G. S. Laha

Phenotyping Rice for Molecular Plant Breeding

Rice is an important food crop, has the plasticity in growing in different ecologies in many countries around the world, which makes this crop to expose to many diseases and pests. The recent development in the genomics has led to the intensive efforts in molecular breeding for improvements of some of the qualitative traits. To make the successful molecular breeding programme, accurate phenotyping techniques need to be coupled with high-throughput genotyping. The chapter discusses the various phenotypic methods available for different diseases, pests and abiotic stress like drought.

Identification and agro-morphological characterization of rice genotypes resistant to sheath blight

Rice is the most widely consumed staple food for a large part of the world’s human population, especially in Asia. Sheath blight caused by Rhizoctonia solani, is one of the most serious diseases of rice. Research progress is limited due to non availability of absolute resistant sources to sheath blight in rice. A total of 1013 germplasm comprising of mutants, introgression lines from wild species, A, B, R lines, Tropical japonica accessions, land races from north eastern India, wild rice accessions of O. nivara and O. rufipogon and gall midge biotype differentials maintained at ICAR-IIRR were evaluated with artificial inoculation for resistance to sheath blight disease during 2012 under field conditions. The germplasm identified as resistant/moderately resistant were further tested both under glass house and field conditions in the subsequent years (2013 and 2014) and seasons (Both Kharif and Rabi). Based on three years of testing, seven genotypes such as SM 801 (N 22 mutant), 10–3 (Introgression line), Ngnololasha, Wazuhophek, Gumdhan and Phougak (land races from north east) and RP 2068-18-3-5 (gall midge biotype differential) were identified as moderately resistant to sheath blight. Agro-morphological characterization using 62 DUS descriptors for two years (2013 and 2014) revealed that disease resistance positively correlated with stem thickness and negatively with tiller number. No correlation was observed with traits like plant height and heading date with sheath blight resistance. Resistant genotypes with medium to semi dwarf stature should be used in breeding programmes in the development of sheath blight resistant cultivars.

Importance and Management of Rice Diseases: A Global Perspective

Plant diseases are one of the major constraints in achieving the potential yield. Rice, the most important food crop in the world, is attacked by a number of fungal, bacterial and viral diseases. Due to hanged cultivation practices, reduced varietal diversity resulting in narrow genetic base and apparent climatic changes, the dynamics of rice diseases has changed over time. The major diseases have become more aggressive and spread to new areas. Many diseases which were earlier considered as minor have become economically important in many regions. For example, false smut of rice which was earlier considered as a sign of a bumper harvest has become widespread and a threatening problem in many areas. Many new disease problems (e.g. red stripe disease of rice) have been reported from different rice-growing regions. Varietal resistance has become short lived due to appearance of new and more virulent forms of the pathogen, making disease management more challenging. The present chapter includes global distribution, economic importance and management strategies of major and emerging rice diseases.

Incorporation of the novel bacterial blight resistance gene Xa38 into the genetic background of elite rice variety Improved Samba Mahsuri

Bacterial blight (BB) in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major global production constraint, particularly in irrigated and rain-fed lowland areas. Improved Samba Mahsuri (ISM) is an elite, high-yielding, fine-grain type, BB-resistant rice variety possessing three BB-resistant genes (Xa21, xa13 and xa5) and is highly popular in the southern parts of India. As the BB pathogen is highly dynamic and the evolution of pathogen virulence against the deployed resistance genes is common, we added a novel BB-resistant gene, Xa38, into ISM through marker-assisted backcross breeding (MABB) to increase the spectrum and durability of BB resistance. The breeding line PR 114 (Xa38) was used as the donor for Xa38, whereas ISM was used as the recurrent parent. Foreground selection was conducted using PCR-based gene-specific markers for the target genes, whereas background selection was conducted using a set of polymorphic SSR markers between the parents and backcrossing that continued until the third generation. Eighteen homozygous BC3F2 plants possessing all four BB-resistant genes in the homozygous state and with a recurrent parent genome (RPG) recovery of more than 92% were identified and advanced to the BC3F6 generation. These 18 backcross-derived lines (BDLs) exhibited very high level of resistance against multiple Xoo strains and displayed agro-morphological traits, grain qualities and yield levels similar to or better than those of the recurrent parent ISM.

Breeding strategies for hybrid rice parental line improvement

In our country, significant progress has been made in the development of hybrid rice parental lines both female and male parents with good floral traits. An array of hybrids with good grain quality (both unique Basmati type and medium slender (MS) grain type) is developed. Efforts are focussed on the development of restorer lines through exploitation of inter sub-specific hybridization, recurrent selection and population improvement that will help in the development of highly heterotic rice hybrids. The female parental lines are being improved for traits like stigma exsertion and high outcrossing that helps in higher seed yields, thereby reducing the hybrid seed cost. Efforts are on to develop appropriate parental lines that can help in the development of suitable hybrids in late duration group and also for unfavourable ecologies. Many newly developed parental lines are being fortified with resistance genes for major diseases such as BB, blast and insect pests such as BPH that will help in the development of hybrids having resistance to major pests and diseases.

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.

Expression Profile of Defense Genes in Rice Lines Pyramided with Resistance Genes Against Bacterial Blight, Fungal Blast and Insect Gall Midge

BackgroundRice, a major food crop of the world, endures many major biotic stresses like bacterial blight (BB), fungal blast (BL) and the insect Asian rice gall midge (GM) that cause significant yield losses. Progress in tagging, mapping and cloning of several resistance (R) genes against aforesaid stresses has led to marker assisted multigene introgression into elite cultivars for multiple and durable resistance. However, no detailed study has been made on possible interactions among these genes when expressed simultaneously under combined stresses.ResultsOur studies monitored expression profiles of 14 defense related genes in 11 rice breeding lines derived from an elite cultivar with different combination of R genes against BB, BL and GM under single and multiple challenge. Four of the genes found implicated earlier under combined GM and BB stress were confirmed to be induced (≥ 2 fold) in stem tissue following GM infestation; while one of these, cytochrome P450 family protein, was also induced in leaf in plants challenged by either BB or BL but not together. Three of the genes highlighted earlier in plants challenged by both BB and BL were also found induced in stem under GM challenge. Pi54 the target R gene against BL was also found induced when challenged by GM. Though expression of some genes was noted to be inhibited under combined pest challenge, such effects did not result in compromise in resistance against any of the target pests.ConclusionWhile R genes generally tended to respond to specific pest challenge, several of the downstream defense genes responded to multiple pest challenge either single, sequential or simultaneous, without any distinct antagonism in expression of resistance to the target pests in two of the pyramided lines RPNF05 and RPNF08.

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.

Improved versions of rice maintainer line, APMS 6B, possessing two resistance genes, Xa21 and Xa38, exhibit high level of resistance to bacterial blight disease

APMS 6B is the stable maintainer of the CMS line APMS 6A, which is the female parent of the popular Indian rice hybrid DRRH 3. APMS 6B has good combining ability and plant stature but is highly susceptible to bacterial blight (BB) disease. In order to improve the BB resistance of APMS 6B, we pyramided two major, dominant BB resistance genes, Xa21 and Xa38, through marker-assisted backcross breeding (MABB). Improved Samba Mahsuri (ISM) was used as the donor for Xa21 while PR 114 (Xa38) served as the donor for Xa38. Individual crosses [APMS 6B/ISM and APMS 6B/PR 114 (Xa38)] were performed, and true F1 plants were then backcrossed with APMS 6B and the MABB process was continued till BC3. A single positive BC3F1 plant identified from both the crosses with maximum genotypic and phenotypic similarity with APMS 6B was selfed to generate BC3F2s. At BC3F2 generation, plants homozygous for either Xa21 or Xa38 were identified and further confirmed for the absence of two major fertility restorer genes, Rf3 and Rf4. A single such homozygous BC3F2 plant, each from both the crosses, was then inter-mated to generate ICF1s (inter-cross F1s). Selected ICF1 plants possessing both the BB resistance genes were selfed to generate ICF2s. A total of 42 ICF2 plants homozygous for both Xa21 and Xa38 were identified and screened with parental polymorphic SSR markers to identify the best F2 plants having the maximum recurrent parent genome recovery. Twelve best ICF2 plants were advanced up to ICF5. The ICF5 lines displayed very high level of BB resistance and were similar to APMS 6B in terms of agro-morphological characters. Further, most of these lines also showed complete maintenance ability and such lines are being advanced for conversion to WA-CMS lines.

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 > 90% 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.