小麦—长穗偃麦草杂种后代的分子细胞遗传学分析及种质材料的筛选鉴定
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摘要
长穗偃麦草（Elytrigia elongata (Host) Nevisk.=Syn. Thinopyrum ponticum (Podp.)Barkworth and D. R. Dewey）是小麦的野生近缘植物之一，具有生长繁茂、多花多实、抗多种小麦病害（三锈、白粉、赤霉、黄矮病等）、抗寒冷、耐盐碱、耐干旱、较耐湿、耐贫瘠以及高蛋白等优良性状，是小麦遗传改良中具有重要价值的优异外源基因供体。本研究利用分子细胞遗传学和分子生物学等方法对小麦与长穗偃麦草不同杂种世代中偃麦草染色体的传递特点、主要细胞学及性状特点进行了分析；综合利用形态学、分子细胞遗传学和分子标记等方法对选育的优异小偃麦种质材料进行了鉴定，对白粉病抗性基因进行了分子标记定位。获得以下主要结果：
1.以拟鹅观草总基因组DNA（St）作探针，对长穗偃麦草根尖细胞有丝分裂染色体进行基因组原位杂交（GISH），可以将长穗偃麦草根尖细胞染色体分为5种不同的类型。利用覆盖普通小麦A、B、D基因组的4560对SSR引物和1200个RAPD引物，对二倍体长穗偃麦草（Ee）、比萨偃麦草（Eb）、拟鹅观草（St）和十倍体长穗偃麦草进行了分子标记分析结果表明，长穗偃麦草的染色体组间具有较近的亲缘关系，但同二倍体供体种染色体组已有较大差异，Ee、Eb和St染色体组应为十倍体长穗偃麦草的基本染色体组，其染色体组组型公式应为StStEeEbEx。
2.运用形态学和分子细胞遗传学等方法，分析长穗偃麦草和小麦杂种F1、F2、BC1F1、BC1F2、BC2F1等不同杂种世代的染色体组组成及构型，明确了小麦与长穗偃麦草不同杂种世代的染色体分离特点，以及不同杂交方式对杂种后代染色体及性状分离的影响。
3.对在普通小麦与长穗偃麦草杂种后代中选育的3个八倍体小偃麦（SN19、SN20和SN122）、11个小偃麦渐渗系山农87074-513、-526、-541、-551、-554、-555、-557、-565、-576、NX22、NX28和3个小偃麦易位系NX5、NX24、NX25进行了鉴定，明确了其主要性状和细胞学特点。
4.利用基因组原位杂交（GISH）和荧光原位杂交（FISH）技术，确定了19个小偃麦异染色体系的染色体构成特点。以拟鹅观草总基因组DNA为探针的GISH分析结果表明，八倍体小偃麦SN19染色体总数为54条，含12条长穗偃麦草染色体；SN20和SN122染色体总数均为56条，各含14条长穗偃麦草染色体；小偃693和小偃7430染色体总数也均为56条，但小偃693含16条长穗偃麦草染色体，小偃7430含12条长穗偃麦草染色体。5个八倍体小偃麦的外源染色体构成分别为：2"St+4"Ee、1"St+5"Ee+1"Eb、1"St+4"Ee+2"Eb、4"Ee+4"Eb和1"St+4"Ee+1"Eb。对NX5、NX24和NX25的原位杂交鉴定证明，它们均是小麦-长穗偃麦草染色体小片段易位系，易位片段均在小麦1BS染色体端部。以荧光绿标记的pAsl和德克萨斯红标记的pHvG38为探针，对11个小偃麦渐渗系（87074-513、-526、-541、-551、-554、-555、-557、-565、-576、NX22、NX28）和3个小偃麦易位系（NX5、NX24、NX25）的荧光原位杂交鉴定证明，14个小偃麦种质材料的部分染色体与普通小麦亲本均有不同程度的结构变异。
5.在对获得的稳定小偃麦种质SN0224进行细胞学和原位杂交鉴定的基础上，利用优选小群体方法，筛选获得3个与小偃麦易位系SN0224的抗白粉病基因PmSn0224紧密连锁的SSR标记（Barc212、Xwmc522和Xbarc1138），并将抗白粉病基因定位在小麦2A染色体上。
Elytrigia elongata (Host) Nevisk.[Syn. Thinopyrum ponticum (Podp.) Barkworth](2n=10x=70) was determined to have many agronomical useful traits for wheat improvement,such as high protein, tolerance to salt and drought, resistance to stripe rust, leaf rust, Fusariumhead blight and wheat streak mosaic virus.These genes have been transferred into wheat andextensively used in improved resistance to various pests and tolerance to abiotic stresses.Inthe present study, identified and analysed by methods of morphology, cytology, biochemistryand molecular biology, analyze the genome composition and configuration of wheat-E.elongata hybrids plants; identified Trititrigia alien chromosome lines from progenies betweenE. elongata and Triticum aestivum; and molecular marker analysis powdery mildew resistancegene. The main results were as follows:
1. GISH was conducted using St-genomic DNA from Pseudoregnaria strigosa as theprobe determined that5types chromosome composition was of E. elongata was exhibited. By4560pairs of SSR markers and1200RAPD markers analyze E. elongata, Agropyronelongatum (Ee), Thinopyrum Bessarabicum (Eb) and Ps. strigosa (St). The sesults indicate thatEe、Eband St had a close genetic relationship, and were basic chromosome complement of E.elongata. The genomic formulas for E. elongata were StStEeEbEx.
2. By methods of morphology andmolecular cytogenetics, analyze the genomecomposition and configuration of wheat-E. elongata F1、F2、BC1F1、BC1F2and BC2F1hybridsplants. To discuss the characteristics of chromosome separation in different hybrid generationsand affect of chromosome segregation with different pattern of crossing.
3. By methods of morphology and cytology,17Trititrigia alien chromosome lines wereidentified from progenies between E. elongata and Triticum aestivum, including3octoploid Trititrigia (SN19、SN20and SN122),11introgression lines (87074-513、-526、-541、-551、-554、-555、-557、-565、-576、NX22and NX28), and3translocation line (NX5、NX24andNX25).
4. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH)were used to establish the genomic constitutions of the19Trititrigia alien chromosome lines.Octoploid Trititrigia SN19has54chromosomes and other partial amphiploids have56chromosomes. SN19and XY7430both had intact wheat genome chromosomes plus12E.elongata chromosomes, SN20and SN122both had14chromosomes from E. elongata,XY693has16chromosomes from E. elongata, and their constitutions of alien chromosomeswere:2"St+4"Ee、1"St+4"Ee+1"Eb、1"St+5"Ee+1"Eb、1"St+4"Ee+2"Eband4"Ee+4"Eb. NX5、NX24and NX25were3translocation line and their translocation chromosomewas1BS. Probing of other14Trititrigia alien chromosome lines with pAsl and pHvG38revealed that their chromosome structure were different with their wheat parents.
5. SN0224, derived from crosses between E. elongata and common wheat Yannong15,was analyzed with cytological procedures, genomic in situ hybridization, powdery mildewresistance screening and molecular marker analysis.A single dominant Pm gene, derived fromE. elongata and temporarily designated as PmSn0224, was found on the wheat chromosome2A of SN0224. Based on data from205F2plants of SN0224/Huixianhong, the resistance genewas linked to three polymorphic microsatellite markers Barc212, Xwmc522and Xbarc1138with genetic distances of5.1,5.3and7.0cM.
1.以拟鹅观草总基因组DNA（St）作探针，对长穗偃麦草根尖细胞有丝分裂染色体进行基因组原位杂交（GISH），可以将长穗偃麦草根尖细胞染色体分为5种不同的类型。利用覆盖普通小麦A、B、D基因组的4560对SSR引物和1200个RAPD引物，对二倍体长穗偃麦草（Ee）、比萨偃麦草（Eb）、拟鹅观草（St）和十倍体长穗偃麦草进行了分子标记分析结果表明，长穗偃麦草的染色体组间具有较近的亲缘关系，但同二倍体供体种染色体组已有较大差异，Ee、Eb和St染色体组应为十倍体长穗偃麦草的基本染色体组，其染色体组组型公式应为StStEeEbEx。
2.运用形态学和分子细胞遗传学等方法，分析长穗偃麦草和小麦杂种F1、F2、BC1F1、BC1F2、BC2F1等不同杂种世代的染色体组组成及构型，明确了小麦与长穗偃麦草不同杂种世代的染色体分离特点，以及不同杂交方式对杂种后代染色体及性状分离的影响。
3.对在普通小麦与长穗偃麦草杂种后代中选育的3个八倍体小偃麦（SN19、SN20和SN122）、11个小偃麦渐渗系山农87074-513、-526、-541、-551、-554、-555、-557、-565、-576、NX22、NX28和3个小偃麦易位系NX5、NX24、NX25进行了鉴定，明确了其主要性状和细胞学特点。
4.利用基因组原位杂交（GISH）和荧光原位杂交（FISH）技术，确定了19个小偃麦异染色体系的染色体构成特点。以拟鹅观草总基因组DNA为探针的GISH分析结果表明，八倍体小偃麦SN19染色体总数为54条，含12条长穗偃麦草染色体；SN20和SN122染色体总数均为56条，各含14条长穗偃麦草染色体；小偃693和小偃7430染色体总数也均为56条，但小偃693含16条长穗偃麦草染色体，小偃7430含12条长穗偃麦草染色体。5个八倍体小偃麦的外源染色体构成分别为：2"St+4"Ee、1"St+5"Ee+1"Eb、1"St+4"Ee+2"Eb、4"Ee+4"Eb和1"St+4"Ee+1"Eb。对NX5、NX24和NX25的原位杂交鉴定证明，它们均是小麦-长穗偃麦草染色体小片段易位系，易位片段均在小麦1BS染色体端部。以荧光绿标记的pAsl和德克萨斯红标记的pHvG38为探针，对11个小偃麦渐渗系（87074-513、-526、-541、-551、-554、-555、-557、-565、-576、NX22、NX28）和3个小偃麦易位系（NX5、NX24、NX25）的荧光原位杂交鉴定证明，14个小偃麦种质材料的部分染色体与普通小麦亲本均有不同程度的结构变异。
5.在对获得的稳定小偃麦种质SN0224进行细胞学和原位杂交鉴定的基础上，利用优选小群体方法，筛选获得3个与小偃麦易位系SN0224的抗白粉病基因PmSn0224紧密连锁的SSR标记（Barc212、Xwmc522和Xbarc1138），并将抗白粉病基因定位在小麦2A染色体上。
Elytrigia elongata (Host) Nevisk.[Syn. Thinopyrum ponticum (Podp.) Barkworth](2n=10x=70) was determined to have many agronomical useful traits for wheat improvement,such as high protein, tolerance to salt and drought, resistance to stripe rust, leaf rust, Fusariumhead blight and wheat streak mosaic virus.These genes have been transferred into wheat andextensively used in improved resistance to various pests and tolerance to abiotic stresses.Inthe present study, identified and analysed by methods of morphology, cytology, biochemistryand molecular biology, analyze the genome composition and configuration of wheat-E.elongata hybrids plants; identified Trititrigia alien chromosome lines from progenies betweenE. elongata and Triticum aestivum; and molecular marker analysis powdery mildew resistancegene. The main results were as follows:
1. GISH was conducted using St-genomic DNA from Pseudoregnaria strigosa as theprobe determined that5types chromosome composition was of E. elongata was exhibited. By4560pairs of SSR markers and1200RAPD markers analyze E. elongata, Agropyronelongatum (Ee), Thinopyrum Bessarabicum (Eb) and Ps. strigosa (St). The sesults indicate thatEe、Eband St had a close genetic relationship, and were basic chromosome complement of E.elongata. The genomic formulas for E. elongata were StStEeEbEx.
2. By methods of morphology andmolecular cytogenetics, analyze the genomecomposition and configuration of wheat-E. elongata F1、F2、BC1F1、BC1F2and BC2F1hybridsplants. To discuss the characteristics of chromosome separation in different hybrid generationsand affect of chromosome segregation with different pattern of crossing.
3. By methods of morphology and cytology,17Trititrigia alien chromosome lines wereidentified from progenies between E. elongata and Triticum aestivum, including3octoploid Trititrigia (SN19、SN20and SN122),11introgression lines (87074-513、-526、-541、-551、-554、-555、-557、-565、-576、NX22and NX28), and3translocation line (NX5、NX24andNX25).
4. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH)were used to establish the genomic constitutions of the19Trititrigia alien chromosome lines.Octoploid Trititrigia SN19has54chromosomes and other partial amphiploids have56chromosomes. SN19and XY7430both had intact wheat genome chromosomes plus12E.elongata chromosomes, SN20and SN122both had14chromosomes from E. elongata,XY693has16chromosomes from E. elongata, and their constitutions of alien chromosomeswere:2"St+4"Ee、1"St+4"Ee+1"Eb、1"St+5"Ee+1"Eb、1"St+4"Ee+2"Eband4"Ee+4"Eb. NX5、NX24and NX25were3translocation line and their translocation chromosomewas1BS. Probing of other14Trititrigia alien chromosome lines with pAsl and pHvG38revealed that their chromosome structure were different with their wheat parents.
5. SN0224, derived from crosses between E. elongata and common wheat Yannong15,was analyzed with cytological procedures, genomic in situ hybridization, powdery mildewresistance screening and molecular marker analysis.A single dominant Pm gene, derived fromE. elongata and temporarily designated as PmSn0224, was found on the wheat chromosome2A of SN0224. Based on data from205F2plants of SN0224/Huixianhong, the resistance genewas linked to three polymorphic microsatellite markers Barc212, Xwmc522and Xbarc1138with genetic distances of5.1,5.3and7.0cM.
引文
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