Marker-Assisted Improvement of Wheat for Biotic Stress Tolerance
Niharika Mallick, Scientist (Plant Breeding) at ICAR-IARI, New Delhi, Plant Breeder
Shailendra K Jha, Scientist (Plant Breeding) at ICAR-IARI, New Delhi, Plant Breeder
Niranjana M, Scientist (Plant Breeding) at ICAR-IARI, New Delhi, Plant Breeder
Lekshmy S, Scientist (Plant Physiology) at ICAR-IARI, New Delhi, Plant Physiologist working in the area of nitrogen use efficiency of crop plants.
Kumar Durgesh, Scientist (Plant Breeding) at ICAR-IARI, New Delhi, Plant Breeder

Wheat (Triticum aestivum L.) is the most important winter cereal crop of India, which is grown during November to April. Two main factors which destabilize wheat yields are biotic and abiotic stresses. Abiotic stresses are those associated with the environment whereas, biotic stresses are induced by other living organisms such as pathogens, insects and weeds. Wheat (Triticum aestivum L.) suffers from several diseases like rusts, alternaria leaf blight, loose smut, Karnal bunt and powdery mildew. Among these diseases rusts have great economic importance since the losses caused by these diseases have been widespread. Stem rust (Black rust) is caused by Puccinia graminis f. sp. tritici Eriks & Henn, leaf rust (Brown rust) by Puccinia triticina Eriks. (Syn: Puccinia recondita) and stripe rust (Yellow rust) is incited by Puccinia striiformis Westend. Although, rust can be controlled by chemical fungicides but it is inefficient, expensive and cannot be adopted by all the farmers. Therefore, the development of genetic resistance to rusts in wheat is advocated because it is economical, effective and environment friendly approach to prevent the damage caused by rust epidemics.

Two different approaches are normally followed to transfer two or more effective resistance genes into an adapted cultivar. In the first approach, targeted genes are assembled first in one line followed by backcrossing while in the second approach, individual target genes are transferred first to develop backcross lines in the genetic background of recipient variety by repeated backcrossing, followed by intercrossing of these backcross lines (NILs) to assemble the target genes. Ishii et al. (2008) demonstrated that the later approach is much more effective.

Development of near isogenic lines (NILs) is the first step to incorporate multiple rust resistance genes in the background of a leading variety which is susceptible to all the three rusts. Developing near-isogenic lines through using marker assisted selection takes only 2-3 years. Also combining two or more resistance genes using classical host-parasite infection methods is highly time consuming and needs specific virulent pathotypes that are often not available or too risky to use.

Molecular markers linked closely with rust resistance genes have been identified and are being used by breeders for indirect selection of rust resistance in breeding populations. Also a large number of microsatellite markers distributed across the 21 chromosomes of wheat are available (Somers et al., 2004) to select the background of the recurrent variety through the help of marker assisted background analyses. Thus marker assisted selection provides a useful tool to accelerate the process of combining two or more targeted genes in the genetic background of susceptible but otherwise superior variety.

Marker assisted backcross breeding (MAS): Marker assisted backcrossing was extensively used to incorporate rust resistance genes in the background of high yielding but susceptible wheat varieties. Steps in marker assisted backcross breeding includes, (i) Parental polymorphism survey, (ii) Marker assisted foreground selection and (iii) Marker assisted background selection.

Parental polymorphism survey: DNA of parents, donor of rust resistance gene and recipient variety has to be assessed for identifying polymorphic regions using SSR markers covering entire 21 chromosomes of wheat. AS SSR markers are co-dominant markers polymorphic markers will produce different product sizes in both the parents. These polymorphic markers used in background selection.

Foreground selection: Foreground selection has to be done in each backcross generation using validated molecular markers linked to the rust resistance genes for identifying plants carrying targeted genes.

Background selection: Polymorphic markers identified in parental polymorphism survey coving all the chromosome arms of wheat will be identified and used in background analysis for recovering recurrent parent genome.

Steps in marker assisted backcross breeding:

  • Crossing of donors (With resistance genes) and recipient (High yielding but susceptible) parents to generate F1 seeds.
  • Survey of parental polymorphism between donor and recipient genotype using SSR markers
  • Raising of F1s and backcrossing with recipient parent to get BC1F1 seeds.
  • Raising of BC1F1 generations of respective crosses
  • Foreground selection for resistance genes in the BC1F 1 generations
  • Background selection in gene positive plants for identifying plants with maximum similarity with recurrent parent using polymorphic markers.
  • Crossing of selected plants with recurrent parent to get BC 2F1 seeds
  • Raising BC2F1 generation
  • Foreground selection for resistance genes in BC 2F1 generations
  • Background selection in gene positive plants for identifying plants with maximum similarity with recipient parent
  • Selfing of plants with targeted resistance genes and with RPG (Recurrent Parent Genome) of more than 95% to get BC2F 2 seeds.
  • Raising BC2F2 plants and conducting foreground and background selection.
  • Intercrossing homozygous gene positive plants with different resistance genes to combine more than one gene in a common background. This will produce NILF1 seeds.
  • Raising NILF1 generation and selfing to get NILF 2 seeds.
  • Raising NILF2 generation and carrying out foreground and background selection to identify plants with two gene combinations in homozygous state plus maximum RPG.
  • Similarly, more than two genes can be combined in the common background by carryout simultaneous backcrossing for different genes and finally combining them together.

Important points:

  • In BC1F1 generations all the polymorphic markers will be used for background selection. BC1F 1 plants with recurrent parent allele will get a score of “1”, plants with both the alleles (recurrent parent allele and donor parent allele) (or) heterozygous will get a score “0.5”. Typically, there should be no donor allele in BC 1F1 or any other backcross progeny but if you get any allele give it a score “0”. Per cent genomic similarity was calculated as number of homozygous loci corresponding to recurrent parent allele+ half the number of heterozygous loci divided by the total number of polymorphic SSR markers used.
  • In BC2F1 generation use only those polymorphic markers which are still with score “0.5” and “0’. In this way number of polymorphic markers to be used in background selection in each backcross generation are reduced.
Markers for different rust resistance genes were identified for their use in marker assisted selection. Some important genes for different rust resistance and their linked molecular markers were given in the following Table.

Gene Location Marker type Reference Origin
Leaf rust resistance genes
Lr9 6BL SCAR (SCS5550) Gupta et al., 2005 Ae. Umbellulata
Lr19 7DL STS(GbF/GbR) Xwmc221 Prins et al., 2001 Gupta et al., 2006 Th. Elongatum
Lr24 3DL SCAR (SCS1302607) Prabhu et al., 2004 Th. Elongatum
Lr28 4AL SCAR (SCS421570) Cherkuri et al.,2005 Ae. Speltoides
Lr34 7DS STS (csLV34) Lagudah et al., 2006 Thatcher
Lr46 1B STS (XSTS1BL2/XSTS1BL9) Mateos-Hernandez et al., 2006 T. aestivum
Lr52 5BS SSR (Xgwm443)* Hiebert et al., 2005 Thatcher
Lr67 4D SSR (Xcfd71, Xcfd23) Hiebert et al., 2010 Thatcher
Lr68 7BL CAPS marker cs7BLNLRR or the dominant STS marker csGS Herrera-Foessel et al., 2012 Frontana
Stem rust Resistance genes
Sr 2 3BS SSR (Xgwm533) CAPS (csSr2) Spielmeyer et al., 2003 McNeil et al., 2008 Yaroslav emmer wheat
Sr 24 3DL SSR (BARC71) AFLPs (Sr24#12 and Sr24#50) Mago et al., 2005 Thinopyron ponticum
Sr25 7DL SCAR (Gb) SSR (BF145935) Yu et al., 2010 Liu et al., 2010 Thinopyrum ponticum
Sr26 6AL SCAR (Sr26#43, BE518379) (Mago et al., 2005) (Liu et al., 2010) Thinopyrum ponticum
Sr36 2BS SSR (Xstm773-2 (a Xstm773 derivative), Xwmc477 and Xgwm319) Tsilo et al., 2008 Triticum timopheevi
Stripe rust resistance genes
Yr5 2BL STS/CAPS Chen et al.,2003 T. spelta album
Yr9 1BL-1RS Microsatelite Shi et al.,2001 Secale cereale
Yr10 1BS SSR (Xpsp3000) Wang-Lanfen et al.,2002 Triticum spelta
Yr15 1BS RFLP Microsatellite/RAPD SSR Sun et al.,1997 Chague et al.,1999 Peng et al.,2000 T. dicoccoides


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