S. M. Balachandran

Identification and Fine-Mapping of Xa33, a Novel Gene for Resistance to Xanthomonas oryzae pv. oryzae

Broadening of the genetic base for identification and transfer of genes for resistance to insect pests and diseases from wild relatives of rice is an important strategy in resistance breeding programs across the world. An accession of Oryza nivara, International Rice Germplasm Collection (IRGC) accession number 105710, was identified to exhibit high level and broad-spectrum resistance to Xanthomonas oryzae pv. oryzae. In order to study the genetics of resistance and to tag and map the resistance gene or genes present in IRGC 105710, it was crossed with the bacterial blight (BB)-susceptible varieties TN1 and Samba Mahsuri (SM) and then backcrossed to generate backcross mapping populations. Analysis of these populations and their progeny testing revealed that a single dominant gene controls resistance in IRGC 105710. The BC(1)F(2) population derived from the cross IRGC 105710/TN1//TN1 was screened with a set of 72 polymorphic simple-sequence repeat (SSR) markers distributed across the rice genome and the resistance gene was coarse mapped on chromosome 7 between the SSR markers RM5711 and RM6728 at a genetic distance of 17.0 and 19.3 centimorgans (cM), respectively. After analysis involving 49 SSR markers located between the genomic interval spanned by RM5711 and RM6728, and BC(2)F(2) population consisting of 2,011 individuals derived from the cross IRGC 105710/TN1//TN1, the gene was fine mapped between two SSR markers (RMWR7.1 and RMWR7.6) located at a genetic distance of 0.9 and 1.2 cM, respectively, from the gene and flanking it. The linkage distances were validated in a BC(1)F(2) mapping population derived from the cross IRGC 105710/SM//2 × SM. The BB resistance gene present in the O. nivara accession was identified to be novel based on its unique map location on chromosome 7 and wider spectrum of BB resistance; this gene has been named Xa33. The genomic region between the two closely flanking SSR markers was in silico analyzed for putatively expressed candidate genes. In total, eight genes were identified in the region and a putative gene encoding serinethreonine kinase appears to be a candidate for the Xa33 gene.

Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas

RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of chemically synthesized 5′ FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12–15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach.

Transgenic Approaches for Abiotic Stress Tolerance in Crop Plants

Abiotic stresses including drought, salinity and cold are a major challenge for sustainable food production as they may decrease the potential yields in crop plants by 70 %. Success in breeding for better adapted varieties to abiotic stresses depends upon intensive efforts using novel biotechnological approaches, including molecular biology, genetics, plant and cell physiology and breeding. Many abiotic stress-induced genes have been identified and some have been cloned. The use of current molecular biology tools to reveal the control mechanisms of abiotic-stress tolerance, and for engineering stress-tolerant crops is based on the expression of specific stress-related genes. Hence, plant genetic engineering and molecular-marker approaches allow development of abiotic stress-tolerant germplasm. Transgenic plants carrying genes for abiotic stress tolerance are being developed, mainly by using Agrobacterium and biolistic methods; transgenics carrying different genes relating to abiotic stress tolerance have been developed in crop plants like rice, wheat, maize, sugarcane, tobacco, Arabidopsis, groundnut, tomato and potato. This chapter focuses on recent progress in using transgenic technology for the improvement of abiotic-stress tolerance in plants. It includes discussion of metabolic engineering for biosynthesis and accumulation of compatible osmolytes (i.e. proline, glycine betaine, ectoine and polyols), reactive oxygen species formation under abiotic stress, ROS scavenging and detoxification in plant cells, single gene transgenic versus multiple genes and transcription factors and their roles in management of abiotic stresses.

Development of Gene-Pyramid Lines of the Elite Restorer Line, RPHR-1005 Possessing Durable Bacterial Blight and Blast Resistance

RPHR-1005, the stable restorer line of the popular medium slender (MS) grain type rice hybrid, DRRH-3 was improved in this study for resistance against bacterial blight (BB) and blast diseases through marker-assisted backcross breeding (MABB). In this study, four major resistance genes (i.e., Xa21 and Xa33 for BB resistance and Pi2 and Pi54 for blast resistance) have been transferred to RPHR-1005 using RPBio Patho-1 (possessing Xa21 + Pi2), RPBio Patho-2 (possessing Xa21 + Pi54) and FBR1-15EM (possessing Xa33) as the donors. Foreground selection was carried out using PCR-based molecular markers specific for the target resistance genes and the major fertility restorer genes, Rf3 and Rf4, while background selection was carried out using a set of parental polymorphic rice SSR markers and backcrossing was continued uptoBC2 generation. At BC2F2, plants possessing the gene combination- Xa21 + Pi2, Xa21 + Pi54 and Xa33 in homozygous condition and with >92% recovery of the recurrent parent genome (RPG) were identified and intercrossed to combine all the four resistance genes. Twenty-two homozygous, pyramid lines of RPHR-1005 comprising of three single-gene containing lines, six 2-gene containing lines, eight 3-gene containing lines, and five 4-gene containing lines were identified among the double intercross lines at F3 generation (DICF3). They were then evaluated for their resistance against BB and blast, fertility restoration ability and for key agro-morphological traits. While single gene containing lines were resistant to either BB or blast, the 2-gene, 3-gene, and 4-gene pyramid lines showed good level of resistance against both and/or either of the two diseases. Most of the 2-gene, 3-gene, and 4-gene containing pyramid lines showed yield levels and other key agro-morphological and grain quality traits comparable to the original recurrent parent and showed complete fertility restoration ability, with a few showing higher yield as compared to RPHR-1005. Further, the experimental hybrids derived by crossing the gene-pyramid lines of RPHR-1005 with APMS6A (the female parent of DRRH-3), showed heterosis levels equivalent to or higher than DRRH-3. The results of present study exemplify the utility of MABB for targeted improvement of multiple traits in hybrid rice.

Marker-assisted improvement of the elite restorer line of rice, RPHR-1005 for resistance against bacterial blight and blast diseases

This study was carried out to improve the RPHR-1005, a stable restorer line of the popular medium slender grain type rice hybrid, DRRH-3 for bacterial blight (BB) and blast resistance through marker-assisted backcross breeding (MABB). Two major BB resistance genes, Xa21 and Xa33 and a major blast resistance gene, Pi2 were transferred to RPHR-1005 as two individual crosses. Foreground selection for Xa21, Xa33, Pi2, Rf3 and Rf4 was done by using gene-specific functional markers, while 59 simple sequence repeat (SSR) markers polymorphic between the donors and recipient parents were used to select the best plant possessing target resistance genes at each backcross generation. Backcrossing was continued till BC 2F2 and a promising homozygous backcross derived line possessing Xa21 + Pi2 and another possessing Xa33 were intercrossed to stack the target resistance genes into the genetic background of RPHR-1005. At ICF 4, 10 promising lines possessing three resistance genes in homozygous condition along with fine-grain type, complete fertility restoration, better panicle exertion and taller plant type (compared to RPHR-1005) were identified.

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

AbstractKey messagePollen-specific expression.AbstractPromoters 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 (1504xa0bp), OSIPP3-∆2 (968xa0bp), OSIPP3-∆3 (388xa0bp) and OSIPP3-∆4 (286xa0bp) 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 thatnthey 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 388xa0bp 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.

Marker-assisted pyramiding of bacterial blight and gall midge resistance genes into RPHR-1005, the restorer line of the popular rice hybrid DRRH-3

Bacterial blight (BB) of rice caused by the pathogen Xanthomonas oryzae pv. oryzae and the insect gall midge (GM) (Orseolia oryzae) are two major constraints of rice production. The present study was carried out to improve RPHR-1005, a stable restorer line of the fine-grain-type rice hybrid DRRH-3, for BB and GM resistance through marker-assisted backcross breeding (MABB). Two major GM resistance genes, Gm4 and Gm8, and a major BB resistance gene, Xa21, were selected as target genes for transfer to RPHR-1005. Two sets of backcrosses were carried out to combine either Xa21 + Gm4 or Xa21+ Gm8 into RPHR-1005 using breeding lines in the genetic background of ISM possessing either Gm4 or Gm8 along with Xa21. Foreground selection was performed for Xa21, Gm4, Gm8, and the major fertility restorer genes Rf3 and Rf4 using gene-specific markers, while 61 polymorphic simple sequence repeat (SSR) markers were used for background selection and marker-assisted backcrossing was continued until BC2 generation. A promising homozygous backcross-derived plant at the BC2F2 generation possessing Xa21 + Gm4, and another possessing Xa21 + Gm8, were intercrossed to stack the target resistance genes. At ICF4 (inter-crossed F4) , three promising lines possessing the three target resistance genes in a homozygous condition along with fine-grain type, complete fertility restoration, and better panicle exsertion than RPHR-1005 have been identified. Among these, a single line, # RPIC-16-65-125, showed better yield, was highly resistant to BB and GM, was of medium–slender grain type, and had complete fertility restoration along with better panicle exsertion and taller plant type than RPHR-1005. This is the first report of combining resistance against BB and GM in the genetic background of a hybrid rice parental line.

Physical interaction of RTBV ORFI with D1 protein of Oryza sativa and Fe/Zn homeostasis play a key role in symptoms development during rice tungro disease to facilitate the insect mediated virus transmission

Rice tungro disease is caused by the combined action of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The RTBV is involved in the development of symptoms while RTSV is essential for virus transmission. We attempted to study the mode of action of RTBV in the development of symptoms. The tungro disease symptoms were attributed to viral interference in chlorophyll and carotenoids biosynthesis, photosynthesis machinery, iron/zinc homeostasis, and the genes encoding the enzymes associated with these biological processes of rice. The adverse effects of virus infection in photosystem II (PSII) activity was demonstrated by analyzing the Fv/Fm ratio, expression of psbA and cab1R genes, and direct interaction of RTBV ORF I protein with the D1 protein of rice. Since ORF I function is not yet known in the RTBV life cycle, this is the first report showing its involvement in regulating host photosynthesis process and symptoms development.

Proteomic and transcriptomic approaches to identify resistance and susceptibility related proteins in contrasting rice genotypes infected with fungal pathogen Rhizoctonia solani

The devastating sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) causes major yield loss in most rice growing regions of the world. In this study, two moderately tolerant and four susceptible genotypes of rice were selected for R. solani induced proteome analysis using two-dimensional polyacrylamide gel electrophoresis. Forty five differentially expressed proteins (DEPs) were identified and analyzed by Mass Spectrometry. Based on their functions, these proteins were classified into different groups, viz., photosynthesis, resistance and pathogenesis, stress, cell wall metabolism and cytoskeleton development associated proteins, and hypothetical or uncharacterized proteins. Expression of 14 genes encoding DEPs was analyzed by quantitative PCR which showed consistency in transcripts and genes expression pattern. Furthermore, the expression of 16 other genes involved in diverse biological functions was analyzed. Up-regulation of these genes in the tolerant genotype Pankaj during sheath blight disease suggested efficient genetic regulation of this cultivar under stress. Also, expression analysis of conserved microRNAs (miRNAs) and their target genes revealed important role of miRNAs in post-transcriptional gene regulation during development of rice sheath blight disease. Genome-wide discovery of miRNAs and further characterization of DEPs and genes will help in better understanding of the molecular events during sheath blight disease development in rice.

Improvement of blast resistance of the popular high-yielding, medium slender-grain type, bacterial blight resistant rice variety, Improved Samba Mahsuri by marker-assisted breeding

Improved Samba Mahsuri (ISM) is a popular, high-yielding, bacterial blight resistant rice variety possessing medium-slender grain type. As ISM is highly susceptible to blast disease of rice, through the present study we have transferred two major blast resistance genes, Pi2 and Pi54 into the elite variety by marker-assisted backcross breeding. The two blast resistance genes were transferred to ISM through sets of backcrosses. In every backcross generation, PCR-based markers, specific for the blast resistance genes (Pi2 and Pi54) and bacterial blight resistance genes (Xa21, xa13 and xa5) were utilized for foreground selection, while a set of 144 parental polymorphic SSR markers were used for background selection and backcrossing was carried out until BC2 generation. A solitary BC2F1 plant possessing Pi2 or Pi54 along with Xa21, xa13 and xa5 and >u200990% recovery of ISM genome was selected from the two sets of backcrosses were crossed and the intercross F1s (ICF1s) thus obtained were selfed to generate ICF2s. Homozygous ICF2 plants carrying all the five resistance genes were identified through markers and advanced through selfing till ICF5 generation by adopting pedigree method of selection. Three best lines at ICF5, possessing excellent resistance against bacterial blight and blast and closely resembling or superior to ISM in terms of grain quality: yield and agro-morphological traits have been identified and advanced for multi-location trials.