Identification Of Aegilops Geniculata-Wheat Derivatives

Wheat is an important food crop.However,the narrow genetic basis has limited the breakthrough of wheat breeding.The genetic basis of wheat includes the nuclear genome inherited from both parents,and the cytoplasm genome(mitochondrial and chloroplast genomes)from maternal inheritance.Presently,a huge number of new wheat lines with various outstanding traits have been created by introducing nuclear genes from wild relatives of wheat via distant hybridization.Nevertheless,there is a relative lack of research on and utilization of the plastid cytoplasmic genomes of wild relatives.Aegilops geniculata(2n=4x=28,U~gU~gM~gM~g)is a tetraploid specie,which harbors many valuable genes for disease,insect,drought,and saltness resistance.Furthermore,its cytoplasm has great potential valuable in wheat breeding.In this study,the derivatives of Ae.geniculata×wheat were identified and evaluated by using in situ hybridization,molecular markers analysis,and agronomic traits measurement.In addition,the cytoplasmic genetic effect of Ae.geniculata introduced into common wheat was also evaluated.The primary results are below:1.Fourteen alloplasmic wheat lines were obtained by using Ae.geniculata as female parent to cross with common wheat,followed by back-crossing and selfing.All of them showed stripe rust resistance,and late heading,suggesting these traits contributed by the cytoplasm of Ae.geniculata.Some of them also showed abnormal spike development,where the spikelet of upper spike was sterile.2.To establish the karyotype of Ae.geniculata,its two genome chromosomes in root tip cells were firstly distinguished by GISH method,using the genomic DNA of Aegilops comosa(2n=2x=14,MM)as the probe while the genomic DNA of Aegilops umbellulata(2n=2x=14,UU)as the block.Then,the same root tip cells were re-hybrided by multiple repeat sequences probes,including Oligo-p Sc119.2,Afa family,Oligo-p Ta71,and(CTT)5.The results showed that most of the chromosomes can be identified just based on these four probes,except for the 3U~gand 4M~g which have the same FISH signals pattern.Thus,cannot be identified without extra GISH information.Hence,new FISH probe combination was screened.The results showed that the 14 chromosomes pair can be unequivocally recognized by using a cocktail of three probes,namely p Ta-713,(AAC)5,and p Ta-71.Finally,a new FISH karyotype of Ae.geniculata was established.3.GISH,FISH and molecular markers were used to characterize the chromosomal constitution of the fourteen alloplasmic wheat lines.One of them was 1M~g disomic addition line,three 4M~g(4B)substitution lines.No detectable introgression of Ae.geniculata were identified of the rest 10 derivatives.All 14 derivatives were significantly later heading than their common wheat parents.However,the three 4M~g(4B)substitution lines were significantly earlier heading than other 10 derivatives,suggesting chromosome 4M~g may carry gene(s)controlling heading,which can be used to improve the late heading trait of wheat with the cytoplasm of Ae.geniculata.In terms of plant height,the 1M~g disomic addition line was significantly shorter than the wheat parents and other 13 derivatives,suggesting 1M~g carried dwarf gene(s).The number of spikelets per spike in the 10derivatives without alien chromatin identified were significantly more than its wheat parents,4M~g(4B)substitution lines and 1M~gaddition line,which may be related to their late heading phenotype(more time for spikelets developing).There were no obvious patterns about tiller number and spikelet length.These derivatives can be served as a valuable bridge material for wheat improvement.4.Glu-M~g1,the high molecular weight subunit locus on the chromosome 1M~g of Ae.geniculata,has been considered as a key potential valuable gene for wheat quality improvement.Based on the single nucleotide polymorphisms(SNPs)among 1Mx,1My and other HMW-GS encoding genes from 1A,1B,1D and 1U~g,1Mx and 1My subunits-specific PCR markers were successfully developed.The Glu-M~g1 specific molecular markers are useful for developing of small fragment translocation lines and tracking wheat lines containing Glu-M~g1.5.The reciprocal F1s are suitable materials for cytoplasmic genetic effect analysis,because they have identical nucleus genome while different cytoplasmic genome.Three major genetic effects(delaying heading,decreasing fertility and reducing plant height)of Ae.geniculata cytoplasmic were confirmed by comparing the agronomic traits of reciprocal F1between the derivatives of Ae.geniculata-wheat and six different wheat cultivars.We sequenced and assembled the cytoplasmic genome(mitochondrial and chloroplast genomes)of Ae.geniculata AS6.It was found the chloroplast genome of AS6 is relatively conservative compared to the chloroplast genome of Chinese Spring.However,the mitochondrial genome of AS6 showed many structural variations,such as inversion,translocation,inversion plus translocation and specific genes.Thus,the differential of mitochondrial genomes between Ae.geniculata and common wheat may be the key factor responding for phenotypic differences,such as heading date.Among the 207 mitochondrial genes annotated in AS6,15genes showed missense mutations that were compared to the mitochondrial genome of CS.Thirty-three AS6-specific genes were also identified.These genes were important candidate gene for further studying the genetic effects of Ae.geniculata cytoplasm.In addition,co-dominant molecular markers for cytoplasm genomes of AS6 and common wheat were designed,which may provide a basis for the identification and screening of wheat lines with Ae.geniculata cytoplasm.