A comprehensive understanding of CMS/Rf system enabled by modern omics tools and technologies considerably improves our ability to harness hybrid technology for enhancing the productivity of field crops.
 Harnessing hybrid vigor or heterosis is a promising approach to tackle the current challenge of sustaining enhanced yield gains of field crops. In the context, cytoplasmic male sterility (CMS) owing to its heritable nature to manifest non-functional male gametophyte remains a cost-effective system to promote efficient hybrid seed production. The phenomenon of CMS stems from a complex interplay between maternally-inherited (mitochondrion) and bi-parental (nucleus) genomic elements. In recent years, attempts aimed to comprehend the sterility-inducing factors (orfs) and corresponding fertility determinants (Rf) in plants have greatly increased our access to candidate genomic segments and the cloned genes. To this end, novel insights obtained by applying state-of-the-art omics platforms have substantially enriched our understanding of cytoplasmic-nuclear communication. Concomitantly, molecular tools including DNA markers have been implicated in crop hybrid breeding in order to greatly expedite the progress. Here, we review the status of diverse sterility-inducing cytoplasms and associated Rf factors reported across different field crops along with exploring opportunities for integrating modern omics tools with CMS-based hybrid breeding.

Cytoplasmic male sterility (CMS) in hybrid breeding in field crops

Plants and microbes are co-evolved and interact with each other in nature. Plant-associated microbes, often referred to as plant microbiota, are an integral part of plant life. Depending on the health effects on hosts, plant–microbe (PM) interactions are either beneficial or harmful. The role of microbiota in plant growth promotion (PGP) and protection against various stresses is well known. Recently, our knowledge of community composition of plant microbiome and significant driving factors have significantly improved. So, the use of plant microbiome is a reliable approach for a next green revolution and to meet the global food demand in sustainable and eco-friendly agriculture. An application of the multifaceted PM interactions needs the use of novel tools to know critical genetic and molecular aspects. Recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing (GE) tools are of great interest to explore PM interactions. A systematic understanding of the PM interactions will enable the application of GE tools to enhance the capacity of microbes or plants for agronomic trait improvement. This review focuses on applying GE techniques in plants or associated microbiota for discovering the fundamentals of the PM interactions, disease resistance, PGP activity, and future implications in agriculture.

Exploration of Plant-Microbe Interactions for Sustainable Agriculture in CRISPR Era.

Future Foods: How Modern Science Is Transforming the Way We Eat

Applications of CRISPR/Cas to Improve Crop Disease Resistance: Beyond Inactivation of Susceptibility Factors

Black rot caused by Xanthomonas campestris pv. campestris (Pam.) Dowson is the most destructive disease of cauliflower causing huge loss to the farmers throughout the world. Since there are limited sources of resistance to black rot in B. oleracea (C genome Brassica), exploration of A and B genomes of Brassica was planned as these were thought to be potential reservoirs of black rot resistance gene(s). In our search for new gene(s) for black rot resistance, F2 mapping population was developed in Brassica carinata (BBCC) by crossing NPC-17, a susceptible genotype with NPC-9, a resistant genotype. Out of 364 Intron length polymorphic markers and microsatellite primers used in this study, 41 distinguished the parental lines. However, resistant and susceptible bulks could be distinguished by three markers At1g70610, SSR Na14-G02 and At1g71865 which were used for genotyping of F2 mapping population. These markers were placed along the resistance gene, according to order, covering a distance of 36.30 cM. Intron length polymorphic markers At1g70610 and At1g71865 were found to be linked to black rot resistance locus (Xca1bc) at 6.2 and 12.8 cM distance, respectively. This is the first report of identification of markers linked to Xca1bc locus in Brassica carinata on B-7 linkage group. Intron length polymorphic markers provided a novel and attractive option for marker assisted selection due to high cross transferability and cost effectiveness for marker assisted alien gene introgression into cauliflower.

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Genetics and Molecular Mapping of Black Rot Resistance Locus Xca1bc on Chromosome B-7 in Ethiopian Mustard (Brassica carinata A. Braun)

Improving plant-resistance to insect-pests and pathogens: The new opportunities through targeted genome editing.

Oilseed crops play an important role in the agricultural economy. Apart from being an integral component of human diet and industrial applications, they are also gaining importance as replacement to fossil fuels for meeting the energy needs. The last two decades have been marked by several important events in genetic engineering and identification of gene targets for enhancing seed oil content in oilseed crops, and will aid the successful development of new generation high yielding oil crops. Specifically, genetic engineering has shown real breakthrough in enhancing oil content in oilseed rape, camelina, soybean and maize. Moreover, ongoing research efforts to decipher the possibilities of genetic modifications of key regulators of oil accumulation along with physiological and biochemical studies to understand lipid biosynthesis will set a platform to produce transgenic oilseed crops with enhanced oil content. In this review, we briefly describe different genetic engineering approaches explored by different researchers for enhancing oil content. Further, we discuss a few promising and potential approaches and challenges for engineering oil content in oilseed crops.

Genetic engineering approaches to enhance oil content in oilseed crops

Cytoplasmic male sterility (CMS) system based on Moricandia arvensis cytoplasm is being used to breed hybrid Brassica juncea varieties. The male fertility restorer gene for this CMS was introgressed into B. juncea from M. arvensis and has been tagged with a dominant SCAR marker. The present study was undertaken to fine map the Rf locus and to assign it to specific B. juncea linkage group. We used information obtained from genome walking, anther transcriptome of the male sterile and the fertility restorer lines and synteny relationship with B. rapa for designing markers for targeted fine mapping. Two BC1F1 populations derived from the cross between CMS and fertility restorer lines were used for mapping. Sequence information obtained through genome walking from the SCAR3 indicated that the Rf gene is located in a genomic region having synteny with B. rapa chromosome A09. Screening of more than 550 SSR markers designed based on B. rapa A09 sequence information identified 12 markers flanking the Rf gene and confirmed its location on B. juncea A09 linkage group. Two (one EST-SSR and one STS) out of 67 markers designed based on differential gene expression between anther transcriptomes were found to be informative and were linked to the Rf gene. The markers were validated in a second BC1F1 population. The closest flanking markers BjESSR01 and BjEST01 were at 0.6 and 1.4 cM from the Rf locus.

Molecular mapping of fertility restorer gene of an alloplasmic CMS system in Brassica juncea containing Moricandia arvensis cytoplasm.

Improving oil yield of Indian mustard (Brassica juncea) is exigent as it is a major oilseed crop of Indian subcontinent, which has severe shortage of vegetable oil production in the world. Some of the regulators of seed development have been shown to improve oil yield in Arabidopsis. Arabidopsis KLUH (AtKLUH), a maternal regulator of seed size, has been shown to control seed oil content. In this study, we identified three homologs of AtKLUH in B. juncea, BjKLUH1, BjKLUH2-1 and BjKLUH2-2. We observed that BjKLUH1 differentially expresses in developing seeds in B. juncea accessions with varying seed size and oil content. Further, analyses for seed oil content in B. juncea transgenics carrying AtKLUH demonstrated an increase in seed oil up to 8.3% compared to wild-type plants. The results of this study suggest that KLUH may have a role in seed development and is a good candidate for engineering seed oil accumulation in B. juncea.

Deepak Singh Bisht

Papers

Improving plant-resistance to insect-pests and pathogens: The new opportunities through targeted genome editing.

Genetic engineering approaches to enhance oil content in oilseed crops

Molecular mapping of fertility restorer gene of an alloplasmic CMS system in Brassica juncea containing Moricandia arvensis cytoplasm.

Isolation and expression analyses of KLUH gene in developing seeds and enhanced seed oil in KLUH overexpressing Brassica juncea transgenics