Research Article

Construction of SSR Based Genetic Linkage Map and Identification of QTLs Conferring CMD Resistance in Cassava

P. Vidya, Aswathy G. H. Nair, C. Mohan, J. Sreekumar and Ambu Vijayan

  • Page No:  387 - 397
  • Published online: 31 Aug 2020
  • DOI : HTTPS://DOI.ORG/10.23910/1.2020.2135

  • Abstract

Cassava mosaic disease (CMD) is one of the most devastating Gemini viral diseases that challenge the cassava production with great economic loss. Utilizing host plant resistance through marker assisted breeding is the best sustainable way to tackle this geminiviral disease. High disease severity and increasing yield loss demands the identification of new quantitative trait loci (QTLs) for CMD resistance. In the present study, an attempt was made to construct SSR based linkage map using bi-parental mapping population developed from a cross of cassava cultivars viz., MNga-1 and CI-732 and identified new QTLs using interval mapping. Thirteen CMD associated marker alleles were revealed through single marker analysis (SMA) and two new QTL regions were identified in linkage groups viz., chrom7 and chrom22 using composite interval mapping (CIM). An epistatic interaction viz., additive-dominant (AD) and dominant-additive (DA) between QTLs in 0.1cM and 60.4cM of chrom7 was identified using multiple interval mapping (MIM). The heritability of resistance viz., broad sense heritability (H2) and narrow sense heritability (h2) were found to be 93% and 15% respectively. The expected gain from breeding population was found to be 0.4.

Keywords :   Cassava mosaic disease, linkage, QTL, SSR, interval mapping

  • Introduction

    Cassava (Manihot esculenta) belonging to the family Euphorbiaceae, is one of the most important tuber crops cultivated extensively in tropical and subtropical regions of Africa and Asia. It is a cross pollinating crop with a diploid number of chromosome 2n=36 and DNA content of 1.67pg per cell. It is highly heterozygous, monoecious and protogynous in nature. The tuberous crop is propagated vegetatively by means of stem cuttings and sexually through seeds.  According to Food and Agriculture Organization Corporate Statistical Database (FAOSTAT, 2017), the global cassava production was estimated to be 291992646 tonnes in which 61% is accounted by Africa, 29.3% contributed by Asia and remaining 9.6% by America. Indian cassava production was accounted to be 42 mt from an area of 199 mha with a productivity of 2,09,598 hg ha-1.

    Cassava is mainly used as food and also as an important raw material for various industrial purposes. The wide acceptability of cassava in the world is increasing due to the broad agro-ecological adaptability, its ability to mitigate unfavourable environmental condition and its abilities to produce reasonable yield. Cassava production is always challenged by various biotic and abiotic stresses. One of the major constraints that affected the cassava production drastically is cassava mosaic disease (CMD), caused by geminivirus which are characterized by small, geminate particles containing circular, single-stranded DNA molecules (Briddon and Markham, 1995) encapsidated in paired icosahedral particles (Fauquet et al., 2008). CMD is prevalent in many parts of African and Indian sub-continent that causes serious yield losses (Sseruwagi et al., 2004). In Africa, the estimated yield losses ranging from 25 to 95% (Fauquet and Fargette, 1990), whereas in India 20 to 80% of yield losses were reported (Nair and Unnikrishnan, 2007).

    CMD was first reported in India by Abraham (1956), but a detailed study was carried out by Alagianagalingam and Ramakrishnan (1966). The important geminivirus causing CMD were isolated from different countries includes African Cassava Mosaic Virus (ACMV), East African Cassava Mosaic Virus (EACMV), East African Cassava Mosaic Virus-Ugandan strain (EACMV-UG), South African Cassava Mosaic Virus (SACMV), East African Cassava Mosaic Cameroon Virus (EACMCV), East Africa Cassava Mosaic Malawi Virus (EACMMV) and East African Cassava Mosaic Zanzibar Virus (EACMZV), East African Cassava Mosaic Kenya Virus (EACMKV), African Cassava Mosaic Burkina Faso Virus (ACMBFV) and Cassava Mosaic Madagascar Virus (CMMGV), Indian Cassava Mosaic Virus (ICMV) and Sri Lankan Cassava Mosaic Virus (SLCMV). Two cassava mosaic geminivirus (CMGs) reported from Indian sub-continents were ICMV and Sri Lankan cassava mosaic virus (SLCMV) (Saunders et al., 2002). CMD is characterized by chlorotic mosaic of leaves, premature leaf abscission, leaf distortion and severe stunting of plant ultimately affected the photosynthesis of the plant and thereby yield. Variation in symptom expression might be due to sensitivity of host genotype, season, stage of crop growth, difference in virus strain (Fargette et al., 1990). Whitefly, Bemisia tabacia is the vector and is transmitted in acirculative, non-propagative mechanism (Harrison, 1985). The direct effect of CMD epidemic spread is serious crop failure thereby yield losses and the severity of losses depends upon the viral strains, crop varieties, age of the crop and varying environmental conditions.

    Development and deployment of resistant cultivars through breeding was the most effective control method for CMD (Thottappilly et al., 2004). CMD resistance breeding studies using inter-specific and intra-specific crosses were initiated in the early 1940s led to the development of highly CMD resistance breeding materials such as TMS (Hahn et al., 1980) and TME series (Okogbenin et al., 2012) in Africa. MAS is a method whereby a phenotype was selected based on the genotype of a marker (Collard et al., 2005). Identifying resistance genes using molecular markers was the basic pre-requisite for performing MAS in resistance breeding programs (Pinheiro et al., 2012). The two main strategies used to identify molecular markers associated with traits of interest were QTL mapping andbulk segregant analysis (BSA) (Tanksley et al., 1989; Michelmore et al., 1991; Giovannoni et al., 1991). Genome mapping or genetic mapping is the process of constructing linkage maps and conducting QTL analysis to identify genomic regions associated with a particular trait (McCouch and Doerge, 1995; Mohan et al., 1997; Paterson, 1996). Till now, three major sources of resistance were reported in cassava against CMD. The first source of resistance identified was designated as CMD1 and was introgressed from wild cassava Manihot glaziovii. The marker associated with source of resistance was SSRY40 and cassava lines developed was termed as Tropical Manihot Selection (TMS). The CMD1 resistance was considered as polygenic and recessive (Fregene et al., 1997). The second source of resistance was monogenic dominant gene (Qualitative resistance), reminiscent of plant resistance genes (R-genes) whose product played a key role in recognizing a hypersensitive response (HR), a localized cell death and tissue necrosis at the site of pathogen ingress and was designated as CMD2 (Akano et al., 2002). This source of resistance was identified in Nigerian landraces were later termed Tropical Manihot Esculenta (TME). The nearest markers associated with CMD2 identified were SSRY28, NS158, NS169 and RME-1. The third source of resistance was quantitative trait loci (QTL) designated as CMD3 and the marker NS198 were found to be associated with this resistance (Okogbenin et al., 2012).

    Resistant cultivars are much less readily infected than the susceptible cultivars with the localized distribution and low concentration of virus in resistant cultivars made them a less potent source of inoculum from which spread could occur (Fargette et al., 1988). But with the emergence of new viral strains, its high virulence and increasing adaptability with vector became a huge challenge to conventional breeding programs. Classical breeding in cassava was also complicated due to strong heterozygosity, inbreeding depression coupled with lengthy breeding cycles and the need for making large number of crossings to obtain the first generations progenies, which were then screened for desired traits (Ceballos et al., 2004). Moreover, most of the agronomically important traits studied in cassava were polygenic (Hahn et al., 1989) and were very difficult to analyze using classical genetic analysis. Identifying and pyramiding different virus resistance genes will therefore provide stable resistance against a broad spectrum of CMGs. Gene pyramiding in cassava require molecular marker assisted selection (MAS) to expedite the breeding process. Important molecular markers particularly promising in assisting the selection for desirable characters are Restriction Fragment Length Polymorphisms (RFLP), Random Amplified Polymorphic DNA (RAPD), Sequence Characterized Amplified Regions (SCAR), Sequence-Tagged Sites (STS), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphic DNA (AFLP), Simple Sequence Repeat (SSR), Single Nucleotide Polymorphism (SNP), Diversity Arrays Technology (DArT).

    Genome mapping or genetic mapping is the process of constructing linkage maps and conducting QTL analysis to identify genomic regions associated with a particular trait (Mohan et al., 1997). Linkage map construction requires a segregating mapping population derived from parents that differ for one or more traits of interest. In cross pollinating crop, the F1 mapping populations were successfully developed by pairing two heterozygous parents that were distinct in trait of interest (Barrett et al., 2004). Cassava being out crossing crop, the heterozygous nature of mating types made the QTL detection more complex and less efficient compared to inbreds (Sliwka et al.,2008).  The entire mapping population were partitioned in to different genotypic groups based on the presence or absence of a particular marker locus and to determine whether significant difference exist between groups with respect to the trait being measured (Tanksley, 1993; Young,1996). Loci associated with quantitative traits have commonly been referred to as QTL and the procedures used to find out QTLs are called QTL mapping. Cassava is a highly heterozygous and cross pollinating crop; thus F1 mapping population was generally used for genetic linkage map construction. Allelic determination and its segregation in mapping population could be analyzed using a special class of markers known as single dose restriction fragments (SDRF) (Wu et al.,1992). SDRFs are DNA markers that are present in one parent and absent in the other and segregate in a 1:1 ratio (absence:presence) in the progeny. Another important factor in linkage map construction is the selection of molecular markers in mapping studies. Microsatellites or SSR markers are highly reproducible and show co-dominant inheritance makes it a simple, cheap and reliable marker for MAS. The high polymorphism of SSR markers were due to its high mutation rate and random occurrence in the genome that makes SSR the most promising class of markers for construction of saturated maps.

    The two major type of plant defense mechanism were quantitative and qualitative mode of resistance (Toojinda et al., 2000), of which qualitative resistance was mediated by major genes and quantitative resistance by multiple genes (Kousik and Ritchie, 1999). CMD is a viral disease showing varying symptom expression from resistance to very severe symptom. The varying disease symptom expressions of the plant were explained by both quantitative and qualitative mode of resistance. The quantitative resistance in cassava was derived from M. glaziovii through inter-specific crosses and was found to be recessive with a heritability of about 60% (Nichols, 1947; Jennings, 1976). The second source of resistance identified was qualitative conditioned by single dominant gene in landraces from Nigeria and other West African countries (Akano et al., 2002). But the limited genetic base for the dominant resistance implies potential vulnerability if the cassava mosaic geminivirus could evolve to overcome it. Qualitative resistance was thought to be R-mediated resistance which provides high level of resistance and is segregated in 3:1 ratio. On the other hand, quantitative resistance (QR) is a highly durable broad-spectrum resistance mediated by multiple genes with minor effects. These kinds of resistance are very effective against biotrophs such as virus and it resulted in the continuous distribution of disease severity score.  Though different sources of resistance had been identified, the exact gene responsible for CMD resistance is still unknown. Several studies were carried out to dissect the genetic basis of qualitative resistance. Recently Wolfe et al. (2016) conducted genome wide association mapping (GWAM) to study the CMD2 locus and identified candidate genes viz., two peroxidase and one thioredoxin gene for CMD resistance. A clear understanding of the CMD resistance mechanism particularly at genomic level will be useful in breeding process and also helpful in the proper selection of plants by MAS. The objective of the current study was to develop a full-sib mapping population for CMD resistance and the identification of QTL markers conferring resistance to CMD.

  • Materials and Methods

    2.1.  Selection of parental lines

    In this study, two cassava genotypes were selected viz., CI-732 and MNga-1 as female and male parent to develop full-sib mapping population which were indexed as score 5 and score1 respectively. CI-732 is a CMD susceptible local genotype and MNga-1 (TMS30001) is CMD resistant genotype developed by the International Institute for Tropical Agriculture (IITA), Nigeria. This Study was conducted at ICAR-Central Tuber Crops Research Institute (ICAR-CTCRI) from 2011 to 2015. In this study, both MNga-1 and CI-732 were planted in four rows with 25 mounts in each row. The parental lines were crossed and produced 114 F1 mapping population. The phenotypic evaluations of the F1 mapping population for CMD resistance were done based of 1-5 symptom severity index (SSI) (Hahn et al., 1980; Table 1).

    In order to avoid bias, clones of each genotype were prepared using five cuttings from each genotype and were planted in a row. Two clonal evaluation trials for CMD response were carried out and the response of each F1 seedlings to CMD were recorded at 3 month after planting (3 MAP), 5 MAP and 8 MAP after planting based on 1-5 SSI.Maximum symptom expression was found to be at 5MAP. All managemental practices were followed as per the recommended package of practices.

    2.2.  Detection of geminivirus in parental lines using ICMV and SLCMV specific primer

    The geminivirus present in the parental lines viz., MNga-1 and CI-732 were detected using ICMV and SLCMV specific primers (Table 2).

    The total DNA was extracted from two parental lines using the same procedure as employed in chapter 3. Standard PCR was carried out by using specific primers designed from replicase gene sequence of both the viruses (Raghu et al., 2012). A 20 µl PCR reaction was carried out in Bio-Rad C1000TM Thermal Cycler containing 50ng genomic DNA, 0.2µM each of forward and reverse primers, 50 µM dNTPs, 1X buffer (10mM Tris-HCl (pH 8.3) 50 mM KCl, 1.5 mM MgCl2, 0.3U of Taq DNA polymerase. PCR parameters were 94 ºC for 2 min then 40 cycles of 1 min at 94 °C, 1 min at 63 °C and 1 min at 72 °C, followed by the final extension of 10 min at 72 °C. The amplified PCR products were checked on 1.5% agarose gel stained with 10 mg ml-1 of ethidium bromide and images were photographed in gel documentation system (Alpha Imager).

    2.3 Construction of genetic linkage map and QTL mapping

    The genomic DNA was isolated from both parents and F1 progenies using Cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1990). The quality and purity of the DNAs were checked using 0.8% agarose gel and stained with ethidium bromide solution. The DNA bands were visualized using UV illumination system and gel images were taken using Gel documentation system (Alpha Imager). The quantification of DNA was determined on the basis of optical density readings (ODs) read at wavelengths of 260nm and 280nm. The concentration of DNA samples were then adjusted to 50ng/μl (vidya et al., 2019). A total of 117 SSR primer pairs were used to detect the parental polymorphism. PCR conditions were maintained as described by Mba et al. (2001). A 20µl PCR reaction was carried out in Bio-Rad C1000TM Thermal Cycler containing 50 ng genomic DNA, 0.2µM each of forward and reverse primers, 50 µM dNTPs, 1X buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2), 0.3U of Taq polymerase. A PCR profile of  94 °C for 5 min then 30 cycles of 1 min at 94 °C, 2 min at 58 °C and 2 min at 72 °C, followed by the final extension of 5 min at 72 °C. A volume of 8 µl of loading dye was added to each of the amplified product which was then denatured at 95 °C for 5 min, snap cooled using ice and separated on 6% denaturing polyacrylamide gel electrophoresis (PAGE) containing 7M urea at a constant current of 100W. Then the amplified products were resolved using silver stain as described by Panaud et al. (1996). The identified polymorphic markers were used in the genotyping of parental lines along with F1 mapping population. The true hybrid analysis of F1 mapping population was carried out using size specific polymorphic markers especially markers produced single alleles in parental lines. Seven single allelic size specific polymorphic markers (SSRY95, SSRY33, SSRY83, SSRY339, SSRY47, MeSSR10 and NS890) obtained from parental analysis were used in true hybrid evaluation (Vidya et al., 2019).

    The genotypic scoring was performed as SDRF as described by Wu et al. (1992). The entire genotypic data was used to construct linkage map using a computer software program MAPMAKER v3.0 (Lincoln et al., 1993) and markers were sorted into different linkage groups based on a LOD threshold of 3.0 with a maximum recombination fraction of 50cM. The marker alleles were tested for goodness-of-fit for Mendelian segregation using Chi-square (χ2) analysis. QTL mapping were carried out by three different methods viz., single marker analysis (SMA), Composite interval mapping (CIM) and multiple interval mapping (MIM) using Windows QTL Cartographer version 2.5 (Basten et al., 2003). Single marker analysis (SMA) was performed by both one way ANOVA in Microsoft EXCEL and simple linear regression analysis in Windows QTL Cartographer version 2.5. In composite interval mapping, LOD value was determined by the genome-wide significant threshold level which itself was calculated by permutation test carried out at 1000 replicates. CIM was performed at 5% level of significant using the parameters viz., forward and backward regression, Model-6 standard model, 10 control markers and a walk speed of 1cM (Jansen, 1993; Jansen and Stann, 1994; Zeng, 1993, 1994). Multiple interval mapping was using performed using the parameters viz., MIM forward scan at 5% significant levels and at a walk speed of 1cM to find out the main QTLs, its exact positions and interaction of QTLs for a particular trait (Kao et al., 1999). 

    2.4.  Heritability and expected genetic advance

    The Broad-sense heritability (H2) and narrow sense heritability (h2) of the trait were calculated using the formula                

    H2=(VG÷VP)×100    h2=(VA÷VP)×100                                                           

    (VG is the total genetic variance (additive, dominant and epistatic), VA is the total additive variance)

    The expected advance or gain from the current breeding population were calculated using the formula GC=(k)(√VP)h2 (GC=genetic advance or gain, √VP= square root of phenotypic variance, k = Selection intensity (1.76)).

  • Results and Discussion

    3.1.  Detection of geminivirus in parental lines using ICMV and SLCMV specific primer

    CMD is one of the most devasting diseases that cause a serious yield losses in major cassava growing countries of tropics and sub-tropical regions. In Africa, CMD related yield losses was estimated to be 20 to 95% (Fauquet and Fargette, 1990) and a severe losses of more than 19 million tons of cassava valued above US$1.9 billion was reported in 2003 (Legg and Fauquet, 2004). In India, yield losses of 16 to 80% was reported (Nair and Unnikrishnan, 2007). The causative agent of CMD was geminivirus that was transmitted either through the use of infected stem cuttings or by whitefly vector B. tabaci. The major geminivirus prevalent in Africa is ACMV and in India, two geminivirus has been reported viz., ICMV and SLCMV. In the present study, an attempt was undertaken to identify the causative agent of CMD using specific primer pairs designed from ICMV and SLCMV replicase gene in parental lines viz., MNga-1 (resistant to CMD and CI-732 (susceptible to CMD) collected from the fields of ICAR-CTCRI. In this study, amplification in specific band size of 1050bp confirmed the presence of SLCMV and no amplification was recorded for ICMV in both samples indicated the absence of ICMV. It was also found that a very thick band was observed in susceptible variety (CI-732) and a very light band in resistant variety gave qualitative account on the amount of virus in the susceptible and resistant samples (MNga-1; Figure 1).

    But previous study conducted by Mohan et al. (2013a) using the parental lines viz., MNga-1 and CO2 detected the presence of ICMV but not SLCMV. Studies of Raghu et al. (2012) reported the presence of both ICMV and SLCMV in cassava variety H-226 using specific primers pairs designed from ICMV and SLCMV replicase gene. But the above mentioned two studies were carried out using the infected leaf samples collected from fields of TNAU campus, Coimbatore. The virulence of SLCMV and ICMV to cause disease in host plant was revealed through the work of Saunders et al. (2002) and found that SLCMV was more virulent in host than ICMV. It was also found that the DNA B of SLCMV was originated from DNA B of ICMV by recombination event involving DNA A of SLCMV.

    3.2.  Construction of genetic linkage map and QTL mapping

    Cassava is an important starchy tuberous root crop cultivated for more than a decade which provides food for all the tropical countries of the world. It is highly heterozygous and cross pollinating crop with long breeding cycle render the conventional breeding tedious. Hence to understand many agronomically important traits in cassava, MAS using DNA markers has become an important choice. DNA markers tightly associated with a particular trait whose presence or absence is used in MAS to assist phenotypic selection. The prime most steps in MAS are the construction of linkage map followed by the identification of QTLs for a particular trait. Linkage map is the road map which shows the position and relative distance between the markers along the chromosome. Three important steps involved in linkage map construction were development of mapping population, identification of polymorphism and finally linkage analysis of markers (Collard et al., 2005). A segregating mapping population with 50 to 250 individuals from two genetically divergent parents for one or two traits is generally employed in linkage map construction (Mohan et al., 1997). Due to the high inbreeding depression in cassava, F1 mapping population derived from highly heterozygous parents are generally used in linkage mapping. The present study made an attempt to develop 114 F1 mapping population from MNga-1 (male parent) and CI-732 (female parent) which were contrasted in CMD resistance. The parental analysis with 117 SSR primer pairs identified 65.8 per cent polymorphism (Figure 2). The polymorphic SSR primer pairs were used in the genotyping of F1 mapping population and their segregation among mapping population were scored using SDRF producing 113 segregating loci.

    A genetic linkage map comprising 24 linkage groups spanned a genetic distance of 1165.9cM was constructed with 70 polymorphic SSR markers (Table 3, Figure 3). The number of linkage group exceeds above the expected number of linkage group in cassava (18 linkage group) explained the incomplete coverage of genome with SSR markers and similar situation was observed in studies of Okogbenin et al. (2006), Sraphet et al. (2011). One of major problem encountered in mapping studies were segregation distortion. Cassava being an out-breeding crop showed a very high segregation distortion and was reported to be common in all out crossing crops (Hanley et al., 2002; Liebhard et al., 2002).

    The usual way to assess segregation distortion for marker data is by χ2 tests (Haitham et al., 2002; Lu et al., 2002; Ruiz and Asins, 2003). In the current study, Ninety three (82.3%) primers showed 1:1 disomic segregation pattern and twenty marker alleles (17.69%) showed deviations from Mendelian segregation pattern. The reason for segregation distortion might be due to the presence of gametophytic selection for sub-lethal genes (Yan et al., 2005) or due to the high recombination event (Nordborg, 2000).

    CMD is a geminiviral showing varying symptom expressions. When disease resistance is inherited in a complex manner, progenies will exhibit quantitative mode of resistance showing low to high disease severity resembling a normal distribution (Miedaner and Korzun, 2012) and thus the identification of QTLs holding resistance is a necessity to achieve durability resistance against CMD. In the present study, two clonal phenotypic evaluation trials for CMD resistance were carried out in two consecutive years (2013-14, 2014-15). Disease severity expression was recorded at 3MAP, 5MAP and 8MAP and the maximum disease severity was observed in 5MAP which was recorded as the final CMD score.  Fourty five F1 progenies showed symptom severity score of 1 followed by 22 individuals with a score 4, 20 individuals with score 3, 17 individuals with score 2 and 10 individuals showed a very severe symptom score of 5. The mean symptom severity value was found to be 2.42 with a variance of 1.96 (Figure 4).

    When disease resistance is inherited in a complex manner, progenies will exhibit quantitative mode of resistance showing low to high disease severity resembling a normal distribution (Miedaner and Korzun, 2012) and thus the identification of QTLs holding resistance is a necessity to achieve durability resistance against CMD. In the present study, four different methods have been employed in QTL mapping studies viz., SMA, SIM, CIM and MIM. Thirteen marker alleles (SSRY28a, SSRY28b, SSRY324d, SSRY59a, SSRY59b, SSRY43d, SSRY32c, SSRY32d, SSRY10b, SSRY30c, NS97c, NS185a and NS185b) revealed through SMA have been found to be significantly associated with CMD resistance (p<0.01), consisting twelve linked markers and one unlinked marker (SSRY324d) (Table 4).

    The distribution of currently identified CMD associated markers in different linkage groups and the contribution from female parent reconfirmed the polygenic means of resistance in cassava which was already well established by Hahn et al. (1980); Lokko et al. (2004); Mohan et al. (2013).

    CIM was performed and identified two QTLs in chrom7 and chrom22 (QTL1 and QTL2) respectively at a LOD score above 8.0. The QTL1 were flanked by NS97a and SSRY38a showed a PVE of 4% and a positive additive effect of 0.38. The QTL2 identified by CIM was strongly associated with CMD resistance with an additive effect -0.76 and PVE of 17%, (Table 5).

    The flanking markers viz., NS97c and SSRY30c associated with QTL2 were found to be significant by SMA. This suggested that QTL present in Chrom7 was a minor gene and QTL on chrom22 was a major gene. Phenotypic variance <10% was considered as minor QTL and several such minor QTLs was reported to be associated with disease resistance (Li et al., 2001; Lindhout, 2002; Pilet-Nayel et al., 2002) which signified the present study. Also minor genes have small effects on the expression of phenotype for resistance showing quantitative segregation (Vidhyasekaran, 2008).

    With multiple interval mapping, the main QTLs, its position and interactions of the QTLs were identified. Two main QTLs were identified in chrom7 at a position of 0.1cM and 60.4cM position. The QTL located at 0.1cM position showed an additive effect of 1.1528 and dominant effect of -2.52 and was associated with marker NS97a. The second QTL located at a position of 60.4cM showed an additive effect of 0.3474 and dominant effect of -0.0325 and was associated with SSRY38a. There was prominent dominant by additive variance (DA) was identified with a value of 0.6962 (Table 6).

    The total R2 value explained by this QTL interaction was found to be 0.95 (95%) indicated a strong phenotypic effect. This is consistent with the studies of Wolfe et al. (2016), identified a major QTL on chromosome 8 and but on further dissection of major gene revealed the presence of two possible epistatic loci which accounted for the difference between moderate and strong disease resistance in germplasm.

    In this study, heritability of the CMD resistance was also analyzed. The total phenotypic variance and the genetic variance explained by CMD resistance trait were found to be 1.96 and 1.85 respectively. Using the phenotypic and genetic variance, the H2 of CMD resistance was calculated and was found to be 93%. Using the additive variance (0.29141), the h2 of CMD resistance was calculated and was found to be 15%. When compared the narrow sense heritability with broad sense heritability (93%), the value obtained was very low (15%) as it considered only additive variance which is the only transferrable variance from parents to progeny. Also the genetic gain from selection was found to be 0.4. The main interactions identified in this study were AD and DA and was due to the heterozygous nature of the crop. Low narrow sense heritability and genetic gain in this study was explained by the epistatic interactions. This emphasis more of the interaction is genotype x environment and additive in nature rather than that of single dominance. Some studies reported that epistatic interactions of QTLs limit their usefulness in MAS programmes (Tan et al., 2001). But Govindaraj et al. (2009) reported that the individual additive effects of these QTLs were sufficient enough to recruit them for MAS. Because of the interaction between different loci, the offspring phenotype will be largely influenced by the genetic background of the receptor line when marker-directed selection is carried out (Tan et al., 2001. The search for new resistance to CMD through RNA silencing was carried out by Vincent (2017). Differential responses of different genotypes to cassava geminiviral infection were studied by Paul et al. (2017). The identification of QTLs and the elucidation of their genetic control (main effects and their epistatic effects) are essential for the development of efficient MAS for improving breeding efficiency.

  • Conclusion

    CMD resistance in cassava was contributed by both qualitative and quantitative disease resistance mechanism through SSR based genetic linkage map and QTL mapping. Heritability and expected gain from breeding were also calculated. This study reinforces the importance of epistasis in marker trait association studies, as the individual effect of a marker at locus depends on the marker genotype at other interacting loci.

  • Acknowledgement

    The authors are grateful to the Director, Head, Division of Crop Improvement, ICAR-CTCRI, Thiruvananthapuram, for providing the laboratory facilities to do the work and University Grant Commission (UGC) for providing the financial support.

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Vidya P, Nair AGH, Mohan C, Sreekumar J, Vijayan A. Construction of SSR Based Genetic Linkage Map and Identification of QTLs Conferring CMD Resistance in Cassava IJBSM [Internet]. 31Aug.2020[cited 8Feb.2022];11(1):387-397. Available from:

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