Virus resistance. Barley Yellow Dwarf Virus (BYDV)

Bdv2

Detection of the Thinopyrum intermedium segment carrying Bdv2

The T. intermedium fragment does not normally recombine with wheat chromosomes, so only one marker can be used. There are different types of markers that can used: RFLP, genome-specific clones and PCR-based.

Collaborators backcrossing this gene with the dominant marker BYAgi found that in some lines its segregation departed from the 1:1 expected ratio in backcrosses. When this deviation occurred, the proportion of plants carrying the Bdv2 gene was lower than expected. In these cases additional crosses are recommended to compensate for lower transmition of the Bdv2 gene.

Ayala et al. (7) determined that the microsatellite marker Xgwm37 cosegregates with Bdv2 derived from Thinopyrum intermedium. However, marker-assisted selection (MAS) with Xgwm37 is not always feasible. H. Ohm reported an improved protocol using BSA and PVP in the PCR reaction, which is described below, but is complicated and time-consuming. More recently, Ohm and Kong developed a SSR [(AG)36] marker based on the identical sequences derived from the critical bands amplified from T. intermedium using the improved PCR protocol. The linkage relationship with Bdv3 was subsequently confirmed in F₂ populations segregating for Bdv3.

Bdv2 and Bdv3 are transferred from apparently similar positions on homoeologous chromosomes, thus it is unlikely that they can be pyramided, given the current germplasm lines that are available.

SSR marker for detecting Bdv2 and Bdv3

Primers

      Bdv2/Forward: 5'- CTT AAC TTC ATT GTT GAT CTT A -3'      Bdv2/Reverse: 5'- CGA CGA ATT CCC AGC TAA ACT AGA CT -3'

Final concentrations of the products used in the PCR reaction:

• 1 X PCR buffer
• 200 µM dNTP each
• 0.2 µM primers each
• 1.5 mM MgCl₂
• 1 U Taq polymerase (Promega)
• 100 ng DNA template

Total volume: 25 µl reaction

PCR conditions

• Denaturing step: 94°C, 2 min
• Amplification step (35 cycles)
  • 94°C, 30 sec
  • 52°C, 40 sec
  • 72°C, 60 sec
• Extension step: 72°C, 7 minutes.

Expected PCR products:

Figure 1. The top and middle arrows show the fragments (290 bp and 198 bp respectively) linked to the Bdv resistance genes, Bdv2 (on chromosome 7St) and Bdv3 (on 7E) from T. intermedium. The bottom arrow shows the band (175 bp) amplified in the non Bdv (C.S.). M= 100 bp DNA ladder; 1= C.S.; 2= P961341, derived from P29 and has Bdv3; 3= P98134 and has Bdv3; 4= Mackellar (has Bdv2 from Line L1; 5= L1 - a wheat-T. intermedium addition line; 6= P29 substitution line (has Bdv3).

Figure 2. The top and middle arrows show the fragments (290 bp and 198 bp respectively) linked to the Bdv3 resistance gene from T. intemedium. The bottom arrow shows the band (175 bp) amplified in the non Bdv3 (C.S.). M= 100 bp DNA ladder; 1= P98134 having Bdv3; 2= Chinese Spring; 3-26: F2 population from P98134 x Chinese Spring.

Microsatellite marker Xgwm37 :

This information was updated in January 2005 by Herb Ohm.

Primers

      WMS37-F: 5'- ACT TCA TTG TTG ATC TTG CAT G -3'      WMS37-R: 5'- CGA CGA ATT CCC AGC TAA AC -3'  

Final concentrations of the products used in the PCR reaction:

• H₂O: 1.3 µl
• PVP-10 (10%): 2 µl
• BSA (Sigma Cat# B-4287) (10 mg/ml): 2 µl
• 10X PCR buffer: 2.5 µl
• dNTPs (2mM): 2.5 µl
• MgCl₂ (2.5 mM): 1.5 µl
• Primer 1 (2.5 µM): 3 µl
• Primer 2 (2.5 µM): 3 µl
• Taq 0.2 µl
• DNA: 2 µl (~50-100 ng)

Final volume: 20 µl

PCR conditions

1. 35 cycles of: [94°C for 30 sec; 52°C for 45 sec; 72°C for 60 sec]

Expected PCR products:

Xgwm37 marker analysis of backcross (BC) F1 plants (susceptible wheat line (lane 28)/resistant line, P961341 (lane 29)//susceptible wheat line). Band A (170 bp) is specific to wheat. Band B (290 bp) is specific to the 7E chromosome segment carrying YDV resistance.

Clone specific for the T. intermedium chromosome carried by P29:

pAW161 is a member of the telomere-specific repetitive sequences of rye, but it also hybridizes to the 7EL telomere and has none homologous sequence in wheat. Experimental details for using this marker and typical results can be found in Ref. 6.

RFLP marker Xpsr129 :

The locus Xpsr129 is polymorphic for wheat and the different translocations of T. intermedium that carry the BYDV resistance (1,4,5). When the probe PSR129 is hybridized to wheat DNA digested with the restriction enzyme Bgl II, unique bands for the 7A, 7B, 7D chromosomes can be observed. For wheats derived from the TC lines (except TC7) and homozygous for the translocation or derived from P29, the 7D band is replaced by another one from the 7Ai-1 chromosome.

SCAR marker BYAgi

This information has been extracted from Ref.8.

      BYAgi Forward: 5'- CAT GGA TAA TTC AGG GAG CAT TCT G -3'      BYAgi Reverse: 5'- CTG AAC ACG AAT TTG CTG AGG TTG -3'   

PCR conditions

1. Denaturing step: 95°C, 15 min
2. 30 cycles of: [94°C 45 s, 56°C 30 s min, 72°C 45 s]
3. Extension step: 72°C 10 min

Final volume: 20 ul

Final concentrations of the products used in the PCR reaction:

• HotStar Taq polymerase, Qiagen (includes buffer and dNTPs)
• primers: 0.5 uM each
• DNA input: 40 ng/reaction

Expected PCR product sizes:

• T. intermedium translocation on chromosome 7DL : 566 bp
• Wheat chromosome 7DL: no amplification.

Conditions presented here should be consider only as a starting point of method optimization for individual laboratories.