利用EST序列对二倍体棉种染色体物理序号的FISH定位
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摘要
棉花染色体物理序号的确定大多以染色体全长、着丝粒位置、臂长、臂比等形态学数据为依据。但由于棉花染色体形体较小，很难从形态上区分各条染色体，尽管有许多学者报道了棉花四倍体和二倍体核型的研究结果，并对其进行了顺序编号，但不同学者之间依然存在着很大的分歧。
本实验以四倍体棉公认的起源二倍体种之一雷蒙德氏棉为材料，利用荧光原位杂交技术(FISH)，选取较长的单拷贝EST序列作为探针，旨在对雷蒙德氏棉13对粗线期染色体进行FISH定位，从而把染色体区分开。主要研究结果如下：
1．在国内首次在棉花的花粉母细胞中制出了粗线期染色体制片。从D组野生棉遗传图谱每个连锁群上选取3个EST序列，共选取39个EST序列作为探针，对每个探针设计引物，以雷蒙德氏棉基因组为模板进行PCR扩增、回收、纯化、测序，最终在每条染色体上筛选出一个单拷贝探针，共十三个探针对雷蒙德氏棉十三条粗线期染色体进行FISH定位，探针长度在866bp—1375bp之间。并最终将十三个EST探针分别定位到十三条雷蒙德氏棉粗线期染色体上。
2．rDNA基因是高度保守的串联重复序列，通过FISH技术对其在染色体上分布的位置、位点以及拷贝数多少进行比较研究，是确定棉花起源与进化的重要手段之一。根据棉花18SrDNA、5S rDNA基因序列设计引物，以陆地棉标准系TM-1基因组为模板进行PCR扩增、回收、纯化、测序。雷蒙德氏棉是棉花四倍体栽培种公认的起源种之一，利用18SrDNA和5S rDNA基因为探针，对雷蒙德氏棉粗线期染色体进行双色荧光原位杂交的物理定位。并通过SOUTHEN杂交确定5S rDNA基因在陆地棉(TM-1)、海岛棉(新海7号)、草棉和雷蒙德氏棉基因组中的拷贝群的数量，对5S rDNA基因在四倍体和二倍体棉种中的同步进化进行了研究。
3．首次对雷蒙德氏棉十三条粗线期染色体进行了核型分析，根据染色体的全长值从高到低确定了染色体的物理序号，并对雷蒙德氏棉粗线期染色体与体细胞中期染色体进行了核型比较。
Numbering of cotton chromosome is given by chromosome length, position of centromere, elative lengths of chromosome, rm ratios. As cotton chromosome is very small, it is difficult to identify Morphologically the individual chromosome from cotton chromosome complement.Although many studies have been conducted on the karyotype of tetraploid and diploid cotton,numbered chromosome order by lengths ,but controversies still exist among different researchers.In this study,we select D-genome diploid cotton Graimondii as materials.And single copy ESTs are used as probe, distinguish thirteen pachytene chromosomes of raimondii by fluorescent in situ hybridization (FISH).The main results are as follows:1.We make slides of cotton pachytene chromosomes from pollen mother cell at first in china.We choose three ESTs from every linkage group,and thirty nine ESTs as probe aggregately.Every EST is designed prime, PCR with template DNA of raimondii genome, reclaimed, purificated and sequenced.At last,we select thirteen ESTs(866bp-1375bp) as probe together.The probes we selected are located thirteen pachytene chromosomes of raimondii respectively by FISH.2.The rDNA genes of cotton are tandemly repeated DNA.It is an important study method on the origin and evolution of tetraploid.Many researchers study its localities and copies by FISH. We design primes with sequence of 18srDNA and 5srDNA.We perform PCR with template DNA of TM-1 genome,reclaim, purification and sequence . 18SrDNA and 5S rDNA as probe located the pachytene chromosomes of raimondii.We confirm copy groups of Ghirsutum, Gbarbadense, Graimondii, G.herbaceum,and study concerted evolution of 5SrDNA in tetraploid and diploid cotton.3.We analyse karyotype of the thirteen pachytene chromosomes of raimondii first,and number chromosomes via the length of thirteen pachytene chromosomes. We compare the karyotype of pachytene and metaphase chromosomes.
本实验以四倍体棉公认的起源二倍体种之一雷蒙德氏棉为材料，利用荧光原位杂交技术(FISH)，选取较长的单拷贝EST序列作为探针，旨在对雷蒙德氏棉13对粗线期染色体进行FISH定位，从而把染色体区分开。主要研究结果如下：
1．在国内首次在棉花的花粉母细胞中制出了粗线期染色体制片。从D组野生棉遗传图谱每个连锁群上选取3个EST序列，共选取39个EST序列作为探针，对每个探针设计引物，以雷蒙德氏棉基因组为模板进行PCR扩增、回收、纯化、测序，最终在每条染色体上筛选出一个单拷贝探针，共十三个探针对雷蒙德氏棉十三条粗线期染色体进行FISH定位，探针长度在866bp—1375bp之间。并最终将十三个EST探针分别定位到十三条雷蒙德氏棉粗线期染色体上。
2．rDNA基因是高度保守的串联重复序列，通过FISH技术对其在染色体上分布的位置、位点以及拷贝数多少进行比较研究，是确定棉花起源与进化的重要手段之一。根据棉花18SrDNA、5S rDNA基因序列设计引物，以陆地棉标准系TM-1基因组为模板进行PCR扩增、回收、纯化、测序。雷蒙德氏棉是棉花四倍体栽培种公认的起源种之一，利用18SrDNA和5S rDNA基因为探针，对雷蒙德氏棉粗线期染色体进行双色荧光原位杂交的物理定位。并通过SOUTHEN杂交确定5S rDNA基因在陆地棉(TM-1)、海岛棉(新海7号)、草棉和雷蒙德氏棉基因组中的拷贝群的数量，对5S rDNA基因在四倍体和二倍体棉种中的同步进化进行了研究。
3．首次对雷蒙德氏棉十三条粗线期染色体进行了核型分析，根据染色体的全长值从高到低确定了染色体的物理序号，并对雷蒙德氏棉粗线期染色体与体细胞中期染色体进行了核型比较。
Numbering of cotton chromosome is given by chromosome length, position of centromere, elative lengths of chromosome, rm ratios. As cotton chromosome is very small, it is difficult to identify Morphologically the individual chromosome from cotton chromosome complement.Although many studies have been conducted on the karyotype of tetraploid and diploid cotton,numbered chromosome order by lengths ,but controversies still exist among different researchers.In this study,we select D-genome diploid cotton Graimondii as materials.And single copy ESTs are used as probe, distinguish thirteen pachytene chromosomes of raimondii by fluorescent in situ hybridization (FISH).The main results are as follows:1.We make slides of cotton pachytene chromosomes from pollen mother cell at first in china.We choose three ESTs from every linkage group,and thirty nine ESTs as probe aggregately.Every EST is designed prime, PCR with template DNA of raimondii genome, reclaimed, purificated and sequenced.At last,we select thirteen ESTs(866bp-1375bp) as probe together.The probes we selected are located thirteen pachytene chromosomes of raimondii respectively by FISH.2.The rDNA genes of cotton are tandemly repeated DNA.It is an important study method on the origin and evolution of tetraploid.Many researchers study its localities and copies by FISH. We design primes with sequence of 18srDNA and 5srDNA.We perform PCR with template DNA of TM-1 genome,reclaim, purification and sequence . 18SrDNA and 5S rDNA as probe located the pachytene chromosomes of raimondii.We confirm copy groups of Ghirsutum, Gbarbadense, Graimondii, G.herbaceum,and study concerted evolution of 5SrDNA in tetraploid and diploid cotton.3.We analyse karyotype of the thirteen pachytene chromosomes of raimondii first,and number chromosomes via the length of thirteen pachytene chromosomes. We compare the karyotype of pachytene and metaphase chromosomes.
引文
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