus.  We developed polymerase chain reaction (PCR) assays using primers designed from pBLT65 for detecting variation between SCN-resistant and -sensitive soybean at this locus (Fig 1) and designed primers designed to detect variations in the I locus using PCR.

Primer 548       GCA GAT ATC AAC AGT TGG GAC

Primer 563       GGA ATG GAC AGC TCG TAA AGC C

Image of a gel showing DNA bands, products from PCR amplification, more details in text.


Figure 1: Products from PCR amplification of genomic DNA from PI290136 (Noir), BARC-2 (Rj4) (lanes 1 and 2) and from F2/F3 plants segregating from a cross of Noir x BARC-2 (Rj4) (lanes 3-7). Lane M contains a molecular weight marker with 1.0 kb indicated.

Bacterial artificial chromosome (BAC) libraries made from genomic DNA of Williams (susceptible) and PI437.654 were screened in collaboration with the Shoemaker and Knap laboratories, respectively, using these markers. The PCR assay is useful using a number of genotypes (Fig. 2) and Table 1.  A map of one BAC in the region is under construction.

Picture of a gel image showing DNA bands after amplifying fragments using primers 548 and 563, more details in text.

Figure 2: The fragments amplified by primers 548 and 563 are also amplified using DNA from other soybean cultivars, indicating that these primers will be of general use for marker-assisted selection in breeding programs. Genomic DNA from 20 cultivars was subjected to PCR amplification with primers 548 and 563 and separated on a 2% agarose gel. Lane M denotes a 1-kb DNA marker. Lane numbers and cultivars are listed in Table 1.

Lane Variety SCN Response Seed coated Pegmentation Banding Pattern      
1
Peking
R S 1a 1b   3  
2
NC55
R S 1a 1b   3  
3
P188788
R S 1a 1b   3  
4
P189772
R S 1a 1b   3  
5
P190763
R S 1a 1b   3  
6
P1437654
R S 1a 1b   3  
7
Custer
R N 1a 1b   3  
8
Pickett
R N 1a 1b   3  
9
Bedford
R N 1a 1b   3  
10
Hartwig
R N 1a 1b   3  
11
S88-1608
R N 1a 1b   3  
12
Essex
S N     2 3 4
13
Lee
S N     2 3 4
14
Hill
S N     2 3 4
15
Kent
S N     2 3 4
16
Scott
S N     2 3 4
17
Bass
S N     2 3 4
18
Williams82
S N     2 3 4
19
Century
S N     2 3 4
20
BARC2 (Rj4)
S N     2 3 4
21
D62-7818
S N     2 3 4
22
D49-2491
S N     2 3 4
23
Blackhawk
S N     2 3 4
24
P1290136 (Noir)
S S 1a 1b   3  

Table 1: Soybean cultivar response to SCN race 3, seed-coat pigmentation and PCR banding pattern using primers 548 and 563.