用于河南省小麦品种特异性和一致性鉴定的SSR分子标记研究
摘要
小麦是我国重要的粮食作物之一,随着科学技术的发展,使得新育成品种的数量呈快速增长的趋势,每年申请参加国家或地方审定的小麦新品种多达上百个。
新品种必须具备特异性、一致性和稳定性(即Distinctness、Uniformity和Stability,简称DUS)。目前,中国及国际上通行的小麦品种DUS鉴定标准主要是建立农艺性状的基础上,鉴定结果容易受环境因素的影响,而且周期较长。而基于现代分子标记技术则具有不受季节限制,简便省时的优点。其中SSR (Simple Sequence Repeat简单序列重复)分子标记技术具有高水平的多态性,技术简便、成本低廉、易于操作、重复性和稳定性好等优点。本文探讨了适于进行DUS鉴定的SSR分子标记应具有的特点,并根据这些特点筛选出适合用于河南省小麦品种进行DUS鉴定的SSR分子标记。
本研究以18份河南省亲缘关系较近的小麦品种构成A组材料(表2-1),选用252对SSR标记进行筛选,分析可用于小麦特异性和一致性鉴定的SSR分子标记的特点,对鉴别力较高的标记进行稳定性分析,确定出可用于河南省小麦品种DUS测定的骨干引物,并以10份来源于周麦13和周麦16亲缘关系较近的高代品系构成B组材料(表4-1)和41份河南省60年以来的大面积推广品种构成C组材料(表4-2),对骨干引物分辨能力作进一步验证。获得主要研究结果如下：
1.所选252对标记涉及整个染色体组,每个标记产生的多态性变异数(扩增出的条带数)存在着很大的差异,最少的有1个多态性变异,最多的产生31个多态性变异。标记之间的鉴别力(Discrimination power,简称DP)也有很大的差异,最小的DP值是0(带型模糊或杂乱),最大的DP值是13。
2.在对SSR标记的相关性分析中,标记的多态变异数与DP值存在着极显著的正相关,相关系数是0.64；标记涉及染色体数与DP值的相关性不显著,相关系数是0.15,这说明SSR标记产生的多态变异数对SSR标记的鉴别力有着重要的正向影响。
3.根据SSR标记的多态位点数与SSR分子标记鉴别力之间的相关性,对252对引物进行筛选得到6对标记,分别是：Xwmc574、Xwmc500、Xgwm637、Xwmc679 Xwmc388、Xgwm497,产生的多态性变异数分别为：9、11、14、6、31、18,而每个标记的鉴别力DP值分别为9、11、10、9、13、13(鉴别的材料数≥50%)。
4.利用6对标记对A组材料的一致性分析表明,单个品种对单个标记的一致性比率r介于0.667～1.000,单个品种对所有标记的平均一致性比率介于0.861-0.993,初步确定评价品种一致性好坏的指标以及分级标准。评价品种一致性高低与单个品种对单个标记的一致性比率r以及单个品种对所有标记的平均一致性比率R有关,并根据分级标准,得出品种太学838、周9823、P9805和周16的一致性最好,属于一级一致性；品种淮麦0566、浚麦2号、许科1018、洛麦23、豫研麦10号、豫展9914、平麦97108-1、偃师819、豫研麦11号、郑麦7449、洛麦21和周麦13的一致性次之,属于二级一致性；品种濮02060的一致性稍差,一致性一般,属于三级一致性,只有品种国麦201的一致性最差,属于四级一致性。
5.利用B组材料和C组材料对骨干引物进行验证表明,6对引物在这两组材料中同样具有高的DP值,在B组材料(10份材料)中,最小的DP是4,最大的DP是7；在C组材料(41份材料)中,最小的DP值是18,最大的DP是21。并且利用这6对标记能够将B组材料和C组材料的各个品种完全区分开。
Wheat is one of China's important food crops, combined with scientific and technological development, making selection and promotion of new varieties was the fast-growing trend, the annual participation in national or local validation of the new wheat species as many as hundreds.
New varieties must have the distinctness uniformity and stability. At present, China and internationally accepted standards for wheat varieties DUS testing is mainly based on some morphological traits, developmental traits, quality traits and resistance-shaped, and identification of the results susceptible to environmental factors, and the cycle longer. The technology is based on modern molecular markers have not season, time-saving. SSR technology has a high level of polymorphism, technical simplicity, low cost, easy to operate, and good repeatability and stability. This paper discusses the characteristics of the SSR is suitable for DUS. Screening out SSR molecular markers which were suitable for DUS identification in Henan Province varieties of wheat.
In this study, using 18 closer genetic relationship wheat varieties make up group A (Table 2-1), This paper discusses the characteristics of the SSR is suitable for distinctness and uniformity. The SSR molecular markers had high power on discriminating power was analyzed stability. The main molecular markers for DUS evaluation of Henan provinces' wheat varieties were screened out. The discernibility of these molecular markers were tested in 10 variety series from Zhoul3 and Zhou16 make up group B and 41 wheat varieties during the recent 60 years in Henan provinces make up group C. The main contents are as follows:
1.252 pairs of SSR markers involved in the whole genome, there were significant differences in polymorphism loci between each marker. At least there was also more than one polymorphic locus, and the most was 31 polymorphic loci. There were significant differences in discrimination power between each marker, the least DP was 0 (band was fuzzy or messy), the most DP is 13.
2. Correlation analysis to SSR molecular marker, there was significant positive correlation between polymorphism loci and discrimination power, correlation coefficient was 0.64; the relativity between number of chromosome and discrimination power was not prominent, correlation coefficient was 0.15. These show that polymorphism loci have significant positive impact to discrimination power.
3. Based on the relativity between polymorphism loci of SSR molecular and discrimination of SSR molecular, six pairs of markers were screened out from 252 pairs of primers, respectively:Xwmc574, Xwmc500, Xgwm637, Xwmc679, Xwmc388, Xgwm497, the number of polymorphic loci were9,11,14,6,31,18respectively, the DP value of each marker were 9,11,10,9,13,13 respectively (identified material number>50%).
4. The uniformity analysis of the group A by six pairs makers show that, the uniformity rate of species (r) was ranging from 0.667～1.000, the average uniformity rate of species was ranging from 0.861-0.993. Preliminary determine the indicators to estimate uniformity of species and grading standards. The uniformity of species was high or low has a relationship with the single uniformity rate(r) and average uniformity rate(R), According to the standard classification, the best uniformity was species Taixue838, Zhou9823, P9805 and Zhou16, it was first level; the uniformity of species Huaimai0566, Junmai5, Xuke1018, Luomai23, Yuyanmai10, Yuzhan9914, Pingmai97108-1,Yanshi891, Yuyanmai11, Zhengmai7449, Luomai21 and Zhoumail3 were second level; the uniformity of Pu02060 was ordinary, it was third level; only the uniformity of species Guomai201 was worst, it was fourth level.
5. In the use of group B material and group C material on the backbone of primers to verify, The six primers had a high DP value in these two group materials in the same way, in the group B material (10 kinds of material), the smallest of the DP was 4, the largest DP was 7; in the group C material (41 kinds of material), the smallest of the DP was 18, the largest DP was 21. Material B and material C could be distinguished completely by the 6 pair markers.
新品种必须具备特异性、一致性和稳定性(即Distinctness、Uniformity和Stability,简称DUS)。目前,中国及国际上通行的小麦品种DUS鉴定标准主要是建立农艺性状的基础上,鉴定结果容易受环境因素的影响,而且周期较长。而基于现代分子标记技术则具有不受季节限制,简便省时的优点。其中SSR (Simple Sequence Repeat简单序列重复)分子标记技术具有高水平的多态性,技术简便、成本低廉、易于操作、重复性和稳定性好等优点。本文探讨了适于进行DUS鉴定的SSR分子标记应具有的特点,并根据这些特点筛选出适合用于河南省小麦品种进行DUS鉴定的SSR分子标记。
本研究以18份河南省亲缘关系较近的小麦品种构成A组材料(表2-1),选用252对SSR标记进行筛选,分析可用于小麦特异性和一致性鉴定的SSR分子标记的特点,对鉴别力较高的标记进行稳定性分析,确定出可用于河南省小麦品种DUS测定的骨干引物,并以10份来源于周麦13和周麦16亲缘关系较近的高代品系构成B组材料(表4-1)和41份河南省60年以来的大面积推广品种构成C组材料(表4-2),对骨干引物分辨能力作进一步验证。获得主要研究结果如下：
1.所选252对标记涉及整个染色体组,每个标记产生的多态性变异数(扩增出的条带数)存在着很大的差异,最少的有1个多态性变异,最多的产生31个多态性变异。标记之间的鉴别力(Discrimination power,简称DP)也有很大的差异,最小的DP值是0(带型模糊或杂乱),最大的DP值是13。
2.在对SSR标记的相关性分析中,标记的多态变异数与DP值存在着极显著的正相关,相关系数是0.64；标记涉及染色体数与DP值的相关性不显著,相关系数是0.15,这说明SSR标记产生的多态变异数对SSR标记的鉴别力有着重要的正向影响。
3.根据SSR标记的多态位点数与SSR分子标记鉴别力之间的相关性,对252对引物进行筛选得到6对标记,分别是：Xwmc574、Xwmc500、Xgwm637、Xwmc679 Xwmc388、Xgwm497,产生的多态性变异数分别为：9、11、14、6、31、18,而每个标记的鉴别力DP值分别为9、11、10、9、13、13(鉴别的材料数≥50%)。
4.利用6对标记对A组材料的一致性分析表明,单个品种对单个标记的一致性比率r介于0.667～1.000,单个品种对所有标记的平均一致性比率介于0.861-0.993,初步确定评价品种一致性好坏的指标以及分级标准。评价品种一致性高低与单个品种对单个标记的一致性比率r以及单个品种对所有标记的平均一致性比率R有关,并根据分级标准,得出品种太学838、周9823、P9805和周16的一致性最好,属于一级一致性；品种淮麦0566、浚麦2号、许科1018、洛麦23、豫研麦10号、豫展9914、平麦97108-1、偃师819、豫研麦11号、郑麦7449、洛麦21和周麦13的一致性次之,属于二级一致性；品种濮02060的一致性稍差,一致性一般,属于三级一致性,只有品种国麦201的一致性最差,属于四级一致性。
5.利用B组材料和C组材料对骨干引物进行验证表明,6对引物在这两组材料中同样具有高的DP值,在B组材料(10份材料)中,最小的DP是4,最大的DP是7；在C组材料(41份材料)中,最小的DP值是18,最大的DP是21。并且利用这6对标记能够将B组材料和C组材料的各个品种完全区分开。
Wheat is one of China's important food crops, combined with scientific and technological development, making selection and promotion of new varieties was the fast-growing trend, the annual participation in national or local validation of the new wheat species as many as hundreds.
New varieties must have the distinctness uniformity and stability. At present, China and internationally accepted standards for wheat varieties DUS testing is mainly based on some morphological traits, developmental traits, quality traits and resistance-shaped, and identification of the results susceptible to environmental factors, and the cycle longer. The technology is based on modern molecular markers have not season, time-saving. SSR technology has a high level of polymorphism, technical simplicity, low cost, easy to operate, and good repeatability and stability. This paper discusses the characteristics of the SSR is suitable for DUS. Screening out SSR molecular markers which were suitable for DUS identification in Henan Province varieties of wheat.
In this study, using 18 closer genetic relationship wheat varieties make up group A (Table 2-1), This paper discusses the characteristics of the SSR is suitable for distinctness and uniformity. The SSR molecular markers had high power on discriminating power was analyzed stability. The main molecular markers for DUS evaluation of Henan provinces' wheat varieties were screened out. The discernibility of these molecular markers were tested in 10 variety series from Zhoul3 and Zhou16 make up group B and 41 wheat varieties during the recent 60 years in Henan provinces make up group C. The main contents are as follows:
1.252 pairs of SSR markers involved in the whole genome, there were significant differences in polymorphism loci between each marker. At least there was also more than one polymorphic locus, and the most was 31 polymorphic loci. There were significant differences in discrimination power between each marker, the least DP was 0 (band was fuzzy or messy), the most DP is 13.
2. Correlation analysis to SSR molecular marker, there was significant positive correlation between polymorphism loci and discrimination power, correlation coefficient was 0.64; the relativity between number of chromosome and discrimination power was not prominent, correlation coefficient was 0.15. These show that polymorphism loci have significant positive impact to discrimination power.
3. Based on the relativity between polymorphism loci of SSR molecular and discrimination of SSR molecular, six pairs of markers were screened out from 252 pairs of primers, respectively:Xwmc574, Xwmc500, Xgwm637, Xwmc679, Xwmc388, Xgwm497, the number of polymorphic loci were9,11,14,6,31,18respectively, the DP value of each marker were 9,11,10,9,13,13 respectively (identified material number>50%).
4. The uniformity analysis of the group A by six pairs makers show that, the uniformity rate of species (r) was ranging from 0.667～1.000, the average uniformity rate of species was ranging from 0.861-0.993. Preliminary determine the indicators to estimate uniformity of species and grading standards. The uniformity of species was high or low has a relationship with the single uniformity rate(r) and average uniformity rate(R), According to the standard classification, the best uniformity was species Taixue838, Zhou9823, P9805 and Zhou16, it was first level; the uniformity of species Huaimai0566, Junmai5, Xuke1018, Luomai23, Yuyanmai10, Yuzhan9914, Pingmai97108-1,Yanshi891, Yuyanmai11, Zhengmai7449, Luomai21 and Zhoumail3 were second level; the uniformity of Pu02060 was ordinary, it was third level; only the uniformity of species Guomai201 was worst, it was fourth level.
5. In the use of group B material and group C material on the backbone of primers to verify, The six primers had a high DP value in these two group materials in the same way, in the group B material (10 kinds of material), the smallest of the DP was 4, the largest DP was 7; in the group C material (41 kinds of material), the smallest of the DP was 18, the largest DP was 21. Material B and material C could be distinguished completely by the 6 pair markers.
引文
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55. Vaccino P. Metakovsky. E. V. RFLP patterns of gliadin alleles in Triticum aestivum L. implications for analysis of the organization and evolution of complex loci[J]. Theor Appl Genet, 1995 (90):173-181.
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64. Temnykh S, Park W. D, Ayres N, et al. Mapping and genome or ganization of microsatellite sequences in rice (Oryza sativa L.) [J]. Theor App 1 Genet,2000,100:697-712.
65. Troyer A F, Rocheford T R. Germplasm ownership:related corn inbreds[J]. Crop Science,2002,42: 3-11.
66. Wang XR, Chhatre VE, Nilsson MC, Song W, Zackrisson O, Szmidt AE. Island Population Structure of Norway Spruce (Picea Abiles) in Northern Sweden[J]. Inter. J. lant Sci.2003, 164(5):711-717.
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