Identification And Genetic Analysis Of Wheat Pyramiding Substitution Lines Carrying Hordeum Vulgare 2H And Thinopyrum Intermedium 2Ai-2 Chromosomes

Thinopyrum intermedium and barley are two close relatives of wheat and carry many useful genes,including tolerance to abiotic stresses,high disease resistance and so on,which are potentially valuable for the improvement of various wheat traits.Distant hybridization,in which desirable chromosomes or fragments are transferred from genetically similar plant species to cultivars,is an effective technique for developing new germplasm or varieties.This study was designed to cultivate wheat pyramiding substitution lines by hybridizing different wheat-Th.intermedium and wheat-barley disomic alien substitution lines and induce the whole-arm translocation line between chromosome 2H of barley and chromosome 2Ai-2 of Th.intermedium in wheat background through the technique of somatic variation population,using molecular markers,in situ hybridization,cytology,agronomic examination,tissue culture and other methods.Based on above information,genetic behavior of homologous chromosomes and homeologous chromosome before and during meiosis were further analysed by in situ hybridization technique.The main findings of this investigation are as follows:Hybrids between wheat-Th.intermedium and wheat-barley disomic alien substitution lines were made and the progeny were repeatedly selfed.Plants containing target chromosomes in generations F₁ to F₅ were identified using chromosome-specific molecular markers,and two types of homozygous substitution lines,2D2Ai-2?2B?2H?2A?and 2A2Ai-2?2B?2H?2D?,were preliminarily obtained.In addition,agronomic traits indicated,compared to parent materials,the wheat pyramiding substitution lines behave differently in flowering date,plant type,and other agronomic characteristics.Genomic in situ hybridization?GISH?using total genomic DNA of Th.intermedium and barley as probes was performed,and the result showed that there were 42 chromosomes in wheat pyramiding substitution lines,with two red hybridization signals and two green hybridization signals.The fluorescence in situ hybridization?FISH?analysis using different probes indicated that there were red hybridization signals in the centromeric regions of 38 wheat chromosomes,green hybridization signals in the centromeric regions of 2 barley chromosomes,and green hybridization signals on both arms of 2Th.intermedium chromosomes.Using molecular markers specific to the two alien chromosomes to genotype plants in the F₁generation derived from reciprocal crosses between wheat cultivar CS and the three substitution lines2Ai-2?2D?/2H?2A?,2Ai-2?2B?/2H?2D?,and 2Ai-2?2B?/2H?2A?,the result showed that the average transmission rates for alien chromosome 2H via the male and female gametes were 24.7%and 32.7%respectively,whereas those for the alien chromosome 2Ai-2 were 19.3%and 21.8%.The alien chromosome 2H thus appeared to be transmitted to the next generation at a higher rate than the alien chromosome 2Ai-2 and the two alien chromosomes were preferentially transmitted via the female gametes.Crossing wheat-Th.intermedium with wheat-barley disomic alien substitution lines,the F₁ hybrid immature embryo for 14-15 days after pollination were cultured on callus induction medium,then two times of subculture was conducted to induce whole-arm translocation between chromosome 2H of barley and chromosome 2Ai-2 of Th.intermedium in wheat background.Ultimately,a total of 102 regenerated plants were obtained.Using molecular markers specific to 2HL,2HS,2Ai-2L,and 2Ai-2S,accompanied by in situ hybridization,a heterozygous translocation line between barley 2H and Th.intermedium 2Ai-2 was successfully obtained.Using drop method to observe PMCs of wheat pyramiding substitution lines,GISH and FISH analysis using different probes was also carried out to identify genetic behavior of the alien chromosomes before and during meiosis.The FISH result revealed that centromeres grouped at the opposite pole with respect to telomeres,which conformed to the basic Rabl configuration,then centromere pairing or clustering appeared to occur prior to telomere bouquet formation,both of them facilitated chromosome pairing.The GISH analysis using labeled total genomic DNA of Th.intermedium and barley with different fluorescent labels indicated that the two types of alien homologous chromosome initially separated,and then associated during the pre-meiotic stages of meiocyte development,while they behaved similarly to wheat chromosomes during the prophase of meiosis.The observation of PMCs at meiotic metaphase I showed that part chromosomes happened structural changes,such as chromosome breakage,number variation and so on.