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MTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F1 and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC2F1, the best backcross plant was selfed to generate BC2F2s. Among them, the plants homozygous for Pup1 (n = 22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC2F6 generation. A total of five elite BC2F6 lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (> 390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.

Bacterial blight (BB) of rice caused by the pathogen Xanthomonas oryzae pv. oryzae and the insect gall midge (GM) (Orseolia oryzae) are two major constraints of rice production. The present study was carried out to improve RPHR-1005, a stable restorer line of the fine-grain-type rice hybrid DRRH-3, for BB and GM resistance through marker-assisted backcross breeding (MABB). Two major GM resistance genes, Gm4 and Gm8, and a major BB resistance gene, Xa21, were selected as target genes for transfer to RPHR-1005. Two sets of backcrosses were carried out to combine either Xa21 + Gm4 or Xa21+ Gm8 into RPHR-1005 using breeding lines in the genetic background of ISM possessing either Gm4 or Gm8 along with Xa21. Foreground selection was performed for Xa21, Gm4, Gm8, and the major fertility restorer genes Rf3 and Rf4 using gene-specific markers, while 61 polymorphic simple sequence repeat (SSR) markers were used for background selection and marker-assisted backcrossing was continued until BC2 generation. A promising homozygous backcross-derived plant at the BC2F2 generation possessing Xa21 + Gm4, and another possessing Xa21 + Gm8, were intercrossed to stack the target resistance genes. At ICF 4 (inter-crossed F4) , three promising lines possessing the three target resistance genes in a homozygous condition along with fine-grain type, complete fertility restoration, and better panicle exsertion than RPHR-1005 have been identified. Among these, a single line, # RPIC-16-65-125, showed better yield, was highly resistant to BB and GM, was of medium–slender grain type, and had complete fertility restoration along with better panicle exsertion and taller plant type than RPHR-1005. This is the first report of combining resistance against BB and GM in the genetic background of a hybrid rice parental line.

Two major nuclear genes, Rf3 and Rf4, are known to be associated with fertility restoration of wild-abortive cytoplasmic male sterility (WA-CMS) in rice. In the present study, through a comparative sequence analysis of the reported putative candidate genes, viz. PPR9-782-(M,I) and PPR762 (for Rf4) and SF21 (for Rf3), among restorer and maintainer lines of rice, we identified significant polymorphism between the two lines and developed a set of PCR-based codominant markers, which could distinguish maintainers from restorers. Among the five markers developed targeting the polymorphisms in PPR9-782-(M,I), the marker RMS-PPR9-1 was observed to show clear polymorphism between the restorer (n = 120) and maintainer lines (n = 44) analyzed. Another codominant marker, named RMS-PPR762 targeting PPR762, displayed a lower efficiency in identification of restorers and maintainers, indicating that PPR9-782-(M,I) is indeed the candidate gene for Rf4. With respect to Rf3, a codominant marker, named RMS-SF21-5 developed targeting SF21, displayed significantly lower efficiency in identification of restorers and non-restorers as compared to the Rf4-specific markers. Validation of these markers in a F2 mapping population segregating for fertility restoration indicated that Rf4 has a major influence on fertility restoration and Rf3 is a minor gene. Further, the functional marker RMS-PPR9-1 was observed to be very useful in identification of impurities in a seed lot of the popular hybrid, DRRH3. Interestingly, when RMS-PPR9-1 and RMS-SF21-5 were considered in conjunction with analysis, near-complete, marker–trait co-segregation was observed, indicating that deployment of the candidate gene-specific markers both Rf4 and Rf3, together, can be helpful in accurate identification of fertility restorer lines and can facilitate targeted transfer of the two restorer genes into elite varieties through marker-assisted breeding.

Depending on the position on the panicle, grain filling differs for spikelets on primary branches and secondary branches of upper and lower portions in rice (Oryza sativa L.). With the objective of identifying candidate genes associated with the grain-filling process across the panicle, a candidate gene (CG)-based mapping approach was attempted in an F2 population derived from the cross between Rasi, a rice variety known for its good grain filling, and IC114927, a local landrace. The grain filling (%) was characterized for 444 F2 individual plants across the panicle. Within the F2 population, thirteen plants were identified with >90% grain filling across the panicle. For 120 markers designed from 1 kb upstream and within the candidate genes reported to be involved in translocation process of sugars, 18.3% polymorphism was observed. Using a selective genotyping approach comprising 24 individuals from eight groups based on their filling and position, two associated CG markers were identified. Further analysis of the mapping population showed a CG marker based on the sucrose phosphate synthase gene on chromosome 2 to be significantly associated with filling of grains on primary branches of the upper half of the panicle and another CG marker based on the transporter gene on chromosome 11 to be associated with filling of grains on primary branches of the lower half; secondary branches of the upper half and lower half of the panicle. Both the positive alleles were contributed by the variety Rasi. With a candidate gene-based marker mapping approach, two genes involved in sugar translocation and transport were identified to be directly associated with grain filling in rice.

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pikh) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site–leucine-rich repeat (NBS–LRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant–susceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.

The major wide-compatibility gene locus S5 in rice (Oryza sativa L.) located on chromosome 6 has been recently cloned and a 136-bp deletion in the candidate gene encoding aspartyl protease has been characterized to be specific for wide-compatible varieties, while many single nucleotide polymorphisms have been identified at S5 between indica and japonica rice types. In the present study, we designed a PCR-based multiplex functional marker system targeting the deletion and the SNPs for precise determination of the allelic status at S5. By deploying the marker system, the allelic status at the S5 locus in 584 rice genotypes has been assayed. A total of 116 genotypes, including 11 cultivars, two known wide-compatible varieties, 48 IRRI germplasm lines, 12 Indian aromatic rice genotypes, 37 restorer lines and six breeding lines, have been identified to possess the 136-bp deletion specific for the neutral allele at S5. The marker system was able to clearly distinguish indica and japonica alleles from the neutral allele and has been validated in a mapping population derived from the three-way cross IR36/Dular//Akihikari, which segregated for spikelet sterility/fertility. The functional marker system targeting S5 developed in the present study will be very useful in rapid identification of wide-compatible genotypes, in predicting the success of inter-subspecific crosses and in targeted introgression of the wide-compatible allele of S5 into elite indica and japonica rice varieties.

Fragrance development in rice has been reported due to a 8-bp deletion in the exon 7 of badh2 gene located on Chromosome 8S. Multiplex markers targeting the functional InDel polymorphism was earlier reported for genotyping fragrance trait, but the marker was observed to be inconsistent and difficult to use. We have developed a simple, co-dominant, functional marker for fragrance trait, which can be resolved in an agarose gel and validated in Basmati and non-Basmati aromatic rice varieties and in a mapping population segregated for fragrance trait. The marker targets the InDel polymorphism in badh2 gene and amplifies 95 and 103 bp fragments in fragrant and non-fragrant genotypes, respectively. The newly developed marker was highly efficient in discriminating all fragrant and non-fragrant genotypes and showed perfect co-segregation with the trait of fragrance in the mapping population. We recommend the use of this simple, low-cost marker in routine genotyping for fragrance trait in large scale breeding materials and germplasm.

DRR17A is a stable wild-abortive cytoplasmic male sterile line with medium-slender grain type. DRR17A and its maintainer line DRR17B are highly susceptible to two of the major rice diseases, bacterial blight (BB) and blast. To improve DRR17B for resistance against BB and blast, we have introgressed a major dominant gene each conferring resistance against BB (Xa21) and blast (Pi54) into the maintainer line through marker-assisted backcross breeding using RP-Bio-Patho-2 (a near-isogenic line of Samba Mahsuri possessing Xa21 and Pi54) as the donor parent. PCR-based molecular markers tightly linked to Xa21 and Pi54 were used for foreground selection of the resistance plants at each backcross generation, while molecular markers tightly linked to the major fertility restorer genes, Rf3 and Rf4, were used for negative selection (i.e. selection of plants possessing non-fertility-restoring alleles at the two loci) at BC1 generation. After foreground selection for the target genes at each backcross generation, the ‘positive’ plants were screened with parental polymorphic markers for identifying backcross plants possessing maximum recovery of DRR17B genome. Marker-assisted backcrossing was continued till BC3 generation, and a single BC3F1 plant possessing the target genes with ~94 % recovery of recurrent parent genome was identified and selfed to generate BC3F2s. A total of six homozygous BC3F2 plants were identified and advanced. At BC3F5, six promising, stable, backcross-derived lines possessing high level of resistance against BB and blast, high yield, short plant stature, fine-grain type, have been identified; their maintenance ability and heterotic potential validated through test crosses and these lines are being converted to CMS lines through marker-assisted breeding.