Rusts resistance genes Lr19-Sr25 and color gene Y

Leaf rust resistance gene Lr19, Semolina color gene Y, stem rust resistance gene Sr25

Tetraploid durum wheat (Triticum turgidum L. var. durum) is the source of the semolina used for manufacturing pasta products. As with bread wheats, there are many quality requirements that the durum wheats must meet. One of these is the yellow color of the semolina, which is correlated with the color of pasta. Consumers have traditionally associated a yellow color with higher quality. Current regulations in many countries ask for pasta products with a yellow color that originates from the wheat and not from any other additive.

Previous works showed that the chemical substances responsible for the yellow pigment(s) in endosperm were carotenoids of the xanthophylls type, mainly lutein in free form and sterified. However, more recently Hentschel et al. (1) applying more sophisticated separation techniques found that the chemical nature of the yellow pigment in semolina is quite complex: they found that the carotenoids fraction accounted for only 30-50 % of the yellow pigment weight, while the rest were unidentified compounds.

Few QTL studies for the flour yellow pigment are available for tetraploid wheat. Elouafi et al. (2) identified a QTL with a large effect on yellow pigment in the distal region of the long arm of chromosome 7B in a cross between the cultivated durum variety Omrabi5 and T. dicoccoides. Microsatellite markers Xgwm344-7B explained 53% of the variation in yellow pigment in this cross. A QTL with smaller effect was found on the distal region of chromosome arm 7AL. Microsatellite marker Xgwm63e explained 13% of the variation in yellow pigment. Omrabi5 contributed the alleles for high yellow pigment. Hessler et al. (3) identified a minor QTL for yellow pigment on chromosome 5A in the cross Jennah Khetifa x Cham 1. RFLP marker Xbcd926 explained 10.4% of the variation, and Cham 1 contributed the allele for high yellow pigment.

QTLs for yellow pigment in hexaploid wheat

A high level of yellow pigment is desirable in durum wheat but is an undesirable trait in bread wheat. Several studies have identified QTLs for yellow pigment in crossed between bread wheat varieties. The main results are summarized below.

Arm Cross Explained variation Marker Ref.
7AL Schomburgk x Yaralinka 60 % STS from AFLP Xwua26.4 5-6
7BL CD87 x Katepwa 10 % Xpsr680 4
2D CD87 x Katepwa 12 % Xwmc25a 4
3A CD87 x Katepwa 17 % XksuB8 4
5D Cranbrook x Halberd (LOD 4.2) -- 4
7A Cranbrook x Halberd (LOD 4.2) Xfba349-Xpsr121 4
3B Sunco x Tasman 20 % Xgwm285-Xcdo583 4
5B Sunco x Tasman 12 % Xgwm499 4
7A Sunco x Tasman 27 % distal Xwmc346 4

The Y, Lr19 and Sr25 genes on chromosome arm 7EL

Map of Lr19

The distal region of the chromosome arm 7EL from Lophopyrum ponticum carries a gene designated Y that increases yellow pigment in the endosperm, and the leaf rust resistance gene Lr19 and the stem rust resistance gene Sr25. A PCR marker is available to trace this alien chromosome segment (7). Lr19 is effective against the new leaf rust race detected in Mexico and California, which is highly virulent to durum wheat (8). However, an Lr19-virulent leaf rust isolate was found in Mexico (9). Sr25 confers resistance to the new African race Ug99. [link to the Sr25 page]

The 7EL segment was reduced by homoeologous recombination (10), and different common wheat and durum wheat lines were obtained that can be used for breeding (see Available germplasm section).

Semolina and flour color

The colors of semolina and flours are expressed using the L* a* b* color system. L* is a measure of brightness, it can ranges from 0, completely non-reflective or black and 100, perfect white or total reflection. Bread wheat flours have reading values around 90, while semolina has lower values. The b* value is the blue-yellow chromaticity coordinate, it can go from -60, pure blue, to +60, pure yellow. Usual b* values for bread wheat flours are around 9.5. For semolina the higher the b* value the more yellowness. Good quality durums has a b* of aprox. 27.3 or more. The b* vlaue is the red-green coordinate

Cooked pasta has higher b* values, more than 41 for good quality durums. There is another quality parameter related to pasta color, the color score, which combines L* and b* in one index. A spaghetti sample with a color score of 9.0 or higher is good. The range of the color score is 1 - 12.

Semolina colors
L* (brightness) and b* (blue <--> yellow) values for flour from Hard Red Winter wheat grown in the US Midwestern and semolina from Durum grown in the US Great Plains. The more intense the yelowness the greater the b* number. The higher L* values of the bread flour is likely due to the finer milling of this kind of products. These color samples are for indication only, to see the colors with the real L*a*b* values calibrated equipment is required.

Other factors that affect pasta color are ash content and the lipoxygenase activity, which has a negative effect being responsible for browning, and industrial factors such as milling conditions, extraction rate and pasta processing.

Markers for Lr19

Prins et al. developed a PCR marker for the 7EL segment containing Y and Lr19 (7). The co-dominant marker, BF145935 derived from a wheat EST (11) was validated by Liu et al.(12) using 42 wheat lines and indicated that two DNA fragments amplified in most lines that differentiates Sr25 and non-Sr25 lines.

Note: you can find more details about the validation of these markers in Sr25 lines here.

PCR-based marker for Lr19, Sr25 and Y

The Gb primers were developed by Prins et al (7). The germplasm used for that publication was an allosynthetic recombinant produced several years earlier from the original translocation on 7D. An intercalary piece of the translocation was retained that only had Lr19 on it; both Y and Sr25 were lost during recombination, making it a suitable line for breeding in hexaploid lines because the white color of flour would be unaffected. Also the shorter translocation was relocated to 7BL during pairing induction. In spite of these rearrangements, this marker also detects the original Lr19 translocation on 7D (Frans Marais, personal communication).

The PCR marker based on EST BF145935 was originally designed for stem rust resistance gene Sr25, and you can find more details about these gene and marker validation here.

Primer sequences

BF145935

BF145935-F       5'- CTT CAC CTC CAA GGA GTT CCA C -3’

BF145935-R       5'- GCG TAC CTG ATC ACC ACC TTG AAG G -3’

Gb

Gb-F       5'- CAT CCT TGG GGA CCT C -3’

Gb-R       5'- CCA GCT CGC ATA CAT CCA -3’

PCR conditions for BF145935 (times for each step might vary according to the thermocycler model):

  • Denaturing step: 94°C, 4 min
  • Amplification step (35 cycles):
    • 94°C, 45 sec
    • 50°C, 30 sec
    • 72°C, 45 sec
  • Extension step: 72°C, 7 min

PCR conditions for Gb (times for each step might vary according to the thermocycler model):

  • Denaturing step: 5 minutes at 94°C.
  • Amplification step (40 cycles)
    • 94°C, 30 sec
    • 60°C, 30 sec
    • 72°C, 1 min
  • Extension step: 5 minutes at 72°C.

Final concentrations of the reagents used in the PCR amplification of Gb.

  • 50-100 ng template DNA
  • 5 pmol each primer
  • 1 unit of Taq DNA polymerase
  • 2 µl 15 mM MgCl2
  • 2 µl 10X buffer
  • 2 µl 1mM dNTPs

Total volume: 25 µl reaction

Expected products

Following amplification for Gb, the products are separated in 2% agarose gels at 65 V constant voltage. The diagnostic fragment is 130-bp in length.

Lr19 marker with Gb primers
PCR products for Gb primers. 1: Opata (-), 2: 7Ag/7D (+), 3: Pavon (-). 4-12: recombinant lines. Lines 4-6 and 10-11: marker present, lines 7-9 and 12: marker absent.

Marker BF145935 produces fragment sizes of 198 and 180 bp in Sr25 lines and 202 and 180 bp in wheat lines without Sr25 (sse figure below).

Lr19 marker with BF145935
Haplotyping Sr25 in CIMMYT spring lines using marker BF145935. “>” indicates the 198 bp fragment associated with Sr25. CIMMYT entry #11, 12, 42 and 70 showed the 198 fragment, indicating the Sr25-haplotype. CIMMYT #69 shows both 202 and 198 bp fragments, suggesting it is heterozygous.

Available germplasm

Zhang et al. (10) produced a set of recombinant durum and bread wheat lines containing translocated fragments of different sizes derived from the original Lophopyrum ponticum(L. elongatum) translocation.

Recombinant tetraploid (durum) lines Ag 1-22 and Ag 1-23 produced by A. Lukaszewski (10) have short translocated terminal 7EL fragments carrying Y and Lr19. The first line has a recombinant event between Lr19 and RFLP locus Xwg420, while the second is slightly larger and has a recombinat event between RFLP loci Xpsr547 and Xwg420. Both lines have normal segregation rates. Recombinant hexaploid line Ag 1-96 carries a translocated 7EL fragment including Lr19 but not Y. This line is appropriate for transferring Lr19 into bread wheat lines without negatively affecting flour color. The tetraplod line is available upon request from Adam Lukaszewski, and the hexaploid line will be available by 2006 after elimination of the ph1b mutation by backcrossing.

Conditions presented here should be considered only as a starting point of the PCR optimization for individual laboratories.

References

1. Spectrophotometric determination of yellow pigment content and evaluation of carotenoids by high-performance liquid chromatography in durum wheat grain. Hentschel V, Kranl K, Hollmann J, Lindhauer MG, Bohm V, Bitsch R. In: Journal of Agricultural & Food Chemistry, 2002, 50(23):6663-6668. [abstract]

2. Identification of a microsatellite on chromosome 7B showing a strong linkage with yellow pigment in durum wheat (Triticum turgidum L. var. durum). Elouafi I, Nachit MM, Martin LM, In: Hereditas, 2001, 135(2-3):255-261. [abstract]

3. Association of a lipoxygenase locus, Lpx-B1, with variation in lipoxygenase activity in durum wheat seeds. Hessler TG, Thomson MJ, Benscher D, Nachit MM, Sorrells ME. In: Crop Science, 2002, 42(5):1695-1700, 2002. [abstract]

4. Mapping components of flour and noodle colour in Australian wheat. Mares DJ, Campbell AW. In: Australian Journal of Agricultural Research, 2001, 52(11-12):1297-1309. [abstract]

5. Mapping Loci Associated with Flour Colour in Wheat (Triticum Aestivum L.) . Parker GD, Chalmers KJ, Rathjen AJ, Langridge P. In: Theoretical & Applied Genetics, 1998, 97(1-2):238-245. [abstract]

6. Development of a STS marker linked to a major locus controlling flour colour in wheat (Triticum aestivum L.). Parker GD, Langridge P. In: Molecular Breeding, 2000, 6(2):169-174. [abstract]

7. AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat. Prins R, Groenewald JZ, Marais GF, Snape JW, Koebner RMD. In: Theoretical & Applied Genetics, 2001, 103(4):618-624. [abstract]

8. Occurrence and impact of a new leaf rust race on durum wheat in northwestern Mexico from 2001 to 2003. Singh RP, Huerta-Espino J, Pfeiffer W, Figueroa-Lopez P. In: Plant Disease, 2004, 88:703–708. [abstract]

9. First report of virulence to wheat with leaf rust resistance gene Lr19 in Mexico. Huerta-Espino J, Singh RP. In: Plant Disease, 1994, 78:640.

10. Molecular characterization of durum and common wheat recombinant lines carrying leaf rust resistance (Lr19) and yellow pigment (Y) genes from Lophopyrum ponticum. Zhang W, Soria MA, Goyal S, Dubcovsky J, Lukaszewski AJ, Kolmer J. In: Theoretical and Applied Genetics, 2005, 111: 573-582. http://dx.doi.org/10.1007/s00122-005-2048-y [abstract]

11. Trigenomic chromosomes by recombination of Thinopyrum intermedium and Th. ponticum translocations in wheat.Ayala-Navarrete L, Bariana HS, Singh RP, Gibson JM, Mechanicos AA, Larkin PJ. In: Theoretical and Applied Genetics, 2007, 116:63-75. DOI:10.1007/s00122-007-0647-5.

12. Diagnostic and co-dominant PCR markers for wheat stem rust resistance genes Sr25 and Sr26. Liu S, Yu L-X, Singh RP, Jin Y, Sorrells ME, Anderson JA. In: Theoretical and Applied Genetics, 2010, 120:691–697. DOI:10.1007/s00122-009-1186-z.

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