Apurva Khanna

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.

Understanding Host-Pathogen Interactions with Expression Profiling of NILs Carrying Rice-Blast Resistance Pi9 Gene

Magnaporthe oryzae infection causes rice blast, a destructive disease that is responsible for considerable decrease in rice yield. Development of resistant varieties via introgressing resistance genes with marker-assisted breeding can eliminate pesticide use and minimize crop losses. Here, resistant near-isogenic line (NIL) of Pusa Basmati-1(PB1) carrying broad spectrum rice blast resistance gene Pi9 was used to investigate Pi9-mediated resistance response. Infected and uninfected resistant NIL and susceptible control line were subjected to RNA-Seq. With the exception of one gene (Pi9), transcriptional signatures between the two lines were alike, reflecting basal similarities in their profiles. Resistant and susceptible lines possessed 1043 (727 up-regulated and 316 down-regulated) and 568 (341 up-regulated and 227 down-regulated) unique and significant differentially expressed loci (SDEL), respectively. Pathway analysis revealed higher transcriptional activation of kinases, WRKY, MYB, and ERF transcription factors, JA-ET hormones, chitinases, glycosyl hydrolases, lipid biosynthesis, pathogenesis and secondary metabolism related genes in resistant NIL than susceptible line. Singular enrichment analysis demonstrated that blast resistant NIL is significantly enriched with genes for primary and secondary metabolism, response to biotic stimulus and transcriptional regulation. The co-expression network showed proteins of genes in response to biotic stimulus interacted in a manner unique to resistant NIL upon M. oryzae infection. These data suggest that Pi9 modulates genome-wide transcriptional regulation in resistant NIL but not in susceptible PB1. We successfully used transcriptome profiling to understand the molecular basis of Pi9-mediated resistance mechanisms, identified potential candidate genes involved in early pathogen response and revealed the sophisticated transcriptional reprogramming during rice-M. oryzae interactions.

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.

Identification and validation of rice blast resistance genes in Indian rice germplasm

Blast disease caused by Magnaporthe oryzae is a major constraint in rice production. Identification of new donors for blast resistance is a pre-requisite for effective utilization of diverse germplasm for marker assisted incorporation of blast resistance into improved varieties. Therefore, in the present study, a set of 100 diverse rice germplasm accessions were evaluated for 11 blast resistance genes namely Pikm, Pik, Pikh, Pi1, Pi5, Pi54, Pib, Piz5, Piz, Pi9 and Pish, both at genotypic and phenotypic level. Genotyping with gene based/gene linked markers could identify six genotypes from the germplasm possessing as many as six resistance specific alleles. A total of 34 and 67 germplasm lines were found to possess resistance alleles for two genes, Pikm and Pik, respectively. Phenotypic validation using artificial inoculation in the germplasm was carried out with 4 diverse isolates under controlled conditions. The congruence between marker genotype and disease phenotype on a set of monogenic lines for blast resistance in the LTH background was used to compute Disease Resistance Index (DRI) in the germplasm. Cumulative DRI for each genotype was computed over all the marker loci. The genotypes Heibao, Kalinga-I, Vijetha, Anjali, Bhaubhog, Sada Kaijam, Kala Jeera had high cumulative resistance score. Allelic Cumulative Disease Resistance Index (ACDRI), a measure for comparing the effectiveness of markers was calculated and markers linked to Pikm, Pik, Piz5, Pi1 were found to possess higher accuracy and better correlation with expected patterns of resistance under artificial inoculation. Based on disease resistance index, 25 germplasm accessions were found carrying blast resistance specific alleles at different loci and were fully validated for disease phenotype, which are valuable in breeding for resistance, allele mining and functional genomics studies.

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.

Development and validation of co-dominant gene based markers for Pi9, a gene governing broad-spectrum resistance against blast disease in rice

Rice production and grain quality are severely affected by blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Incorporation of genes that confer broad-spectrum resistance to blast has been a priority area in rice breeding programs. The blast resistance gene Pi9 sourced from Oryza minuta has shown broad spectrum and durable resistance to blast world-wide. In the present study co-dominant gene-based markers were developed for the precise marker-assisted tracking of Pi9 in breeding programs. The developed markers were validated across a diverse set of cultivars including basmati, indica and japonica varieties. Two markers, Pi9STS-1 and Pi9STS-2, effectively differentiated Pi9 donors from all the indicas and commercial basmati varieties tested. However, these markers were monomorphic between Pi-9 donors (IRBL9-W and Pusa 1637) and japonica type varieties. An additional gene-derived CAPS marker Pi91F_ 2R was developed to differentiate Pi9 donors from japonicas and traditional basmati lines. The co-dominant markers developed in the present study will be of immense utility to rice breeders for precise and speedy incorporation of Pi-9 into susceptible rice varieties through marker-assisted selection.