Characterization Of Wheat-Dasypyrum Breviaristatum Introgression Lines And Physical Localization Of The Genes Encoding Important Agronomic Traits

As an important relative of wheat,Dasypyrum contains three species,namely the annual diploid D.villosum(2x=2n=14,VV),the perennial diploid D.breviaristatum(2x=2n=14,V^bV^b)and the perennial tetraploid D.breviaristatum(4x=2n=28,V^bV^bV^bV^b).Dasypyrum species carry many agronomically useful genes such as resistance to diseases,resistance to pests,cold tolerance,drought adaptation and high protein quality,all of which could enrich the increasingly narrow genetic variability of the wheat gene pool.Great progress has been made in the studies on D.villosum,and researchers have characterized and analyzed some beneficial genes originated from D.villosum,including Pm21 widely used in wheat breeding.However,the research on D.breviaristatum is relatively fall behind.It is urgent to promote the creation of wheat-D.breviaristatum introgression lines,the analysis of V^bchromosomes and the development of genetic resources of D.breviaristatum.TDH-2,a wheat-D.breviaristatum partial amphiploid,was developed in our laboratory,laying a foundation for the cytological analysis of V^bchromosomes and the study of genetic resources.In order to identify seven pairs of V^bchromosomes effectively and map the excellent genes on V^bchromosome,we conducted the following studies:(1)An efficient Oligo-FISH painting system was used to complete the cytological analysis of 1V^b-7V^bchromosomes.(2)A large number of oligo probes and molecular markers were developed for identifying and tracking small chromosomal segments of D.breviaristatum.(3)A plenty of wheat-D.breviaristatum chromosome2V^bdeletion and translocation lines were screened from the hybrid population and the radiation induction population by using the 2V^b(2D)substitution line D11-5.We mapped some elite genes by newly developed 2V^bchromosome deletion lines,and identified several potential candidate genes related to excellent traits by transcriptome analysis.The main results are as follows:1.The standard karyotype of seven pairs of V^bchromosomes were constructed by using Oligo-FISH painting system with Synt1-Synt7 and non-denaturing fluorescence in situ hybridization(ND-FISH)analysis in the wheat-D.breviaristatum partial amphiploid TDH-2.2.The ND-FISH with multiple oligo probes was conducted on the durum wheat-D.villosum amphiploid TDV-1 and the perennial tetraploid D.breviaristatum PI564517,the results were as follows:(1)there is a relative close genetic relationship between V genome and R genome,as well as between V^bgenome and J^Sgenome;(2)there are obvious differences between V-and V^b-chromosomes in the distribution of repeat sequences,which were mainly distributed at the end and centromeric region of chromosomes;(3)the perennial tetraploid D.breviaristatum is an autotetraploid,directly derived from the perennial diploid D.breviaristatum;(4)highly polymorphic of seven linkage group chromosomes in PI564517 was showed,and several noticeable changes occurring in chromosomes V^bbecame stable after being introduced into wheat background.3.Based on the reported genomic sequences,eight novel oligo probes for ND-FISH were designed,of which the probe Oligo-7E-744 could specifically recognize seven pairs of V^bchromosomes,and the probe Oligo-7E-430 only produced hybridization signals on chromosome 6V^b.The detailed FISH map of all of seven D.breviaristatum chromosomes with 27 oligo probes enabled effective identification of 12new wheat-D.breviaristatum introgression lines,including T6V^bS.2V^bL and T3V^bS.6V^bL translocation lines,4V^baddition line,3V^bS and 3V^bL-deletion line,etc.A total of 27,115,7,53 D.breviaristatum-specific markers were assigned to 3V^bS,4V^b,6V^bS and 6V^bL,respectively.4.The seeds of D11-5(2V^b/2D)and new developed D2230(2V^b-1/2D)substitution lines were induced by gamma ray radiation with different dosage rate,and the seeds of the generation M₀-M₂were analyzed by using ND-FISH,the results were as follows:(1)The study defined 120 types of wheat chromosome rearrangements,of which B-genome chromosomes showed the highest translocation frequency(47.77%);(2)a new probe Oligo-k288 can produce signals covering the AB-chromosomes of wheat,which can be applied to detect the translation generated between the AB-genomes and the D-genome combined with the probe Oligo-D;(3)26non-Robertsonian translocation lines were characterized,and these breakpoints were mainly distributed at the telomeric region of these chromosomes;(4)17 types of 2V^bchromosomal structure variation were identified,and a high frequency breakpoint was targeted in the region of 2V^bL with two Oligo-713 signals;(5)The study selected 13stable lines carrying multiple translocation chromosomes and displaying excellent agronomic traits.5.Chromosome 2V^b-1 of D2230 showed a higher transmission ratio in progenies from background Sumai3(28.7%)than in those of Mianyang11 and Chuanmai62,and less than chromosome 2D;2V^b-1 chromosome deletion was commonly appeared in Mianyang11 and Taichang29,while the recombination involving chromosome 2V^b-1was clearly observed in population of Sumai3 and Chuanmai62;Four new types of 2V^bdeletion lines and six types of W-2V^btranslocation lines,including T2DS.2V^bL,T2V^b-1S.2DL and T2V^bL.2BL were obtained.6.A total of 183 D.breviaristatum-specific molecular markers,including 25 pairs of primers developed by RNA-seq,were located in nine region of chromosome 2V^b;transferring chromosomes 2V^bor 2V^b-1 into wheat could significantly increase the spike length and spikelets per spike;Three types of transcription factors related to growth were identified through transcriptome analysis,which could be related to the difference in plant height between D11-5 and D2230;field evaluation of stripe rust reactions about the wheat-2V^bdeletion lines indicated that the stripe rust resistance gene(s)were located on FL 0.47-0.80 of D.breviaristatum chromosome 2V^bL,and two homologous candidate genes Thint.05G0328100 and Thint.05G0340800 were screened by transcriptome analysis according to the reported 2J^S-genome sequence.In this present study,I completed the sorting of V^bchromosomes by using Oligo-FISH painting system with Synt1-Synt7,and built a high-resolution FISH karyotype of 1V^b-7V^bchromosomes for identifying small segment of D.breviaristatum chromosomes in common wheat background,and created a set of wheat-D.breviaristatum introgression lines.The study also analyzed the difference between chromosomes V and V^bwith multiple probes,and confirmed the phylogenetic relationship of the perennial tetraploid D.breviaristatum in Dasypyrum.The genetic stability of D.breviaristatum chromosome 2V^bin different wheat background was explored,and some excellent agronomic traits that chromosome 2V^bcarried were verified,of them the stripe rust resistance gene(s)were located and analyzed precisely.