Marker assisted improvement of low soil phosphorus tolerance in the bacterial blight resistant, fine-grain type rice variety, Improved Samba Mahsuri.

Improved-Samba-Mahsuri (ISM), a high-yielding, popular bacterial blight resistant (possessing Xa21, xa13, and xa5), fine-grain type, low glycemic index rice variety is highly sensitive to low soil phosphorus (P). We have deployed marker-assisted backcross breeding (MABB) approach for targeted transfer of Pup1, a major QTL associated with low soil P tolerance, using Swarna as a donor. A new co-dominant marker, K20-1-1, which is specific for Pup1 was designed and used for foreground selection along with functional markers specific for the bacterial blight resistance genes, Xa21, xa13, and xa5. A set of 66 polymorphic SSR marker were used for the background selection along with a pair of flanking markers for the recombination selection in backcross derived progenies and in BC2F2 generation, 12 plants, which are homozygous for Pup1, all the three bacterial blight resistance genes and possessing agro-morphological traits equivalent to or better than ISM were selected and selfed to produce BC2F3s. They were evaluated in plots with low soil P and normal soil P at ICAR-IIRR, Hyderabad for their low soil P tolerance, and bacterial blight resistance and superior lines were advanced to BC2F6. One of the lines, when tested at multiple locations in India was found promising under both normal as well as low soil P conditions.

Pectin induced transcriptome of a Rhizoctonia solani strain causing sheath blight disease in rice reveals insights on key genes and RNAi machinery for development of pathogen derived resistance.

KEY MESSAGE: RNAi mediated silencing of pectin degrading enzyme of R. solani gives a high level of resistance against sheath blight disease of rice. Rice sheath blight disease caused by Rhizoctonia solani Kuhn (telemorph; Thanatephorus cucumeris) is one of the most devastating fungal diseases which cause severe loss to rice grain production. In the absence of resistant cultivars, the disease is currently managed through fungicides which add to environmental pollution. To explore the potential of utilizing RNA interference (RNAi)-mediated resistance against sheath blight disease, we identified genes encoding proteins and enzymes involved in the RNAi pathway in this fungal pathogen. The RNAi target genes were deciphered by RNAseq analysis of a highly virulent strain of the R. solani grown in pectin medium. Additionally, pectin metabolism associated genes of R. solani were analyzed through transcriptome sequencing of infected rice tissues obtained from six diverse rice cultivars. One of the key candidate gene AG1IA_04727 encoding polygalacturonase (PG), which was observed to be significantly upregulated during infection, was targeted through RNAi to develop disease resistance. Stable expression of PG-RNAi construct in rice showed efficient silencing of AG1IA_04727 and suppression of sheath blight disease. This study highlights important information about the existence of RNAi machinery and key genes of R. solani which can be targeted through RNAi to develop pathogen-derived resistance, thus opening an alternative strategy for developing sheath blight-resistant rice cultivars.

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica.

KEY MESSAGE: Pollen-specific expression. Promoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 5' deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 5' promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3-∆1 (1504 bp), OSIPP3-∆2 (968 bp), OSIPP3-∆3 (388 bp) and OSIPP3-∆4 (286 bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated that they were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504- and 968-bp fragments directed GUS expression in roots and anthers, while the 388- and 286-bp fragments restricted the GUS expression to only pollen, of which 388 bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1-P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-Δ1 followed by OSIPP3-Δ2 (eightfold) and OSIPP3-Δ3 (threefold) when compared to OSIPP3-Δ4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.

Pyramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna) lectin genes impart enhanced resistance against major sap-sucking pests.

We have developed transgene pyramided rice lines, endowed with enhanced resistance to major sap-sucking insects, through sexual crosses made between two stable transgenic rice lines containing Allium sativum (asal) and Galanthus nivalis (gna) lectin genes. Presence and expression of asal and gna genes in pyramided lines were confirmed by PCR and western blot analyses. Segregation analysis of F2 progenies disclosed digenic (9:3:3:1) inheritance of the transgenes. Homozygous F3 plants carrying asal and gna genes were identified employing genetic and molecular methods besides insect bioassays. Pyramided lines, infested with brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH), proved more effective in reducing insect survival, fecundity, feeding ability besides delayed development of insects as compared to the parental transgenics. Under infested conditions, pyramided lines were found superior to the parental transgenics in their seed yield potential. This study represents first report on pyramiding of two lectin genes into rice exhibiting enhanced resistance against major sucking pests. The pyramided lines appear promising and might serve as a novel genetic resource in rice breeding aimed at durable and broad based resistance against hoppers.