| Wheat(Triticum aestivum L.,AABBDD,2n=6x=42)is one of the most important crops,1RS.1BL translocation wheat lines,in which the 1RS.1BL chromosome was formed through rye 1RS replacing wheat 1BS,possess lots of outstanding features,such as high yield and diseases/pests resistance.Therefore,1RS.1BL translocation chromosome is widely used in wheat breeding program.So far,researchers focus on either broadening the genetic basis of 1RS.1BL translocation chromosome or developing specific molecular markers in 1RS,but for the studying on the mechanism of interaction between 1RS and its wheat genetic background are not enough.Hence,seeking genes that have 1RS-specific expression in wheat genetic background is conducive to study on this mechanisms.Besides,utilizing 1RS.1BL deletion lines can map 1RS specific genes on a given 1RS chromosome region,and this is helpful for identifying the function of different 1RS chromosome regions.Using repeated sequences to analyze structure character of 1RS is also helpful for investigating the function of 1RS.In this paper,non-denaturing fluorescence in situ hybridization(ND-FISH)was used to analyze the offspring derived from common wheat Mianyang 11(MY11)×rye Kustro,and some 1RS.1BL translocation lines and 1RS.1BL deletion lines were obtained.Using SLAF-Seq and RNA-Seq technologies to develop new 1RS-specific makers.These new markers combining with some previously reported 1RS makers were located on three regions of 1RS arm using 1RS.1BL deletion lines.Additionally,PCR amplification using cDNA as templates was conducted to select some 1RS-specific expression genes,and the expression of these genes were investigated in different growth stages of 1RS.1BL translocation lines.Besides,qRT-PCR was used to study the expression of tandem repeats pSc200 and pSc250 in different growth stages of 1RS.1BL deletion lines.Finally,the agricultural traits of 1RS.1BL deletion lines were also investigated.Some results were obtained as following:1.Oligo-1162,Oligo-pSc200,Oligo-pSc250 and Oligo-pSc119.2-1 were used as probes for ND-FISH analysis of the offspring derived from MY11×Kustro.A 1RS.1BL translocation line in which the telomeric region of 1RS was absent,a 1RS.1BL translocation line in which the satellite of 1RS was absent,and a 1RS.1BL translocation line in which the 1RS arm was intact were identified.According to these 1RS.1BL deletion lines,the 1RS arm is divided into three regions including S.1-S.3,S.3-S.5,and S.5-S.7,these are new types of 1RS deletion lines that has not been reported before.Besides,some other 1RS.1BL translocation lines and their corresponding non-translocation lines were also obtained.2.Using SLAF-Seq and RNA-Seq technologies,a total of 2550 pairs of primer were designed,and 114 new 1RS-specific makers were selected.Eleven of the 114 makers can be used to distinguish the 1RS derived from Kustro and Insave,and the remaining markers show no polymorphism.From the previously reported 1RS makers,33 markers were verified to be 1RS-specific.The 147 markers were physically located using the 1RS.1BL deletion lines,and 93 makers were mapped on S.1-S.3 region,40 makers were mapped on S.3-S.5,and 14 makers were mapped on S.5-S.7 region.In addition,the break point of 1RS in deletion line 15T-2 was located between the markers Xtsm587 and SCM9,and the break point of 1RS in 15T-1 was located between the markers Sec-1 and P6M12-P.3.Using cDNA of six growth stages including tillering,elongation,booting,heading,flowering and filling stages of translocation and non-translocation lines as templates,3 of the 147 markers(G-1RS-140,G-1RS-143,PE005)amplified 1RS-specific products only from the six growth stages of translocation lines indicating their corresponding genes are 1RS-specific expression in wheat backgrounds.The G-1RS-140 related gene has 94.07% similarity with the mechanosensitive ion channel protein gene located wheat 1D chromosome,the G-1RS-143 related gene has 94% similarity with the heat shock protein gene located on wheat 1A chromosome,and the PE005 related gene has 92% similarity with the glutathione S-transferase gene located on wheat 1A chromosome,this has not been reported before.Using the cDNA of 1RS.1BL deletion lines,both the G-1RS-140 and G-1RS-143 related genes are located on the S.5-S.7 region of 1RS,and PE005 related gene has been located on the S.3-S.5 region.4.RNA-Seq results indicate that the G-1RS-140 related gene had the highest expression level in all the six growth stages of T-3 line.In tillering stage,elongation stage and booting stage,the expression level of G-1RS-140 related gene was higher in T-2 line than that in T-1 line,and in heading stage,flowering stage and filling stage,the expression level of this gene in T-1 line was higher than that in T-2 line.Except filling stage,in the other five growth stages,the expression level of G-1RS-143 related gene was the highest in T-3 line.In the first three stages,the expression level of G-1RS-143 related gene was higher in T-2 line than that in T-1 line,and in the last three stages,the expression level of this gene was higher in T-1 line than that in T-2 line.5.Using qRT-PCR,the expression level of tandem repeats pSc200 and pSc250 in the six growth stages of 1RS.1BL deletion lines was analyzed,and the results displayed that in tillering,booting,heading and flowering stages,the expression level of pSc200 was: 15T-2>15T-1>15T-3,in elongation stage,the expression level of pSc200 was: 15T-3>15T-2>15T-1,in filling stage,the expression level of pSc200 was: 15T-1>15T-2>15T-3.In tillering,heading and flowering stages,the expression level of pSc250 was: 15T-2>15T-1>15T-3,in elongation stage,the expression of pSc250 was: 15T-3>15T-2>15T-1,in booting stage,the expression of pSc250 was: 15T-3>15T-1>15T-2,in filling stage,the expression of pSc250 was: 15T-1>15T-2>15T-3.Therefore,the expression of pSc200 and pSc250 had no obvious rule in the six growth stages of 15T-1,15T-2 and 15T-3 lines.6.The agronomic traits of 1RS.1BL deletion lines,including plant height,spike length,tiller number,panicle number,spikelet number and thousand-kernel weight were investigated.The following are the results: plant height and thousand-kernel weight showed significant difference among the three lines,displayed that 15T-3>15T-2>15T-1,thousand-kernel weight showed highly significant difference in all materials;spike length and spikelet number showed that 15T-1>15T-3>15T-2,and the significant differences were also observed between 15T-3 and 15T-1,and between 15T-2 and 15T-1.However,the difference between 15T-3 and 15T-2 is not significant;tiller number and panicle number did not display great differences among the 1RS.1BL deletion lines.These results are more likely to indicate that different 1RS regions have differently contributed to yield.In the present study,we acquired part of sequences of three 1RS-specific expression genes and relevant markers,these results will provide a necessary basis for studying the interaction between 1RS.1BL translocation chromosome and wheat genetic background.Additionally,the investigated results that we received from 1RS.1BL deletion lines provides new thoughts about the development 1RS small-segment translocation. |
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