Combined strategy employing MutMap and RNA-seq reveals genomic regions and genes associated with complete panicle exsertion in rice.

Complete panicle exsertion (CPE) in rice is an important determinant of yield and a desirable trait in breeding. However, the genetic basis of CPE in rice still remains to be completely characterized. An ethyl methane sulfonate (EMS) mutant line of an elite cultivar Samba Mahsuri (BPT 5204), displaying stable and consistent CPE, was identified and named as CPE-110. MutMap and RNA-seq were deployed for unraveling the genomic regions, genes, and markers associated with CPE. Two major genomic intervals, on chromosome 8 (25668481-25750456) and on chromosome 11 (20147154-20190400), were identified to be linked to CPE through MutMap. A non-synonymous SNP (G/A; Chr8:25683828) in the gene LOC_Os08g40570 encoding pyridoxamine 5'-phosphate oxidase with the SNP index 1 was converted to Kompetitive allele-specific PCR (KASP) marker. This SNP (KASP 8-1) exhibited significant association with CPE and further validated through assay in the F2 mapping population, released varieties and CPE exhibiting BPT 5204 mutant lines. RNA-seq of the flag leaves at the booting stage, 1100 genes were upregulated and 1305 downregulated differentially in CPE-110 and BPT 5204. Metabolic pathway analysis indicated an enrichment of genes involved in photosynthesis, glyoxylate, dicarboxylate, porphyrin, pyruvate, chlorophyll, carotenoid, and carbon metabolism. Further molecular and functional studies of the candidate genes could reveal the mechanistic aspects of CPE. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01412-1.

Revealing the effect of seed phosphorus concentration on seedling vigour and growth of rice using mutagenesis approach.

The harvested plant products, specifically, the grains of cereals are major drivers of soil phosphorus (P) depletion. However, the breeding or biotechnology efforts to develop low P seeds have not been attempted because of possible adverse effects on seedling vigour and crop establishment. Several studies have contradictory observations on influence of seed P on seedling vigour. Lack of appropriate genetic material has been the major bottleneck in reaching the consensus. In this study, we used 30 EMS induced mutants of rice cultivar Nagina22 to understand the role of seed P on seedling vigour and associated physiological processes. Seedling vigour, morpho-physiological characteristics, acid phosphatases, alpha-amylase, and expression of P transporter genes were analyzed in seedlings obtained from seeds of high and low grain P mutants. The study suggests that seed P has a significant role on seedling vigour, chlorophyll content and photosynthesis process of young seedlings, and P transport from roots. Notably, we identified few mutants such as NH4791, NH4785, NH4714, NH4663, NH4614, and NH4618 which showed least influence of low seed P on seedling vigour and other metabolic processes. Therefore, these mutants can be used in breeding programs aiming for development of low P grains. Also, these and other identified mutants can be used to decipher the genetic and molecular mechanisms regulating the differential response of seed P on germination, seedling vigour and several other physiological processes influencing the crop growth and establishment.

Grain Fe and Zn content, heterosis, combining ability and its association with grain yield in irrigated and aerobic rice.

Genetic improvement of rice for grain micronutrients, viz., iron (Fe) and zinc (Zn) content is one of the important breeding objectives, in addition to yield improvement under the irrigated and aerobic ecosystems. In view of developing genetic resources for aerobic conditions, line (L) × tester (T) analysis was conducted with four restorers, four CMS lines and 16 hybrids. Both hybrids and parental lines were evaluated in irrigated and aerobic field conditions for grain yield, grain Fe and Zn content. General Combining Ability (GCA) effects of parents and Specific Combining Ability (SCA) effects of hybrids were observed to be contrasting for the micronutrient content in both the growing environments. The grain Fe and Zn content for parental lines were negatively correlated with grain yield in both the contrasting growing conditions. However, hybrids exhibited positive correlation for grain Fe and Zn with grain yield under limited water conditions. The magnitude of SCA mean squares was much higher than GCA mean squares implying preponderance of dominance gene action and also role of complementary non-allelic gene(s) interaction of parents and suitability of hybrids to the aerobic system. The testers HHZ12-SAL8-Y1-SAL1 (T1) and HHZ17-Y16-Y3-Y2 (T2) were identified as good combiners for grain Zn content under irrigated and aerobic conditions respectively.

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.