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Development of monoecious lines in muskmelon (Cucumis melo L.) through marker assisted selection
(Punjab Agricultural University, 2024) Nidhi; Sharma, Sat Pal
In heterosis breeding, monoecious sex expression offers significant advantages over andromonoecy in muskmelon, as it eliminates emasculation and produces fruits with desirable traits, such as earliness, high TSS, thick flesh, small scar size, and large fruit size. Thus, development of stable monoecious lines possessing desirable horticultural traits is critical for hybrid breeding in this crop. This investigation was aimed to identify molecular markers linked to monoecious trait and to transfer this trait to a popular melon cultivar through marker assisted selection. For this purpose, stability of monoecism was assessed under varying environments and identified 18 stable monoecious using the andromonoecious index. A set of identified lines was used to develop KASP markers for the sex-determining gene, CmACS-7, which revealed two mutations in exons 1 and 2, with one SNP at 170th, followed by insertion of ‘GA’ at 222nd nucleotide position. Upon validation, KASP-CT-2 accurately separated homozygous andromonoecious and monoecious parents and was also successfully validated on F2 population segregating for monoecious trait. Subsequently, marker-assisted backcross breeding was carried out to transfer the dominant gene 'A' controlling the monoecious trait into the Punjab Sunheri, a popular muskmelon cultivar. Heterozygous plants in BC1F1 and BC2F1 and homozygous plants in BC2F2 with high recurrent parent genome recovery and monoecious trait were advanced using the cleaved amplified polymorphic sequence (CAPS) marker. In the BC2F2 population, homozygous monoecious plants were identified using the CAPS marker. These plants were self pollinated to generate a BC2F3 progenies, of which 11 out of 13 lines had phenotypic similarities with the recurrent parents. These 11 lines were self pollinated to generate 34 BC2F4 progenies. Eight BC2F4 lines, viz. Mono-6R2, Mono-IOB1, Mono-2OB3, Mono-7R3, Mono-2OV2, Mono-1OB2 were selected based on TSS, TSS/TA ratio, β-carotene, low titratable acidity, pH, flesh thickness, fruit shape index, and yield. The information and developed associated molecular marker will be useful in developing new stable monoecious lines. Furthermore, the generated monoecious lines can potentially be utilized as a female parent for F1 hybrid development in muskmelon.
Open Access
Inheritance and molecular mapping of gene(s) associated with yellow vein mosaic virus disease (YVMVD) resistance in pumpkin (Cucurbita moschata Duch. ex Poir)
(Punjab Agricultural University, Ludhiana, 2022) Verma, Neha; Dhatt, Ajmer Singh
Worldwide begomoviruses causative diseases are major reason of quantitative and qualitative losses in pumpkin. The three types of viral symptoms were molecularly characterized as SqLCCV, ToLCNDV and their mixed infection (MI-Sq/To). Artificial screening, histopathological study and PCR confirmed PVR-1343 as a novel resistant source against both begomoviruses. To understand the inheritance and mapping of begomovirus resistant gene(s), segregating populations between PVR-1343 and five susceptible lines (P-135, P-6711, Punjab Samrat, MBN-6242 and P-278) were developed and artificially inoculated with Bemisia tabaci Asia II strain against both viruses and their mixed infection in enclosed structures for three consecutive years (2018-2020). Disease scoring at 75DAS rendered all F1 progenies as susceptible indicating recessive nature of resistance with no maternal or cytoplasmic effects. Chi-square analysis showed best fit of 9(S):3(MS):3(MR):1(R) and 1(S):1(MS):1(MR):1(R) ratio in F2 and backcross with resistant parent respectively. Phenotypic segregation ratio of F2:3 confirmed the F2 genotypic ratio of 1:2:2:4:1:2:1:2:1. This substantiated that multiple begomovirus resistance in PVR-1343 is controlled by a digenic recessive gene. Moreover, F2:3 progenies resistant to SqLCCV were also resistant to ToLCNDV and MI-Sq/To and vice versa, suggesting that common or tightly linked gene(s) are involved in resistance to both viruses. To map these genes, WGRS coupled with QTLseq of two extreme bulks (resistant and susceptible) of cross PVR-1343×P-135 along with parents were employed and two genomic regions viz., qMI-Sq/To7.1 on chromosome 7 (3.18 to 4.70Mb) and qMI-Sq/To17.1 on chromosome 17 (7.30 to 8.17Mb) were identified. Nine polymorphic SNPs identified within the highly significant qMI-Sq/To7.1 region were converted into KASP marker. The KASP genotyping of 177 F2 individuals narrowed down the qMI-Sq/To7.1 interval to a 103Kb region flanked by Cmo3914729 and Cmo4018182 KASP markers. One of the flanking SNP (Cmo4018182 KASP marker) imparting resistance against MI-Sq/To, accurately predicted the disease reaction of 91 per cent of diverse Cucurbita genotypes. This SNP showed nonsynonym substitution in the coding region of putative candidate syntaxin-121 protein and might be responsible for diminishing or abolishing the viral infections. This study has thus, seeded the path for marker assisted selection for efficient introgression of resistance into elite C. moschata lines and providing a viewpoint for gene cloning.
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Generation of frost tolerant potato (Solanum tuberosum L.) clones through intervarietal hybridization and expression analysis of putative frost tolerance genes
(Punjab Agricultural University, Ludhiana, 2022) Angmo, Dechen; Sharma, Sat Pal
The present study was carried out with an overall aim to generate frost tolerant potato clones and optimization of phenotyping techniques in order to complement the natural field selection criteria. The physiological responses to low temperature stress were explored using four potato genotypes viz. Kufri Anand, J-2/19, MS/7-645 and Kufri Pukhraj under in vitro controlled conditions. Significant variation was observed amongst genotypes for parameters associated with frost tolerance in potato such as cell membrane stability, activity of PSII, photosynthetic efficiency and leaf morpho-anatomical traits. The genotypes J/2-19 and Kufri Anand exhibited higher PN, gs and ΔF/Fm’values and lower electrolyte leakage as compared to the sensitive genotype Kufri Pukhraj upon cold acclimation and during recovery days after freezing exposure. Similarly, J-2/19 genotype also recorded higher adaxial trichome density than the sensitive genotype MS/7-645. In order to identify the fold changes in the expression of three candidate genes involved in cold stress responses in potato viz. dehydrin, hsp70 and SAD a qRT-PCR study was also performed. Overall an increase in gene expression was observed in cold acclimated plants of all the genotypes compared to non-acclimated plants. However, the increase was more pronounced in tolerant genotype (J-2/19) as compared with susceptible genotypes (MS/7-645 and Kufri Pukhraj). In the summer 2018, the segregating progenies were developed through hybridization among contrasting parents viz. J-2/19  MS/7-645 (PAU3), MS/7-645  J-2/19(PAU7) and MS/7-645  CP-3765 (PAU9) at Keylong, HP. In F1C1 and F1C2 generations, the characterization of the generated potato clones was accomplished on the basis of leaf morpho-anatomical traits and electrolyte leakage assay. To determine the association between the key traits and potential frost tolerant potato clones, various statistical approaches, such as cluster analysis, principal component analysis and genotype by trait biplot analysis were performed. Total 11, 10 and 8 clones from PAU3, PAU7 and PAU9 crosses, respectively were identified exhibiting potential frost tolerant characteristics with desirable horticultural traits for their further evaluation. Thus, the findings of this study concluded that in addition to electrolyte leakage the chlorophyll fluorescence and leaf gas exchange parameters can be effectively utilized for screening frost tolerant potato genotypes, but with certain limitations. Furthermore, the leaf morpho-anatomical traits have been confirmed to be associated with frost tolerance through decrease in foliage damage and electrolyte leakage. Therefore, these traits can potentially be used for screening of large segregating populations for freezing tolerance in the early clonal generations in potato.
Open Access
Introgression of yellow vein mosaic disease resistance in okra
(Punjab Agricultural University, Ludhiana, 2021) Gagandeep Singh; Pathak, Mamta
The present investigation was undertaken with the objectives to screen okra wild relatives, study inheritance of yellow vein mosaic disease, identification of marker(s) linked to YVMD resistance and to evaluate introgressed lines for economic traits. Two year consecutive artificial YVMD screening of sixteen entries of seven wild species and two varieties of cultivated okra (Punjab Padmini and Pusa Sawani) revealed Abelmoschus angulosus accessions IC-470751 and IC-203833 and Hibiscus acutetus accessions SBT-12557 and SBT-12592 highly resistant to YVMD. A. moschatus accession IC-140986, A. caillie and A. manihot (PAUAcc-1) exhibited moderately resistant reaction at 90 DAI. Whereas, A. moschatus accession IC-141056, A. manihot accession EC-329394, A. tuberculatus and cultivated okra variety Punjab Padmini manifested susceptible and cultivated okra variety Pusa Sawani highly susceptible reaction to YVMD up to 90 DAI. Deng Begomovirus specific universal primer was used to confirm the YVMD resistance. The YVMD resistant plants of A. angulosus (IC-203833) and A. manihot PAUAcc-1 were used as pollen parent for the development of F1, BC1F1, BC1F2 and BCIF3 population using Punjab Padmini as the female parent. Two recessive genes were found to govern resistance to YVMD as revealed by the appearance of susceptible and resistant plants in the ratio of 9:7 in the BC1F2 generation. Out of 200 SSR markers, 31.5% were found polymorphic between the two parents Punjab Padmini and A. manihot (PAUAcc-1). Four primer pairs viz. Okra 032, Okra 049, Okra 129 and Okra 270 differentiated the two contrasting YVMD resistant and susceptible bulks in bulk segregant analysis. A set of 137 BC1F3 progenies of Punjab Padmini x A. manihot (PAUAcc-1) were evaluated along with five checks (Punjab Padmini, Pusa Sawani, PORL, POL-44 and Punjab Suhawani) for yield and its contributing traits. Fifty seven progenies were found to be free from YVMD up to 90 DAI. Seventy four progenies having marketable yield in the range of 112.5 to 189.6 g/plant were observed to be significantly superior to standard check Punjab Suhawani (111.9 g/plant). Progeny PAUMIOL-56 was at top for yield (189.62 g/plant) followed by PAUMIOL-36, PAUMIOL-138, PAUMIOL-105 and PAUMIOL-142 and had no YVMD incidence upto 90 DAI. Similarly, 88 BC1F3 progenies of Punjab Padmini x A. angulosus (IC203833) were accessed along with seven checks (Punjab Padmini, Punjab Suhawani, PORL, POL44, POL-78 and Pusa Sawani) for economic traits. Six progenies recorded marketable yield in range of 108.2 to 130.2 g/plant significantly higher than standard check Punjab Suhawani (97.9 g/plant). Forty one progenies were free from YVMD up to 90 DAI. Progeny PAUAIOL-324 yielded maximum (130.2 g/plant) followed by progeny PAUAIOL-251, PAUAIOL-301, PAUAIOL-256 and PAUAIOL-283 and had no YVMD incidence up to 90 DAI.
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Molecular mapping and interspecific hybridization for hull-less seed trait in pumpkin for value addition in food processing industry
(Punjab Agricultural University, Ludhiana-, 2020) Barinder Kaur; Dhatt, Ajmer Singh
The present investigation entitled ‘Molecular mapping and interspecific hybridization for hull-less seed trait in pumpkin for value addition in food processing industry’ was conducted in the Department of Vegetable Science, Punjab Agricultural University, Ludhiana from 2016 to 2020. Segregating populations were developed using hulled and hull-less genotypes of Cucurbita pepo for understanding the genetics and molecular mapping of hull-less seed trait. Chi-square analysis of segregating populations revealed that single recessive gene is responsible for controlling this trait. The sequence based bulked segregant analysis of two extreme bulks (hulled and hull-less) of cross HP111 × HLP36 along with their parental lines identified one QTL on chromosome 12 (Cp4.1LG12) between 1891261 to 3863267 bp covering 1.97 Mb genomic region with 396 number of SNPs for hull-less seed trait. In interspecific hybridization, total 916 crosses between C. pepo (HLP36, HLP44, HLP53, HLP72) and C. moschata (HM1404, HM108, HM1343, HM1022, HM2211, HM6711) were attempted to transfer hull-less seed trait, however success was achieved in 5.57% crosses only. The fruit setting, number of seeds/fruit, seed germination and survival of F1 seedlings were high when C. pepo was used as female parent in cross HLP36 × HM1343 (66.67%, 15.00, 45.24% and 49.48%), followed by HLP36 × HM1022 (22.73%, 23.00, 44.35%, 47.06%) and HLP44 × HM1022 (13.79%, 12.00, 45.85%, 45.46%), respectively. However, in reciprocal crosses (C. moschata × C. pepo) success rate was very poor, which was overcome through in vitro ovule culture after 17 to 19 days of pollination in cross HM1343 × HLP53 and HM6711 × HLP72. The hybridity of putative Cucurbita interspecific hybrids was confirmed through molecular, morphological and micromorphological markers and their results were found to be highly consistent. The phenotyping of F2 populations of interspecific crosses confirmed the transfer of hull-less seed trait. Findings of the present study will facilitate in genetic dissection of candidate gene(s) and to enhance productivity, improve quality and multi-season cultivation of hull-seeded pumpkin in India.
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Marker assisted breeding for introgression of genetic male sterile ms10 gene from hot pepper to heat tolerant bell pepper
(Punjab Agricultural University, Ludhiana-, 2020) Menisha Rani; Jindal, Salesh Kumar
In the present investigation, marker assisted selection (MAS) strategy was employed to introgress genetic male sterile (GMS) ms10 gene from the donor parent MS-12 (Capsicum annuum var. annuum), popular GMS line, into the genetic background of eight heat tolerant bell pepper (C. annuum var. grossum) inbreds, to develop new GMS lines in bell pepper. The chi-square test (χ2) results of six F2 and BC1F2 populations showed that the ratio well fitted to the Mendelian ratio i.e. 3:1, validated the non-discriminated segregation of the male sterility (MS) trait possessing ms10 gene. The analysis confirmed that the MS trait is inherited by a single recessive gene. Further, the recently identified molecular marker ‘AVRDC-PP12’ linked to the ms10 gene was employed to test the segregation in backcross generations of bell pepper incorporated with the ms10 gene. A total of 576 and 360 individuals in BC1F1 and BC2F1, respectively, 72 plants of each BC1 and 60 plants of each BC2 were screened (foreground selection) for the ms10 gene by using SSR marker. Among the eight backcrossed populations screened, six were showed compatibility with the ms10 gene. The results were analyzed by χ2-tests to know whether the observed number of plants in each class fitted well to the anticipated genetic ratio of 1:1. The molecular marker in each backcrossed series displayed a good fit in-to the expected ratio of Mendelian segregation i.e. 1:1 for one gene in backcross population(s), which was non-significant at a probability level of 0.05, indicating the relationship with pepper male sterility ms10 gene. Simultaneously, targeted gene positive (Ms10ms10) non-pungent (pun1) heterozygous plant(s) were identified in BC2F1 generations using BF7 and BF9-SCAR marker. A total of 27 BC2F1 Ms10ms10 plants of MSSM-2 population were segregated in a 5: 22 non-pungent to pungent ratio, and 26 Ms10ms10 plants of MSSM-3 segregated in 3 (non-pungent): 23 (pungent) ratio. The rest of the four BC2F1 populations, MSSM-1, MSSM-9, MSSM-17 and MSSM-21 have pungent heterozygous male fertile (Ms10ms10 Pun1pun1) plant. The background recovery rate of the recurrent parent phenome (RPP) in BC2F1 progeny was estimated by measuring the shift in means for horticultural traits. Based on a paired 't' test analysis, out of six, the MSSM-1 and MSSM-2 generation was shown to be non-significant for all 19 studied traits followed by MSSM-3 (18), indicating a successful recovery of the recipient genome through marker-assisted backcross breeding after successful introgression of the ms10 gene. Finally, from this work six gene positive (ms10) GMS lines namely, MSSM-1, MSSM-2, MSSM-3, MSSM-9, MSSM-17 and MSSM-21 were obtained. The newly developed GMS lines will provide great economic value for non-pungent hybrid bell pepper seed production in the near future.
Open Access
Imparting root knot nematode resistance through grafting in tomato (Solanum lycopersicum L.)
(Punjab Agricultural University, Ludhiana, 2020) Sumeet Singh; Kulbir Singh
The present investigation was conducted at Vegetable Research Farm, Department of Vegetable Science, Punjab Agricultural University, Ludhiana for “Imparting root knot nematode resistance through grafting in tomato (Solanum lycopersicum L.)” during Autumn season of 2015-2016 and 2016-17. The work plan was divided into three experiments based on different grafting methods which further divided into two different sites i.e. infested with root knot nematode and non-infested soil. Ten rootstocks with combination of two different scions (Punjab Sartaj and Punjab Ratta) were used as experiment material. For infested plots pure culture of nematodes were raised by taking galled roots of tomato plants from infested plots than galls were teased and freshly hatched second stage juveniles were raised and used for inoculums. It was observed that plant and fruit characters like plant height, fruit weight, harvest duration, marketable yield as well as total yield increased in grafted plants over nongrafted plants both in nematode free as well as Nematode infested plots. Further it was also revealed that plants grafted with Tube graft method observed highest success rate and was even responsible for earliness as well as high marketable yield in tomato plants. On evaluation of rootstocks plants grafted on EC-119197 recorded highest plant height, higher total and marketable yield, higher fruit weight and prolonged harvest duration both in Nematode infested and nematode free plots. Whereas plants grafted on rootstock PNR-7 recorded early flowering as well as early picking in infested plots. Rootstock L-097 recorded maximum fruit TSS while plants grafted on rootstock EC-119197 recorded maximum pericarp thickness. No significant effect was observed when grafted and non-grafted plants were examined for lycopene content. Overall rootstock EC-119197 can be used as a resistant rootstock against root knot nematode in addition it can fetch higher yield and better fruit quality.
Open Access
Identification and characterization of salt tolerant genotypes and salt tolerant gene in brinjal (Solanum melongena L.)
(Punjab Agricultural University, Ludhiana, 2020) Talwar, Dilpreet; Kulbir Singh
The present investigation was conducted at vegetable research farm, Department of Vegetable Science, Punjab Agricultural University, Ludhiana for “Identification and characterization of salt tolerant genotypes and salt tolerant gene in brinjal (Solanum melongena L.)” during the year 2016-18. The experiment was laid out in split plot design with four salinity levels (control, 2 dS/m, 4 dS/m and 6 dS/m) as main factor and 102 genotypes of brinjal as sub main factor. The desired levels of salinity were induced using sodium chloride, magnesium sulphate and calcium chloride in ratio 2:1:1 in field as well as in pot study. These genotypes were categorised as 19 genotypes under tolerant, 39 as semi-tolerant and 44 were susceptible to salinity on the basis of nursery and morphological screening trials. Tolerant genotypes were further evaluated for physico-chemicals characters. It has been observed from the investigation that plant growth and fruit characters of brinjal i.e. plant height, leaf area, root fresh and root dry weight; number of fruits per plant, fruit yield decreased as the salinity level increased. The results showed that brinjal genotypes SL-8-PB-1-3-1-4 followed by KBSR-343-1 and BL-215 performed better upto 4 dS/m levels of salinity with a slight decline in yield traits, while at 6 dS/m yield reduction was high, which was 12%, 28% and 28.5% respectively as compared to control. The total chlorophyll and carotenoid pigments of brinjal leaves decreased as the salinity level increased. Whereas, proline, protein, phenols and MDA content increased as the salinity level increased with maximum increase in SL-8-PB-1-3-1-4 genotype. Brinjal genotype SL-8-PB-1-3-1-4, KBSR-343-1 and BL-215 performed better for germination and survival rate at all salinity levels. By using NCBI database, SOS-3 gene sequence for brinjal was identified. Designed primer was amplified and the resulted PCR product was sent to Europhin Company for identification of haplotypes in brinjal. In the present investigation, SL-8-PB-1-3-1-4 (Small Long), KBSR-343-1 (Small round) and BL-215 (Long) emerged as ideal genotypes for growing under high salinity in field as well as pot conditions. Quality of these genotypes found least affected under salt stress. Thus, three brinjal genotypes (SL-8-PB-1-3-1-4 (Small Long), KBSR-343-1 (Small round) and BL-215 (Long)) may be recommended for future studies in salt affected area of Punjab.
Open Access
Assessment of genetic diversity in carrot genotypes using morphological, biochemical and molecular markers
(Punjab Agricultural University, Ludhiana, 2018) Davinder Singh; Dhillon, Tarsem Singh
The study entitled “Assessment of genetic diversity in carrot genotypes using morphological, biochemical and molecular markers” was carried out at Vegetable Research Farm, Department of Vegetable Science, Punjab Agricultural University, Ludhiana. Eighty one genotypes of carrot were characterized by morphological, biochemical and molecular markers during 2015-16 and 2016-17. Analysis of variance showed significant differences for 19 quantitative traits with respect to treatment, year and treatment x year. Maximum total root yield was obtained from genotypes viz. PC-161, PC-15, PC-43 and PC-103 (red), PCO-30 and PCO-5 (orange) and PCP-2 and PCP-1 (purple). On the basis of morphological diversity, 81 genotypes were distributed into ten clusters. Cluster X was the best for root weight and anthocyanin content, cluster III for days to 1st root harvest and root girth, while dry matter content, total sugar content and carotene content were maximum in cluster V. The highest intra cluster distance has been observed from the cluster VII (162.9), while inter cluster distance was maximum between II and cluster X (43678.5). The cumulative proportion of variation explained by the four PC-axes was 85.12 %. Seventeen polymorphic SSR markers differentiated genotypes into three groups where cluster I consisted of 48 genotypes followed by cluster II with 28 genotypes and cluster III had 5 genotypes. The morpho-biochemical and SSR markers were effective tools to identify closeness among genotypes, so future strategy involves utilizing more SSR markers culled from various linkage mapping could be effectively and efficiently used to fasten the breeding programs and to design trait specific carrot breeding.

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