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MOLECULAR CHARACTERIZATION OF A BEGOMOVIRUS AND ASSOCIATED SATELLITES FROM COTTON (GOSSYPIUMHIRSUTUM FROM DERA GHAZI KHAN DISTRICT OF PAKISTAN.

Byline: A. Akram, K. Hussain, N. Nahid, Mahmood-ur-Rahman, A. Nasim and S. Shaheen

ABSTRACT

Cotton leaf curl disease is a major threat to cotton production in tropical and subtropical regions of the world. Begomoviruses are whiteflies-transmitted single-stranded DNA viruses belonging to the family Geminiviridae that cause leaf curl disease in cotton. In present study, a strain of tomato leaf curl betasatellite and Xanthium strumarium alphasatellite have been found associated with Cotton leaf curl Kokhran virus in Gossypium hirsutum. A begomovirus and associated molecules were amplified, cloned and sequenced. Partial sequence analysis for begomovirus showed 98.2% sequence identity with an isolate of Cotton leaf curl Kokhran virus (HQ257374). The partial sequence of betasatellite showed highest sequence identity of 96.4% with tomato leaf curl betasatellites (EF068245, FR819710). The complete sequence for alphasatellite showed 98.2% sequence identity with Xanthium strumarium alphasatellite (HF547408).

This is the first report that tomato leaf curl betasatellite have been found in cotton and after second epidemic of cotton leaf curl disease this is the first report of parent strain of Cotton leaf curl Kokhran virus in the field.

Keywords: Cotton, Begomovirus, Betasatellite, Alphasatellite, Cotton leaf curl disease.

INTRODUCTION

Geminiviruses are plant viruses having circular single-stranded DNA (ss-DNA) genome of size 2.6-2.8Kb transmitted by insect vectors. Members of family Geminiviridae has been classified into seven genera (Begomovirus, Mastrevirus, Curtovirus, Topocuvirus, Becurtovirus, Turncurtovirus and Eragrovirus) based on their genome arrangement, insect vectors, sequence relatedness and host range (Brown, 2012). Among all seven genera only two of them (Begomovirus and Mastrevirus) have been reported to have associated satellite DNAs with them (Briddon et al., 2001; Kumar et al., 2014). Begomovirus is the largest genus having more than 280 species and have wide host range. They infect dicotyledonous plants and are transmitted by whiteflies (Bemisia tabaci). Begomovirus genome can be monopartite (single component genome) or bipartite (two component genome). In bipartite genome both components (DNA A and DNA B) are essential for bonafied infection.

In the New World, begomoviruses are exclusively bipartite with one recently reported exception of a monopartite Tomato leaf curl deformation virus [ToLDeV; (Melgarejo et al., 2013)]. In Old World mostly monopartite begomoviruses are found but a few bipartite begomoviruses have also been reported (Ha et al., 2008).

Approximately 15 years ago, some ss-DNA satellite molecules were discovered associated with monopartite begomoviruses named as betasatellite (previously known as DNA[beta]) and alphasatellite (previously known as DNA-1) that are around half the size (approximately 1.3-1.4 Kb) of a begomovirus genome. Betasatellite-begomovirus complexes cause many economically important diseases in Africa and Asia, resulting in yield losses estimated at millions of US dollars (Mansoor et al., 1999, Mansoor et al., 2003). Betasatellites encodes only a single protein (betaC1) involved in the pathogenicity. They are dependent on the helper begomovirus for their replication and encapsidation. Begomovirus-associated alphasatellites are autonomously replicating satellite-like molecules. They encode one protein, the replication associated protein (Rep) that is replication initiator protein. They rely on the helper virus for movement, encapsidation and vector transmission.

Therefore they are called as satellite-like since, by definition, satellites are not capable of independent replication (Briddon et al., 2004). The elective advantage of alphasatellites to begomovirus-betasatellite complexes remains uncertain. Satellites have shown to be associated with mostly Old World monopartite begomoviruses-betasatellite complexes but recently been identified with bipartite begomoviruses in the New World (Fiallo-Olive et al., 2012) as well as with a mastrevirus in India (Kumar et al., 2014). Begomoviruses have found associated with leaf curl and mosaic diseases in many crop plants as well as in weed plants such as croton, sweet potatoes and Xanthium strumarium (Hussain et al., 2011; Albuquerque et al., 2012; Mubin et al., 2012).

The economically most important disease caused by monopartite begomovirus-betasatellite complexes in Pakistan is cotton leaf curl disease (CLCuD) affecting Gossypium hirsutum L. CLCuD is characterized by typical symptoms of leaf curling, vein darkening, vein swelling and enations on undersides of leaves (Briddon and Markham, 2001). The disease was sporadic problem throughout southern Asia during early 1980s and it became epidemic in 1986 near Multan region and spread rapidly nearly all cotton growing regions of Punjab, Pakistan and moved eastwards to the east Punjab and Rajasthan provinces of India (Briddon et al., 2001). During late 1990s plant breeders developed cotton varieties showing resistance against Cotton leaf curl Multan virus and Cotton leaf curl Khohkran virus, the most prevailing begomovirus complex causing CLCuD (Rahman et al., 2005).

But the resistant varieties succumbed to CLCuD in the area of Burewala near Multan Pakistan during 2001 and this for the beginning of a second epidemic (Mansoor et al., 2003). A new strain of begomovirus was associated with these con epidemic; Cotton leaf curl Kokhran virus strain Burewala (CLCuKoV-Bur; previously called Cotton leaf curl Burewala virus; Amrao et al., 2010 b; Rajagopalan et al., 2012). This new resistance breaking strain is dominant in all cotton growing regions of Punjab Pakistan as well as western India. Economic losses due this disease in cotton are significant and a challenge to modern agricultural biotechnology. The new resistance breaking virus complex associated with CLCuD is recombinant a of two species of begomovirus Cotton leaf curl Multan virus and Cotton leaf curl Kokhran virus (Amrao et al., 2010 b).

This resistance-breaking recombinant strain was found associated with recombinant strain cotton leaf curl Multan betasatellite (Amrao et al., 2010 b) but no alphasatellite was found associated with this recombinant begomovirus complex (Amrao et al., 2010 b). In present study we survey cotton fields in Dera Ghazi Khan district of Punjab Pakistan and found begomovirus likely to be Cotton leaf curl Kokhran virus associated with a betasatellite likely to be tomoto leaf curl betasatellite and Xanthium strumarium alphasatellite (Hussain, 2013). Significance of this new complex in cotton has been discussed.

MATERIALS AND METHODS

Sampling and DNA isolation: Infected cotton leaves showing typical disease symptoms of begomovirus infection were collected from different cotton fields in D G Khan district (Fig1). Leaves showing symptoms like leaf curling, vein swelling and yellowing and enations under side of the leaf were given particular importance for total plant genomic DNA extraction by using cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987).

Amplification and cloning of viral components: For amplification of begomovirus components rolling circle amplification (RCA) technique using phi29 DNA polymerase and random hexamer primers was used (TempliPhi TM, GE Healthcare). Amplified fragments were digested with restriction enzymes and ligated in to vector pTZ57R (Fermentas/Thermo Fisher Scientific, Massachusetts, USA) as per recommendations of the supplier.

Sequencing and Sequence analysis: Cloned molecules were sequenced completely using M13 based primers by Macrogen (Seoul, SouthKorea). Sequences were assembled and analyzed with the help of Lasergene package (DNASTAR, Madison, Wisconsin). Multiple sequence alignments were performed and phylogenetic trees were constructed using Clustal X. Trees were displayed and manipulated using Tree view software. Percentage identity was determined using MegAlign application. The reference sequences used in analyses were obtained from National Center for Biotechnology Information (NCBI) databases (https: //www. ncbi.nlm. nih.gov/).

RESULTS AND DISCUSSION

Pakistan is a hub of begomoviruses infection since decades, which was a big reason of cotton crop destruction in terms of millions and billions dollars (Sanz et al., 2000). As Pakistan is an agriculture dependent country and its economy is based on such economical important crops so it is necessary at prior basis to save such crops (Wilkins et al., 2000). Cotton leaf curl disease is a major viral constraint faced by cotton growers in sub-continent. In 2002 on-wards disease reappeared in epidemic form in cotton varieties which were showing some tolerant behavior. During this second epidemic of single species of begomovirus Cotton leaf curl Kokhran virus-Burewala strain (CLCuKV-Bu) is dominating throughout Punjab Pakistan.

In this study the presence of parent strain of Cotton leaf curl Kokhran virus has been shown in infected cotton plants. The partial sequence of a begomovirus AA55 was done. The sequence of clone was submitted to database and accession number KX599186 assigned to it. The clone of begomovirus was partially sequenced for replication associated protein(Rep) gene and intergenic region. Rep is conserved gene in function and position among geminiviruses except in Mastrevirus which is indispensable for rolling circle replication of virus genome (Hanley-Bowdoin et al.,2004). The clone was determined to 786 nucleotides in length (coordinates: nucleotides 1967-2748 of Cotton leaf curl Kokhran virus; HQ257374).

Comparison of the sequence of clone AA55 with sequences of same regions of begomoviruses available in data bases revealed the higher level of nucleotide sequence identity to different isolates of Cotton leaf curl Kokhran virus (CLCuKV) and Cotton leaf curl Rajasthan virus ranging from 97.6-98.2% (Table 1). The highest identity value was shown with Cotton leaf curl Kohkran virus; HQ257374 isolated from cotton in India. To all other begomovirus sequences already available in the data bases this partial sequence showed less than 90% nucleotide sequence identity. We can say our clone AA55 is likely to be an isolate of Cotton leaf curl Kokhran virus present in cotton in D.G. Khan district of Punjab Pakistan. In phylogenetic dendogram our clone has been segregated along with different isolates of Cotton leaf curl Kokhran virus (CLCuKV) and Cotton leaf curl Multan virus Rajasthan strain (CLCuMV-Rajstrain; Fig1).

This is a first report of CLCuKV and CLCuMV-Raj strain in cotton after second epidemic caused by CLCuKV-Bu. As CLCuKV-Bu is a recombinant strain of two parental viruses Cotton leaf curl Multan virus (CLCuMV) and Cotton leaf curl Kokhran virus. The partial sequences we are discussing here can be the counter part of CLCuKV-Bu. But it is not the case as the CLCuKV-Bu has higher sequence identity with CLCuKV in its virion-sense genes whereas complementary-sense genes have higher sequence identity with CLCuMV (Amrao et al., 2010 b). The partial sequence covering Rep gene sequence (a complementary-sense gene) are showing highest sequence identity with CLCuKV.

The partial sequences of a betasatellite molecule, which was cloned in vectorpTZ57R and designated as AA30 was obtained. The sequence is available in the sequence databases under accession number KX599188. The sequence of clone AA30 encompasses [beta]C1 gene region and some downstream sequences towards the satellite conserved region (SCR) of betasatellite. The clone was determined to be 652 nucleotides in length. Alignment of the sequence of the clone AA30 with betasatellite sequences available in database (of same regions) revealed highest sequence identity 96.4% with two isolates of tomato leaf curl betasatellite (EF068245 and FR819710) and second highest nucleotide sequence identity is 95.2% with another isolate of tomato leaf curl betasatellite (HM989847; Table2).

In the tropical and sub-tropical countries, chilli-infecting begomoviruses have emerged as a major threat by infecting economically important crops such as potato, bitter gourd, papaya, tomato and tobacco leading to complete crop loss (Mubin et al., 2009). The result shows betasatellite associated with Cotton leaf curl Kokhran virus may be tomato leaf curl betasatellite. In phylogenetic dendogram our clone AA30 has been grouped together with some isolates of tomato leaf curl betasatellite and tomato yellow leaf curl betasatellite in a separate clade (Fig 2).

The complete nucleotide sequence of clone AA33 of alphasatellite was found to be 1361 nt and showed the characteristics of a typical alphasatellite. It encodes a single 311 amino acids long protein on virion sense similar to nanovirus encoded rep (coordinates of gene76-1011 nt). Usually alphasatellite Rep protein is 315 amino acids long but in this case, there is deletion of four amino acids. Pairwise protein sequence alignment was performed with Rep protein of Gossypium darwinii symptomless alphasatellite (GoDSA) using Emboss Water on line tool (http: //www.ebi.ac.uk/Tools/ psa/emboss_water/). It showed that four amino acids (Lue, Gln, Gly, Try) present on position 50-53 of GoDSA Rep were deleted in Rep of AA33 alphasatellite. The complete sequence of the clone AA33 (accession number KX599187) was analyzed via phylogenetic dendogram and sequence identity with 29 different alphasatellites from a variety of hosts.

It made a distinct clade with GoDSA, (FR877535), Xanthium symptomless alphasatellite (XSA, HF547408) and Cotton leaf curl Burewala alphasatellite (CLCuBuA, FN658730) as in Fig 3. Highest nucleotide identity was observed with Xanthium symptomless alphasatellite (XSA, HF547408) which is 98.2%, second highest identity is 97.8% with GoDSA and it has 96.6% identity with CLCuBuA. Other sequences showed less than 77.9% similarity (Table 3). The alphasatellites found in this complex is an isolate of Xanthium symptomless alphasatellite previously reported from Xanthium strumarium a weed growing in cotton fields (Mubin et al., 2012).

Cotton is the backbone of economy of Pakistan and CLCuD is a major threat to cotton production in the country Monopartite begomovirus complex is responsible for epidemics of CLCuD. Genetic changes by recombination and pseudorecombination in the complex are driving force for the development of new complexes, which could be more virulent. In present study, we are reporting CLCuKoV, ToLCuB and XSA as complex for the first time. Further experimentation is needed for cloning of full genome of begomovirus and betasatellite which lead to infectivity efficiency of the complex.

Table 1. Nucleotide sequence identity percentage of begomovirus clone AA55 with begomoviruses from databases

AA30 Betasatellite###***

AF104036 SPLCV###3.7###***

AF259896

###6.4###63###***

EACMCamV

AJ002448 CLCKV-

###4###69.1###65.5###***

Fai

AJ002449 CLCKV-

###4###69.1###65.7###99.3###***

Man

AY029750 ToSRV###4.7###66.4###63.7###64.7###64.5###***

AY309241 DoYMV###3.5###55.5###47.9###48.7###48.9###56.5###***

DQ026296 SLCCV###5.5###65.2###65.2###73.4###73.8###64.2###52.8###***

DQ887537 ToLCBV###7.7###69.4###65.9###70.9###71.3###66.7###49.4###73.3###***

EF459696 ToYVSV###8.9###7.9###4.5###4.2###4.2###2.7###4.5###10.1###6.1###***

GU181356 OYVMV###4.5###67.1###65.4###76.8###76.8###65.9###36.1###68.4###75.3###3###***

JQ411237 ToYLCV###5.5###68.7###68.7###74.1###73.9###66.6###55.6###72.9###80.5###4###77.5###***

JQ424826 CLCuMV###3.9###64.2###64.7###83.5###83.4###68.2###48.1###69.4###70.6###2.9###76.8###72.3###***

JQ793786 PLCV###6.6###69.1###68.6###78.7###78.8###59.3###50.6###74.5###73.9###5.2###73.1###78.5###79.2###***

JX415195 SiMMV###4###63.9###62.4###63.4###63###89.4###48.2###62.4###64.2###3.7###63.7###66.4###67.7###60###***

JX415200 EuYMV###3.5###19###35###43###43.2###31.3###21.7###19.3###34.5###3###45###35.5###44.2###18.7###33.9###***

JX416174 YYVV###6.4###68.7###66.1###72.3###72.6###60###49.9###70.6###80###4.2###75.8###79.7###72.4###78.7###65.9###33.3###***

JX416177 ToLCV###6.4###68.9###66.9###77###77.5###60###49.6###72.8###74.1###2.9###72.4###78###77###83.4###61###33.4###78###***

KC424490 SrCMV###6.4###66.1###55.1###73.8###73.3###61###49.4###69.2###68.7###6.4###67.1###71.9###70.4###77.5###59.8###26.4###70.8###76.8###***

KC589699 AgEV###6.1###69.2###61.7###70.9###71.4###72.6###49.2###69.2###71.9###6.9###73.3###73.6###74.8###74.1###67.7###46.2###71.8###74.8###66.2###***

KC683374 LeMV###9.7###59###57.1###63.2###63.2###69.7###49.1###58.2###61.3###6.6###63.4###61.5###66.2###64.2###75.3###51.4###61.5###65.2###59.8###67.1###***

KC852204 MYMIV###3.4###62###64.4###66.1###66.1###59.3###47.2###66.2###70.1###7.2###66.9###71.3###61.5###66.2###58.3###42.5###71.6###68.4###64.2###59.8###57.8###***

KC878472 EYVV###5.7###63###66.9###69.6###69.9###66.9###53.8###70.8###77.3###3.7###80.7###79.2###72.8###74.3###66.9###35.6###80.2###72.9###65.5###73.8###59.8###68.1###***

KF312364 ChLCV###3.4###69.4###63.4###65.5###65.9###62###55.8###65.5###67.2###5.5###65.5###66.6###62.9###68.7###59.2###44.2###66.9###68.4###67.4###68.7###58.5###61.5###65.2###***

KF429251 HUYMV###2.9###71.3###65.2###78.5###78.5###63.9###48.6###74.8###73.3###3.5###75.1###77###78.7###84.2###63.7###42.9###76###82.7###75.6###76.3###65.2###68.2###73.4###70.4###***

KF444948 CLCuMV###3.9###67.1###64.4###82.7###82.5###67.9###48.2###69.2###69.9###2.9###76.3###71.6###99.2###78.5###66.7###44.2###72.1###77###70.3###74.1###66.1###60.8###71.8###63.4###78###***

KX599186 AA55###4###69.2###65.7###97.8###98###64###48.7###74.3###71.1###4###77.3###73.8###84.4###78.5###62.9###42.9###72.3###77.1###73.1###71.3###64###66.1###68.1###66.6###78.7###84###***

NC_001439 BGYMV###2.4###67.6###63.2###66.6###66.6###68.1###58.7###68.6###66.1###2.5###63###68.9###68.1###67.9###62.9###27.4###66.9###67.9###63.2###66.9###63.7###63.7###55.1###62.7###68.9###67.7###66.2###***

NC_008373

###6.4###75.6###67.4###72.4###72.9###71.6###48.9###71.8###76.1###5.7###73.4###77.1###71.8###76.6###68.1###29.2###77.5###75.6###70.8###77.3###64###65.9###75.6###70.9###77.3###71.4###72.4###69.7###***

ToLCGuV

NC_008374

###3.4###67.6###68.2###73.1###73.6###56.5###53.4###73.1###81.7###3.7###79.7###80.2###70.3###75.8###62.9###32.8###81.8###75.1###70.1###72.6###57.5###70.9###84.5###68.2###75.6###69.6###72.9###64###78###***

ToYLCGuV

NC_010947 AlLCV###2.9###75.3###68.1###73.6###73.8###67.9###53.6###72.8###75.1###4###72.4###77.3###73.4###78###67.2###42.9###77.5###75.3###69.9###78###65.2###65.9###76###71.9###79###72.9###73.4###70.1###90.6###77.3###***

NC_011309 JYMIV###6.1###69.9###62.5###79.2###79.7###67.1###57###72.3###75###8.1###77###77###74.8###77.5###63.7###43###76.1###80.2###73.4###73.6###61.5###66.4###70.8###69.9###77.6###74.1###79.3###67.4###74.1###75.5###74.8###***

NC_014446 SGMFV###4###52.4###52.4###52.1###52.3###60.3###46.1###52.4###51.3###3.9###55.8###53.3###57.1###55###64.4###51.4###53.8###52.6###50.1###60###64.7###50.4###54.8###48.6###55.8###56.5###52.3###52.9###58.5###52.6###58.5###52.8###***

NC_022073 HYMV###9.2###67.4###62.4###69.1###68.7###53.9###50.9###65###67.7###3.7###65.5###70.6###67.2###73.1###60.3###28.2###68.4###72.6###73.9###64.5###55###61.7###62.9###66.9###73.3###66.9###68.9###64.7###69.1###68.2###71.1###71.1###52.6###***

###AA30###AF259896 AJ002448 CLCKV- AJ002449 CLCKV-###AY309241###DQ887537###EF459696ToYVS###GU181356###JQ411237###JQ424826###JX415195###JX415200EuYM###JX416177###KC852204###KF312364###KF429251###KF444948###NC_001439###NC_008373###NC_008374###NC_014446

###AF104036 SPLCV###AY029750 ToSRV###DQ026296 SLCCV###JQ793786 PLCV###JX416174 YYVV###KC424490 SrCMV KC589699 AgEV KC683374 LeMV###KC878472 EYVV###KX599186 AA55###NC_010947 AlLCV NC_011309 JYMIV###NC_022073 HYMV

###Beta###EACMCamV###Fai###Man###DoYMV###ToLCBV###V###OYVMV###ToYLCV###CLCuMV###SiMMV###V###ToLCV###MYMIV###ChLCV###HUYMV###CLCuMV###BGYMV###ToLCGuV###ToYLCGuV###SGMFV

Table 2. Nucleotide sequence identity percentage of betasatellite clone AA30 with betasatellites from databases

KX599188 AA30###***

AA33 Alphasatellite###4###***

EF068245 ToLCB###96.2###2.6###***

EF095958 TYLCTHB###93.4###3.1###95.4###***

EF614161 LuLDB###18.4###2.8###48.3###20.4###***

FJ469629 CLCuGB###40.5###2.6###41.7###52.1###38.3###***

FN658737

CLCuMuB###35###4.3###36.2###17.5###63.2###19.6###***

FN678779 KLCuB###56###4.3###52###56###79.6###46.6###68.6###***

GQ478344 DiAYVB###71.6###1.8###72.5###72.7###36.5###55.8###45.6###54.4###***

HM101173 PaLCuB###54.9###2.3###55.5###54.9###23.2###13.8###23.6###20.4###58.1###***

JF502376 CLCuMuB###32.5###4.3###44.5###45.6###78.7###37.4###73.2###87###48.6###23.9###***

JN663873 RaLCB###73.2###2###74.2###73.6###35.1###43.3###34.7###56.1###84.8###56.1###33###***

JQ728545 HYVB###42.2###4.6###38.7###41###17.8###27.1###24.8###21.9###33.7###19.2###34.4###36.3###***

JX678964 SLCB###57.8###2.3###58.4###58.1###19.5###54###43.6###43.3###66.6###91.9###19.6###61.3###14.7###***

KC222955 BYVB###56.7###4.6###49.2###50.5###45.7###43.6###31.9###45.4###39.9###11.7###42.3###58.7###17.3###22.7###***

KC305092 AgYLCB###12.1###2.9###11.7###11.5###3.2###6.3###7.1###10###10.9###4.3###9.8###11.5###5.4###4.8###4###***

KC545814

CroYVMB###56.6###3.1###56.4###57.7###5.2###15.2###16.7###4.9###53.1###56.3###19.9###54###26.1###62.6###21###10.4###***

KC959935 PaLCuB###57.1###2.5###56.9###57.4###19.9###47.7###20.7###19.5###61###91.9###20.7###59.4###12.7###94.9###21.9###10.3###63.3###***

KJ605115 ToLCB###94###3.1###96.3###98.8###17.9###36.7###35.4###56.9###72.1###54.8###44.6###73.6###42###57.2###49.4###12.1###57.2###56.4###***

NC_012557 AgLCB###53.4###2.5###51.5###57.8###21.9###56.7###19.6###19.2###52.6###13.2###19.5###32.5###28.1###39.1###41.1###8###11.2###54.9###50.5###***

NC_013423 VYVB###36###2.3###36.8###36.5###27.1###16###16.6###35###35.7###11.8###15.2###40.3###26.7###28.7###35.6###4.3###15.2###26.4###36.5###35.4###***

NC_015928

VYVFujB###29.3###3.2###26.5###26.8###20.2###8.7###3.4###21.8###28.5###19.5###21.2###30.2###31###27.1###37.3###3.8###24.8###17.3###26.8###38.5###63.2###***

NC_018869 MYMIB###53.4###2.9###53.8###39.9###19.2###15.6###5.5###26.1###57.7###54.4###23.2###33.4###29.4###53.8###20.2###4.6###52.8###36.5###54.6###13.8###25.9###32.2###***

NC_020475

CorYVMB###54.8###2.5###54.8###55.7###27.1###26.7###17.2###49.7###54###54.1###26.5###53.4###32.1###55.5###6###5.1###53.2###54.9###55.8###13###26.7###27.1###72.1###***

###KX599188###AA33###EF068245###EF095958###EF614161###FJ469629###FN658737###FN678779###GQ478344###HM101173###JF502376###JN663873###JQ728545###JX678964###KC222955###KC305092###KC545814###KC959935###KJ605115###NC_012557###NC_013423###NC_015928###NC_018869###NC_020475

###AA30###Alphasat###ToLCB###TYLCTHB###LuLDB###CLCuGB###CLCuMuB###KLCuB###DiAYVB###PaLCuB###CLCuMuB###RaLCB###HYVB###SLCB###BYVB###AgYLCB###CroYVMB###PaLCuB###ToLCB###AgLCB###VYVB###VYVFujB###MYMIB###CorYVMB

Table 3. Nucleotide sequence identity percentage of alphasatellite clone AA33 with alphasatellites from databases

KX5 99188

AA33###***

FJ956707

AYVSGA###23.1###***

FN554583

CLCuGA###45.5###26.2###***

FN658716

BYVMA###69.6###27.4###42.3###***

FN658717

BYVMA###69.6###27.4###42.3###10.0###***

FR772085

AgCSA###79.7###21.9###40.3###79.3###79.3###***

FR772086

HYVSA###43.3###15.1###73.5###44.1###44.1###44.8###***

FR772087

GoMSA###52.5###25.4###54.4###47###47###53###72.9###***

FR873573

CLCuMA###62###14.6###49###82.7###82.7###64.6###52.1###53.4###***

FR877532

CLCuMA###76.8###16.7###48.9###83.2###83.2###65.2###51.7###55###88.9###***

FR877534

CrYVM A###49###25.8###56.3###48.5###48.5###48.6###65.2###82.4###54.9###55.2###***

FR877535

GoDSA###96.1###21.5###47###69.9###69.9###77.9###44.9###52.7###61.9###63.6###48.5###***

GU385877

CyTLCuA###75.3###21.6###44.7###71###71###85.2###45.9###51.1###70.1###78###49.5###75.6###***

HE984149

CMA###61.6###14.2###39.4###66.5###66.5###57.1###37.9###40.7###64.5###65.9###42.3###62.1###63.1###***

HQ343234

CLCuA###60.8###15.6###40.8###70.3###70.3###72.4###53###54.8###73.3###71.6###54.7###60###68.3###64.3###***

JQ041697

ToLCuND

A###16.8###48.6###13.1###28.5###28.5###30.2###25.3###6.4###16.1###17###23.6###23.3###29.1###14.5###17.5###***

KC 305095

GLCuA###75.1###15.9###47.4###69.2###69.2###83.5###47.3###52.6###71.9###81.5###51.6###74.1###93.8###62.8###73.6###17.7###***

KC959932

VYVFujA###42.8###26.2###57.1###50.6###50.6###44.6###65.1###75.9###50.3###53.3###75.3###45.1###46.4###40.5###53.2###12.6###54.3###***

NC_018628

CMMA###63.8###25.6###47.9###68.9###68.9###67.5###46.4###47.6###66.5###67.6###49.4###63.5###64.1###68.4###68.2###15.9###63.8###50.9###***

AJ298903

CLCuMB###9###8.5###2.2###7.2###7.2###8.5###2.1###3.6###5.8###4.7###1.8###8.7###8.5###5.7###3.8###8.2###4.2###2.7###1.7###***

###JQ041697

###KX599188###FJ956707###FN554583###FN658716###FN658717###FR772085###FR772086###FR772087###FR873573###FR877532###FR877534###FR877535###GU385877###HE984149###HQ343234###KC305095###KC959932###NC_01862###AJ298903

###ToLCuND

###AA33###AYVSGA###CLCuGA###BYVMA###BYVMA###AgCSA###HYVSA###GoMSA###CLCuMA###CLCuMA###CrYVMA###GoDSA###CyTLCuA###CMA###CLCuA###GLCuA###VYVFujA###8CMMA###CLCuMB

###A

Acknowledgments: This research was supported through a start-up research grant no. PM-IPFP/HRD/HEC/2012/3543 funded by Higher Education Commission (HEC) Pakistan awarded to Khadim Hussain.

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Publication:Journal of Animal and Plant Sciences
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Date:Aug 31, 2017
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