6.4.1.1 Understanding of the molecular mechanism of leaf rust resistance in wheat
MicroRNA of wheat that suppressed important genes of wheat host and its pathogen (Puccinia triticina)
were identified to understand the role of miRNA in host pathogen interaction for leaf rust disease.
Differentially expressed miRNA were identified in wheat NILs carrying leaf rust resistant gene Lr28.
6.4.1.2 Exploration of heat stress-responsive markers in wheat
A total of 243 novel SSRs, developed from heat stress-associated genes identified through RNA- seq, were used for marker-trait associations. Out of 37 SSRs which exhibited polymorphism, 27 SSR loci were significantly associated with component traits of heat stress (HS) tolerance. Expression analysis revealed that six out of seven selected genes were induced under HS in the wheat cultivars WH 1021 and Raj 3765 (thermotolerant) and repressed in HD 2009 (thermosusceptible). The information on candidate genes based SSR markers will help the breeders in precise development of heat tolerant genotypes through MAS.
6.4.2 Maize
6.4.2.1 Validation of candidate genes for retention of carotenoids during storage
Expression level of the CCD1 gene in two genotypes viz, V335PV (low retention) and HKI161PV (high retention) showed an increasing trend in the CCD1 experession first three months of storage, followed by a decreasing trend in the subsequent two months. The correlation between the expression level of CCD1 gene and retention potential for pro-vitamin A was significant, with higher expression of CCD1 associated with the loss of proA. Lipoxygenase activity did not correlate with loss of pro-vitamin A. Transcript expression level of LOX3 and putative Orange gene though varied between V335PV and HKI161PV, the expression level did not show association with retention of pro-vitamin A.
6.4.2.2 Development of breeder-friendly gene- based marker for sugary1 gene
Sugary1 (su1) based sweet corn cultivars are popular worldwide. The entire su1 gene was sequenced among
six sugary type and five wild type inbreds using 27 overlapping primers. A 36-bp InDel (at position 1247) in the promoter region and a 6-bp InDel (at position 6456) in intron-10 were predicted to have SRp40 exon- splicing enhancer. Nucleotide substitution in exon-2 at position 2703 (SNP-2703) was involved in C to G mutation leading to conversion of phenylalanine to leucine. The 6-bp and 36-bp InDels and SNP-2703 were used to develop three breeder-friendly codominant markers, (i) SuDel6-FR, (ii) SuDel36-FR and (iii) SNP2703-CG-85/89. All three markers were validated in five F2 populations, and SuDel36-FR and SNP2703- CG-85/89 were validated in a set of 230 diverse inbreds.
This is the first report of development and validation of universal functional markers for su1. These markers assume great significance in marker-assisted breeding programme.
6.4.2.3 Screening and identification of inbred lines for nutrient use efficiency
Forty-eight maize inbred lines were exposed nitrogen and phosphorous starvation in hydrophobic condition. The inbreds LM 13 and MG 13 showed contrasting morpho physiological responses for nitrogen starvations. Whereas, the inbred lines LM 14 and PML 47 showed contrasting responses under phosphorus starvation. Further, MG 13 and PML 47 showed higher nitrate accumulation and increased root acid phosphatase activity, respectively.
6.4.3 Diversity analysis and allele mining of DHN gene from chickpea
Candidate gene based mapping was used to comprehend the genes governing drought tolerance.
Seven candidate genes viz., ASR, ERECTA, SPS, MYB, AMADH, AKIN and DHN were sequenced across forty chickpea genotypes using the BigDye terminator v3.1 kit. Sequencing data was analysed in Sequencing Analysis v5.4. The sequence similarities were confirmed using NCBI-BLAST (BLASTN) and aligned using CLUSTALW (http://www.ebi.ac.uk/
Tools/clustalw2/index.html). nSequence variants were analyzed using Sequencer 5.4.6 software and 1079 SNPs
were identified in total. Highest numbers of SNPs (517) were identified in SPS gene while the lowest numbers of SNPs (14) were identified in DHN gene.
6.4.4 Development of novel EST-SSR markers in lentil
Lentil is nutritionally important crop for human diet and enriched with quality protein, complex carbohydrates, fibres, essential minerals and vitamins.
However, genetic improvement of lentil is hampered largely due to limited use of genetic and genomic resources. To administer genomic resources in lentil, we identified 9949 EST-SSR loci from lentil RNASeq data and validated 50 of them using 234 genotypes representing various Lens species and 34 accessions of 12 different legumes. Out of 50 EST-SSRs, 46 were polymorphic with polymorphic information content (PIC) ranging from 0.16-0.74. The transferability of these markers exhibited varied levels from 45.1 to 71.3%
across the cultivated/wild species of lentil and from 10.8 to 54.3% across the twelve legume genera. On the basis of total identified EST-SSRs, mononucleotide (51%) repeat proportion was high followed by trinucleotide (30%) and dinucleotide (14%) repeat. Population structure and cluster analysis classified all the studied genotypes into 4 groups. Principal component analysis (PCA) grouped the genotypes based on their area of collection. Annotation of all the 46 polymorphic marker sequences revealed that most of the markers linked to genes involved in metabolism of plants. Further, polymorphic markers were also used for linkage
mapping in F3 population where 4 markers were found to be linked with a map distance of 72.5 cM. The newly developed markers will be useful for characterization of germplasm, genetic linkage mapping, phylogenetic studies, as well as to determine disparity in taxonomic status of subspecies of the genus Lens.
6.4.5 Mustard
6.4.5.1 Trait improvement and creating genetic variability through re-synthesis of Brassica juncea
To infuse genetic variability from related species B. juncea derived B. carinata introgression lines were developed. These fixed ILs were again crossed with improved B. juncea genotypes and a total of 191 progenies from F6 generations were raised under both irrigated and rainfed conditions for characterization.
Multiple/ three way interspecific crosses involving D. erucoides/ B. rapa//B. juncea derived introgression lines were used to improve B. juncea genotypes and F3/ BC1F1 generation were raised. For re-synthesis of B.
juncea six crosses were attempted involving B. rapa acc.
NRCPB rapa8 and six different accessions of B. nigra and a total of 53 progenies were raised.
6.4.5.2 Development of STS marker for glucosinolates
Markers associated with phenotypic variation in total glucosinolate were GER1, GER5, At5g101, At5gAJ30, At5g41, At5g67, Myb28A9, Myb28B1 and CYP79F1 were reported. Out of these markers viz.,
Marker GER1 and GER5 showing polymorphism between low and high glucosinolates content genotypes (1- PM 21, 2- PM 22, 3- PM 29, 4- PM 30, 5- Heera, 6- PDZ 1, 7- PDZ 4, 8- PDZ 11, 9- Pusa Vijay, 10- Pusa Jagannath, 11- Laxmi, 12- RLC 2, 13- RLC 3, 14- RLC 6, 15- EC 597325, 17- Donskaja, 18- BioYSR)
GER1, GER5 Myb28A9, Myb28B1 and CYP79F1 were found to be controlling maximum phenotypic variance of total glucosinolates trait. The primers for these markers were developed using the orthologs from Arabidopsis. These primers either amplified multiple alleles or at times failed to amplify the desired allele in the individuals. To overcome this limitation, we developed STS markers for GER1 and GER5 by in-silico analysis and re-sequencing the genes from RLC3 and Pusa Jagannath. These STS markers were validated with the previous reported markers and no recombination frequency was observed.
6.4.5.3 Introgression of Genes/QTLs governing stress tolerance, yield and quality traits into elite cultivars
Several molecular markers linked with total glucosinolates, low erucic acid and white rust resistance in Brassica have been reported. To introgress white rust resistance using Donskaja as a donor, marker WR360360 was used for foreground screening, while FAE1.1P and FAE1.2P markers were used to track the genes for low erucic acid trait. Markers contributing major phenotypic variation for glucosinolates content were GER1, GER5 Myb28A9, Myb28B1 and CYP79F1.
These markers were found to be polymorphic between high glucosinolate containing recipient parents and low glucosinolate donors and used for foreground selection in back cross progenies to introgress low glucosinolates in elite nuclear backgrounds.
The BC3F2 population generated from cross PDZ1 X Pusa Jagannath was genotyped for glucosinolates and erucic acid linked molecular markers. Total 150 plants with all the desirable markers were then phenotyped for glucosinolates and erucic acid content in the seeds.
Total glucosinolates in the selected progenies ranged from 22 to 125 µmol/g of defatted seed meal cake.
Plants with double low quality traits, viz., less than 30 µmol/g glucosinolates and less than 2% erucic acids in oil were grown in plant to progeny rows to select desirable progenies for further yield trials.
6.4.7 Cauliflower
6.4.7.1 Expression analysis of anthocyanin genes in purple cauliflower ‘PC-1’
The expression of BoMYB1 gene was upregulated in both the lines with purple cauliflower, viz. PC-1 and ‘Graffiti’ as compared to DC-466, the white curded line. The expression of BoMYB2 gene was however, found to be slightly upregulated in PC-1’ but was downregulated in Graffiti. Both BoMYB3 and BoMYB4 genes were found to be substantially upregulated (29.14 and 9.80 fold, respectively) in Graffiti, while the former was down regulated in PC-1 by 0.47 fold BoMYB4 however, showed only 0.83 fold higher expression in PC-1 as compared to the white curded line DC-466. These observations indicate that different modes of regulations of anthocyanin pathway in PC-1 and Graffiti.
Screening of BC2F2 population derived from cross Pusa Jagannath (P2) X Donskaja (P1) for white rust resistant using markers WR360360.
6.4.7.2 Development of mapping population for Xcc black rot resistance in alien Brassicas
The F1 crosses in Brassica napus, viz., GSL-1 × Bn-2, GSL-15 × Bn-2, Zhang Suang × Bn-2 were raised and artificially challenged with Xcc race 1, 4 and 6. The all F1 s were found highly resistant indicating dominant nature of resistance gene. Single plant in each F1’s were selfed to produce F2 seed, Besides, 106 RILs of Brassica carinata (NPC-17 × NPC-9) were advanced to F8 generation for black rot resistant trait. These populations will be useful for mapping a novel A and B genome specific R gene(s) in alien Brassicas.
6.4.7.3 Marker-assisted backcross breeding
Through marker assisted breeding, black rot resistance gene (Xca1bo) and downy mildew resistance gene (Ppa3) were successfully transferred to Pusa Meghna background and five double gene homozygous backcross derived NILs were identified.
The genome recovery of five selected lines ranged from 74.3% to 92.3%. Among these lines, BC2F2:3-7-33 (Xca1boXca1bo/Ppa3Ppa3) had the highest yield with highest vitamin C content.
6.4.7.4 Development of haploids through isolated microspore culture
A large number of (>100) plants were developed through isolated microspore culture in Indian cauliflower. Ten F1 hybrids with diverse parents for wider adaptability, resistance to back rot (race-4) and
interspecific hybrids (B. cretica x B. oleracea var. botrytis) were used as donor plant for this purpose. Among the different developmental stages, bud size of 3.5-4.5 mm was found to be most responsive for microspore embryogenesis. Isolated microspore embryogenesis was successful using the earlier optimized NLN based medium with a shock treatment of 32.5 0C for 48 hours.
6.4.8. Cucumber
6.4.8.1 Development of haploids through gynogenesis
A tissue culture-based protocol for development of haploids through gynogenesis has been standardized in cucumber. Two gynoecious x gynoecious and three gynoecious x monoecious F1 hybrids were used as donor parent for gynogenesis. Using the earlier developed nutrient media composition and unopened female flower buds on the day of anthesis or one day after anthesis, we were able develop a large number of embryo like structures (ELS). We have also optimized a temperature shock treatment (2-4 days of cold at 4oC and 4-6 days of heat at 32.5oC) for generating large number of ELS.
6.4.8.2 Mapping population for extended shelf- life and high β-carotene content
Four F2 and Back-cross progenies were developed by utilizing novel genotypes, DC-48 (staygreen trait with extended shelf life) and AZMC-1 (high carotene content with orange flesh).
Expression of different BoMYB genes in DC-466’ (white curd), ‘PC-1’ (intense purple curd) and Graffiti (bright purple curd)
14
Expression of different BoMYB genes in DC-466’ (white curd), ‘PC-1’ (intense purple curd) and Graffiti (bright purple curd)
6.4.7.2 Development of mapping population for Xcc black rot resistance in alien Brassicas
The F1 crosses in Brassica napus viz., GSL-1 × Bn-2, GSL-15 × Bn-2, Zhang Suang × Bn-2 were raised and artificially challenged with Xcc race 1, 4 and 6. The all F1‟s were found highly resistant indicating dominant nature of resistance gene. Single plant in each F1‟s were selfed to produce F2 seed, Besides, 106 RILs of Brassica carinata (NPC-17 × NPC-9) were advanced to F8 generation for black rot resistant trait. These populations will be useful for mapping a novel A and B genome specific R gene(s) in alien Brassicas.
6.4.7.3 Marker assisted backcross breeding
Through marker assisted breeding, black rot resistance gene (Xca1bo) and downy mildew resistance gene (Ppa3) were successfully transferred to Pusa Meghna background and five double gene homozygous backcross derived NILs were identified. The genome recovery of five selected lines ranged from 74.3% to 92.3%. Among these lines, BC2F2:3-7-33 (Xca1boXca1bo/Ppa3Ppa3) had highest yield with highest vitamin C content.
6.4.7.4 Development of haploids through isolated microspore culture
A large number of (>100) plants were developed through isolated microspore culture in Indian cauliflower.
Ten F1 hybrids with diverse parents for wider adaptability, resistance to back rot (race-4) and interspecific hybrids (B. cretica x B. oleracea var. botrytis) were used as donor plant for this purpose. Among the different developmental stages, bud size of 3.5-4.5 mm was found to be most responsive for microspore embryogenesis. Isolated microspore embryogenesis was successful using the earlier optimized NLN based medium with a shock treatment of 32.5 0C for 48 hours.
6.4.8. Cucumber
6.4.8.1 Development of haploids through gynogenesis in cucumber
A tissue culture-based protocol for development of haploids through gynogenesis has been standardized in cucumber. Two gynoecious x gynoecious and three gynoecious x monoecious F1 hybrids were used as donor parent for gynogenesis. Using the earlier developed nutrient media composition and unopened female flower buds on the day of anthesis or one day after anthesis, we were able develop a large number of embryo like structures (ELS). We have also optimized a temperature shock treatment (2-4 days of cold at 4oC and 4-6 days of heat at 32.5oC) for generating large number of ELS.
1
2.05 1.86
0 0.5 1 1.5 2 2.5
DC-466 PC-1 Graffiti
Fold change in BoMYB1 expression
1 1.09
0.17 0
0.5 1 1.5
DC-466 PC-1 Graffiti
Fold change in BoMYB2 expression
1 0.53 29.14
0 5 10 15 20 25 30 35
DC-466 PC-1 Graffiti
Fold change in BoMYB3 expression
1 1.83
9. 80
0 2 4 6 8 10 12
DC-466 PC-1 Graffiti
Fold change in BoMYB4 expression
152
6.4.9 Carotenoid biosynthesis in bitter gourd
The expression of three different carotenoid biosynthetic genes viz. McPSY, McPDS and McCYP was studied in two the bitter gourd varieties (Pusa Rasdar and Pusa Vishesh) at 8, 12, 16, 20 and 24 DAP.
The expression of PSY gene was after 24DAP 57.9 fold higher in Pusa Rasdar at 24 days after pollination (DAP) in comparison to only 17.1 fold in Pusa Vishesh.
Similarly PDS gene expression also showed a sharp increase (8.6 fold) at 24 DAP in Pusa Raasdar as against only 2.7-fold in Pusa Vishesh. CYP gene expression was increased to 8.3 and 19.7-folds after 20 and 24
DAP respectively in Pusa Rasdar and 4.3 and 11.4- fold increase at these time points respectively in Pusa Vishesh.
6.4.10 Heat tolerance in chilli
Biochemical as well as gene expression analysis of heat tolerant genotypes (DLS-152-1, DLS-161-1, DLS- 10-2 and DLS-20-11) and susceptible genotypes (Chilli Kashmir Long, Jwalamukhi, Anugraha and Pusa Jwala) was carried out. Five biochemical characters like protein content, lipid peroxidation (malonaldehyde content), activities of enzymes like superoxide dismutase (SOD) and guaiacol peroxidase and proline accumulation as well as expression of seven different Heat shock protein genes, viz. CaHSP832, CaHSP703, CaHSP90, CaHSP3, CaHSP2272, CaHSP2271 and CaHSP was studied. Protein content and proline accumulation was higher in heat tolerant genotypes along with higher SOD and GPX activities as compared to heat susceptible genotypes. Significant difference between heat tolerant and susceptible group was found for the expression of HSP832, HSP703 and HSP2272. Expression of these genes was significantly upregulated in the highly heat tolerant lines under heat stress.
6.4.11 Molecular diversity in onion
A total of 96 onion accessions from 17 countries were genotyped with 145 microsatellite markers.
Seven SSR markers (46.7%) showed PIC values higher than 0.5 and eight markers (53.4%) had PIC values less than 0.5. The microsatellite marker (ACM091) had the highest PIC value (0.715) and ACM463 had lowest PIC value (0.203). Ninety-six accessions were clustered into three main groups based on cluster analysis. Structure analysis also clustered the onion accessions into three groups. Group I was made up of thirty six (75%) exotic accessions and 25% mixtures of Indian genotypes. In group II, 32 accessions were grouped together out of which 21 (65.6%) accessions were of Indian origin and eleven (34.3%) were exotic accessions. Twenty-eight accessions formed group III out of which 20 accessions 6.4.8.2 Development of mapping population for extended shelf-life and high β -carotene content in cucumber.
Four F2 and Back-cross progenies were developed by utilizing novel genotypes, DC-48 (staygreen trait with extended shelf life) and AZMC-1 (high carotene content with orange flesh).
6.4.9 Carotenoid biosynthesis in bitter gourd
The expression of three different carotenoid biosynthetic genes viz. McPSY, McPDS and McCYP was studied in two the bitter gourd varieties (Pusa Rasdar and Pusa Vishesh) at 8, 12, 16, 20 and 24 DAP. The expression of PSY gene was after 24DAP 57.9 fold higher in Pusa Rasdar at 24 days after pollination (DAP) in comparison to only 17.1 fold in Pusa Vishesh. Similarly PDS gene expression also showed a sharp increase (8.6 fold) at 24 DAP in Pusa Raasdar as against only 2.7 fold in Pusa Vishesh. CYP gene expression was increased to 8.3 and 19.7 folds after 20 and 24 DAP respectively in Pusa Rasdar and 4.3 and 11.4 fold increase at these time points respectively in Pusa Vishesh.
Expression of Carotenoid biosynthetic genes in Pusa Rasdar and Pusa Vishesh at different DAP 6.4.10 Heat Tolerance in Chilli
Biochemical as well as gene expression analysis of heat tolerant genotypes DLS-152-1, DLS-161-1, DLS- 10-2 and DLS-20-11 in comparison to susceptible genotypes (Chilli Kashmir Long, Jwalamukhi, Anugraha and Pusa Jwala) was carried out. Five biochemical characters like protein content, lipid peroxidation (malonaldehyde content), activities of enzymes like superoxide dismutase (SOD) and guaiacol peroxidase and proline accumulation as well as expression of seven different Heat shock protein genes viz. CaHSP832, CaHSP703, CaHSP90, CaHSP3,
0 10 20 30 40 50 60 70
8DAP 12DAP 16DAP 20DAP 24DAP
Relative expression of Psy
Days after pollination Pusa Rasdar
Pusa Vishesh
0 5 10 15 20 25
8DAP 12DAP 16DAP 20DAP 24DAP
Relative expression of Cyp
Days after pollination Pusa Rasdar
Pusa Vishesh
0 2 4 6 8 10
8DAP 12DAP 16DAP 20DAP 24DAP
Relative expression of PDS
Days after pollination Pusa Rasdar
Pusa Vishesh
B
Expression of carotenoid biosynthetic genes in Pusa Rasdar and Pusa Vishesh at different DAP
(71.4%) were Indian genotypes and eight accessions (28.5%) belong to exotic origin. It was observed that grouping of genotypes was irrespective of the any morpho-agronomic traits.
6.4.12 Molecular diversity in carrot
Molecular diversity analysis of twenty four carrot genotypes (4 CMS lines and 20 inbreds) was carried out using 34 SSR markers. Maximum number of alleles were amplified by the primer GSSR101 (11) followed by GSSR130 (10) and BSSR43 (9). The average number of alleles obtained was 5.82. The mean value of observed heterozygosity and expected heterozygosity was 0.05 and 0.74, respectively. Highest observed heterozygosity (0.50) was noted in the primers GSSR 130 and BSSR 43 and highest expected heterozygosity (0.87) was observed in the primer BSSR 43, GSSR 35 and GSSR 51. Polymorphic information content (PIC) with a population mean of 0.83 was recorded highest in the primer BSSR 43, followed by GSSR 35 and GSSR 51.
6.4.13 Molecular diversity in okra
To study the genetic diversity among 96 okra genotypes, 65 pairs of microsatellite markers (SSRs) were used. Polymorphism among 96 okra genotypes was revealed by 50 primers and all these primer pairs were considered for this study. Analysis of allelic frequency revealed that overall 168 alleles were amplified through these 50 SSR primer pairs with a mean value of 3.36 alleles per locus. Number of alleles per locus varied from 2 to 7 (for primers AVRDC okra 21 and AVRDC okra 64). PIC value ranged from 0.05 (okra 105) to 0.71(AVRDC okra 64). Further, neighbor joining (NJ) cluster analysis revealed the formation of three major clusters and two outgroups (only IC- 218893 of A. esculentus in one group and IC- 265268 and EC- 762130 of A. esculentusin another group. Among the three major clusters, cluster-I comprising of 22 okra genotypes out of which all were cultivated types.
However, three genotypes EGR, DOV- 37 and Pusa Makhmali were clearly distinct from the rest of the genotypes. Two red fruited types (IC-1610753 and IC- 685583) of A. esculentus species were grouped together in a single clade.
6.4.14 Drosophila
6.4.14.1 Characterization of DWnt4 mutant alleles
To detect mutant lesions causing DWnt4 embryonic lethality, Next Generation Reseqencing of all seven mutations has been done. Putative SNPs were categorized as synonymous and non synonymous SNPs, which are further classified into Missense mutations, located in coding region or regulatory regions of the gene through bioinformatics analysis.
6.4.14.2 Expression of Patched protein in DWnt4AL7 embryos
Drosophila embryos is made up of para segments (PS) Patterning of PS borders in two adjacent cell rows is a function of interaction between wingless and hedgehog signalling pathways. Expression of other segment polarity genes depends upon crosstalk between these two signalling pathways. Disruption of any one of the gene results in change in expression of wingless in embryonic ectoderm. We checked expression patched protein in DWnt4AL7 homozygous null embryos.
Patched protein acts upstream of wingless pathway and after hedgehog reception patched act negatively on hedgehog expressing cells and causes activation of wingless expression. We previously reported expansion of wingless domain upon loss of DWnt4, but DWnt4AL7 mutation did not show change in expression pattern of patched, indicating that Patched is acting upstream of DWnt4 and hence no change in expression of patched is seen upon loss of DWnt4.
Expression pattern of DWnt4AL7 embryos in Drosophila