Prolay K. Bhowmick

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.

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.

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.

Development of high throughput screening protocol and identification of novel sources of resistance against bakanae disease in rice (Oryza sativa L.)

Bakanae or foot rot disease caused by Fusarium fujikuroi (teleomorph: Gibberella fujikuroi, Sawada, Wollenweber) is emerging as a serious disease of rice. A simple, reliable and high-throughput method for screening the disease would enable rapid screening of germplasm aimed at identifying resistance sources, mapping QTLs/genes and developing resistant rice cultivars. In the present study, a highthroughput, reliable bioassay to screen rice germplasm for resistance to bakanae disease was developed and compared with the conventional screening technique. This technique involves soaking of rice seeds in fungal spore suspension (1.0x106 spores ml–1) for 24 hours at room temperature. Seedling growth at 30°/25° (±3)°C day/night temperature and 60/80(±10)% day/night relative humidity in glasshouse gave the best results. The new protocol described here produces consistent and reproducible bakanae disease symptoms and enables screening of hundreds of rice germplasm within 15 days without any loss of precision in screening of rice genotypes against bakanae disease. The resistant and susceptible genotypes can be used for developing mapping population and identification of QTLs/genes conferring resistance to bakanae disease.

Genetic Improvement of Basmati Rice: Transcendence Through Molecular Breeding

Basmati rice from the foothills of Himalayas is a connoisseurs’ delight. Basmati rice improvement began with germplasm collection and pureline selection among the landraces. Systematic Basmati rice improvement program at ICAR-Indian Agricultural Research Institute, New Delhi (ICAR-IARI), led to a breakthrough with the release of first semidwarf high-yielding variety Pusa Basmati 1. Subsequently, the release of Pusa Basmati 1121, unique for its cooked kernel length and volume expansion, brought a significant change in Basmati cultivation. The total volume of Basmati rice exports quadrupled leading to 6.8 times increase in foreign exchange earnings during 2013–2014 (Rs. 29,299 crores). Furthermore, the development of Pusa Basmati 1509, the first early maturing Basmati rice variety, demonstrated that undesirable linkage between duration and yield can be broken, thus achieving better per day productivity while increasing profitability. Although better productivity and quality were achieved, these varieties were susceptible to various biotic and abiotic stresses. The beginning of the twenty-first century witnessed rice genome sequencing, which enabled the use of molecular tools for precision breeding in Basmati rice. Molecular marker-assisted selection has been effectively used in combination with phenotypic selection to develop Basmati rice varieties with resistance to diseases like bacterial blight and blast. The bacterial blight resistance genes xa13 + Xa21 were incorporated into the genetic background of Pusa Basmati 1, Pusa Basmati 1121, and Pusa Basmati 6 to develop Improved Pusa Basmati 1, Pusa Basmati 1718, and Pusa Basmati 1728, respectively. Similarly, Pusa Basmati 1637 carrying blast resistance gene Pi9 in the genetic background of Pusa Basmati 1 was also developed. In addition to the release of varieties, molecular breeding has helped in creating several genetic stocks in Basmati rice with resistance to biotic stresses such as BB, blast, sheath blight, and bakanae and tolerance to abiotic stresses such as salinity and drought. The high-yielding Basmati rice varieties developed have brought in a paradigm shift in Basmati rice cultivation, domestic consumption, and export to the tune of Rs. 25,000 crores annually, bringing prosperity to millions of Basmati rice farmers in the Indo-Gangetic Plains (IGP); this success story can be appropriately described as “Basmati revolution.” This chapter describes five decades of concerted research efforts on genetic improvement of Basmati rice at the ICAR-IARI, India.

Marker-Assisted Introgression of Saltol QTL Enhances Seedling Stage Salt Tolerance in the Rice Variety “Pusa Basmati 1”

Marker-assisted selection is an unequivocal translational research tool for crop improvement in the genomics era. Pusa Basmati 1 (PB1) is an elite Indian Basmati rice cultivar sensitive to salinity. Here, we report enhanced seedling stage salt tolerance in improved PB1 genotypes developed through marker-assisted transfer of a major QTL, Saltol. A highly salt tolerant line, FL478, was used as the Saltol donor. Parental polymorphism survey using 456 microsatellite (SSR)/QTL-linked markers revealed 14.3% polymorphism between PB1 and FL478. Foreground selection was carried out using three Saltol-linked polymorphic SSR markers RM8094, RM493, and RM10793 and background selection by 62 genome-wide polymorphic SSR markers. In every backcross generation, foreground selection was restricted to the triple heterozygotes of foreground markers, which was followed by phenotypic and background selections. Twenty-four near isogenic lines (NILs), with recurrent parent genome recovery of 96.0–98.4%, were selected after two backcrosses followed by three selfing generations. NILs exhibited agronomic traits similar to those of PB1 and additional improvement in the seedling stage salt tolerance. They are being tested for per se performance under salt-affected locations for release as commercial varieties. These NILs appear promising for enhancing rice production in salinity-affected pockets of Basmati Geographical Indication (GI) areas of India.

Molecular mapping of rice root-knot nematode (Meloidogyne graminicola) resistance gene in Asian rice (Oryza sativa L.) using STMS markers

Rice root-knot nematode, Meloidogyne graminicola is one of the major pests of rice cropping system and is responsible for considerable yield reductions. Out of various management options tried so far, the resistance against M. graminicola in rice could be the most valuable in alleviating this problem. In present study a rice cultivar Abhishek exhibited a strong resistance with least number of galls (2galls/plant) and a kind of necrotic browning of roots which is typical of R-gene mediated resistance response. In order to map the gene governing resistance to root-knot nematode, the cultivar Abhishek was crossed with the Bangla Patni, a highly susceptible genotype to generate F2 mapping population. Using bulked segregant analysis, a marker HvSSR10–21 was identified to be putatively linked with the resistant locus in cv. Abhishek with significant LOD score. The significant LOD score value indicates the linkage between identified marker and the resistant locus against M. graminicola. We designate this gene as Mg1(t).

Development and evaluation of iso-cytoplasmic rice restorer lines for different agro-morphological traits

A set of 390 iso-cytoplasmic restorer lines were developed from 25 popular rice hybrids grown in India through pedigree breeding exercising selection for spikelet fertility, yield and extent of panicle exsertion were evaluated for various agromorphological traits. Correlation analysis showed that yield per plant was significantly correlated with number of tillers (0.208), panicle length (0.127) and spikelet fertility (0.134). Principal component analysis indicated that the first four principal components had eigenvalue of > 1.0 and cumulatively explained 74.3% of total variance. Grain characters namely, test weight (0.451), kernel length before cooking (0.418), kernel breadth before cooking (0.136) and spikelet fertility (0.402) contributed positively towards principal component I. Yield per plant showed a positively skewed distribution with panicle length and a nearly linear relationship with spikelet fertility. A core set of 21 genotypes capturing the entire range of phenotype for different traits, was identified from 390 genotypes using advanced M (maximization) strategy and statistically validated. Besides this, promising iso-cytoplasmic restorer lines originating from different hybrids were identified for various agromorphological traits. This is the first report on developing iso-cytoplasmic restorers from popular rice hybrids, which can be used in developing heterotic hybrids when crossed with diverse non-parental CMS lines.