G. hirsutum (American Cotton) is susceptible to Cotton Leaf Curl Disease (A-DNA and Beta DNA of virus).

Cotton (Gossypium hirsutum L.) is the most important fiber crop in the world and is also called “white gold”. . It adds to export revenue and GDP of the country substantially. Both cotton and textile exports account for nearly one- third of total foreign exchange earnings of the country, crossing Rs. 60,000 crores. India is the only country in the world where all the cultivated species of genus Gossypium namely G. arboreum, G. herbaceum (both diploids), G. hirsutum and G. barbadense (both tetraploids) are grown on fairly commercial scale.

The viral disease that threaten cotton production the most is cotton leaf curl disease (CLCuD) which has been reported from a number of countries across Africa and from southern Asia, specifically Pakistan and northwestern India.It was first reported 1912 from Nigeria on Gossypium peruvianum and Gossypium vitifolia and then affecting Gossypium barbadense in Sudan in 1924 and subsequently from Tanzania in 1926. 

In Pakistan CLCuD was first reported in 1967 and became a minor sporadic problem until 1986, when a plot of a newly introduced cotton variety (G. hirsutum) became infected. In India, the disease was first reported on G. hirsutum near Sriganganagar in Rajasthan in 1993. While in Punjab, it was observed at a few locations in Hindumal Kot area of Abohar, during 1994.

 Bamicia tabaci) transmitted Gemini virus, which interacts in a persistent circulative manner with its vector and is commonly known as CLCuD-associated begomoviruses (CBVs), belonging to genus Begomovirus and family Geminiviridae. It infects only dicotyledonous plants. Previously, it was thought that only CLCuV was responsible for causing cotton leaf curl disease, but recent investigations have suggested the involvement of a virus complex in causing the disease.

CLCuD is caused by a whitefly (

 Mansoor and coworkers (1999) isolated and identified it as a novel circular single stranded DNA (DNA-1) associated with CLCuD in Pakistan. Similarly, Briddon and coworkers (2001) identified that cotton leaf curl disease is a causative complex comprising DNA-A molecule of CLCuV and DNA-β or betasatellite, which is responsible for the production of typical symptoms of this disease. Genetic resistance to CLCuD is the most viable, environmentally safe, ecologically sound and less expensive approach for the management of this menace. During the late 1990s, losses were gradually reduced, returning Pakistan’s cotton output to above pre-epidemic levels, by the introduction of CLCuD-resistant cotton varieties. 

Four distinct symptom types namely leaf curl with prominent enations, severe leaf curl without prominent enations, upward and downward curling of leaves have been documented. Sequences of DNA A and β DNA components of the isolates associated with different symptoms showed existence of significant variation and recombination with other strains of CLCuD. However, correlation of specific sequence or recombination event with specific symptom needs to be established. More than one thousand lines have been screened and many lines resistant/tolerant to CLCuD were identified. However, most of these lines, of late, have been knocked out. Among other factors, it may be due to the mutation(s) in DNA A/β DNA.  Thus, the present study was proposed, in the light of above discussion, to study the sequence of β DNA for occurance of any mutation, from the lines which were earlier resistant but the resistance has been broken down recently. Considering this, the present study was carried out with the objectives to identify and characterize the β DNA of the Cotton Leaf Curl Disease Complex. 

β DNA is a single stranded, circular, satellite DNA molecule of about 1.3 Kb size that induces typical symptoms of cotton leaf curl disease including leaf scurling, vein swelling, vein darkening and enations on the underside of leaves. It relies on helper begomovirus for replication, encapsidation, movement within plants and transmission between plants. It has highly conserved structure consisting of an A-rich region, hairpin structure with a loop containing motif TAATATTAC which is conserved among all  β-DNA and is called as Satellite Conserved Region (SCR) and a single ORF for βC1 protein, in complementary sense and is conserved in both position and sequence. Briddon and coworkers (2002) compared the sequences of two available β DNA molecules and identified a highly conserved region upstream the predicted hairpin structure. 

Primers specific to this conserved region were designed which allowed PCR-mediated amplification of the full-length β DNA component from the total nucleic acid extracts isolated from infected plants originating from a variety of geographically distinct sources and host plants. To accomplish the mentioned objectives, various plants showing symptoms of CLCuD were collected from Cotton Research Station, Abohar and were subjected to molecular analysis to check for the presence of β DNA in them using universal primers specific for β DNA. Two of the cotton varieties susceptible to CLCuD viz. F846 and RS 921were selected for further sequencing analysis of β DNA. Total genomic DNA was extracted from the diseased leaf samples using CTAB method. The β DNA of there two varieties was amplified and was run on the 1% agarose gel. It was then purified out of the gel using Wizard® SV Gel and PCR Clean-Up System from Promega (A9281). The purified PCR product was A-tailed to introduce single A residue at the ends of the DNA insert. The tailed DNA product was then ligated to the vector using a cloning kit namely, pGEM®-T and pGEM®-T Easy Vector Systems from Promega (A 3610). The ligated plasmids were then transformed into competent cells of JM 109 strain and DH5 Alpha strain and subjected to overnight incubation at 37 °C. The blue and white colonies were obtained on LB-agar plates supplemented with ampicillin, X-gal and IPTG. Screening of white colonies for the presence of positive clones containing DNA insert of interest was done by colony PCR using M13 forward primer.

The plasmids from clones giving positive results were isolated from bacterial cells by Alkaline Lysis Method and were subjected to RNase treatment. The plasmids were sequenced using M13 universal primers with ABI sequencer version 3730xl. The sequences obtained for both the samples were compared with the already available sequences of β DNA associated with Cotton Leaf Curl Multan Virus (CLCuMuV) and Cotton Leaf Curl Burewala Virus (CLCuBuV). Both CLCuMuV and CLCuBuV are the most prevalent strains of the begomoviruses causing Cotton Leaf Curl Disease. CLCuBuV is the result of recombination between CLCuMuV and CLCuKoV and has been emerged out as the resistance breaking begomoviral strain which has knockout all the resistant cultivars of cotton in Pakistan. The sequences of β DNA obtained from F846 (2010) and RS 921(2011) showed a sequence identity ranging between 98-100 % with the β DNA associated with CLCuBuV. This result established that CLCuBuV is the prevalent strain in Indian cotton growing states.