Role of Functional Markers in Plant Breeding

Author: Dr. SV Amitha Mithra and Dr. Amolkumar U. Solanke
NRC on Plant Biotechnology, LBS Building, IARI, Pusa Campus, New Delhi

Molecular markers are now increasingly used in crop breeding as selection tools in all major crops like rice, wheat, maize, sugarcane, cotton, pigeonpea and in other cereal, oilseed and vegetable crops. There are a variety of markers known in plants, for example, Restriction Fragment Length Polymorphisms (RFLP), Random Amplified Polymorphic DNA (RAPDs), Simple Sequence Repeats (SSRs), Amplified Fragment Length Polymorphism (AFLP) and Cleaved Amplified Polymorphic Sequences (CAPS) and Single Nucleotide Polymorphisms (SNPs). Generally they are classified on the basis of their technical and genetic aspects - how do they generate polymorphism and how much polymorphism do they generate, how to handle them, cost of genotyping, whether they are bi or multiallelic, come from single or multiple loci, codominant or dominant etc. For plant breeding purpose, markers which are easy to genotype, belong to single locus, show codominance and tightly linked with the trait of interest are preferred. It is now possible to generate thousands of random DNA markers which are phenotypically neutral in any plant species which can be used in genetic diversity analysis, mapping, F1 purity testing, transgene testing, selection etc. However, when such markers are used for marker assisted selection in plant breeding they may have some limitations owing to genetic recombination giving rise to false positives (Frisch et al. 1999).

Functional markers - Definition and development:
Functional markers in contrast are developed from polymorphic sites within genes that causally affect target trait variation i.e based on functional characterization of the polymorphisms (Anderson and Lubberstedt 2003). Hence they are more meaningful in plant breeding. It is comparatively easier to develop functional markers in plants such as rice, tomato, and Medicago where either compete or nearly complete genome sequence information is available than in others in which little or no genomic information is available. Owing to the advent of Next Generation Sequencing (NGS) technologies, in many plants structural and functional genomic information is being generated. Establishing the identity and function of the gene using both forward and reverse genetic tools (Map based cloning, Insertional mutagenesis using transposons or T-DNA, TILLING) and complementation by cloning or RNAi and comparative genomics is a pre-requisite for developing functional markers. Once candidate genes are known for the trait of interest, allelic polymorphism across contrasting sets of genotypes can also be mined and the functional motif identified through association analysis. However it is recommended that the association be verified using a segregating population.

Once known and established, functional markers are the most reliable ones to use in MAS circumventing the recombination issue thereby getting rid of false positives. They also do not require additional calibration across mapping populations, which random genetic markers demand, as long as the parents have the required polymorphism. Thus they are highly useful for most efficient fixation of alleles in populations. They can be directly used in natural as well as breeding populations and thus an invaluable tool for targeted search in Germplasm collections.
Since crop improvement is expected to go designer way in the future, combining useful functional markers in a controlled cross for varietal development is expected to become an integral part of breeding. Genetic diversity analysis is another important exercise plant breeders take up in order to identify appropriate parents for making crosses to select transgressive recombinants. However what constitutes the genetic diversity has always been a subject of intense debate. The only consensus that could be arrived here is that whatever is important for a genotype to fare well, satisfying growers and consumers alike in terms of stability, yield and cooking and nutritional quality should constitute genetic diversity. Given this consensus, the most accurate and useful description of diversity in plant breeding can be provided by functional markers.

The most important limitation of functional markers is their development which is time and resource consuming and laborious. However, with more countries and researchers involved in structural and functional genomic research and with varied techniques being used for the same, more and more functional markers will be available in all important crop species. There is also a limitation to how many markers can be bred for simultaneously in a cross with a reasonable population size. This can be confounded by having more number of functional motifs within a gene, for example GS3 in rice which has four functional domains one of the domains being positive regulator and the other three being negative regulators of grain size (Fan et al. 2006; Takano-Kai et al. 2009) . Another important drawback is that once more and more such markers are developed, their interaction effect would be very important in order to put them together in a single genotype. Decoding such interaction effects though would be very laborious, will reward us with explanations for positive and negative correlations in molecular terms.

Anderson J and Lubberstedt T (2003) Functional Markers in Plants. Trends Plant Sci. 8(11): 554-560.

Fan CC, Xing YZ, Mao HL, Lu TT, Han B, Xu CG, Li XH, Zhang QF (2006) GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor. Appl. Genet. 112, 1164-1171.

Frisch, M, Bohn M and Melchinger TE. (1999) Minimum sample size and optimal positioning of flanking markers in marker-assisted backcrossing for transfer of a target gene. Crop Sci. 39, 967-975.

Takano-Kai N, Jiang H, Kubo T, Sweeney M, Matsumoto T, Kanamori H, Padhukasahasram B, Bustamante C, Yoshimura A, Doi K, McCouch S (2009) Evolutionary history of GS3, a gene conferring grain length in rice. Genetics 182:1323-1334.

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