Screening of new isolates of Bacillus thuringiensis (Bt) and cloning of the cry genes

Nine new indigenous isolates of Bacillus thuringiensis (Bt) were characterized for their colony type, crystal inclusion and toxicity analysis with Helicoverpa armigera Hubner and Spodoptera litura Linn. Genomic deoxyribonucleic acid (DNA) isolated from all the new isolates were subjected to screening for cry1, cry2, cry4, cry10 and cry11 genes and predicted possible potential DNA amplicons were cloned and sequenced. Partial cry1 gene fragment (~1.5 kb) amplified by degenerate primers and about 450 bp DNA fragment amplified by cry10 gene specific primers from two isolates T109 and T136 were cloned in to T/A cloning vector. DNA sequencing of about 1.5 kb amplicon showed 99% homology to the holotype sequence of cry1Ac1. Nucleotide sequence of about 450 bp fragments of isolate T109 and T136 showed homology to a hypothetical protein and serine/threonine phosphatase respectively.Keywords: Bacillus thuringiensis (Bt), cloning, cry genes, polymerase chain reaction (PCR), toxicity analysi

Prospecting true ScYLV resistance in Saccharum hybrid parental population in India by symptom phenotyping and viral titre quantification

Not AvailableIn sugarcane (Saccharum spp. hybrids) cultivation, viral diseases pose a great challenge across the globe. Yellow leaf (YL) disease is one of the important viral diseases caused by Sugarcane yellow leaf virus (ScYLV), a positive-sense ssRNA virus, genus Polerovirus, family Solemoviridae. The disease symptoms appear in later stages of crop growth during grand growth to maturity phase with intense midrib yellowing in the abaxial leaf surface. At present, this disease is managed through tissue (meristem) culture and healthy seed nurseries in India. However, the virus-free plants are infected quickly by secondary inoculum from aphid vectors in the field, which necessitates the importance of developing YL-resistant varieties. We screened about 600?625 sugarcane parental clones to identify true YL resistance based on 0?5 disease rating scale since 2015 and categorised them as resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible. Leaf samples were collected from all these categories of plants during 2018?20 for the viral titre estimation through absolute quantification method (qRT-PCR assay). The viral load was invariably high in all categories of susceptible samples that ranged from 4.40 ? 102 to 8.429 ? 106, whereas in YL-free asymptomatic clones, the viral load ranged from 82.35 ? 5.90 to 5.121 ? 104. The results clearly indicated that highest viral titre of 105?107 copies was present in all the susceptible clones irrespective of their disease severity grades. Our results clearly established that about 22.85% of apparently resistant sugarcane clones remained free from YL symptoms with significantly low ScYLV titre although we could not find a significant correlation between virus titre and symptom expression. The identified resistant parents will serve as sources of YL resistance to develop virus resistant sugarcane varieties

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Not AvailableBacterial blight (BB) disease reduces the yield of rice varieties and hybrids considerably in many tropical rice growing countries like India. The present study highlights the development of durable BB resistance into the background of an elite maintainer of rice, DRR17B, by incorporating two major dominant genes, Xa21 and Xa33 through marker-assisted backcross breeding (MABB). Through two sets of backcrosses, the two BB resistance genes were transferred separately to DRR17B. In this process, at each stage of backcrossing, foreground selection was carried out for the target resistance genes and for non-fertility restorer alleles concerning the major fertility restorer genes Rf3 and Rf4, using gene-specific PCR-based markers, while background selection was done using a set of 61 and 64 parental polymorphic SSR markers respectively. Backcross derived lines possessing either Xa21 or Xa33 along with maximum genome recovery of DRR17B were identified at BC3F1 generation and selfed to develop BC3F2s. Plants harboring Xa21 or Xa33 in homozygous condition were identified among BC3F2s and were intercrossed with each other to combine both the genes. The intercross F1 plants (ICF1) were selfed and the intercross F2(ICF2) plants possessing both Xa21 and Xa33 in homozygous condition were identified with the help of markers. They were then advanced further by selfing until ICF4 generation. Selected ICF4 lines were evaluated for their resistance against BB with eight virulent isolates and for key agro-morphological traits. Six promising two-gene pyramiding lines of DRR17B with high level of BB resistance and agro-morphological attributes similar or superior to DRR17B with complete maintenance ability have been identified. These lines with elevated level of durable resistance may be handy tool for BB resistance breeding.Department of Biotechnology, Government of India Grant number: BT/PR11705/AGR/02/646/200

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Not AvailableImproved-Samba-Mahsuri (ISM), a high-yielding, popular bacterial blight resistant (possessing Xa21, xa13, and xa5), fne-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 specifc for Pup1 was designed and used for foreground selection along with functional markers specifc 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 fanking 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 ICARIIRR, 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.Not Availabl

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Not AvailableMTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F1 and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC2F1, the best backcross plant was selfed to generate BC2F2s. Among them, the plants homozygous for Pup1 (n = 22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC2F6 generation. A total of five elite BC2F6 lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (> 390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.Not Availabl

Novel major QTLs associated with low soil phosphorus tolerance identified from the Indian rice landrace, Wazuhophek

Not AvailableWith an objective of mapping novel low soil P (Phosphorus) tolerance loci in the non-Pup1 type donor rice line, Wazuhophek, we screened a recombinant inbred line (RIL) mapping population consisting of 330 lines derived from the cross Wazuhophek x Improved Samba Mahsuri (which is highly sensitive to low soil P) in a plot with low soil P for tolerance associated traits. Molecular mapping with SSR markers revealed a total of 16 QTLs (seven major and nine minor QTLs), which are associated with low soil P tolerance related traits. Interestingly, a QTL hotspot, harbouring 10 out of 16 QTLs were identified on the short arm of chromosome 8 (flanked by the makers RM22554 and RM80005). Five major QTLs explaining phenotypic variance to an extent of 15.28%, 17.25%, 21.84%, 20.23%, and 18.50%, associated with the traits, plant height, shoot length, the number of productive tillers, panicle length and yield, respectively, were located in the hotspot. Two major QTLs located on chromosome 1, associated with the traits, total biomass and root to shoot ratio, explaining 15.44% and 15.44% phenotypic variance, respectively were also identified. Complex epistatic interactions were observed among the traits, grain yield per plant, days to 50% flowering, dry shoot weight, and P content of the seed. In-silico analysis of genomic regions flanking the major QTLs revealed the presence of key putative candidate genes, possibly associated with tolerance.Council of Scientific & Industrial Research (CSIR)-Human Resource Development Group (HRDG), URL of funder website:- https://www.csirhrdg.res.in/Home/Index/1/Home/1459/