Transferrence Of Resistances To Cotton Leaf Curl Disease From Two Diploid Species Into Gossypium Hirsutum Via Interspecific Hybridization

Cotton crop is a leading cash crop considered for almost 100 countries.The Cotton leaf curldisease that started in early 1990's has been the most important limitation on cottonproduction in Pakistan and India.The disease spread throughout the cotton belt of Pakistan,India,and now reported also in South China.It is pertinent to mention that since 2001 novariety with extreme tolerance against the disease is available.Wild cotton speciesparticularly Gossypium arboreum and G.stocksii have rich potential sources for breeding,including strong fibre and resistances against cotton leaf curl disease.The vast sources inwild,diploid cotton species are available but very few reports are available about theidentification of DNA markers linked to sources of resistance to CLCuD.Identification ofquantitative trait loci(QTL)conferring resistance to CLCuD will facilitate breeding forresistance to cotton leaf curl disease.In this study,firstly,we developed an interspecifichybrid by crossing G.hirsutum with two diploid species,i.e.G.arboreum and G.stocksii,and then evaluated these hybrids for resistance against cotton leaf curl disease and thenidentify the DNA markers linked to CLCuD.1.Transferrence of resistances to cotton leaf curl disease from diploid species into G.hirsutum via interspecific hybridization(1)Development of backcross populations from an interspecific cross between G.arboreum and G.hirsutumThe plant materials used to develop BC1?BC3 population from an interspecific crossbetween G.arboreum and G.hirsutum consist of G.hirsutum cv CRSM-38(2n=4x=52),G.arboreum cv 15-Mollisoni(2n=2x=26),and an artificial autotetraploid of G.arboreum(2n=4x=52).The F1 CLCuD-resistant populations involving these parents,comprising direct cross 2(G.arboreum)×G.hirsutum and its reciprocal G.hirsutum ×G.arboreum,were backcrossed with G.hirsutum to produce up to BC4 populations.Thelimitation of interspecific hybrids was overcome by application of plant hormones,andintensive crossing.Development of BC1 was more challenging after F1 that required 12890 numbers ofpollinations that yielded only 57 seeds,followed by BC2 and BC3 with number ofpollinations 1295 and 980,respectively.Direct cross offered more arduous effort for gettingBC1?BC3 seeds as compared with its reciprocal cross.Similarly,reciprocal cross showedrapid advancement towards normal behavior regarding presence of more bivalentchromosomes,good boll setting percentage and seed germination rate.Phenotypicresemblance of BC1?BC3 progenies to G.arboreum confirmed the success of cross betweenthe two species and aided great for selection of suitable BC progenies.(2)Evaluation of cotton leaf curl virus resistance in backcross populations from aninterspecific cross between G.arboreum and G.hirsutumThe BC1?BC3 interspecific progenies from direct cross[2(G.arbore(G.×G.hirsutum]andits reciprocal[G.hirsutum × G.arboreum]was evaluated for their resistance to CLCuD.The F1 progenies were completely resistant to this disease,while a decrease in resistancewas observed in all backcross generations.As backcross progressed,the disease index(DI)increased in BC1(1.3?1.6%),BC2(1.8?4.0%),and BC3(4.2?7.0%).However,DI wasmuch lower than that of the check variety CIM-496,with a CLCuD index of 96%.Theassessment of CLCuD through grafting showed that the BC1?BC3 progenies were highlyresistant to this disease.The latent period(days)to acquire disease symptoms were longerfor 2(G.arboreum)×G.hirsutum cross as compared with G.hirsutum ×G.arboreum.Thus,this study successfully demonstrates the possibility of introgressing CLCuDresistance genes from G.arboreum to G.hirsutum.2.Association analysis between SSR markers and tratits from G.arboreum in G.hirsutum(1)Identification of donor chromosome segments from G.arboreum in G.hirsutum viaSSR markersA GGT version 2.0 tools was used to analyze the proportion of chromosomal segmenttransferred from G.arboreum in G.hirsutum.The graphical genotyping analysis revealedthat genomic constitution was contributed majorly by allele A(G.hirsutum)(76.2%)withlittle contribution of allele B(G.arboreum)(4.3%).Similarly the marker located onchromosome 9 showed maximum contribution of allele B(G.arboreum)(24.4%)and contribution of allele A(G.hirsutum)was 55.5%.Introgression line 2,8,10,and 60 showed more than 10%contribution of allele B(G.arboreum).Introgression lines like 1,2,8 and 10 showed good resistance to CLCuD with desirable agronomic and fibre traits.These ILs can be helpful for MAS.The information about the donor chromosome segment present in introgression lines will be very expedient for appropriate identification and assortment of right progenies for breeding programs,including gene mapping,and ultimately for emphasizing the importance of MAS in cotton improvement worldwide.(2)Association analysis between traits and molecular markersAssociation analysis between markers and phenotypic values analysed by SMA resulted in the identification of six significant SSR loci associated at 0.1%significance level for cotton leaf curl disease related traits.For fibre and agronomic traits 20 and 27 loci were associated at 0.01%significance level,respectively.These number of associated markers are more as compared to detected QTLs.This showed that false positive markers are also identified by SMA.Our results demonstrated that using association analysis to examine important traits is an efficient way of identifying significant loci associated with these traits.Simultaneous associations of markers with other traits can also be identified.This can be potentially useful for plant breeding programs.(3)QTLs for cotton leaf curl diseaseThe results obtained from IciMapping analysis detected five QTLS for cotton leaf curl disease;two for disease index(DI)and three for percent disease tolerance(PDT).The percent variation explained range for these five QTLs was 7.6-14.6%with LOD 2.0-2.9.Five markers BNL3347-140,NAU3695-280,dc40134-370,NAU3317-225 and JESPR290-125 found to be associated for these seven QTLs.Single marker analysis also confirmed these markers associated significantly with disease related traits.(4)QTLs for agronomic traitsA total of 26 QTLs was identified by QTL Ici Mapping for 12 different traits;five QTLs for SCW,three QTLs each for FBpP and LM;two each for LpB,BS,SpL,SpB,and SM;and one QTL each for MPpP,BpP,SpB,SCpS and SCpL.The maximum and minimum phenotypic variation explained for these agronomic traits was 3.8 and 29.0%.Totally 13 markers were expressed for QTLs involved for agronomic traits i.e.BNL3347-140,Gh369-145,Gh594-100,JESPR274-105,NAU1014-185,NAU1246-200,NAU2108-350,NAU2508-150,NAU2887-450,NAU3022-235,NAU3401-400,NAU462-650,NAU6993-130.However,five markers were found to be overlapping and helpful for MAS.Forexample,on chromosome 5,two markers BNL3347-140 for traits LpB,BS and markerGh594-100 for five different traits i.e.SpL,SpB,SCW,LM,SM was found to be importantfor expression of these agronomic traits.Similarly,marker NAU462-650 was activelyparticipated for expression of MPpP,FBpP,LpB,BS;marker NAU2508-150 for SpB,SCpL;and marker NAU3401-400 for FBpP,SpL,SCW,LM,SM.(5)QTLs for fibre quality traitsFor fibre quality parameters,a total of 15 QTLs was identified for six different traits;fourQTLs for LP,three QTLs each for FU and Rd;two each for FS and SFI;and one for FL.The maximum and minimum phenotypic variation explained for fibre QTL was 4.0 and19.6%.Totally 11 markers were expressed for QTLs involved for fibre traits i.e NAU2494-215,Gh594-100,NAU2002-600,JESPR290-125,NAU2686-1250,NAU3695-280,NAU1278-270,NAU3401-400,cgr6812-150,BNL1438-120,BNL3479-220.Four markerswere found to be overlapping that were found to be stable for expression of different fibretraits.For example,on chromosome 5 markers NAU2494-215 was found to be importantfor expression of FU,SFI;marker JESPR290-125 for two different traits i.e.FL,FS.Similarly,marker NAU3401-400 was actively participated for expression of FU and SFI;and marker BNL1438-120 for LP and Rd.3.Transferrence of resistances to cotton leaf curl disease and increased fibre strengthfrom G.stocksii into G.hirsutum via interspecific hybridization(1)Development of F1 hexaploid from an interspecific cross between G.hirsutum and G.stocksiiGossypium.hirsutum was crossed with G.stocksii.Hybridization was done by manualemasculation and pollination under field conditions.Intensive number of crossing(438)together with application of plant hormones made it possible to set 15 bolls which yieldedonly 28 F1 seeds.The obtained seeds,treated with 0.03%colchicine for 6 hours,and 12seedlings were successfully obtained,but,only 4 hexaploid(2n=6x=78)plants reachedmaturity,which represented 42.9%germination and a 33.3%success rate of hexaploiddevelopment.F1 plants showed 51.1%pollen viability with intermediate expression formost of the morphological characteristics(stem,leaf,flower,boll).Microscopicexamination of PMCs at metaphase-1 revealed that the hybrid plants had 78 chromosomes and 70.3%genome 2[(AD)1 E1]of hexaploid F1 showed 39 bivalents.The 51.1%,pollen viability of F1 lead to low boll setting on selfing in the hexaploid.(2)Evaluation of cotton leaf curl virus resistance and increased fibre strength in F1 hexaploid from an interspecific cross between G.hirsutum and G.stocksiiF1 hybrid plants of G.hirsutum × G.stocksii were screened against CLCuD by grafting,and under natural field conditions.In field conditions,none of the 4 hexaploid plants showed symptoms of CLCuD compared with variety S-12.All 17 grafts were also remained asymptomatic to CLCuD.The results of fiber traits,i.e.,fiber strength,elongation rate and micronaire,indicated that F1 hexaploid possessed greater fiber strength(54.4 g/tex),fiber elongation(6.7%)and fiber fineness(5.6 ?g/inch)than that of commercial cultivar MNH-886.The fiber strength,elongation rate and micronaire,was increased by 34.9,23.9 and 3.6%over the MNH-786(control),respectively.