【作者】 王青；

【导师】 亓增军；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2020， 博士

【摘要】 百萨偃麦草(Thinopyrum bessarabicum L(?)ve,2n=2x=14,JJ)具有很强的耐盐性并兼抗多种小麦病害,是小麦改良的重要基因资源。前期研究发现百萨偃麦草4J染色体包含蓝色糊粉层基因BaThb,利用电离辐射诱导获得了 19个4J染色体变异体并将BaThb初步定位于4J染色体长臂(4JL)。本研究在此基础上,对百萨偃麦草端体4JL进行流式细胞分拣和基因组测序,在揭示4JL基因组序列特征基础上,开发了新的4JL特异分子标记,结合寡核苷酸探针套荧光原位杂交(Fluoresence in situ hybridization,FISH)和基因组原位杂交(Genomic in situ hybridization,GISH)分析,对小麦-百萨偃麦草4J异染色体系进行了准确鉴定,构建了 4JL物理图谱并精细定位蓝粒基因。此外,构建百萨偃麦草基因组BAC文库,结合转录组分析鉴定BaThb候选基因,取得的主要研究结果如下:1、21个小麦-百萨偃麦草4J异染色体系的细胞学鉴定利用寡核苷酸探针套FISH/GISH分析,对21个小麦-百萨偃麦草4J异染色体系进行了鉴定,这些材料包括5个异附加系和16个易位系,染色体数分别为42、43、44和46,在16个易位系中包括14个纯合和2个杂合,只涉及4JL染色质的易位有4个,同时涉及4JS和4JL染色质的易位12个,其中13个小麦-百萨偃麦草4J易位染色体身份鉴定清楚,分别涉及小麦染色体3A、5A、1B、2B、3B、6B、1D、4D和7D。另外,除小麦和4J易位染色体外,还检测到小麦种内染色体变异,其中8个为染色体结构变异,4个同时涉及染色体结构和数目变异,分别涉及小麦染色体1A～7A、1B～6B、1D～3D、5D和7D,染色体结构变异类型包括易位和缺失。2、百萨偃麦草端体4JL分拣与测序分析在捷克Dolezel教授的帮助下,采用流式细胞学方法分拣了百萨偃麦草端体4JL并通过基因组测序,获得数据质量Q30>85%的基因组reads 70.7 Gb,拼接组装获得1,347,306 个contig,长度约为 588 Mb,contig N50为420 bp,GC含量44.6%;4JL基因组去除重复序列后,进行基因注释,综合Pfam数据库(3,302个基因)和百萨偃麦草转录组(2,896个基因)比较分析,鉴定重叠基因1,643个,涉及多种生物代谢进程。其中,注释到涉及花青素代谢的结构基因和转录因子结构域,将转录组序列支持基因2,896个剔除1D、4D和6D污染,获得高可信度基因1,791个,与普通小麦中国春4A、4B和4D进行比对,其中1,209个具有很好的比对结果,这些基因大部分位于4AS、4BL和4DL,少部分基因位于4AL,推测4J没有发生类似于4A的倒位等结构变异。3、4JL特异分子标记的开发和蓝粒基因区段定位根据4JL基因组序列,设计SSR引物1202对,参考前人构建的4J物理图谱,选取蓝粒基因区段附近的357对引物进行扩增,共获得稳定扩增的特异标记140个,多态率为39.2%。利用182个(包括已经发表42个)4JL特异分子标记对中国春和21个小麦-百萨偃麦草4J异染色体系进行扩增分析,构建了包含182个标记的4JL物理图谱,将4JL划分为8个区段,每一个区段包含的分子标记分别为4、10、20、10、4、18、32 和 84 个。涉及4JL物理作图和蓝粒基因定位的关键变异体7个,分别为16WQ02、16WQ05、16WQ09、16WQ14、16WQ18、16WQ22 和 16WQ27,对应 4JL 断点分别为 FL0.54、FL0.00-1.00、FL0.51、FL0.15、FL0.43、FL0.27 和 FLx-y。通过连续 4 年调查,16WQ02、16WQ14、16WQ22 和 16WQ27 籽粒呈非蓝色,16WQ05、16WQ09 和 16WQ18 籽粒呈蓝色,因此蓝粒基因BaThb位于重叠区域FL0.43-0.51。其中,共定位于该区段的特异标记18个,分别是4EST187、lfz3718、lfz4502、lfz4503、lfz4509、lfz4528、lfz4772、lfz4784、lfz4788、lfz4794、lfz4796、lfz4797、lfz4804、lfz4809、lfz4812、lfz4817、lfz4818和lfz4821,可以用于追踪BaThb。4、4JL蓝粒基因区段与中国春4A、4B和4D共线性验证将8个区段内标记对应的4JL contig序列与中国春4A、4B和4D进行比对以确定共线性区域,circos分析表明,除第6和第8段外,其余6个区段与4BL和4DL有良好的共线性,与4A相比,4JL第2～7区段与小麦4AS有共线性,但在第8段,29个基因中14个对应4A短臂,15个对应4A长臂,表明4J不同于4A,没有发现明显的染色体结构重排。4JL第6段包含BaThb,对应于小麦4AS、4BL和4DL的共线区段分别为29.32 Mb、25.24 Mb和21.19 Mb,这三个区段内共包含514个基因,其中17个涉及花青素代谢合成途径。将该区段内35个基因根据内含子差异设计特异标记,扩增结果显示,18个内含子差异标记定位于第6段,确证蓝粒基因区段与小麦4A、4B和4D相应区段的共线性,同时,BaThb所在区段的特异标记增加到36个,其中2个(lfz4509和lfz4528)标记序列涉及花青素代谢调控转录因子MYB和bHLH。5、蓝粒候选基因百萨偃麦草BAC克隆筛选与序列分析构建了覆盖1×的百萨偃麦草BAC文库,并将与BaThb共定位的36个标记对350个BAC混池质粒DNA进行筛选,其中22个标记扩增出目标条带,将22个标记对应的BAC混池进行菌液PCR验证,发现18个标记扩增稳定。将18个标记进行单克隆菌液PCR分析,最终获得8个阳性克隆,其对应的标记分别为lfz3718、lfz4509、lfz4528、lfz4812、lfz6279、lfz6284、lfz6285和lfz6321,其中涉及花青素代谢途径的两个标记lz4509和lfz4528均获得单克隆。对包含MYB的单克隆测序分析发现,该克隆插入片段178,509 bp,共包含24个基因,含有1个Myb-like-binding结构域基因。包含bHLH的BAC插入序列全长142,429 bp,包含18个基因,含有1个Helix-loop-helix DNA-binding结构域基因。挑选拟南芥涉及不同生物学功能的MYB和bHLH蛋白基因分别与百萨偃麦草BAC中MYB和bHLH进行系统发育分析,发现BAC中MYB蛋白与拟南芥不同功能的蛋白差异较大,而与BAC中bHLH蛋白基因最接近的为长穗偃麦草Ba1候选基因编码蛋白ThMYC4E,其次是大麦中控制花青素代谢的HvMyc2和MYC4H。据此推断筛选得到的MYB和bHLH转录因子可能参与百萨偃麦草花青素代谢调控。6、小麦-百萨偃麦草等臂染色体系I4JL·4JL转录组分析用以上两个百萨偃麦草蓝粒基因候选BAC序列为参考基因组,对小麦-百萨偃麦草等臂染色体系I4JL·4JL及其近等基因系的籽粒转录组分析表明,在两个BAC共预测到的42个基因中,仅有三个基因在开花后21天的籽粒中检测到表达,其中在蓝、非蓝粒中共同表达的基因有两个,均位于lfz4509所在的BAC序列,分别为MYB转录因子和未知功能的基因;第三个基因只在蓝粒中检测到表达,是lfz4528所在BAC中的bHLH转录因子。综合bHLH基因HvMyc2仅在大麦蓝色糊粉层细胞中特异表达,为蓝色糊粉层主要决定基因,推测百萨偃麦草BAC中预测到的bHLH转录因子为百萨偃麦草蓝粒候选基因的关键因子,初步命名为ThbbHLH。7、百萨偃麦草基因组专化BAC克隆鉴定与小麦-百萨偃麦草蓝粒易位系改良筛选得到百萨偃麦草基因组专化BAC克隆一个,序列分析显示插入片段99,814 bp,包含13个基因,该克隆的BAC FISH可以替代基因组GISH用于特异性追踪小麦背景中的百萨偃麦草染色质。为转移和利用百萨偃麦草蓝粒基因,验证蓝粒基因特异分子标记的应用潜力,将4个小麦-百萨偃麦草蓝粒易位系分别与烟农19、扬麦6号等杂交、回交,结合分子标记选择,育成20份农艺性状明显改善的蓝粒株系,为彩色小麦育种提供新材料。更多还原

【Abstract】 Thinopyrum bessarabicum L(?)ve(2n=2x=14,JJ)has a strong salt tolerance and resistance for multiple wheat diseases,representing an important genetic resource for wheat improvement.Previous studies have found that the chromosome 4J of the Th.bessarabicum contains the blue aleurone layer gene BaThb,and 19 mutants of 4J were induced by irradiation.BaThb was preliminary located in the long arm of 4J(4JL).Based on this study,telosomic 4JL of Th.bessarabicum was sorted and sequenced.Based on the specific sequence of 4JL,new molecular markers specific to 4JL were developed and used for identification of 21 wheat-Th.bessarabicum alien lines involving 4J in combination with oligonucleotides probe multiplex(ONPM),FISH(Fluorescent in situ hybridization)and genomic in situ hybridization(GISH).The 4JL physical map was developed and the blue-grained gene BaThb was fine mapped.In addition,a BAC library of Th.bessarabicum was constructed and combined with transcriptome analysis to screen and identify candidate genes of BaThb.The major results are as following:1.Cytological identification of 21 wheat-Th.bessarabicum alien lines of 4JTwenty-one wheat-Th.bessarabicum alien lines involving 4J were identified after ONPM FISH and GISH.These lines included 5 additions and 16 translocations,The chromosome number varied from 42 to 46.Among 16 translocations,14 were homozygous,and 2 were heterozygous.The alien segments of 4 translocation lines involved only 4JL,and the other 12 involved both 4JS and 4JL.The wheat chromosomes translocated with Th.bessarabicum 4J in 13 of 16 translocation lines were clearly identified,which involved 3A,5A,1B,2B,3B,6B,1D,4D and 7D.Additionally,besides the wheat and Th.bessarabicum translocations,intra wheat chromosome variations were detected in 12 lines,where 8 were structural variations(translocations and deletions),and 4 involved both structural and numerical variations,the wheat chromosomes involved 1A～7A,1B～6B,1D～3D,5D and 7D,respectively.2.DNA sequence analysis of 4JL via chromosome sorting using flow cytometryWith the help of Prof.Dolezel,from Czech Republic,chromosome 4JL was sorted using flow cytometry and sequenced.A total of 70.7 Gb genome reads with data quality Q30>85%were obtained.Sequence assembly yielded 1,347,306 contigs of total length approximately 588 Mb.Contig N50 was 420 bp,and GC content was 44.6%.After repeat filtering,coding sequences of 4JL were annotated.Based on the comparison of Pfam database(3302 genes)and the transcriptome database(2896 genes)of Th.bessarabicum,a total of overlapping 1,643 genes were identified,involving various biological metabolic processes.Among them,structural genes and transcription factors involved in metabolism of anthocyanins were identified.In addition,a total of 1,791 high-confidence genes were obtained after eliminating the contamination of chromosomes 1D,4D and 6D from 2,896 transcriptome-supported genes,and 1,209 of them aligned well with those in chromosomes 4A,4B and 4D of the Chinese Spring(CS).Most of these genes distributed in 4AS,4BL and 4DL,while a small part found in 4AL.It was speculated that 4J did not involve structural variations like inversion occurred in 4A.3.Development of 4JL-specific markers and physical mapping of blue-grained geneAccording to the assembled sequences of 4JL,1202 new SSR markers were developed.Based on the previous physical map of 4J,357 SSR markers located in the vicinity of the blue-grained gene were selected for further screening 4JL-specific markers.Among them,140 produced stable and specific amplicons in 4JL with a polymorphism rate of 39.2%.A total of 182(including 42 previously published)markers were used to amplify CS and 214J chromosome alien lines.A physical map of 4JL containing 182 markers was developed,where 4JL was divided into eight segments,and markers mapped onto each segment as 4,10,20,10,4,18,32 and 84,respectively.The seven key chromosome variants involved in 4JL for physical mapping of the blue-grained gene are 16WQ02,16WQ05,16WQ09,16WQ14,16WQ18,16WQ22 and 16WQ27,with breakpoints in 4JL are FL0.54,FL0.00-1.00,FL0.51,FL0.15,FL0.43 FL0.27 and FL x-y,respectively.Based on four years of phenotyping,16WQ02,16WQ14,16WQ22 and 16WQ27 showed stable non-blue grains,however,16WQ05,16WQ09 and 16WQ18 all showed stable blue grains.The blue-grained gene BaThb was thus mapped in the overlapping segments of FL0.43-0.51.Eighteen markers that were collocated in this region are 4EST187,lfz3718,lfz4502,lfz4503,lfz4509,lfz4528,lfz4772,lfz4784,lfz4788,lfz4794,lfz4796,lfz4797,Lfz4804,lfz4809,lfz4812,lfz4817,lfz4818,and lfz4821.4.Collinearity analysis and verification of blue-grained gene segment in 4JLThe 4JL contigs corresponding to the markers in 8 blocks were compared with the sequences of CS 4A,4B and 4D.Circos analysis was performed using 4J and 4A,4B and 4D collinear genes.Except blocks 6 and 8,the remaining six blocks had good collinearity with 4BL and 4DL.However,compared with 4A,most markers in 2～7 segments were collinear with 4AS.In block 8,a total of 29 collinear genes were identified,14 corresponded to the short arm of 4A,while 15 to the long arm,indicating 4J was different from 4A,and no structural rearrangements were found.The block 6 contains BaThb,the collinear segment size in 4AS,4BL and 4DL was 29.32 Mb,25.24 Mb and 21.19 Mb,respectively.A Total of 514 genes in the three regions were annotated.Among them 17 were related with anthocyanin metabolic synthesis pathway.Thirty-five were further used for designing specific markers based on intron differences.Eighteen intron differential markers were identified and further located in block 6,confirming the good collinearity between block 6 and the corresponding segments of 4A,4B and 4D.In total,thirty-six 4JL-specific markers were collocated with BaThb,and two(lfz4509 and lfz4528)involved anthocyanin metabolic transcription factors MYB and bHLH.5.Identification and sequence analysis of BAC clones of Th.bessarabicum with candidate genesA BAC library of Th.bessarabicum covering about 1 X was constructed.Thirty-six markers co-localized with BaThb were screened in 350 BAC bulk pools.Twenty-two markers amplified specific bands in the isolated plasmid DNAs of the pooled BACs,which were consistent with the positive controls.The 22 markers were further amplified and validated in the pooled BACs via PCR amplification.Eighteen markers produced stable amplificons,which were then used for identifying single BAC clones via PCR,and 8 positive clones were finally obtained corresponding markers lfz3718,lfz4509,lfz4528,lfz4812,lfz6279,lfz6284,lfz6285 and lfz6321,respectively.Two positive clones corresponding markers lfz4509 and lfz4528 involving MYB and bHLH were obtained.BAC sequencing revealed that insertion segment of lfz4509 clone(MYB)was 178,509 bp containing 24 genes,including one Myb-like-binding domain gene.The lfz4528 clone(bHLH)with a 142,429 bp containing 18 genes including a Helix-loop-helix DNA-binding domain gene(bHLH).Phylogenetic analysis found that MYB protein in BAC is different from those in Arabidopsis thaliana;however,the bHLH in BAC was the closest to ThMYC4E encoded by the Ba1 candidate gene,followed by HvMyc2 and MYC4H,which also control anthocyanin metabolism in barley.It was inferred that the bHLH transcription factors of Th.bessarabicum might involve regulation of anthocyanin metabolism.6.Transcriptome analysis of wheat-Th.bessarabicum isochromosome I4JL·4JLUsing the two candidate BAC sequences as reference,transcriptomes of I4JL·4JL and its near isogenic line were analyzed.Among 42 genes predicted in the two BACs,only three expressed in the grains after flowering for 21 days.Two genes expressed both in the blue and non-blue grains,which were both from BAC-lfz4509,including MYB transcription factor and an unknown gene.The third gene expressed only in the blue grains,which was bHLH transcription factor from BAC-lfz4528.Considering bHLH transcription factor HvMyc2 only expressed in barley blue aleurone layer and represents the major gene controlling blue grains,it is deduced that the predicted bHLH was the key factor of Th.bessarabicum regulating blue grains,which was preliminary named ThbbHLH.7.Identification of a genome-specific BAC clone of Th.bessarabicum and developing germplasm with blue grainsOne genome-specific BAC clone of Th.bessarabicum was identified.Sequence analysis showed that the inserted fragment was 99,814 bp,containing 13 genes.The BAC clone FISH could replace GISH for tracking the chromatin of Th.bessarabicum in wheat background.In order to transfer and utilize the blue-grained gene of Th.bessarabicum,and to verify the potential of BaThb specific molecular markers,four wheat-Th.bessarabicum blue-grained translocations were respectively crossed with elite cultivars Yannong 19 and Yangmai 6.Twenty blue-grained lines with improved agronomic traits were identified for breeding wheat with colored grains.更多还原