Identification Of Wheat-haynaldia Villosa 6VL Alien Chromosome Structural Aberrants

Haynaldia villosa(2n=2x=14,VV)is a closely related wild species of wheat and a tertiary gene pool for wheat genetic improvement.The excellent genes in H.villosa can be transferred to the background of common wheat using wide hybridization and chromosome engineering.Studies have reported that the 6VL chromosome carries the gene Sr52 for resistance to wheat stem rust race Ug99 and the gene Cre V for resistance to cyst nematode.Based on previous studies,in order to better explore and utilize the excellent genes on the 6VL chromosome of H.villosa,IT molecular markers specific to the certain regions of 6VL chromosome of H.villosa were developed;using sequential GISH/FISH and IT molecular marker analysis,homozygous structural aberrations involving the 6VL chromosome of H.villosa induced by irradiation were screened from the heterozygous structural aberrations;using cytological technology and molecular marker analysis,the homozygous structural aberrations involving 6VL chromosome identified in this study and screened previously were further analyzed.The size and physical location of the 6VL chromosome in the structural aberrations were identified;a physical map of the 6VL chromosome was constructed according to the amplification of 80 IT molecular markers in 53 homozygous structural aberrations,.The main results obtained in this study are as follows:1.Development of IT markers in the 540-554Mb and 566-572Mb regions on the 6VL chromosome of H.villosaThe marker density in the 540-554Mb and 566-572Mb regions of the 6VL chromosome of H.villosa is low,which is not conducive to the fine mapping of excellent genes in these regions in the future.In this study,based on the whole-genome sequencing information of H.villosa(91C43)(unpublished),27 and 24 IT molecular markers were developed in the 540554Mb and 566-572Mb regions of the 6VL chromosome,respectively.Among them,13 and 13 were polymorphic markers with an average polymorphic rate of 51.16%.According to the amplification of these markers,26 IT molecular markers were divided into type Ⅰ,type Ⅱ and type Ⅲ,with 4,15 and 7,respectively.2.Fine identification of the homozygous structural aberrations of the chromosome 6VL of wheat-H.villosa induced by irradiationBased on previous studies,homozygous structural aberrations were screened from 14 heterozygous and 13 complex structural aberrations involving the 6VL chromosome of H.villosa induced by irradiation using sequential GISH/FISH cytological technology.Finally,24 homozygous structural aberrations,73 heterozygous structural aberrations,7 heterozygous telosomics and 34 complex structural aberrations were screened from 423 individual plants.In addition to 13 homozygous structural aberrations identified by previous study,there were 37 homozygous structural aberrations in total.In order to clarify the size and physical location of the 6VL fragments in the structural aberrations and the changes in the background chromosomes of common wheat,sequential GISH/FISH technology was carried out.According to the standard karyotype of common wheat Chinese Spring published,it was found that among 37 homozygous structural aberrations,31 homozygous structural aberrations were related to the 6VL fragment of H.villosa and the 6A chromosome of common wheat;3 homozygous structural aberrations were related to the fragment of 6VL of H.villosa and the 2B chromosome of common wheat;2 homozygous structural aberrations were related to the 6VL fragment of H.villosa and the 3D chromosome of common wheat;1 homozygous structural aberrations was related to the 6VL fragment of H.villosa and the 7B chromosome of wheat.Using 80 6VL chromosomespecific IT molecular markers,37 homozygous structural aberrations were analyzed the size and physical location of 6VL fragments.Combined with the cytological analysis,37 homozygous structural aberrations were divided into 7 different types of structural aberrations3.Fine identification of the homozygous structural aberrations of wheat-H villosa 6VL chromosome induced by CSph1b mutantBased on previous studies,this study divided 23 homozygous structural aberrations into three types using sequential GISH/FISH technology:16 homozygous terminal aberrations,5 homozygous intermediate aberrations and 2 homozygous telosomics.In addition,it was also found that there were 19 and 2 aberrations related to 6VL chromosome and common wheat 6A and 6D chromosomes,respectively.Using 80 6VL chromosome-specific IT molecular markers,23 homozygous structural aberrations were analyzed in detail.23 homozygous structural aberrations were further divided into 8 different types according to the differences in the fragment size of the 6VL chromosome and the recombination site.4.Construction of the physical map of the 6VL chromosome of D.villosumUsing the 64 6VL chromosome-specific IT molecular markers developed previously and 16 6VL chromosome-specific IT molecular markers developed in this study,21 CSph1binduced and 32 irradiation-induced homozygous structural aberrations of the 6VL chromosome of H.villosa were analyzed.According to the amplification of 80 IT markers in 53 homozygous structural aberrations,a 6VL chromosome physical map containing 21 bins was constructed and these 21 bins were named binl-bin21.Each bin had an average of 3.81 molecular markers.The region with the largest number of IT molecular markers was bin4,which contained17 markers;the bin with the lowest number of IT molecular markers had 7 bin,namely bin5,bin9,bin11,bin13,bin14,bin19 and bin20.Each bin had only one IT molecule marker.The marker density of the middle to the terminal region(402.54-572.03Mb,169.49Mb apart)of the 6VL chromosome of H.villosa was relatively high.This region contained 73 pairs of markers,with an average of one marker every 2.32Mb;the mark density of the sub centromere to the middle region(260.91-402.54Mb,141.63Mb apart)was very low.This region contained 7 pairs of markers,and there was an IT molecular marker every 20.23Mb on average.