Plant materials
In this study, we used the herbicide tolerant rice mutant Robin as donor and a mega-Basmati rice variety, PB 1121 as the recurrent parent (RP) for the transfer of HT trait. The mutant Robin was developed from an upland rice variety Nagina 22 (N22) through induced mutagenesis using ethyl methane sulfonate (EMS) (Shoba et al. 2017). PB 1121, developed by our group at ICAR-Indian Agricultural Research Institute (IARI), New Delhi, is a Basmati rice variety par excellence in grain and cooking quality with an exceptionally high cooked kernel length (22 mm) and elongation ratio of > 2.5. This variety is currently grown on approximately 1.2 million ha area (60 percent of the Basmati area in India) and contributes annually 3.41 billion USD to foreign exchange earnings through the export of Basmati rice (Singh et al. 2018).
MABB Strategy For Development Of HT Lines
PB 1121 and mutant Robin were first evaluated for their tolerance to the herbicide “Imazethapyr” at the dose of 2.5 ml/litre. The F1 seeds were produced by crossing PB 1121 as female and Robin as male and the hybridity of the F1 plants was tested using the SSR marker RM6844 linked with AHAS gene, The F1s were designated as Pusa 1979 and single F1 was backcrossed with PB 1121 to generate BC1F1 seeds. One BC1F1 plant with the highest recovery of RPG and recurrent parent phenome (RPP) was backcrossed to PB 1121 to generate the BC2F1 seeds. A similar strategy was employed till BC4F1 generation, wherein in each of the generations, the plant heterozygous for the mutant AHAS allele along with maximum recovery for RPG and RPP were identified. The superior BC4F1 plants were advanced to BC4F2 generation and plants homozygous for the mutant AHAS gene were identified. Further, the selected BC4F2 plants were advanced to BC4F4 generation via pedigree-based phenotypic selection (Fig. 1).
Molecular Analysis
Total genomic DNA from leaf tissues was extracted using Cetyl Trimethyl Ammonium Bromide (CTAB) method (Doyle 1991). The PCR reaction of a total 10 µl volume was set up which included, 25–30 ng of template DNA, 5 pmol each of the forward and reverse primers and 2X red dye PCR master mix (Genei Laboratories Pvt. Ltd., Bangalore). The program of PCR amplification consisted of initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 40 s, annealing at 58 °C for 40 s, extension at 72 °C for1 min; and a final extension at 72 °C for 10 min. The amplified products were resolved on 3.5% Metaphor™ Agarose gel mixed with 0.1 mg/ml ethidium bromide. The amplicons were visualized on ultraviolet trans-illuminator (Gel Doc™ + Imager, Bio- Rad Laboratories Inc., U.S.A).
Foreground And Background Selection
Foreground selection for the identification of plants carrying the mutant AHAS allele was carried out using SSR marker RM6844 linked with AHAS gene at a distance of 1.2 cM in chromosome 2(Shoba et al. 2017), primer details are given in Table S1. For background selection, the primer sequence of genome-wide SSR markers was fetched from the rice marker database of Gramene (http://www.gramene.org). A total of 856 SSR markers were used to identify polymorphic markers between the parents PB 1121 and Robin, for use in background selection (Table S2). During the background selection in backcross generations, the homozygous and heterozygous plants for PB 1121 allele at each marker loci were counted separately. A reductionist strategy was considered for this, markers that were found to be homozygous for PB 1121 allele in a given generation were not included in subsequent generations for background selection. RPG recovery was estimated using the formula:
The RPG recovery was visualized using Graphical GenoTypes (GGT) Version 2.0 software (Van Berloo 1999). Based on molecular marker analysis, similarity of NILs to PB 1121 was computed using Jaccard’s coefficient of similarity for generating a dendrogram following an unweighted pair group method with arithmetic mean (UPGMA). Further, for cluster analysis, NTSYS-PC-2.02f (Rohlf 1998) was used.
Molecular Screening For Aroma Gene
The NILs and the parents were also screened for the presence of badh2 gene, responsible for aroma in Basmati rice, using gene based marker ‘nksbadh2’ (Amarawathi et al. 2008). Primer details are given in Table S3.
Screening For Imazethapyr Tolerance
Twelve HT-NILs of PB 1121 along with PB 1121 and Robin were planted in a randomized complete block design (RCBD) with three replications and sprayed with herbicide, Imazethapyr (commercially available as Pursuit™) @ concentration of 2.5 ml/liter, after 10 days of transplanting. Another set of same experimental material was grown side by side and used as unsprayed control with manual weeding. Visual observation of herbicide tolerance of the HT-NILs was made on 15 days after spray as per the standard protocol in rice (Shoba et al. 2017).
Evaluation Of Agro-morphological, Grain And Cooking Quality Parameters
Agro-morphological evaluation of the HT-NILs and parents was done in RCBD with three replications following recommended agronomic practices. Data on the agro-morphological traits viz, days to 50% flowering (DFF), plant height (PH), number of productive tillers per plant (NPT), panicle length (PL), spikelet fertility (SF %), thousand grain weight (TGW) were recorded on five plants taken at random from the two middle rows of each plot under both sprayed and unsprayed conditions. The plot yield was recorded in kilogram/hectare (kg/ha) from each replication. The data on grain and cooking quality traits such as hulling percentage (HUL%), milling percentage (MIL%), head rice recovery (HRR%), kernel length before cooking (KLBC), kernel breadth before cooking (KBBC), kernel length after cooking (KLAC), kernel breadth after cooking (KBAC), kernel elongation ratio (ER), alkali spreading value (ASV) (Little et al.,1958) and aroma (Sood and Siddiq 1978) was recorded.
The statistical analysis of agro-morphological data was carried out using CropStat 7.2 (IRRI CropStat, 2014). Student’s t-test was performed for statistical significance differences for yield between unsprayed and herbicide sprayed condition at P ≤ 0.05.