Ranjith K. Ellur

Molecular breeding for the development of multiple disease resistance in Basmati rice

Marker assisted backcross breeding for combining three resistance genes (xa13 and Xa21 for Bacterial Blight, Pi54 for blast) and a major QTL (qSBR11-1 for resistance to Sheath blight) in Basmati rice.

Improvement of Basmati rice varieties for resistance to blast and bacterial blight diseases using marker assisted backcross breeding.

Marker assisted backcross breeding was employed to incorporate the blast resistance genes, Pi2 and Pi54 and bacterial blight (BB) resistance genes xa13 and Xa21 into the genetic background of Pusa Basmati 1121 (PB1121) and Pusa Basmati 6. Foreground selection for target gene(s) was followed by arduous phenotypic and background selection which fast-tracked the recovery of recurrent parent genome (RPG) to an extent of 95.8% in one of the near-isogenic lines (NILs) namely, Pusa 1728-23-33-31-56, which also showed high degree of resemblance to recurrent parent, PB6 in phenotype. The phenotypic selection prior to background selection provided an additional opportunity for identifying the novel recombinants viz., Pusa 1884-9-12-14 and Pusa 1884-3-9-175, superior to parental lines in terms of early maturity, higher yield and improved quality parameters. There was no significant difference between the RPG recovery estimated based on SSR or SNP markers, however, the panel of SNPs markers was considered as the better choice for background selection as it provided better genome coverage and included SNPs in the genic regions. Multi-location evaluation of NILs depicted their stable and high mean performance in comparison to the respective recurrent parents. The Pi2+Pi54 carrying NILs were effective in combating a pan-India panel of Magnaporthe oryzae isolates with high level of field resistance in northern, eastern and southern parts of India. Alongside, the PB1121-NILs and PB6-NILs carrying BB resistance genes xa13+Xa21 were resistant against Xanthomonas oryzae pv. oryzae races of north-western, southern and eastern parts of the country. Three of NILs developed in this study, have been promoted to final stage of testing during the ​Kharif 2015 in the Indian National Basmati Trial.

Marker-aided Incorporation of Xa38 , a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21

Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India’s forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region.

Prediction of hybrid performance based on the genetic distance of parental lines in two-line rice (Oryza sativa L.) hybrids

A total of six TGMS (thermosensitive genic male sterile lines) and nine pollinator lines were subjected to molecular characterization using 48 genome-wide SSR (simple sequence repeat) markers. Cluster analysis revealed clear differentiation among the TGMS lines according to their source of origin. The SSR-based genetic distance between the hybrids of the parental lines ranged from 0.36 to 0.79 suggesting a high degree of genetic divergence. Among a set of 54 hybrids generated using parental lines, 32 showed better parent heterosis (+21.7%) while 19 showed mid-parent heterosis (+15.0%). For the trait yield per plant genetic distance (GD) was negatively correlated with F1 performance (r = −0.202), mid-parent heterosis (r = −0.325*; P < 0.05), and better parent heterosis (r = −0.261), while it was positively correlated with specific combining ability (r = 0.042). Based on the grouped genetic distance (GGD), the hybrid combinations were divided into four groups. The GGD showed linear correlation with hybrid performance within the group (GGD = 40–50: r = −0.07; GGD = 70–80: r = 0.32). This information can be utilized in the development of higher yielding, two-line rice hybrids through selection of intermediately diverse parental lines using GGD.

Determination of genetic relationships among elite thermosensitive genic male sterile lines (TGMS) of rice (Oryza sativa L.) employing morphological and simple sequence repeat (SSR) markers

A set of morphological traits and SSR markers were used to determine the genetic relationship among 12 elite thermosensitive genic male sterile (TGMS) lines developed at three different research institutions of India. Agro-morphological data recorded on 20 morphological traits revealed a wide base of genetic variation and a set of four morphological traits could distinguish most of the TGMS lines. Analysis with 30 SSR markers (20 EST-SSRs and 10 genomic SSRs) revealed 27 markers to be polymorphic, amplifying a total of 83 alleles. Each SSR marker amplified 2–6 alleles with an average of 2.76 alleles per marker and a PIC value varying from 0.54 to 0.96. Cluster analysis based on SSR and morphological data clearly differentiated the lines according to their source of origin. Correlation analysis between morphological and molecular data revealed a very poor association (r = 0.06), which could be attributed to selection pressure, genetic drift, sampling error and unknown relationship among related lines. The SSR markers discriminated the genotypes distinctly and quantified the genetic diversity precisely among the TGMS lines. Data on the yield per plant indicated that the genotypes grouping under a similar cluster showed same heterotic behaviour as compared to the genotypes from different clusters when crossed to similar pollinators.

Marker Aided Incorporation of Saltol, a Major QTL Associated with Seedling Stage Salt Tolerance, into Oryza sativa ‘Pusa Basmati 1121’

Pusa Basmati 1121 (PB1121), an elite Basmati rice cultivar is vulnerable to salinity at seedling stage. A study was undertaken to impart seedling-stage salt tolerance into PB1121 by transferring a quantitative trait locus (QTL), Saltol, using FL478 as donor, through marker assisted backcrossing. Sequence tagged microsatellite site (STMS) marker RM 3412, tightly linked to Saltol was used for foreground selection. Background recovery was estimated using 90 genome-wide STMS markers. Systematic phenotypic selection helped in accelerated recovery of recurrent parent phenome (RPP). A set of 51 BC3F2 lines homozygous for Saltol were advanced to develop four improved near isogenic lines (NILs) of PB1121 with seedling stage salt tolerance. The background genome recovery in the NILs ranged from 93.3 to 99.4%. The improved NILs were either similar or better than the recurrent parent PB1121 for yield, grain and cooking quality and duration. Biochemical analyses revealed significant variation in shoot and root Na+ and K+ concentrations. Correlation between shoot and root Na+ concentration was stronger than that between root and shoot K+ concentration. The effect of QTL integration into the NILs was studied through expression profiling of OsHKT1;5, one of the genes present in the Saltol region. The NILs had significantly higher OsHKT1;5 expression than the recurrent parent PB1121, but lower than FL478 on salt exposure validating the successful introgression of Saltol in the NILs. This was also confirmed under agronomic evaluation, wherein the NILs showed greater salt tolerance at seedling stage. One of the NILs, Pusa1734-8-3-3 (NIL3) showed comparable yield and cooking quality to the recurrent parent PB1121, with high field level seedling stage salinity tolerance and shorter duration. This is the first report of successful introgression of Saltol into a Basmati rice cultivar.

Introgression of multiple disease resistance into a maintainer of Basmati rice CMS line by marker assisted backcross breeding

Globally, blast incited by Magnaporthe oryzae and sheath blight (ShB) by Rhizoctonia solani Kuhn forms two major fungal diseases that cause significant yield loss in rice. Pusa 6B, the Basmati quality maintainer line of the popular superfine grain aromatic rice hybrid Pusa RH10, is highly susceptible to both the diseases. The rice cultivar Tetep was used as the donor for transferring a major blast resistant gene, Pi54 and three ShB resistant quantitative trait loci (QTLs) namely, qSBR11-1, qSBR11-2 and qSBR7-1 into Pusa 6B using a marker assisted backcross breeding scheme with restricted number of backcrosses. Plants heterozygous for the alleles of interest and phenotypic similarity to the recurrent parent were used for generating BC1F2 population by selfing selected BC1F1 plants. Selected BC1F2 plants homozygous for Pi54 were selfed to generate BC1F3 families that were subjected to a step-wise reductive screening for the three ShB resistant QTLs. Final selections were advanced to BC1F5 generation through selfing while subjecting to stringent phenotypic selection. The advanced selections carrying blast and ShB resistant genes (Pi54, qSBR11-1, qSBR11-2, qSBR7-1) in the background of Pusa 6B were resistant to highly virulent strains of rice blast as well as ShB isolates without compromising the grain and cooking quality of Pusa 6B. Marker assisted transfer of blast and ShB resistance into Pusa 6B will aid in developing CMS lines with inbuilt resistance to these diseases. When combined with restorer lines possessing resistant genes/QTLs for these diseases, the improved Pusa6A lines will aid in development of improved Pusa RH10 and other novel aromatic hybrids with resistance to blast and ShB diseases. The present study demonstrates successful use of a restricted backcrossing strategy for introgression of multiple loci conferring resistance to two important fungal diseases in rice.

Marker assisted pyramiding of major blast resistance genes Pi9 and Pita in the genetic background of an elite Basmati rice variety, Pusa Basmati 1

Basmati is a premium quality rice of India which is highly priced in the international market. Pusa Basmati 1, an elite Basmati rice variety is highly susceptible to rice blast caused by Magnaporthe oryzae. Therefore, pyramiding blast resistance genes is essential to effectively combat the blast disease and increase the durability of resistance genes. The blast resistance genes Pi9 and Pita have been earlier demonstrated to be effective in Basmati growing regions of the country. Therefore, in the present study, monogenicnear isogenic lines Pusa 1637-18-7-6-20 and Pusa 1633-3-88-16-1 carrying Pi9 and Pita, respectively, were intercrossed to generate pyramided lines through marker assisted foreground, background and phenotypic selection for recurrent parent phenotype. The pyramided lines carrying Pi9+Pita were found to be either at par or superior to the recurrent parent Pusa Basmati 1 for agro-morphological, grain and cooking quality traits. Further, these pyramided lines were also found to show resistance against three virulent pathotypes of M. oryzae namely, Mo-nwi-kash 1, Mo-nwi-lon2 and Mo-ei-ran1, when evaluated under artificial inoculation conditions as well as in the natural epiphytotic conditions of uniform blast nursery at two locations. The developed pyramided lines are the potential sources of blast resistance genes in the Basmati improvement program and can also be released for commercial cultivation after required testing.

Validation of rapid DNA extraction protocol and their effectiveness in marker assisted selection in crop plants

In the present study, a simple and ultra quick protocol for extraction of genomic DNA was successfully validated in four different crops namely rice, wheat, lentil and Indian mustard. The method is simple and involves only two steps, it does not involve use of liquid nitrogen and other expensive and toxic reagents, and is highly suited for analyzing large number of samples within a short span of time. This makes it very attractive for use in MAS even in smaller labs where no sophisticated facilities are available.

An Account of Unclassified Species (Oryza schlechteri), Subspecies (Oryza indandamanica Ellis and Oryza sativa f. spontanea Baker), and Ortho-group Species (Leersia perrieri) of Oryza

The Oryza genus comprises of 24 well-recognized, morphologically distinct species that are classified into four different major complexes, consisting of ten different types of genomes. Apart from that, there are some species which are considered to be subspecies due to some similarities with the existing species and lack of characters that clearly distinguish them from well-defined species. Wild relatives of rice are untapped reservoirs of valuable genes which can combat various stresses and improve the productivity of cultivated rice. Oryza L. and Leersia Sw. are the two largest genera of the Oryzae tribe. Oryza schlechteri Pilger is an allotetraploid (2n = 48) with HHKK genome of 1568 Mb and is distributed throughout the Finisterre Mountains of Papua New Guinea. Leersia perrieri is a diploid species from the nearest out-group of Oryza with an estimated genome size of 323 Mb which is found in wet and marshlands of Africa and Madagascar. The genome sequence of L. perrieri has been assembled. Both these species are perennials and are stoloniferous in plant habit. On the other hand, O. indandamanica is morphologically quite similar to O. meyeriana, while O. sativa f. spontanea is a subspecies of O. sativa. However, the systematic study needs to be carried out to identify the unique characteristics of these species for their effective utilization.