SSR based molecular profiling in chilli (Capsicum annuum L.)

Introduction

Chilli (Capsicum annuum L.), a member of the family Solanaceae with chromosome number 24, is one of the widely cultivated crops throughout the world. It originated from South and Central America, where it is still under cultivation (Pickersgill, 1997) with the secondary centers in Gautemala and Bulgaria. Brazil is the major centre of diversity where representatives at all cited levels are found. Chilli was introduced into India from Brazil in 16th century by Portuguese. Chilli is used as culinary supplement, vegetable, spice, condiment, medicine and as an ornamental plant. Therefore, it is indispensable to the Indian and many African and South American people. Dry chilli, powdered or ground into paste and even green chillies are used everyday in Indian kitchens for curries, sambar, rasam, pickles, sauce, soups, salads and other savory dishes to impart colour, pungency and flavour to the food stuff. Capsaicin is the pungent principle in chilli which produces a highly irritating vapour on heating. Chilli is also called as hot pepper because of its pungency. This volatile acrid crystalline compound present in the pericarp and placenta of the fruit has immense therapeutic and prophylactic uses in allopathic and ayurvedic medications. Chillies are medicinally found to be used in treatment of constipation, indigestion, cold and as pain killer. It is used in tear gas preparation by the security agencies. A non-conventional use of chilli is it's use in self defence sprays which is gaining popularity in USA. The capsicum oleoresin present in the spray released at ultra high emission rate temporarily immobilizes the attacker. Moreover, capsaicin has shown great potential as chemo preventive agent against cancer disease. Green chilli is rich in rutin (Used in pharmaceutical industries). Red colour in chilli is due to capsanthin and capsorubin. The colour extracts find use as colour additives in food industry, poultry and prawn feed industry. In food and beverage industries, chilli is being used in the form of oleoresin which permits better distribution of colour and flavour in food. Chilli is rich in vitamin-A and C than tomato (Vitamin-A: 870IU/100 g and Vitamin-C: 321 mg/100 g). Chilli is sometimes referred as capsule of vitamin C because of rich vitamin C content in the fruit.

Various chilli accessions were developed by different breeding and crop improvement methods. The presence of genetically divergent chilli genotypes made it an impactful source for the introduction of new characteristics but most of them are uninvestigated. Collection of this germplasm and their appropriate characterization and classification is a tenacious need for the improvement of these genotypes. Subjective classification of the chilli germplasm based on the reported morphological data is tedious and also confusing (Costa et al., 2006) [4, 5] . Inadequacy of polymorphism at morphological level limits the differentiation of genotypes in addition to this, they are effected by the environmental fluctuations. This once again hinders the crop improvement. Molecular markers combats these complications and acts as a usefull tool for genotypes identification, organization, and evaluation. DNA markers are widely used and are not affected by the different growth stages and the environment, due to which they are usually preferred than morphological and biochemical markers. Various molecular markers like restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) have been advanced for chilli. Among these molecular markers, SSRs stand for highly polymorphic, co-dominant, reproducible and multi allelic type of variation (Beecher et al., 2000) and are being employed for genome mapping, gene tagging, and estimation of genetic diversity, variety identification, and marker-assisted selection. Hence, in order to devise a sound breeding and crop improvement plan, the present experiment was conducted to determine gentic diversity in forty chilli genotypes using SSR markers.

Material and Methods

Experimental site

The experiment was conducted in Biotechnology laboratory, SKLTSHU, College of Horticulture, Rajendranagar, Hyderabad-500030, Telangana, India.

Plant material

The experimental material comprising of 40 chilli genotypes (Table 1), obtained from NBPGR Regional Station, Rajendranagar, Hyderabad and few local varieties collected from Warangal district of Telangana state, few released varieties from RARS, Lam, Guntur, Andhra Pradesh and AVRDC Taiwan. The details of seed germplasm are presented in Table 1. The genotypes were grown during Kharif, 2018 at PG Research Block, Department of Vegetable Science, Sri Konda Laxman Telangana State Horticultural University, College of Horticulture, Rajendranagar, Hyderabad-500030, Telangana.

D.N.A isolation

Approximately 100 mg of fresh young leaves from each accession of chilli were collected. DNA extraction was done by modified C-TAB method. The quality of DNA was checked using 1% Agarose gel electrophoresis and quantity of DNA was determined with a biophotometer (Eppendorf, India). These samples were diluted to 25 ng/ul for SSR analysis and stored at-20 °C until use.

SSR analysis

Total thirty two SSR markers (Table 2) were tested for PCR amplification. Each primer was amplified according to it's specific annealing temperature. PCR amplified products were subjected to horizontal gel electrophoresis using 3% Agarose gel in 1X TBE buffer at 80V using (Cleaver Scientifics). The ethidium bromide stained gels were documented using gel documentation unit.

Data analysis

Clear amplified fragments of RAPD markers were scored as present (1) or absent (0) and each characteristic state as taken as independent allele. All the polymorphic and no polymorphic bands were calculated for each individual primer and the polymorphic information content (PIC) value was calculated using following formula developed by Roldan-Ruiz et al. The PIC values may range from 0 indicates monomorphic to 1, very highly discriminative with many alleles in equal frequencies. NTSYS-PC program (Rohlf 1998) was used to construct the clustering dendrogram based on the genetic distance matrix. The Unweighted Pair Group Method Arithmetic Mean (UPGMA) was used for cluster analysis.

Results and Discussion

Genetic diversity analysis of 40 chill genotypes was carried out using 32 Simple Sequence Repeats (SSRs) selected from the linkage map of chilli genome covering all the 12 chromosomes to study the polymorphism, diversity and similarity. Results obtained from the study have been presented below under the following headings. PCR analysis of the 40 genotypes using 32 SSRs ensued distinct polymorphism with 29 markers and monomorphism with 3 markers. Using the allelic data of 29 polymorphic markers, binary matrix data was prepared. A total of 110 alleles were observed and the number of alleles varied from 1 (monomorphic) to 12 with an average number of alleles per locus being 3.44. The mean number of alleles per locus furnishes supportive knowledge of polymorphism and adequate information to co-dominant markers (IPGRI, 1999). Highest number of alleles were observed with HpmsE 074 (Fig. 1) and the same marker was used by Nanda et al., (2016) for tagging genomic regions controlling anthracnose resistance and reported polymorphic to F 2 mapping population parents (PBC 80 and SB 1) in Capsicum baccatum followed by HpmsE 022 (10) and HpmsE 114 (10).

Conclusion

SSR markers are assessed to be the most powerful tools for the approximation of genetic diversity and similarity among the studied chilli genotypes. Out of 40 genotypes, two genotypes NIC 19967 was found to be the most diverse, grouping separately with lowest similarity, followed by PBC 80, ST 13837 and ST 13875 and two highly similar genotypes (Hirkani and SRS 13578) were identified which can be deployed in chilli breeding and crop improvement programmes. Those genotypes which showed highest genetic variation can be used as parental lines for breeding to improve chilli varieties. The present effort is a preparatory investigation to distinguish and notice the genetic variation of chilli genotypes under study and had few shortcommings like restricted number of genotypes taken for the study besides the less number of primers used. The results manifested in the current study can be exploited as a guideline for progressing mapping population, advancing marker assisted selection (MAS) and crop improvement of chilli germplasm.

Acknowledgement

AcknowledgementThe author is thankful to SKLTSHU, for providing financial assistance in the form of a fellowship for pursuing the PG program. Authors also thank's the other authors for supporting the research work in relavant field and for proof reading the manuscript. Assistance received from ICAR-NBPGR, RS Hyderabad for providing germplasm is also acknowledged by the author. The studies were conducted with the help of financial support aided by SKLTSHU.

References

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