Castor is an industrial crop with diverse applications, including biodiesel production. However, its cultivation faces significant challenges from both biotic and abiotic stresses, with Fusarium wilt being a major disease. SNP markers linked to Fusarium wilt resistance and genotypic assays based on Kompetitive Allele Specific PCR (KASPTM) method are available in castor. However, the cost of introducing resistance genes into preferred lines through marker-assisted selection (MAS) is prohibitive, particularly when using KASP based genotyping. In this study, we developed an agarose-based Mismatch Amplification Mutation Assay (Agarose-MAMA) as a costeffective SNP genotyping method for selecting wilt resistance in castor breeding programs using standard laboratory equipment and reagents. We selected two SNP markers, Rc_29806-125126 on chromosome 6 and Rc_29706-482910 on chromosome 8, which are linked to wilt resistance in castor for designing and optimizing the MAMA assay. A comparative analysis of KASP and MAMA assays demonstrated similar performance in allele discrimination and genotyping accuracy. Furthermore, we successfully applied the Agarose-MAMA assay for MAS of wilt resistance in three breeding populations segregating for this trait. This approach circumvents the need for expensive equipment for SNP genotyping, thereby enabling broader adoption in research settings with limited resources. Overall, this study highlights the potential of Agarose-MAMA assay as a practical tool for SNPbased MAS, not only in castor but also in other crops.

Gray mold disease caused by Amphobotrys ricini is a prevalent disease in castor, reducing the yield by directly causing damage to the economic parts (inflorescence). Implementing host plant resistance breeding is considered as an effective approach for the management of gray mold. The utilization of molecular markers linked to gray mold resistance will serve as a valuable tool in the breeding program. In this study, a population of 156 F6 recombinant inbred lines (RILs) derived by crossing the susceptible line JC-12 with the moderately resistant line 48-1 was used for identification of Quantitative Trait Loci (QTL) linked to gray mold resistance. A major QTL flanked by the SNP markers Rc_29775-68167 and Rc_2994-41303 was mapped on chromosome-10, which explained 23.5 % of the total phenotypic variance (R2). Additionally, a F2 population derived from the cross RG1673 x 48-1 was used to validate the identified QTL. In the F2 population, the same QTL region was detected with LOD 8.3 and R2 value of 32.5 %. Furthermore, it was discovered that the QTL identified in this study is tightly linked to capsule spininess, which can be used as a surrogate trait for selection. In-silico analysis of genes underlying the QTL region predicted three putative genes namely methyltransferase, pyrroline-5-carboxylate reductase, and pentatricopeptide repeat-containing protein, that may play a potential role in gray mold resistance. These findings provide important insights into the genetic basis of gray mold resistance in castor and opportunities to improve the resistance against gray mold by phenotypic selection.

To understand the castor wax layer's multifaceted contribution to Amphobotrys ricini pathogenesis, the hydrophobic components of cuticular wax were analysed from waxy and non-waxy castor genotypes. Gas chromatography-mass spectrometry (GC-MS) technology enabled the stable detection and quantitative determination of various fatty acids and terpenoids. The investigation revealed a significant presence of triterpenoid compound 'lupeol', accounting for approximately 53.6% of the wax composition in the waxy genotype (DCH-519), which was absent in non-waxy genotype (ICS-324). On exposure to lupeol, about 93.3% of conidia germinated leading to rapid mycelium growth and sporulation of A. ricini. SEM analysis of waxy and non-waxy genotypes infected with A. ricini confirmed faster germination and production of longer germ tubes on waxy genotype compared with non-waxy genotype, which may likely due to early recognition of the suitable host in the presence of lupeol, ultimately aiding in speedy germination and growth of the pathogen setting pace for pathogenesis.

The present study was aimed to screen castor germplasm lines varying in wax content against gray mold disease for the identification of resistance source under in vitro conditions using detached spike and detached capsule technique. Out of thirty-three lines screened against gray mold under in vitro conditions six lines with low wax content viz., RG-1754 (0.24 µg/mg), RG-1875 (0.12 µg/mg), RG-1915 (0.21 µg/mg), RG-1919 (0.24 µg/mg), RG-1972 (0.08 µg/mg) and RG-1926 (0.08 µg/mg) recorded significantly low levels of infection ranging from 10 to 20 per cent 7 days after inoculation. Whereas the susceptible cultivar DCH -519 has recorded disease severity of more than 90 %. The pearman's rank correlation analysis showed a strong positive relation between disease severity and capsule wax content, with p < 0.01 and r = 0.884.

Amplifluor, a genotyping system used to analyze single nucleotide polymorphisms (SNPs), is supplied by Merck-Millipore. Amplifluor is based on polymerase chain reaction (PCR) with two competing allele-specific primers and a SNP specific common reverse primer. Sequence information flanking SNP of interest and fluorescent plate reader for end-point measurement or qPCR machine for real time measurement are required for the execution of the Amplifluor assay. In this chapter, the principle and working protocol of the Amplifluor assay based on end-point fluorescence detection of SNP allele is presented with an example.

The association of morphological features of inflorescence viz., presence/absence of spine on the capsule, presence/absence of bloom and compactness on the incidence of capsule borer was studied in breeding populations segregating for these traits. The spine and bloom on capsules were significantly correlated with per cent capsule damage whereas weak correlation was observed between spike compactness and pest incidence. Castor genotypes with non-spiny capsules and less or no bloom are expected to be less prone to capsule borer damage.

Sex expression of castor pistillate lines and their agronomic traits were studied by evaluating six pistillate lines (DPC-22, IPC-30, IPC-31, DPC-9, SKP-84, and M-574) in four different environments in India (Hyderabad, Bengaluru, SK Nagar, and Junagadh). Stable pistillate expressions even at later-order spikes were observed at all the locations without any sex reversions to monoeciousness. In castor crop, generally, interspersed staminate flowers (ISFs) are produced on pistillate spikes to facilitate pollination and maintenance of pistillate lines. Lesser, or preferably no ISF production in pistillate lines is essential to maintain the genetic purity of castor hybrid seed. Evaluation of genetically diverse pistillate lines in different environments indicated that the expression of ISFs was governed by different temperature-dependent genes. It was found that nitrogen and other environmental factors, excluding temperature, had no role in ISF production or sex expression of pistillate lines. We report the novel observation that genes for the expression of ISFs and sex reversion are different and unrelated, although the expression of both sets of genes was found to be temperature-dependent. There was no sex reversion in any order of racemes in all the evaluated pistillate lines, even at temperatures > 38 °C and only the number of ISFs varied in the pistillate lines at the different locations. We also establish that genetically stable, and non-revertant pistillate lines with little or no ISFs, at temperatures as high as > 32 °C can be developed by evaluating and selecting diverse pistillate lines in different environments. GGE biplot analysis indicated that SK Nagar, Junagadh, and Hyderabad locations were the best testing centres for discriminating the genotypes for stable pistillateness as well as ISF expression, and consequently were ideal locations for the development of the best and most stable pistillate lines. Bengaluru could potentially be the ideal location for quality hybrid seed production, as low to no expression of ISFs was found in all the pistillate lines at this location. IPC-30 had stable pistillate expression with the lowest ISFs across the four test environments, followed by DPC-22 and IPC-31. Phenological evaluation indicated that DPC-22 and IPC-31 were relatively early flowering, with a lower node number.

Reniform nematode (Rotylenchulusreniformis) infects castor and makes it vulnerable to vascular wilt and root rot diseases. In this study, a population consisting of 92 recombinant inbred lines (RILs) derived from the cross between reniform nematode resistant line JC-12 and susceptible line 48-1 was used to identifythe genomic regions linked to reniform nematode resistance. The parents, F1 and RILs were screened against reniform nematode in pot culture with artificial inoculation of nematodes. The scoring for nematode resistance was done on the basis of number of nematodes extracted from the soil at 60 days after inoculation. The mean nematode count in 48-1 and JC-12 was 215.0±9.1 and 77.8±4.8 nematodes/ml of soil wash, respectively. The F1 reaction (217.3±13.2 nematodes/ml of soil wash) was similar to the susceptible parent indicating that nematode resistance in JC-12 is recessive in nature. The nematode count in RILs ranged from 43.8 to 327.3. QTL mapping using a linkage map consisting of 1,090 SNP markers resulted in the identification one QTLeach on chromosome-6 and chromosome-8, linked to resistance. This is the first report on mapping of genomic regions linked to reniform nematode resistance in castor, which form the basis for furthering the research on genetic and molecular biology of nematode resistance in castor.

Castor is an industrially important oilseed crop, which provides raw material to many industries. Despite its industrial importance, the genomic resources available for molecular breeding applications are limited. In the present study, a set of 135 SNP loci polymorphic between two castor inbred lines namely, JC-12 and RG-1963 were targeted to develop competitive allele specific PCR assays for genotyping purposes. The assays were validated in the F2 population of JC-12 × RG-1963 cross. Out of 135 assays designed, 124 were found to be successful in discriminating three expected genotypes in the F2 population. Using the SNP genotyping data, a genetic linkage map representing 10 haploid chromosomes of castor was constructed, which corresponded very well with the physical map. This set of validated SNP markers is a useful resource for application in molecular breeding of castor.

Gray mold is a major disease of castor caused by the pathogen Amphobotrys ricini. It mainly affects the inflorescence resulting in severe yield losses. Role of qualitative characters of inflorescence (spike) and capsule viz. spike compactness, capsule type, waxy bloom intensity on capsules (visual), capsule spine length, spine texture and spine density in predisposing castor crop to gray mold was investigated. A set of 26 castor lines differing for presence and intensity of bloom was characterized for spike and capsule characters. The lines were screened in vitro (detached spike and capsule techniques) and in vivo by raising the plants in poly house and field conditions for disease reaction. Spearman's rank correlation analysis revealed significant positive correlation (r(s) = >0.5; p = <0.05) between disease severity and capsule type, capsule waxy bloom, spine texture, spine length. Principal component regression analysis revealed that waxy bloom intensity is the major factor, among the evaluated, that predisposes castor lines to the fungus infection promoting disease severity. Quantification of epicuticular wax from capsules of different waxy bloom lines revealed that there was an increase in severity of infection with increase in wax quantity on capsules irrespective of nature of bloom substantiating the role of waxy bloom quantity in promoting the infection. High quantities of wax (1.50-3.00 mu g/mg) were extracted from lines showing high per cent disease severity (40%-95%), and low quantities of wax (0.00-1.50 mu g/mg) were extracted from lines showing low per cent disease severity (1%-40%). A disease scoring scale considering differences in waxy bloom quantity as a biochemical marker has been proposed. It may not be a replacement to phenotypic screenings but helps breeders in selecting early generation material for low disease severity when huge populations are to be screened against gray mold by choosing low waxy bloom varieties.

A segregating population was developed by crossing two pistillate (female) lines of castor viz., IPC-23 and IPC-21. Population behavior was studied to understand the variability for major yield and yield components. The distribution of node number, plant height and seed yield were deviated fromnormal distribution and it was positively skewed with significant leptokurtic curve indicating that the two parents with high seed yield were selected for population development. Positive skewness indicates that the plants with exceptionallyhigh seed yield were isolated from the population. Difference between phenotypic co-efficient of variation (PCV) and genotypic co-efficient of variation (GCV) was very low to moderate for major yield components viz., number of effective spikes per plant, number of nodes to primary spike, plant height, total spike length and effective spike length of primary indicating that phenotypic selection can be effectively used for yield improvement. High heritability and genetic advance over mean (GAM) indicated the role of additive gene action for plant height and total/effective primary spike length. Correlation of number of nodes up to the primary spike with plant height up to primary spike, and total/effective spike length was positive indicating the possibility of short-statured, early pistillate selections with long primary spikes in castor.

Host plant resistance is an effective means of managing aphid pests in safflower but its genetic basis is not known. In this study, F-1 and F-6-recombinant inbred lines (RILs) produced from the CO-1 x EC-523368-2 cross were phenotyped for tolerance to the aphid, Uroleucon compositae based on days-to-wilt (DW) after infestation. The F-1 plants showed the dominance effect and RILs exhibited quantitative variation. The RIL population was genotyped with 242 simple sequence repeat (SSR) markers, which included a set of 150 new SSRs designed through next-generation sequencing and bioinformatics approaches. Two quantitative trait loci (QTLs), QUc-Ct3.1 and QUc-Ct5.1, putatively associated with DW after aphid infestation were detected. QUc-Ct3.1 was a major QTL located on linkage group (LG) 3 with the closest marker SafM-290 (LOD = 18.3), which explained 31.5% of the phenotypic variation. QUc-Ct5.1 was a minor QTL located on LG-5 with the closest marker CtDES-237 (LOD = 7.0), which explained 9.1% of the phenotypic variation. This is the first report and a significant lead towards discovering genes for tolerance to aphids in safflower.

Sunflower, safflower, castor, sesame, linseed and niger are theminor oilseed crops having potential to contribute towards achieving self-sufficiency in vegetable oil production in India. Decades of breeding research have resulted in release of high yielding cultivars with resistance to biotic stresses. However, the productivity levels are stagnated; further improvement in genetic gain requires integration of molecular tools in breeding programmes. Molecular markers, genomics andmarker-assisted selection technologies are widely exploited for improvement of crops. In this review, current status of development and application of molecular markers in the oilseed crops viz., sunflower, castor, safflower, sesame, linseed and niger are presented.

Fusarium wilt, caused by Fusarium oxysporum f. sp. ricini, is the most destructive disease in castor. Host plant resistance is the best strategy for the management of wilt. Identification of molecular markers linked to wilt resistance will enhance the efficiency and effectiveness of breeding for wilt resistance. In the present study, genomic regions linked to wilt resistance were mapped using a bi-parental population of 185 F6–RILs and a genetically diverse panel of 300 germplasm accessions. Quantitative trait loci (QTL) analysis performed using a linkage map consisting of 1090 SNP markers identified a major QTL on chromosome 7 with an LOD score of 18.7, which explained 44% of the phenotypic variance. The association mapping performed using genotypic data from 3465 SNP loci revealed 69 significant associations (p < 1 × 10−4) for wilt resistance. The phenotypic variance explained by the individual SNPs ranged from 0.063 to 0.210. The QTL detected in the bi-parental mapping population was not identified in the association analysis. Thus, the results of this study indicate the possibility of vast gene diversity for Fusarium wilt resistance in castor.

A total of 41 new castor pistillate lines along with three checks were evaluated in two sets during rabi and latekharif under irrigated and rainfed conditions to study the variability for nine agro-morphological traits and sex expression. Based on better performance than the checks, both for seed yield and pistillate expression, ten pistillate lines were identified for further studies on heterosis and combining ability

Castor ( Ricinus communis L.) is a highly cross pollinated crop and for the first time breeding efforts through single seed descent (SSD) were done in castor in India. The improvement effects of SSD is compared to pedigree selection (PS) method which is commonly followed. A cross between two diverse parents, VP-1 and 48-1 was attempted and further selections were made both through SSD and PS methods. Sixty lines, 30 each generated by SSD and PS methods were evaluated for agronomic traits and yield potential along with two checks in two separate sets for 2 years under rainfed conditions. The data was subjected to descriptive statistics, genetic parameters, pooled ANOVA using SAS/STAR software, principal component analysis etc. to compare the efficacy of SSD and PS methods. Ten elite lines of SSD and 11of PS method recorded 128% and 89% seed yield increase over the male parent. Two methods of selection per se were not significantly different while effect of selection method as indicated by interaction of methods with lines was significant for pooled mean seed yield. Moderate to low GCV and PCV and high heritability were observed for most of the traits studied in both the methods. The trends of genetic gain from selection was different in both the methods for days to 50% flowering, hundred seed weight and seed yield at 120 DAS. The present study indicated that SSD breeding method is equally effective, efficient and less expensive than PS in producing diverse elite parental lines in highly cross pollinated crop like castor through artificial selfing.

An experiment was conducted to determine the nature and magnitude of heterosis in castor for seed yield and its yield attributing traits. Forty hybrids were synthesized involving five lines and eight testers through line x tester(L×T) mating design and were evaluated for yield and its components. Among 13 parental lines, JP-96, VP-1, DPC-9, RG-2661-1, RG-109 and RG-3160 were identified as good general combiners for seed yield and its components, which can be directly exploited in heterosis breeding. The cross combination, DPC-18 × RG-2661-1 was good specific combiner for early maturity. High seed yield per plant was recorded for hybrids: VP-1×RG-109(105.04 g), DPC-18×RG-1771 (98.26g) and DPC-18×RG-2661-1 (97.97 g). These promising crosses involved parents with high × high and high × low GCA effects and were found promising for high yield potential in castor.

Development of superior cultivars in any crop including castor requires knowledge on inheritance of various traits to handle the segregating generations in various breeding methods. In the present study, inheritance of anthocyanin pigmentation in younger leaves was studied in three F2 populations. The results revealed that the presence of anthocyanin in emerging leaves inherited as monogenic dominant and it showed tight linkage with red stem colour.