小麦—冰草异源染色体易位的原位杂交与分子标记鉴定
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摘要
小麦产量三要素中穗粒数是最具提高潜力的因素。小麦野生近缘植物冰草不仅具有抗逆、抗病等优良性状,而且具有多分蘖、多花多粒等丰产特性。将冰草携带的优异基因转入小麦,是拓宽小麦遗传基础、提高产量的有效途径。本研究利用原位杂交和分子标记对电离辐照和山羊草杀配子染色体诱导产生的小麦-冰草异源染色体易位进行鉴定,研究结果如下:
1、对杀配子染色体诱导小麦-冰草6P附加系产生的异源易位株系进行GISH鉴定,在F3代共获得18个易位株。F3、F4代共获得10个纯合易位株系。利用辐照4844-12/藁城8901杂交种的方法,在M4代中共获得两种大小片段相互易位和端部易位3种易位类型。
2、利用双色GISH-FISH或双色FISH-GISH二次杂交方法对小麦-冰草6P异源易位系中参与易位的小麦染色体进行鉴定,结果表明:冰草6P染色体与小麦染色体的重组涉及小麦A、B、D 3个基因组,其重组频率A组>B组>D组。确定了其中8个异源易位系涉及小麦1A、2A、5A、3B、6B和3D共6条染色体。
3、利用GISH方法筛选,鉴定出18个不同长度的6P染色体缺失系。
4、利用实验室开发的3个冰草P基因组特异SCAR标记和6个冰草6P染色体特异的STS标记对82份具有大穗多粒表型而GISH检测阴性的辐照M4代渐渗系进行检测,共检测出52株(63.41%)阳性植株,证明这些渐渗系中携带有P染色质。SCAR标记的检测频率(36株,43.90%)高于STS标记(29株,35.37%)。
5、利用整臂易位系对本实验室开发的冰草6P染色体特异STS标记进行定位,确定了12个标记所属的染色体臂,其中6个为6PS特异标记,6个位6PL特异标记。利用非整臂易位系和缺失系,对其中7个标记进行了更为细致的定位。
6、通过电离辐照获得的6P易位系农艺性状比杀配子方法诱导的易位(渐渗)系材料农艺性状总体表现较好,其中M4-442、M4-757等19株(23.17%)渐渗系的穗粒数超过100粒,具有应用前景。
7、对杀配子染色体诱导的小麦-冰草2P附加系F2代进行GISH鉴定,获得14个小麦-冰草2P异源易位,易位频率为5.34%。其中包括整臂易位6株、大片段易位1株、端部易位3株、中间插入易位2株和双着丝点易位2株。
本研究获得的小麦-冰草6P异源易位系、缺失系和渐渗系为小麦穗粒数的遗传改良和基础研究提供了新材料。6P特异标记的开发和定位能够辅助快速追踪和鉴定小麦背景的6P异染色质,从而为冰草6P染色体物理图谱的绘制和多粒基因的定位与克隆奠定基础。
The kernel number per spike is the most important factor of the many potential characteristics that determine wheat yield. Agropyron cristatum (L.) Gaertn., a wild relative of wheat, is resist to abiotic stress, major diseases and has many high-yield characteristics, e.g. more tillers, more spikelets and more florets. Transfer the excellent gene(s) from A. cristatum to wheat, is an effective mothed to widen the wheat genetic background and improve the wheat yield. In this research, wheat-A. cristatum alien translocation lines induced by ionizing radiation and gametocidal chromosome were identified by GISH and molecular markers. The results are as following:
1. Eighteen translocation lines were obtained in F3 of wheat-A. cristatum alien translocation lines induced by gametocidal chromosome and ten homozygous translocation lines in F3 and F4 were detected by GISH. There were two types of reciprocal translocation and one terminal translocation of M4 of irradiated hybrid seeds (4844-12/Gaocheng8901).
2. The two-color GISH-FISH or twice hybridization of two color FISH-GISH showed that A. cristatum chromosome 6P could be translocated to wheat ABD genome, and the recombination frequency occurred with A and B genomes was higher than which with D genome. Wheat chromosomes involved in the eight translocaion lines were identified clearly, including 1A, 2A, 5A, 3B, 6B and 3D.
3. We also obtained eighteen deletion lines with different length of chromosome 6P by GISH.
4. Three P genome-specific SCAR markers and six 6P-specific STS markers were used to identify 82 M4 introgression lines with characteristics of more spikelets and more florets but no GISH signals. We selected fifty-two positive plants (63.41%). The positive frequency detected by SCAR markers (36 plants, 43.90%) was higher than STS markers (28 plants, 34.14%).
5. Utilized the whole-arm translocation lines, partial of the STS markers were mapping on the chromosome 6P. The results identified 12 STS markers belong to the short or the long arm. Six of them were 6PS-specific markers, and others were 6PS-specific markers. Seven of them were identified more elaborate utilized the fragment translocation lines and deletion lines.
6. The translocation (introgression) lines induced by irradiate had the better agronomic features. The kernel number per spike of 19 introgression lines (23.17%) exceeds 100. These lines could be utilizaed directly in the wheat inprovement.
7. Fourteen wheat-A. cristatum 2P translocation lines were detected in F2 progeny, and the frequency of translocation was 5.34%, including 6 plants of whole-arm translocation, 1 plant of large fragment translocation, 3 plants of terminal translocation, 2 plants of intercalary translocation and 2 plants of dicentric translocation.
The wheat-A. cristaum 6P translocation, deletion and introgression lines obtained in this study had provided novel material for genetic improvement of kernels number per spike and basic researches of wheat. The development and location of the 6P-specific markers will be useful of tracing quickly and identifying the 6P chromatin in wheat background, which laid the foundation for constructing the physical map of 6P chromosome and locating and cloning the multi-kernel gene(s).
1、对杀配子染色体诱导小麦-冰草6P附加系产生的异源易位株系进行GISH鉴定,在F3代共获得18个易位株。F3、F4代共获得10个纯合易位株系。利用辐照4844-12/藁城8901杂交种的方法,在M4代中共获得两种大小片段相互易位和端部易位3种易位类型。
2、利用双色GISH-FISH或双色FISH-GISH二次杂交方法对小麦-冰草6P异源易位系中参与易位的小麦染色体进行鉴定,结果表明:冰草6P染色体与小麦染色体的重组涉及小麦A、B、D 3个基因组,其重组频率A组>B组>D组。确定了其中8个异源易位系涉及小麦1A、2A、5A、3B、6B和3D共6条染色体。
3、利用GISH方法筛选,鉴定出18个不同长度的6P染色体缺失系。
4、利用实验室开发的3个冰草P基因组特异SCAR标记和6个冰草6P染色体特异的STS标记对82份具有大穗多粒表型而GISH检测阴性的辐照M4代渐渗系进行检测,共检测出52株(63.41%)阳性植株,证明这些渐渗系中携带有P染色质。SCAR标记的检测频率(36株,43.90%)高于STS标记(29株,35.37%)。
5、利用整臂易位系对本实验室开发的冰草6P染色体特异STS标记进行定位,确定了12个标记所属的染色体臂,其中6个为6PS特异标记,6个位6PL特异标记。利用非整臂易位系和缺失系,对其中7个标记进行了更为细致的定位。
6、通过电离辐照获得的6P易位系农艺性状比杀配子方法诱导的易位(渐渗)系材料农艺性状总体表现较好,其中M4-442、M4-757等19株(23.17%)渐渗系的穗粒数超过100粒,具有应用前景。
7、对杀配子染色体诱导的小麦-冰草2P附加系F2代进行GISH鉴定,获得14个小麦-冰草2P异源易位,易位频率为5.34%。其中包括整臂易位6株、大片段易位1株、端部易位3株、中间插入易位2株和双着丝点易位2株。
本研究获得的小麦-冰草6P异源易位系、缺失系和渐渗系为小麦穗粒数的遗传改良和基础研究提供了新材料。6P特异标记的开发和定位能够辅助快速追踪和鉴定小麦背景的6P异染色质,从而为冰草6P染色体物理图谱的绘制和多粒基因的定位与克隆奠定基础。
The kernel number per spike is the most important factor of the many potential characteristics that determine wheat yield. Agropyron cristatum (L.) Gaertn., a wild relative of wheat, is resist to abiotic stress, major diseases and has many high-yield characteristics, e.g. more tillers, more spikelets and more florets. Transfer the excellent gene(s) from A. cristatum to wheat, is an effective mothed to widen the wheat genetic background and improve the wheat yield. In this research, wheat-A. cristatum alien translocation lines induced by ionizing radiation and gametocidal chromosome were identified by GISH and molecular markers. The results are as following:
1. Eighteen translocation lines were obtained in F3 of wheat-A. cristatum alien translocation lines induced by gametocidal chromosome and ten homozygous translocation lines in F3 and F4 were detected by GISH. There were two types of reciprocal translocation and one terminal translocation of M4 of irradiated hybrid seeds (4844-12/Gaocheng8901).
2. The two-color GISH-FISH or twice hybridization of two color FISH-GISH showed that A. cristatum chromosome 6P could be translocated to wheat ABD genome, and the recombination frequency occurred with A and B genomes was higher than which with D genome. Wheat chromosomes involved in the eight translocaion lines were identified clearly, including 1A, 2A, 5A, 3B, 6B and 3D.
3. We also obtained eighteen deletion lines with different length of chromosome 6P by GISH.
4. Three P genome-specific SCAR markers and six 6P-specific STS markers were used to identify 82 M4 introgression lines with characteristics of more spikelets and more florets but no GISH signals. We selected fifty-two positive plants (63.41%). The positive frequency detected by SCAR markers (36 plants, 43.90%) was higher than STS markers (28 plants, 34.14%).
5. Utilized the whole-arm translocation lines, partial of the STS markers were mapping on the chromosome 6P. The results identified 12 STS markers belong to the short or the long arm. Six of them were 6PS-specific markers, and others were 6PS-specific markers. Seven of them were identified more elaborate utilized the fragment translocation lines and deletion lines.
6. The translocation (introgression) lines induced by irradiate had the better agronomic features. The kernel number per spike of 19 introgression lines (23.17%) exceeds 100. These lines could be utilizaed directly in the wheat inprovement.
7. Fourteen wheat-A. cristatum 2P translocation lines were detected in F2 progeny, and the frequency of translocation was 5.34%, including 6 plants of whole-arm translocation, 1 plant of large fragment translocation, 3 plants of terminal translocation, 2 plants of intercalary translocation and 2 plants of dicentric translocation.
The wheat-A. cristaum 6P translocation, deletion and introgression lines obtained in this study had provided novel material for genetic improvement of kernels number per spike and basic researches of wheat. The development and location of the 6P-specific markers will be useful of tracing quickly and identifying the 6P chromatin in wheat background, which laid the foundation for constructing the physical map of 6P chromosome and locating and cloning the multi-kernel gene(s).
引文
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