Sero-RAPD makers based evaluation of chilli pepper germplasm for resistance to chilli veinal mottle virus

2.1 Virus source and maintenance

Pakistani chilli veinal mottle virus (ChiVMV) ATIPK isolate (Accession No. KJ472764) was obtained from M. Ashfaq's Plant Virology Laboratory. The ChiVMV was revived, propagated and maintained on Capsicum annuum cv., Loungi pepper plants and assayed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using ChiVMV specific monoclonal antisera (Ashfaq et al., 2014; Iqbal et al., 2011). The inoculated Loungi pepper plants were retained in screen house (insect free environment) and renewed after two to three weeks. The infection of ChiVMV in Loungi pepper plants was confirmed by using specific DAS-ELISA.

2.2 Plant material

The chilli pepper genotypes consisting of 18 sets of cultivars (Bhakkar Local, Pepper PLSQ, Loungi pepper, CIM-506, Gola Peshawar, CV-7, Red Pepper, Sanam, CV-1, Hot Pepper, Doni Pepper, Ghotki, Yolo wonder, CR-25, Soofi, Royal Wonder, California wonder, Red Top) were obtained from the Federal Seed Certification & Registration Department, Islamabad and Horticultural Research Institute, NARC, Islamabad. Fifteen seeds of each cultivar were sown in germination trays containing peat moss under greenhouse conditions (relative humidity 70 % and temperature 25 °C). The capsicum seedlings at two to three-leaf stage were transplanted @ two plants per plastic pot (3″x3″) filled with sterilized soil mixture (sand, clay and compost @ 1:1:1) under greenhouse conditions and one to two plants per pot were maintained. Each experiment was replicated thrice consisting of 10 plants per replication.

2.3 Mechanical inoculation

Symptomatic and ELISA positive leaf samples of Loungi pepper inoculated with ChiVMV ATIPK isolate at 14 days of post-inoculation (dpi) were used for screening chilli pepper genotypes. One gram infected Loungi pepper leaves were harvested and homogenized in 0.05 M Phosphate buffer (pH 7.2) containing 1 % Na₂SO₃ @ 1/3 w/v and resultant sap extract was subsequently used to mechanically inoculate pepper plants at 2–3 leaf stage (Ashfaq et al., 2014, Ashfaq et al., 2010). The inoculated plants were washed with distilled water to get rid of excess material and placed under greenhouse conditions (relative humidity 70 % and temperature 25 °C) while healthy plants of test genotypes served as control. The genotypes were categorized as highly resistant (symptoms less), resistant (Infected plants showing mild symptoms i.e. 1–10 %), moderately resistant (infected plants showing 11–20 % symptoms), moderately susceptible (infected plants representing 21–30 % symptoms), susceptible (infected plants showing 31–40 % symptoms) and highly susceptible (Infected plants showing severe symptoms i.e. >40 %) based on host reaction (Ashfaq et al., 2007).

2.4 Serological assay

Leaf sample from ChiVMV-inoculated (diseased) plants was taken at 21 dpi and subsequently subjected to ChiVMV-specific DAS-ELISA (Loewe Biochemicals, Germany Cat. No. 07185) as per manufacturer’s instructions. An Automatic ELISA reader (HER-480 HT Company (Ilford) ltd., UK) was used to measure absorbance value at 405 nm and the samples with two times or higher reading compared to the average of healthy tissue and negative control were categorized as ChiVMV positive.

2.5 Molecular analysis

Total DNA from six chilli pepper genotypes (three susceptible and three resistant) was extracted by (i) Proteinase K method (Sambrook and Ruseel, 2001) and (ii) Plant Genomic DNA Purification Mini Kit (Cat No. K0791, Thermo Scientific Gene JET, USA). Twenty RAPD primers were deployed in the current study and primers having polymorphic amplification were used for molecular characterization studies. Polymerase chain reaction (PCR) of extracted DNA was carried out by following the protocol as described by Devos and Gale (1992). Furthermore, the amplified products were separated by electrophoresis and polymorphism was detected by using agarose gel (3 %) pre-stained with ethidium bromide.

2.6 Statistical analysis

UV Transilluminator was used to observe the amplified bands in electrophoresed gels of RAPD markers. SynGene Gel Documentation System was used to take the image. The absence or presence of amplified bands was recorded as 0 or 1, respectively for all tested genotypes with eleven primers. For analysis, data was obtained by the counting of polymorphic bands and subjected to Popgene32 software (Yeh et al., 2000). The value of polymorphism information content (PIC) was measured with the below given equation (Botstein et al., 1980). $$\mathrm{P}\mathrm{I}\mathrm{C}=1-\sum _{i=1}^n{\mathit{pi}}^2-2\sum _{i=1}^{n-1}\sum _{j=i+1}^n{\mathit{pi}}^2{\mathit{pj}}^2=1-\sum _{i=1}^n{\mathit{pi}}^2-\left(\sum _{i=1}^n{\mathit{pi}}^2\right)+\sum _{i=1}^n{\mathit{pi}}^4$$

3.1 Germplasm screening

Eighteen genotypes were screened against ChiVMV ATIPK isolate based on visual symptoms (Fig. 1) by following the disease rating scale and serological assay. All the 18 genotypes of chilli pepper showed characteristic symptoms' viz., dark green vein banding and leaf mottling upon mechanical inoculation with infective sap of ChiVMV. Individual plants of Loungi pepper, Sanam, Red Pepper and CV-1 showed dark green vein banding and leaf mottling at 14 dpi while the rest of the genotypes showed symptoms between 18 and 26 dpi. All the plants of these genotypes exhibited >50 % ChiVMV infection based on 0–5 disease rating scale (DRS) and DAS-ELISA revealed relatively high titer (>1.0), so all of these genotypes were considered as highly susceptible to ChiVMV ATIPK isolate. Similarly, other three genotypes viz., Soofi, Royal Wonder, Pepper PLSQ and four genotypes viz., Yolo wonder, Bhakkar Local, CIM-506 and CR-25 exhibited <50 % infection and DAS-ELISA showed relatively low titer (>0.5 %), were catalogued as susceptible and moderately susceptible, respectively. On the other hand, three genotypes (California wonder, Gola Peshawar, Red top) showed 5–10 % ChiVMV infection based on DRS with relatively low titer (<0.25), therefore, grouped as resistant showing light green banding along veins followed by the rest of the four genotypes viz., CV-7, Hot Pepper, Dane Pepper, and Ghotki with a moderately resistant response showing symptoms like moderate green banding along veins with light mottling (Table 1).

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Fig. 1

Chilli pepper (cv., Loungi pepper) plants showing characteristic symptoms (A, B) upon sap inoculation with ChiVMV isolate ATIPK.

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3.2 Genetic diversity

Six chilli pepper genotypes (Loungi pepper, Sanam, CV1, California Wonder, Gola Peshawar, Red Top) were selected because of their diverse response against ChiVMV to genetically evaluate them using twenty RAPD primers. Eleven primers were used for final analysis out of twenty primers which produced clear, distinct, and readily detectable fragments having a high rate of polymorphism. Considering all the primer amplification in total tested genotypes, 75 bands were amplified in PCR reactions. Eleven primers generated 57 polymorphic fragments which showed 76 % of polymorphism with a maximum polymorphism percentage by primers GLD-19 and GLH-13 (85.71 %), whereas primers GLA-06, GLB-02, and GLC-10 showed the minimum percentage of polymorphism 66.66 % (Table 2). The average statistics of total bands per primer were 6.8, whereas, average amplification of polymorphic bands of each primer was 5.18 (bands) (Fig. 2). The score of amplified fragments per variety varied from 28 to 39, with an average number of 36.33 fragments per variety. The highest score of fragments was generated by Loungi pepper (39), Sanam (39), and CV-1 (39), followed by Red Top (38) and Gola Peshawar (35) genotypes. Whereas, California Wonder genotype produced minimum numbers of bands i.e., 28 (Fig. 3). The fragment amplification varied from 5 to 9; the highest score of bands was recorded for primer GLF-06 i.e., (9), while minimum numbers of fragments (5) were produced by primer GLD-13 (Fig. 4). The PIC value for 11 primers ranged from 0.388 to 0.666 with an average value of 0.512. Out of eleven primers, the maximum PIC value (0.66) was shown by primer GLH-13 while the minimum PIC value (0.38) was shown by primer GLA-06. Whereas, primer GLB-02 showed the PIC value (0.41), followed by GLA-02 (0.42), GLC-10 (0.44), GLB-07 (0.45), GLC-09 (0.54), GLD-13 (0.56), GLD-19 (0.57), GLF-06 (0.57) and GLE-02 (0.58) (Table 2). Ranges of markers PIC values showed that out of eleven markers, 6 were moderately informative having a share of 54.54 % while 5 were highly informative having a share of 45.45 % (Table 3). The size of securable bands produced by all primers ranged between 250 and 1500 bps. Primer GLA-06 produced bands within a range of 1000 to 1500 bps, whereas three primers GLA-02, GLB-02, and GLC-10 produced securable bands ranged from 750 to 1500 bps. Two primers GLC-09 and GLD-19 produced bands within a range of 500 to 1500 bps, primer GLD-13 produced bands between 400 and 1500 bps (Fig. 5), while single primer GLB-07 produced bands between 750 and 1000 bps. Primers GLE-02 and GLF-06 produced bands between a range of 500 to 1000 bps, while primer GLH-13 produced bands ranging from 250 to 1000 bps.

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Fig. 2

Total No of bands and polymorphic bands per RAPD primer.

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Fig. 3

Number of amplified bands per genotypes with 10-base nucleotide RAPD primers.

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Fig. 4

Dendrogram of six chilli pepper genotypes constructed based on genetic distance using 11 RAPD primers.

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Fig. 5

PCR amplification profile of chilli pepper genotypes (L = Loungi pepper, S = Sanam, C = CV-I C.W = California Wonder, G.P = Gola Peshawar, R.T = Red Top) using the primer GLD-13.

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3.3 Genetic similarity matrix

Estimation of genetic similarity and distance of capsicum genotypes was done using multivariate analysis based on Nei’s UPGMA dendrogram. Genetic distance analysis showed the similarity coefficient varied from 44 % to 78 % within tested chilli pepper genotypes. The highest similarity 78.67 % was recorded between two sets of genotypes i.e., CV-1 and Loungi pepper, Sanam and Gola Peshawar, whereas the lowest similarity was noticed within genotypes CV-1 and Sanam. Red Top and California Wonder genotypes showed the similarity of 73.33 %, while California Wonder and Sanam, Red Top and Loungi pepper showed 69.33 % similarity. Three sets of genotypes i.e., Gola Peshawar and California Wonder, CV-1 and Red Top, California Wonder and Loungi pepper revealed 64 % similarity. Gola Peshawar showed 62.67 % similarity with Loungi pepper and CV-1 while 58.67 % similarity was observed between CV-1 and California Wonder. Whereas, similarities of 56 %, 50.67 %, and 49.33 % were evident in Red Top and Sanam, Red Top and Gola Peshawar, and Sanam and Loungi pepper. Maximum mean similarity index (65.86 %) was shown by California Wonder with other five genotypes followed by Loungi pepper with 64.80 %, Gola Peshawar (63.73 %), Red Top (62.66 %), CV-1 (61.60 %), and minimum mean similarity index (59.46 %) was shown by Sanam (Table 4).

Cluster analysis catalogued understudied six genotypes into two main clades. Clade I further comprised of two sub-clades each consisting of two genotypes, sub-clade (Ia) contained Loungi pepper and CV-1 which are closely related to each other, while sub-clade (Ib) contained Red Top and California Wonder which are closely related to each other. Clade II comprises of two genotypes, Sanam, and Gola Peshawar which also showed their close relationship with each other (Fig. 4).