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MOLECULAR PROFILING OF INTROGRESSION LINES DERIVED FROM CROSSES OF O. sativa (L.) X O. longistaminata (A.CHEV. &ROEHR.)
(PAU Ludhiana, 2012) Nguyen Le Van; Kuldeep Singh
Rice (Oryza sativa L.) is staple food for more than a third of the world’s population, and we need to produce 30% more rice to meet the demand in next 25 years. Utilization of wild species for enhancing productivity is one of the several approaches proposed for meeting food requirements. In this study we evaluated a number of BC2F6 introgression lines (ILs) generated from the crosses between O. sativa cv PR114 X O. longistaminata acc IRGC 104301 and using DNA markers identified regions introgressed from the wild species. A set of 45 ILs and three checks were evaluated in a complete randomized block design with three replications for two years. Data were recorded on 11 agronomic traits. Several ILs showed significant increase or decrease over the recurrent parent in yield components like plant height (PH), days to flowering (DF), tiller number (TN), panicle length (PL), spikelet per panicle (SPP), spikelet fertility (SF), grain length (GL), grain width (GW), thousand grains weight (TGW) and plot yield (PY) compared to the recurrent parent PR114. Ten ILs showed significant increase in SPP during both the years over the recurrent parent and the increase ranged from 9.3 – 41.0 per cent. Likewise, five ILs showed significant increase in grain length ranging from 4.95 – 10.1 per cent, two ILs showed significant increase (16.0%) in TGW, and nine ILs showed significant increase (6.4-24.1%) in grain width. Parent polymorphism between PR114 and donor species O. longistaminta was done using a set of 366 SSR markers spanning all the 12 linkage groups and 92 polymorphic SSRs were used for characterizing the ILs. The alien segments introgressed in each of the ILs varied from 1.09% to 13.04%. Alleles which showed positive effect from O. longistaminata were observed in chromosomal regions associated with DF, PL, SPP, GL and GW traits. Alleles associated with negative effects were also observed for PH, TN, SF, PY and grain size traits. The ILs showing significant increase for one or more traits have been identified and these could be used for mapping QTL introgressed for these traits, using bi-parental crosses.
Open Access
MARKER ASSISTED INTROGRESSION OF THE opaque2 (o2) GENE INTO ELITE MAIZE (Zea mays L.) INBRED LINES
(PAU Ludhiana, 2012) Ravneet Kaur; Yogesh, Vikal
Maize (Zea mays L.) is deficient in the essential amino acids, lysine and tryptophan. The low nutritive value of maize endosperm protein is genetically corrected in Quality Protein Maize (QPM), which contains the opaque 2 (o2) gene along with numerous modifiers for kernel hardness. Two normal inbred lines viz. LM12 and LM13 were targeted for conversion into high quality protein versions using gene based SSR markers located within o2 using two generation marker-based backcross breeding program. DMR7 and CML165 were used as the QPM donor parents. In BC2F1 foreground selection for o2 gene was done using SSR marker phi057 in cross 1 (LM12/DMR7//2*LM12) and with SSR marker umc1066 in cross 2 (LM13/CML165//2*LM13). Out of the total 188 BC2F1 plants of cross 1, 87 heterozygous plants were obtained and similarly in cross 2, 114 heterozygous individuals were obtained out of 206 plants. The BC2F1 plants having o2 allele in heterozygous form were further screened using o2 gene flanking SSR marker and the plants either single or double recombinants, were identified. Whole genome background selection on the single or double recombinants using 104 SSR markers identified three plants with 83.7 to 91.0% recurrent parent genome content in each cross and were selfed to generate BC2F2 population. The three BC2F2 families were subjected to foreground selection and phenotypic selection for kernel modification. Fifty six plants in cross 1 and thirty nine plants in cross 2 having o2 allele in homozygous condition were selfed to generate BC2F3 progenies. The kernels from BC2F2 ears were segregated for hardness of endosperm and showed different levels of modification. In BC2F2 kernels the tryptophan concentration ranged from 0.56% to 0.97% for cross 1 whereas for cross 2 it ranged from 0.58% to 0.91%. 50% opaque BC2F3 lines were evaluated for agronomic traits for selection of single plant progenies to generate BC2F4 progenies.
Open Access
CYTOGENETIC CHARACTERIZATION AND MOLECULAR TAGGING OF LEAF RUST RESISTANCE GENE TRANSFERRED FROM Aegilops peregrina INTO HEXAPLOID WHEAT
(PAU Ludhiana, 2012) Deepika Narang; Satinder Kaur
Leaf rust is the most damaging disease of wheat worldwide. Wild germplasm of wheat is known to be a rich source of different useful genes. Ae. peregrina acc. pau 3519, a non-progenitor tetraploid species with UUSS genome, was found to be an excellent source of resistance for various diseases. In the present investigation, WL711-Ae. peregrina introgression lines (ILs) were characterized through Genomic in situ hybridization and no hybridization signal was detected in any of the IL. F2 population derived from the cross of introgression line 973 with WL711, was tested at seedling stage and segregated in a ratio of 191 resistant:63 susceptible plants with 2=0.0052(3:1). At the adult plant stage, the population segregated into 185 resistant: 65 susceptible with 2=0.0053(3:1). The progeny testing in F3 confirmed the transfer of a single gene for leaf rust resistance. Molecular characterization of the IL973 with the SSR markers indicated Ae. peregrina specific introgression on homoeologous groups 1, 2, 5 and 7. Bulked segregant analysis was done using 79 polymorphic markers and one of the SSR marker Xcfd50, detected IL973 specific allele in resistant bulk. This marker has already been reported to be linked with known gene Lr58 from Ae. triuncialis on long arm of chromosome 2B. STS marker Xncw-Lr58 amplified on whole F2 population and it was mapped at a distance of 1.7 cM from leaf rust resistance gene using computer software MapDisto. Nullitetrasomic analysis confirmed the introgression of Ae. peregrina leaf rust resistance gene (LrAP) on the wheat chromosome 2D. LrAP gene studied during the present investigation is either a new gene or an allele of the already known gene Lr58 will be established through allelic tests in subsequent studies.
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Molecular cytogenetic characterization of wheat- Aegilops umbellulata leaf rust and stripe rust resistant introgression lines
(PAU Ludhiana, 2011) Mitaly Bansal; Parveen, Chhuneja
Leaf rust and stripe rust are the most damaging diseases of wheat. Ae. umbellulata, was found to be an excellent source of resistance for various diseases. In the present investigation, characterization of the WL711-Ae. umbellulata introgression lines (ILs) through Genomic in situ hybridization to determine the size of the alien segment and Bulked Segregant Analysis (BSA) for tagging of stripe rust and leaf rust resistance genes introgressed from Ae. umbellulata into Triticum aestivum were undertaken. GISH showed the presence of complete Ae. umbellulata chromosome in ILs T311-5 and T315-5 whereas no hybridization signal was detected in other ILs. BC2F4 population derived from the cross of introgression line T393-4 with PBW343 segregated for a leaf rust (103R:66H:77S; 2=4.102) and a stripe rust resistance gene (108R:64H:74S; 2=6.401). The progeny testing in BC2F5 confirmed the transfer of a single gene for leaf rust resistance. BSA using 31 polymorphic markers indicated the association of short arm of chromosome 5D with rust resistance. The 5DS mapped SSR markers Xgwm190 and Xwmc805 detected parental IL specific alleles in resistant bulk. In order to distinguish the leaf rust and stripe rust resistance gene transferred from Ae. umbellulata (LrU and YrU) from previously mapped Lr57/Yr40 at same location, the Lr57/Yr40 CAPS primer was amplified and the amplicons were sequenced. A number of SNPs were detected which could distinguish two introgressions carrying Lr57/Yr40 and LrU/YrU. Linkage mapping using computer software MapDisto mapped leaf rust gene on 5DS with Lr57/Yr40 CAPS marker at a distance of 2.5cM. Stripe rust resistance gene mapped towards the distal end of 5DS at a distance of 4.1cM from the LrU. LrU/YrU carrying alien introgression was further distinguished from Lr57/Yr40 introgression, through the identification of SNPs in the grain softness protein specific amplicons. The LrU and YrU are proposed to be putatively new genes which provide complete resistance against leaf rust and stripe rust, respectively.
Open Access
MOLECULAR CYTOGENETIC CHARACTERIZATION OF WHEAT- Aegilops umbellulata LEAF RUST AND STRIPE RUST RESISTANT INTROGRESSION LINES
(PAU Ludhiana, 2011) Mitaly Bansal; Parveen, Chhuneja
Leaf rust and stripe rust are the most damaging diseases of wheat. Ae. umbellulata, was found to be an excellent source of resistance for various diseases. In the present investigation, characterization of the WL711-Ae. umbellulata introgression lines (ILs) through Genomic in situ hybridization to determine the size of the alien segment and Bulked Segregant Analysis (BSA) for tagging of stripe rust and leaf rust resistance genes introgressed from Ae. umbellulata into Triticum aestivum were undertaken. GISH showed the presence of complete Ae. umbellulata chromosome in ILs T311-5 and T315-5 whereas no hybridization signal was detected in other ILs. BC2F4 population derived from the cross of introgression line T393-4 with PBW343 segregated for a leaf rust (103R:66H:77S; 2=4.102) and a stripe rust resistance gene (108R:64H:74S; 2=6.401). The progeny testing in BC2F5 confirmed the transfer of a single gene for leaf rust resistance. BSA using 31 polymorphic markers indicated the association of short arm of chromosome 5D with rust resistance. The 5DS mapped SSR markers Xgwm190 and Xwmc805 detected parental IL specific alleles in resistant bulk. In order to distinguish the leaf rust and stripe rust resistance gene transferred from Ae. umbellulata (LrU and YrU) from previously mapped Lr57/Yr40 at same location, the Lr57/Yr40 CAPS primer was amplified and the amplicons were sequenced. A number of SNPs were detected which could distinguish two introgressions carrying Lr57/Yr40 and LrU/YrU. Linkage mapping using computer software MapDisto mapped leaf rust gene on 5DS with Lr57/Yr40 CAPS marker at a distance of 2.5cM. Stripe rust resistance gene mapped towards the distal end of 5DS at a distance of 4.1cM from the LrU. LrU/YrU carrying alien introgression was further distinguished from Lr57/Yr40 introgression, through the identification of SNPs in the grain softness protein specific amplicons. The LrU and YrU are proposed to be putatively new genes which provide complete resistance against leaf rust and stripe rust, respectively.
Open Access
MEAN AND VARIANCES AMONG F4 PROGENIES AND THEIR PREDICTION FROMPARENTAL MEANS AND GENETIC DISTANCES BASED ON MOLECULAR MARKERS IN Gossypium arboreum L.
(Punjab Agricultural University, Ludhiana –, 2011) Jamwal, Navdeep Singh; Gumber, R.K.
The objective of the present study was to see whether genetic distance based on SSR markers (GDSSR) and Mahalanobis D2 statistics (GDDy), parental means ( P1 + P2 /2) and absolute difference of parental means (| P1 - P2 |) can be used for predicting the means and variances of F4 populations derived from 52 crosses of Gossypium arboreum L. Fifty-two F4 populations along with 17 parents were evaluated in a randomized complete block design using three replications during Kharif 2009. Data were recorded for seed cotton yield (g), number of bolls, boll weight (g), seed index (g), halo length (mm), ginning out turn (%) and plant height (cm). Fifty-seven SSR primers belonging to BNL, MUSS, MUCS, MUSB, CIR, NAU and MGHES series detected 74 alleles in 17 arboreum lines. Total number of bands ranged from 1-3 with an average of 1.29 bands per primer. Polymorphism information content (PIC) ranged from 0.00 to 0.88 with a mean of 0.12 for all 57 primers. Maximum genetic distance based on SSR markers was observed between LD866 and AH11(0.059), whereas minimum genetic distance recorded was 0.00 between lines LD327 and MDL2643; LD694 and DLSa1001. The genetic similarity coefficient among 17 genotypes ranged from 1.00 to 0.94 with an average genetic similarity of 0.97. The maximum genetic distance (280.63) based on Mahalanobis D2 statistics was observed between LD694 and RG395, whereas minimum of 2.49 between lines LD866 and RG8. No association was found between parentage and geographical origin of parental lines with genetic distance among parental lines revealed by both the estimates of genetic divergence. Poor correlation (r=0.06) between both estimates of genetic distance GDSSR and GDDy was observed. The means of F4 populations can be predicted successfully from the means of the parents for seed cotton yield (r=0.40) and halo length (r=0.273). Likewise, | P1 - P2 | and GDSSR proved to be good predictor of F4 population means for boll weight (r=0.273) and ginning out turn (r=0.290), respectively. However, the prediction of F4 variance for most of characters except plant height (r=0.277) from different properties of parental lines still remains an unsolved problem.
Open Access
PROFILING AND MOLECULAR TAGGING OF GLUCOSINOLATES AND TOCOPHEROLS IN BRASSICA JUNCEA
(Punjab Agricultural University, Ludhiana, 2011) Shilpa
The present study was aimed at profiling and molecular tagging of glucosinolates (leaves and seeds) and tocopherols (seeds) in B. juncea germplasm collection maintained at PAU, Ludhiana. Recombinant inbred line (RIL) population comprising F6 and F7 progenies, (developed by crossing NUDH-YJ-04; low glucosinolate European juncea and RL-1359; high glucosinolate Indian juncea, were evaluated during years 2008-2011 in Oilseeds section, Department of Plant Breeding and Genetics, for biochemical traits viz. oil quality (i.e. tocopherols) and meal quality (i.e. glucosinolates) which were later used for molecular tagging. In the first year, approximately 366 RILs were screened for total glucosinolate content in the seeds and out of these, 97 lines were selected on the basis of range observed for total glucosinolate content. In these 97 lines, values for total glucosinolates in seeds and leaves, total tocopherol and oil content in seeds ranged from 15.73-127.61 (µmole/g DW seed), 4.33-129.93 (µmole/g DW leaf), 32.29-1250 (ppm) and 34.96-45.00 (%) respectively. Individual components (GNA, SIN, IBE, NAS and NEO) in seeds and (GNA, SIN and EPIPRO) in leaves, obtained from profiling ranged from (2.36-90.87 µmole/g DW seed), (0.194-35.16 µmole/g DW seed), (0.117-10.93 µmole/g DW seed), (0.0-6.10 µmole/g DW seed), (0.0-0.99 µmole/g DW seed) and (0.653-73.04 µmole/g DW leaf), (0.089-4.85 µmole/g DW leaf) and (0.65-16.37 µmole/g DW leaf) respectively. Biochemical data generated from these evaluations was used for molecular tagging. SSR markers (414) were evaluated on parents and out of these, 100 markers were found to be polymorphic which were then screened on 92 RILs. 100 polymorphic markers gave 106 loci, out of which 82 were found to be linked. A framework linkage map of 1718.9 cM length with 20 linkage groups was developed from 82 linked loci. From this linkage map, a QTL map has been developed which gave four QTLs with total glucosinolates as significant QTL (LRS-14.5, R2-15%) and GNA (LRS-11.2, R2-11%), NAS (LRS-12.2, R2-13%) and total tocopherol (LRS-11.0, R2-11%) as the suggestive QTLs. Total glucosinolates and total tocopherols were found to be NUDH-YJ-04 specific alleles and GNA and NAS as RL-1359 specific alleles. More number of significant QTLs with higher R2 values can be obtained by using higher number of polymorphic markers which will generate a highly saturated linkage map. In future, our study can act as a skeleton for tagging and MAS of economically important traits.
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IDENTIFICATION OF MOLECULAR MARKER(S) ASSOCIATED WITH FIBRELESS TRAIT IN Gossypium arboreum L.
(Punjab Agricultural University, Ludhiana, 2011) Baljinder Kaur
The present investigation was carried out with the objectives (i) to study inheritance of fibreless trait in the cotton fibre mutant PAUFL1 and (ii) identification of molecular marker(s) linked to the fibreless trait. The experimental plant material consisted of parental lines viz. fuzzless, lintless and trichomeless mutant PAUFL1 and wild type parent LD902 with normal fuzz, lint and trichome development, F1, F2 population and respective backcross generations. F1 and BC1 (F1 x LD902) plants were observed to possess both fuzz and lint indicating that allele for fibre development was dominant over the allele governing fibreless condition. Of the 296 F2 plants, 230 plants were observed to be fuzzy and linted, whereas rest of the 66 plants was devoid of fibre (lint and fuzz). F2 population segregated into monogenic 3 fibred:1 fibreless ratio. The backcross population with mutant parent PAUFL1 did not deviate from ratio of 1:1 (fibred : fibreless) expected for a monogenic trait. Of the 124 plants, 66 plants were fibre bearing and rest 58 plants were observed to be fibreless. Monogenic recessive control of the fibreless trait in PAUFL1 is suggested. F1 hybrid and its wild type parent LD902 did not differ for span length, uniformity ratio, elongation, fibre stength and micronaire values. PAUFL1 did not bear any trichomes, whereas, in LD902, an average of 92.2 and 155.6 trichomes were counted on mature and young leaves respectively. In case of F1 plants, mean trichome number for young leaf was 118.4 and 53.5 for mature leaf. Mean trichome density for mature and young leaves was 47.7 and 104.2 respectively in F2 generation. A strong positive correlation between trichome density on young and mature leaf was observed. Deviation from normal distribution was recorded in distribution of leaf trichomes in young as well as mature leaf in F2 plants. The trichomeless condition co-segregated with the fuzzless and lintless condition. It is suggested that either the genes controlling fuzzless, lintless and leaf trichomless state are tightly linked or these three traits are under the control of a single gene which is involved in both fibre and leaf trichome development. 351 SSR primers were used for the molecular characterization of fibreless trait. Of the 40 primer pairs observed to be polymorphic between the parental lines, ten primers were found to be uncommon between the F2 bulks. Further, 190 individual F2 plants were genotyped using these primers. None of the primers showed association with the fibreless and trichomeless trait. Desi
Open Access
Somatic embryogenesis and genetic transformation of sugarcane variety CoJ 64 using Cry1A(c) gene
(Punjab Agricultural University, Ludhiana, 2011) Palvi Malik
The present investigation entitled, “Somatic embryogenesis and genetic transformation of sugarcane variety CoJ 64 using Cry1A(c) gene” was undertaken on a high sugared variety of sugarcane viz., CoJ 64. Maximum per cent somatic embryogenesis and regeneration was observed on MS medium supplemented with NAA (5 mgL-1) + Kin (0.5 mgL-1) + sucrose (3%) + agar (8 gL-1), i.e. 79.63 and 75.17, respectively. Changing the agar (8 gL-1) concentration to 16 gL-1 in above medium improved somatic embryogenesis (84.87%) and regeneration (80.68%). Sucrose replaced with maltose significantly increased the frequency of somatic embryogenesis (97.61%) and plant regeneration (93.74%). Use of glutamine also enhanced per cent somatic embryogenesis (89.97) and percent plant regeneration (85.82). Activated charcoal (1.0 gL-1) along with citric acid (100 mgL-1) controlled the problem of browning of medium and also promoted somatic embryogenesis (90.33%) and plant regeneration (86.19%). For genetic transformation, particle gun method was employed. The already standardized parameters were used for carrying out bombardments. The embryogenic regions of cultured leaf rolls were used as the target tissue and co-bombardment was done using two plasmids: one carrying gusA and hpt genes, and the other carrying Cry1A(c) gene. The selection of bombarded embryogenic leaf segments was carried out on MS + NAA (5 mgL-1) + Kin (0.5 mgL-1) + Sucrose (3%) + hygromycin B (40 mgL-1). Out of 860 embryogenic leaf segments bombarded, 96 putative transgenic plants were obtained. Histochemical GUS assay revealed two GUS-positive plants (2.08% expression). PCR analysis of all the putative transgenics with a gene-specific primer confirmed the integration of the gene in 24 plants (25.00% expression). The plants were hardened and successfully transferred to the glasshouse.

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