Importance of Reverse Breeding in the Modern Plant Breeding Era

Abstract 

Reverse breeding is a new technique that produces homozygous parental lines from the heterozygous plant, antagonistic to the conventional Plant breeding varietal development method. Reverse breeding bears huge importance in crop breeding because of its significant applications, like generation of chromosome substitution lines that make it easy to study the effects of an individual gene phenotypically, QTLs identification would be quicker, and help in the interpretation of gene interactions. Reverse breeding also generates new heterozygous germplasm that can be applied in a hybridization program. There were many barriers to breaking the hybrid into parental lines before the discovery of reverse breeding techniques; now, more stable hybrid crop performance is possible. Reverse breeding involves transformation steps, but the products are free from transgenes, similar to conventionally bred plants. Reverse breeding has high possibilities, but it is not commercialized yet. Since Reverse breeding is an emerging technique, still investigations are needed to confirm its applications and limitations. 

Introduction

Humans have always been in search of new tools and techniques that could improve their crop production considering thousands of years. They have developed many techniques to feed the world with better crop varieties (Guan et al., 2015). The intensive research on crop science makes it possible to generate novel crop breeding methods that could secure the food supply system forever. One most innovative ways that produces complementary homozygous parental lines from the hybrid plant is Reverse Breeding (RB), the concept pioneered by Rob Dirks in 2009. RB technique is possible due to the fast growth in molecular biology and crop gene sequencing.

Fig. Rob Driks

 Also read:  Genetic recombination               Speed breeding            Species

Reverse breeding is a novel approach outlined to directly produce complementary parental lines for any heterozygous plant, one of the most sought-after goals in Plant breeding. It is a de novo Plant breeding approach that produces homozygous parental lines from any hybrids. The method of RB is based on engineered meiosis whereby selected heterozygote genetic recombinations are reduced by eliminating meiotic crossing over (Dirks et al., 2009). RB could fundamentally change the future of Plant breeding as classical breeding fails to fix the unknown heterozygous genotypes. Though reverse breeding has wide applications, it has not been commercialized yet. 

Reverse Breeding, Reverse Genetics, and Forward Genetics

Reverse genetics is a procedure of understanding the function of a gene by interpreting the phenotypic effects of specific engineered gene sequences. It seeks to find the result of particular genetic sequences on crop phenotypes, while Forward genetics finds the genetic basis of a phenotype or a trait. Reverse genetics involves functional analysis of phenotype based on genotype, whereas Forward genetics studies the position of a genotype based on phenotype. In the same way, forward breeding is a kind of backcross breeding method that takes advantage of improved cultivars and genetic knowledge that might have been developed during the process of backcross breeding.

Why Reverse Breeding?

Reverse breeding has huge importance in modern Plant breeding as it generates complementary homozygous parental lines from the heterozygous plant. Since the homozygous parental lines are fundamental materials for a stable hybrid, RB is needed to maintain the hybrid stability. RB fixes parental genes and thus improves parental lines genetically to enhance the hybrid performance. RB establishes the breeding lines for uncharacterized heterozygote and also multiplies a highly heterozygous plant from homozygous parental lines (Wijnker et al., 2012).

 

Reverse breeding speeds up the breeding process and increases the number of available genetic combinations, allowing breeders to respond much quicker to the needs of farmers with better crop varieties. It facilitates the selection of superior hybrids, and large populations of plants can be generated, screened, and well-performing plants can be regenerated indefinitely without prior knowledge of their genetic constitutions.

Fig. Reverse Breeding and Traditional Breeding b

 

Mechanism of Reverse breeding

 

Reverse breeding involves mainly three steps

 1) Suppression of meiotic recombination

       2) Doubled haploid formation and

 3) Selection of complementary lines through marker-assisted selection

Suppression of meiotic recombination

Reverse breeding initiates with gamete formation. Start suppressing the crossing over during spore formation at the meiotic stage. This step is known as suppression of meiotic recombination. Suppression is done in two ways: either by suppressing the genes required for meiotic recombination or by exogenous application of chemical compounds. Suppress the genes by RNAi to completely knock down the function of DMC1 homologue to RecA, a meiosis-specific recombinase essential for the formation of crossover. Some genes responsible for meiotic recombination are Disrupted Meiotic cDNA (DMC1), Sporulation Deficient 11 (SPO11), and Rec, whose functions are knocked down by RNAi during spore formation. To proceed with the suppression of genes by RNAi, exogenous chemicals (e.g., Mirin) are also applied, which inhibit the recombination during meiosis would speeding up the application of RB (Dupree, 2008). Mirin causes G2 arrest and inhibits the phosphorylation of ATM (Ataxia Telangiectasia Mutated (ATM).

 

Double haploid formation

Use the tissue culture technique to produce double-haploid plants from the haploid. Obtain haploids from the immature pollens after suppression by RNAi. The special technique of tissue culture is referred to as anther culture and isolated microspore culture, where immature pollen grains grow to produce colonies of cells. Transfer the colonies to a medium with different plant growth regulators and sugars to induce the growth of shoots and then roots. Use colchicine to double the chromosome number of haploids into diploids.

Selection of complementary lines through marker-assisted selection

The plants formed after tissue culture techniques might be aneuploids, haploids, or double haploids. Select only double haploids to run specific primers, quit the haploids and aneuploids. The used primers confirm the complementary homozygous lines, which are compared with the F1. Conduct field-based phenotyping to confirm the results. 

Crossing of appropriate doubled haploid lines to develop superior hybrids

Cross the appropriate double-haploid complementary lines to obtain the parents. Check the level of similarity of synthetic parents with the original parents at the genetic and morphological levels.

Wijnker and Jong (2008) described that at regular meiosis, a chiasma is formed during crossing over. While reverse breeding is based on achiasmatic meiosis, a meiotic phenomenon where there is formation of no crossing over. Thus, the products obtained are homozygous at the double haploid stage. Only those DH plants are selected for reverse breeding that are complementary homologous to each other.

 

The number of DH plants required for reverse breeding might vary depending on crop species. Each crop species has a unique chromosome number, and the number of chromosomes is a key factor in deciding the number of non-recombinant double haploids required for reconstructing the original starting plant at different probability levels in various species. For example, Arabidopsis has a haploid chromosome number of 5; thus, at a 90 % level of probability, the DHs required are 13, while at 95%, 99% and 100% levels of probability, the DHs required are 14, 18, and 47. Similarly, rice, tomato, eggplant, pepper, and melon have 12 haploid chromosome numbers; thus, at a 90% probability level, the 138 DHs are required for reconstructing the original starting plants (Wijnker and Jong, 2008).

 

Comparison of the end products of reverse breeding and conventionally bred plants

Controversy arises with the use of genetic engineering techniques to improve crop plants. One strongly believes GE modified crops are harmful to human beings. Many countries have strong regulations for the entry of GE modified crops. Reverse breeding also uses GE techniques to suppress meiotic crossover during spore formation, but only those double haploids are selected that are free from transgenes. The transgene products (recruit parents without RNA construct) and aneuploids are discarded so that reverse-breeding breds are similar to conventionally bred plants.

The end products of Reverse Breeding are similar to parental lines obtained by Conventional breeding. The RNAi silencing is restricted only to meiotic crossover suppression, but there will be no change in the DNA sequences of reverse bred plants. Thus, the resulting offspring can be regarded as non-genetically modified.

Construction of heterozygous germplasm

Construct heterozygous germplasm from segregating F2 generations by applying reverse breeding methods. Each line from F2 generations is heterozygous, which could be used as a starting hybrid. For those crops where there is a lack of breeding lines, RB could accelerate the development of varieties. Superior heterozygous plants can be propagated without prior knowledge of their genetic constitution.

 

Breeding on the single chromosome level

Chromosome substitution lines differ from the others by at least one chromosome. Reverse breeding explains how chromosome substitution lines can be obtained when it is applied to an F1 hybrid of known parents. These chromosome substitution lines provide novel tools for the study of gene interactions. It also helps to study plants on a genetic basis. For example, offspring of plants in which just one chromosome is heterozygous will segregate for the traits present on that chromosome only. Development of improved breeding lines carrying introgressed traits is also possible (Kumari et al, 2018).

Reverse Breeding and Marker-Assisted Selection

Reverse breeding combined with marker-assisted selection speeds up the identification of complementary homozygous plants from the populations of DHs. Chromosome substitution lines produced through reverse breeding could be integrated with marker-assisted selection that allows the quick identification of QTLs. Gene reporting and gene tagging approaches become easier and simpler. Helps in the study of gene interaction in the heterozygous inbred families, the generation of chromosome-specific linkage maps becomes uncomplicated, and fine mapping of genes and alleles is possible. Reverse breeding with marker-aided selection also helps in studying the nature of heterotic plants.

 

Back Crossing in the CMS background

In several vegetable crops, such as cabbage and carrots, breeders make use of cytoplasmic male sterility (CMS). In these systems, the presence of male sterility presents a special challenge to RB. In these cases, gynogenesis rather than androgenesis can be used to obtain DH plants. This is perfectly compatible with RB in the sense that the chromosomes from the maintainer line can be recovered directly in the cytoplasm of the sterile line in one step.

 

Limitations of RNA-induced Reverse Breeding

•       Development of RB is limited to those crops where DH technology is common practice, eg, cucumber, onion, broccoli, sugarbeet, maize, pea, and sorghum.

•       There are some exceptions , such as soybean, cotton, lettuce, and tomato, where doubled haploid plants are rarely formed or not available at all.

•       The technique is limited to crops with a haploid chromosome number of 12 or less and in which spores cannot be regenerated into DHs.

 

Marker-Assisted Reverse Breeding and RNA-Mediated Reverse Breeding

 

MARB

•       No need for gene silencing.

•       1- 1.5 years for the development of homozygous lines.

•       No limitations in crops with < 12 haploid chromosome no.

•       Not limited to crops where DH is not possible.

•       No need for sophisticated transformation techniques like DHs.

 

RMRB

•       Need for silencing.

•       2-2.5 years for the development of homozygous lines.

•       Limitations in crops with < 12 haploid chromosome no.

•       Limited to crops where DH is not possible.

•       Young technique, hence requires more research to suppress crossover

 

Consequences for food and environmental safety

•       RNA-directed DNA methylation transmitted to the offspring will only have an effect on meiotic recombination and no genetic modification-related DNA sequences.

•       Reverse bred crops are similar to those of parental lines and F1-hybrids obtained by conventional breeding.

 

The organization works in Reverse Breeding

•       CHIC Project (www.chicproject.eu)

•       Rijk Zwaan Breeding bv, Eerste Kruisweg 9, 4793 RS Fijnaart, The Netherlands

•       NBT Platform (www.nbtplatform.org)

•       European Plant Science Organization

 

Conclusion

RB is a novel breeding approach that accelerates the breeding process. Increases the available genetic combinations. A large number of plants are generated, screened, and regenerated without prior knowledge of their genetic constitution. Thus, RB puts this century-long endeavor upside down by starting with superior hybrid selection followed by recovery of parental lines.

 

Future thrust

Mediated Reverse Breeding is a young work that requires extensive study to overcome technical problems. Additional research is required to improve the efficiency of the DH production. Emphasis should be given to the production of hybrids in crops like cucumber, onion, broccoli, and cauliflower, where seed production is problematic.

 

Keywords: Reverse breeding, Forward breeding, Reverse genetics, Forward genetics, Doubled haploids, Homozygous complementary lines

References

Dirks, R., Dun, K.V., Snoo, C.B., Berg, M.V., Cilia, L.C., Lelivelt ,Woudenberg, W.V., Wit, J., Reinink, K., Schut, J.W, Zeeuw, W., Vogelaar, A., Freymark, G., Gutteling, W., Keppel, N.M., Drongelen, P.N, Kieny, M., Ellul, P., Touraev, M., Ma, H., Jong, H.D. and Wijnker, E. 2009. Reverse breeding: a novel breeding approach based on engineered meiosis. Plant Biotechnology Journal.7, pp. 837–8457.

Guan, Yi-Xin, Wang, Bao-hua, Feng, Yan, Li, Ping 2015. Development and application of marker-assisted reverse breeding using hybrid maize germplasm. Journal of Integrative Agriculture, 14(12): 2538–2546.

Kumari, P., Nilanjaya, Singh, N.K. 2018. Reverse breeding: Accelerating innovation in Plant Breeding. Journal of Pharmacognosy and Phytochemistry,SP1: 1811-1813.

Wijnker, E. and Jong, H.D. 2008. Managing meiotic recombination in plant breeding. Trends Plant Sciences.3:640–646.

Wijnker, K.V., Snoo, C.B.D., Lelivelt, C.L.C., Joost, K.B., Naharudin, N.S., Ravi,  M., Chan, W.L., de Jong, H., Dirks, R. 2012. Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant. Nature genetics. DOI: 10.1038/ng.2203.