Brief Analysis On The Structure Of Rye 6RL Minichromosome With Resistance To Powdery Mildew

Wheat is the main food source for human beings in China and even the world.Wheat powdery mildew is one of the most important diseases in wheat production at present.The6 R chromosome of rye has the resistance gene to powdery mildew,and it is still resistance to many powdery races.In our previous study,a wheat-rye 6RL minichromosomal addition line,which is resistant to powdery mildew,was obtained.In this study,the structure and meiosis behavior of the 6RL minichromosomes were analyzed by ND-FISH and molecular biology techniques.Some irradiated progeny of a wheat-rye 6RL minichromosomal addition line were analyzed using molecular cytological method.At the same time,the experimental materials were evaluated for resistance to powdery mildew in the field.All the results were described as below:1.Using SLAF-seq technique,1086 primer pairs were designed,and 19 markers that are 6RL minichromosomes-specific were selected from these primer pairs.2.The products of 16 markers were cloned and sequenced.The length of the 16 sequences is between 200-464 bp long.Using the BLAST tool in GrainGenes database,these 16 sequences were compared with the scaffold sequence of rye.There are eight sequences have mor than 98% similarity with five 6R scaffolds sequences in the database.3.The five sequences of the scaffolds are Lo7_v2_scaffold₄53717 6R,Lo7_v2_scaffold₄84582 6R,Lo7_v2_scaffold₄45202 6R,Lo7_v2_scaffold₄51612 6R,and Lo7_v2_scaffold₄45253 6R,respectively.The base lengths of these 5 scaffolds are1879 bp,954bp,47346 bp,4446bp and 17519 bp,respectively.Lo7_v2_scaffold₄45202 6R,Lo7_v2_scaffold₄51612 6R and Lo7_v2_scaffold₄45253 6R have high homology with the sequences of wheat functional genes?GenBank: CF133726.1,GR504306.1 and HX057614.1?that have been logged in GenBank.4.We studied the meiotic behavior and transmission regularity of 6RL minichromosomes by ND-FISH technique.The experimental results show that minichromosomes are not properly paired,separated early,and lag behind during meiosis in most of the observed cells;The frequency of the transmission of minichromosome andthe frequency of normal pairing are also different among different plants with the same genetic background;Moreover,the transmission of 6RL minichromosome was also different in different wheat genetic background.5.The ND-FISH analysis of the minichromosomes was performed using centromere probes Oligo-CCS1 and Oligo-pAWRC.1.The results showed that the probe Oligo-CCS1 and Oligo-pAWRC.1 signals were detected in the 6RL minichromosome and this indicates that the minichromosome has centromere.6.The seeds of 14T-154,a plant containing a pair of 6RL minichromosomes,were irradiated,and the 19 6RL minichromosome specific markers were used to detect the progeny of the irradiated seeds.A total of 117 radiated progeny plants were obtained,and not all the 19 marker amplified their products from 79 of the 117 plants.This indicates that the structure of 6RL minichromosomes in the 79 plants has changed.7.Using ND-FISH technique to identify 1428 progeny plants of these 79 plants.The results showed that one plant 16T-7-1 contains a structurally changed 6RL minichromosome.6D/6RL small fragment translocation chromosomes were detected in 10 plants including 16T-379-1,16T-39-4,16T-39-6,16T-379-8,16T-379-9,16T-379-11,16T-379-13,16T-39-14,16T-39-14,16T-380-2 and 16T-380-3.All these plants were highly resistant to powdery mildew.In the small fragment translocation chromosome,the 6RL minichromosomes was divided into two smaller fragments,which were respectively translocated to the short and long arm of 6D chromosome.8.The results in this study provides useful materials for the succesful utilization of powdery mildew gene on 6RL in wheat breeding,and also provides foundation for the further study on the structure and function of the 6RL minichromosome.The above results provide a good material basis for the application of powdery mildew resistance gene carried by rye 6RL in wheat breeding,and also lay a foundation for further studying the structure and function of 6RL minichromosome.