Genome Editing in Cereals: Approaches, Applications and Challenges.

Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.

Development and validation of cross-transferable and polymorphic DNA markers for detecting alien genome introgression in Oryza sativa from Oryza brachyantha.

African wild rice Oryza brachyantha (FF), a distant relative of cultivated rice Oryza sativa (AA), carries genes for pests and disease resistance. Molecular marker assisted alien gene introgression from this wild species to its domesticated counterpart is largely impeded due to the scarce availability of cross-transferable and polymorphic molecular markers that can clearly distinguish these two species. Availability of the whole genome sequence (WGS) of both the species provides a unique opportunity to develop markers, which are cross-transferable. We observed poor cross-transferability (~0.75 %) of O. sativa specific sequence tagged microsatellite (STMS) markers to O. brachyantha. By utilizing the genome sequence information, we developed a set of 45 low cost PCR based co-dominant polymorphic markers (STS and CAPS). These markers were found cross-transferrable (84.78 %) between the two species and could distinguish them from each other and thus allowed tracing alien genome introgression. Finally, we validated a Monosomic Alien Addition Line (MAAL) carrying chromosome 1 of O. brachyantha in O. sativa background using these markers, as a proof of concept. Hence, in this study, we have identified a set molecular marker (comprising of STMS, STS and CAPS) that are capable of detecting alien genome introgression from O. brachyantha to O. sativa.

The potentiality of rice microsatellite markers in assessment of cross-species transferability and genetic diversity of rice and its wild relatives.

The main aim of this study is to assess the potentiality of SSR markers for the identification of the cross-species transferability frequency in a large set of the diverse genome types of wild relative rice along with cultivated rice. Here, we used 18 different rice genotypes representing nine different genome types with 70 SSR markers to investigate the potentiality of cross-species transferability rate. The overall cross-species transferability of SSR markers across the 18 rice genotypes ranged from 38.9% (RM280 and RM447) to 100% (RM490, RM318, RM279, RM18877 and RM20033, RM19303) with an average of 76.58%. Also, cross-species transferability across chromosome ranged from 54.4% (chromosome 4) to 86.5% (chromosome 2) with an average of 74.35%. The polymorphism information content of the markers varied from 0.198 (RM263) to 0.868 (RM510) with a mean of 0.549 ± 0.153, showing high discriminatory power. The highest rate of cross-transferability was observed in O. rufipogon (97%), The highest rate of cross-species transferability was in O. rufipogon (97.00%), followed by O. glaberrima (94.20%), O. nivara (92.80%), Swarna (92.80%), O. longistaminata (91.40%), O. eichingeri (90%), O. barthii (88.50%), O. alta (82.80%), O. australiensis (77.10%), O. grandiglumis (74.20%), O. officinalis (74.20%), Zizania latifolia (70.00%), O. latifolia (68.50%), O. brachyantha (62.80%), Leersia perrieri (57.10%) and O. ridleyi (41.40%) with least in O. coarctata (28.50%). A total of 341 alleles from 70 loci were detected with the number of alleles per locus ranged from 2 to 12. Based on dendrogram analysis, the AA genome groups was separated as distinct group from the rest of the genome types. Similarly, principal coordinate analysis and structure analysis clearly separated the AA genome type from the rest of the genome types. Through the analysis of molecular variance, more variance (51%) was observed among the individual, whereas less (14%) was observed among the population. Thus, our findings may offer a valuable resource for studying the genetic diversity and relationship to facilitate the understanding of the complex mechanism of the origin and evolutionary processes of different Oryza species and wild relative rice.

Phenotypic and Marker-Assisted Genetic Enhancement of Parental Lines of Rajalaxmi, an Elite Rice Hybrid.

The cytoplasmic male sterile line system comprising CRMS 32A and its maintainer line CRMS 32B is a popular choice for the development of new hybrids in India as CRMS 32A, having Kalinga 1 cytoplasm (other than WA), is a viable alternative to WA cytoplasm. However, both lines are susceptible to bacterial blight (BB), a major disease on rice. As enhancement of host plant resistance is the most effective and economical strategy to control this disease, four resistance genes (Xa4, xa5, xa13, and Xa21) were transferred from a BB pyramid line of IR64, into the A and B lines using a marker-assisted backcrossing (MAB) breeding strategy. During the transfer of genes into CRMS 32B, foreground selection was applied using markers associated with the genes, and plants having resistance alleles of the donor, are selected. Selection for morphological and quality traits was practiced to select plants similar to the recurrent parent. The four gene and three gene pyramid lines exhibited high levels of resistance against the BB pathogen when challenged with eight virulent isolates. Using genome wide based SSR markers for background selection, pyramids having >95% of the recurrent parent genome were identified. With CRMS 32B gene pyramid as donor, the four resistance genes were transferred into the A line through repeated backcrosses and the A line pyramids also exhibited high level of resistance against BB. Through a combination of selection at phenotypic and molecular levels, four BB resistance genes were successfully introduced into two parental lines (CRMS 32 B and A) of Rajalaxmi, an elite popular hybrid. The pyramided B lines did exhibit high levels of resistance against BB. Selection for morphological and quality traits and background selection hastened the recovery of the recurrent parent genome in the recombinants. Through repeated backcrosses, all the four resistance genes were transferred to CRMS 32A and test crosses suggest that the maintenance ability of the improved CRMS 32B lines is intact. These improved maintainer and CMS lines can directly be used in hybrid rice breeding and the new hybrids can play an important role in sustainable rice production in India.