| Gossypium australe(2n=2x=26,G2G2),possessing valuable characteristics unavailable in the cultivated cotton gene pool,such as cold and drought resistance,resistance to blight,verticillium wilt,resistance to cotton aphid,red spider,increased lint fractions,delayed seed gland development,glandless-seed and glanded-plant,can be used to improve cultivated cotton,which is an important genetic resource.However,G.australe belongs to the tertiary gene pool of cultivated cotton and has a far genetic relationship with G.hirsutum,chromosome exchange between them seldom occurs,making it difficult to use the excellent genes of G.australe in breeding.In order to effectively transfer the excellent genes of G.australe into G.hirsutum,pollen from the sesquidiploid of G.hirsutum-G.australe was irradiated to induce the chromosome aberrations between G.hirsutum and G.australe.The main research results are as follows:1.Effects of irradiation on boll set and seed germinationTo indicate the effect of irradiation dose on boll setting rate and seed viability,preliminary pollen irradiation with three doses(10,12 and 20 Grays,Gy)was performed in 2011.The results indicated that all the boll set rates were>80%and plenty of seeds were obtained from irradiation treatments,implying these three irradiation doses had little influence on boll set.We then planted the seeds and found that as the dose increased,the germination rates and number of seedlings decreased.Even so,the germination rates and seedling percentages were up to 44.85%and 14.71%at the dose of 20 Gy.Based on these initial results,in 2012,higher doses of irradiation(20,30 and 40 Gy)were employed.The results showed that boll set rates were significantly decreased to the half of the control(no irradiation)and seed germination rates and seedling percentages greatly dropped,reaching 22.86-31.69%and 2.86-5.19%,respectively.The results implied that doses of 30 and 40 Gy are too high for pollen irradiation.In 2013,the doses were further adjusted to 15,20 and 25 Gy.To obtain much more Mi progenies,much more flowers were pollinated by irradiated pollen to produce more hybrid seeds.However,most of the obtained seeds were immature and were lower viability because cotton plants suffered from low temperature and frost during the late development.Therefore,cotton seeds harvested in 2013 showed very low rates of germination and viability.The results demonstrated that all the boll set rates were>80%,while only at the lower two doses were slightly more seeds generated and 23.73-35.20%of seeds germinated and produced 16.95-27.20%of seedlings.2.Identification of aberrations of G.hirsutum-G.australe by GISHA total of 632 irradiation-produced hybrid seeds were obtained and analyzed by GISH,among them,170 GISH-positive(with GISH signal(s)on chromosome(s))individuals were identified into three groups.The first one was the chromosome number variation involved in one to four alien chromosomes additions(142/170)respectively.The second was chromosome structure variation involoved in sole translocations(10/170).The third one was complex structure variation involoved in both alien chromosomes addition and translocation(18/170).The control group(pollen without irradiation treatment)identified showed only one group that contained monosomic alien chromosome additions(13/120).In the M1 generation,most aberrations in GISH positive individuals were monosomic alien chromosome additions(106/170).The frequency of induced chromosome translocation is 18.82%(32/170).A total of 6,7 and 7 irradiation-produced chromosome aberration types were obtained under 10,12 and 20 Gy dose in 2011;10,6 and 4 types were obtained under 20,30 and 40 Gy dose in 2012 and 2,1 and 0 types were obtained under 15,20 and 25 Gy respectively in 2013.The CK induced only 1 chromosome mutation type.The dosage of 20 Gy led to the largest number of chromosomal variation types,combined with the boll rate and seed survival rate of irradiation,the dose of 20 Gy was more suitable for cotton pollen irradiation to induce chromosome aberration.Moreover,based on the sizes and translocated positions of the alien chromosomes,translocation could generally be divided into five types,namely,whole arm translocation(WAT),terminal translocation(TT),large alien segment translocation(LAST),small alien segment translocation(SAST),and intercalary translocation with the inserted segment from G.australe(IT).Here,we identified 28 individuals containing translocations,that 32 breakage-fusion events had occurred,containing three types of translocation,most of them being WAT(22/32),followed by LAST(7/32),and SAST(3/32).The other two types of translocations(TT and IT)were not found.3.Identification of alien(G.australe)chromosome aberrationson M1 generations was identified by molecular marker analysisAmong the 70 surviving individuals,59 contained chromosome additions only,3 contained chromosome translocations only,and 8 contained both chromosome additions and chromosome translocations.Among the 67 individuals with alien chromosomes added,chromosomes 5G and 6G had the greatest incidence(7.81%),followed by 12G(6.25%),9G(5.73%),2G and 11G(2.60%),7G(2.08%),3G,4G and 10G(1.56%),and 1G(1.04%).No incidence of added chromosome 13G was found.Among the 11 individuals with chromosome translocations,there were two incidences each of 3G,7G,8G and 13G translocations,and one of 2G,6G,9G and 10G translocations.No translocations of the other five chromosomes(1G,4G,5G,11G and 12G)were found.4.Identification of the G.hirsutum chromosomes translocated by the chromosomes from G.australeWe analyzed somatic cells of 17 translocation lines by multicolor-GISH using the total genomic DNA of G.australe and G.herbaceum as probes,and G.raimondii as a blocker.The multicolor-GISH results showed that all translocations occurred between chromosomes of the A-subgenome in G.hirsutum and those of G.australe and no translocations occurred between chromosomes of the D-subgenome in G.hirsutum and those of G.australe.5.Chromosomal segment introgression from G.australe into G.hirsutum induced by irradiationA total of 192 individuals were analyzed using SSR markers that are evenly distributed on the D-subgenome chromosomes of tetraploid cotton.The results indicated that 107 out of 192 individuals were introgressed,each by a number of alien chromosomes ranging from one to five,of which most of individuals(71/192)were introgressed by one alien chromosome,followed by two(18/192),three(14/192),four(3/192),and five(1/192).The other 85 individuals were not introgressed by any alien chromosomes.The average introgression percentage in the M1 generationas was 55.73%(107/192),which was far higher than that in controls(11.76%).In the control,102 individuals were analyzed using the SSR markers.Only 12 plants were found to be introgressed by alien chromosomes.Among them,nine plants were each introgressed by two alien chromosomes,followed by one(2/12)and three(1/12).The most frequent introgressive chromosomal segments caused by the irradiation were chromosome 5G(20.83%),followed by 1G(9.90%),8G(8.85%),6G and 10G(both 7.81%),2G(7.29%),4G and 9G(both 5.73%),11G and 13G(4.69%),and 3G(0.52%).Chromosome 7G did not give rise to any introgressive chromosomal segments caused by irradiation.In control plants,however,only four chromosomes were found to be introgressed.Chromosomes 5G and 13G were simultaneously introgressed at a rate of 9.80%,2G at a rate of 1.96%and 9G at 0.98%.No other chromosomes were found to be introgressed.6.Identification and location of 45S rDNA and 5S rDNA in G.australeBased on a set of G.hirsutum-G.australe alien addition lines,the chromosomes and arm positions of 45 S rDNA and 5S rDNA in G.australe were identified.The results showed that the G.australe genome had two 45S rDNA loci(on the short arms of 7G and 9G,respectively).The 5S rDNA was located on the G.australe 9G chromosome,which was on the same chromosome with the 45 S rDNA.7.Evaluation of 13 MAALs and two ATLs resistance to Verticillium wiltUsing Verticillium wilt isolate V991,a set MAALs of G.hirsutum-G.australe,two ATLs and their parents were inoculated.The results indicated that MAAL-4G and MAAL-7G were immune,the relative disease index was 0.00 and ATL-7G was high resistant,the relative disease index was 1.42.MAAL-1G,MAAL-6G and ATL-8G showed disease-resistant,the relative disease index was 33.33,32.41 and 33.46,respectively.For BP2,MAAL-4G,MAAL-7G and ATL-7G all showed immune,the relative disease index was 0.00.MAAL-6G was the disease resistant,the relative disease index was 14.06.MAAL-2G,MAAL-3G,MAAL-6G,Not-AC and ATL-8G showed disease-resistant,the relative disease index was 20.28,24.96,14.06,32.21 and 32.50,respectively.This study will provide new data of cotton for Verticillium wilt-resistance breeding. |
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