百萨偃麦草染色体5J特异标记的开发与应用
毕业论文关键词：小麦；共线性；百萨偃麦草；分子标记；转录组
Development and application of molecular markers specific for Thinopyrum bessarabicum chromosome 5J
Abstract： Thinopyrum bessarabicum chromosome 5J contains genes such as hardness, vernalization and dwarf status, representing an important gene resource for wheat improvement. After RNA-Seq, 4 Gb sequences were generated from a Th. bessarabicum transcriptome. De novo assembly produced 47877 sequences. Using bowtie software, 397 sequences of them were identified with high similarity to homoeologous group 5 chromosomes of wheat and 100 EST-SSR primer pairs were designed using these sequences. Amplification in Chinese Spring (CS), CS- Th. bessarabicum amphiploid and CS- Th. bessarabicum disomic substitution lines DS5J(5A) found 19 primer pairs produced specific bands in 5J and could be used as specific markers. Combined with previously identified markers, total 13 markers were applied to identify the chromosome constitution of 12 CS- Th. bessarabicum chromosome aberrations involving 5J which identified two plants and one line were small segmental translocations involving 5JS, one plant was telosome 5JS, three plants and one line involved variation of 5JL, and one homozygous line was T5JS.5JL-W#2. Based on these aberrations, three new developed markers were located to different segments of 5J. This result indicated that transcriptome sequencing combining with marker-assisted selection would facilitate chromosome engineering of wheat and help developing physical maps of alien chromosomes.
Key words: wheat; synteny; Thinopyrum bessarabicum; molecular marker; transcriptome
目录
摘要.3
关键词.3
Abstract....3
Key words ...3
引言..3
1 材料与方法..4
1.1 材料 ...5
1.2 序列比对与引物设计法...5
1.3 分子标记分析...5
2 结果分析..5
2.1 百萨偃麦草5J染色体特异分子标记开发 ....5
2.2 涉及5J变异的分子标记分析 5
3 讨论 .... 7
致谢.10
参考文献.10
百萨偃麦草染色体5J特异标记的开发与应用引言：由于集约化栽培和少数骨干品种的广泛推广，小麦遗传基础日趋狭窄，严重限制了小麦产量和品质的进一步提高，加剧了小麦应对生物和非生物胁迫的脆弱性[1]。小麦野生近缘种中携有丰富的抗逆基因，通过染色体工程可以将这些基因导入普通小麦，丰富小麦的遗传基础，提高小麦的抗逆能力[2]。百萨偃麦草（Thinopyrum bessarabicum Löve, 2n = 2x = 14, JJ）耐盐和兼抗多种麦类病害，是小麦改良的重要基因资源[3]。研究发现，小麦的第5部分同源群染色体上携有抗病、春化、抗寒性、产量和品质等有利基因[4]，推测百萨偃麦草染色体5J可能携有有利的同源基因，对于拓宽栽培小麦的遗传基础具有潜在的应用潜力。诱致涉及不同百萨偃麦草染色体的易位系是精细定位、转移和利用这些基因的前提，而开发简单经济有效的PCR标记，有利于加快百萨偃麦草染色体工程[5]。二代测序技术（NGS）给禾谷类作物发掘其近缘物种的有利基因提供了一个有效的手段[6]，例如，基于转录组测序的EST（expressed sequence tags）的 SSR（EST-SSR）标记开发，与传统的基因组SSR相比，更加简单高效[7]，通过高通量测序，获得百萨偃麦草转录组序列，并通过拼接获得EST contig序列。刘志涛（2015）[8]利用这些EST序列开发了一批百萨偃麦草的EST-SSR标记并用于物理图谱的构建和易位系的鉴定。李晨旭等（2015）利用这些序列与已经公开发表的小麦D基因组框架序列进行比对分析，鉴定百萨偃麦草相对特异的基础序列，利用这些基础序列中的部分序列设计PCR引物、开发并定位一批染色体特异标记，并利用这批特异标记对细胞学鉴定出的中国春-百萨偃麦草易位系进行分析[5],这些标记在鉴定易位染色体身份和缺失作图中的有很大的应用潜力。
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