EMS诱变小麦河农822的SSR及SDS-PAGE鉴定

为给小麦育种提供基础材料,利用SSR技术和高分子量麦谷蛋白SDS-PAGE对由EMS诱导河农822获得的矮秆、短芒、密穗、棒状穗、早熟等8个小麦性状变异株进行鉴定。结果表明：在DNA水平,除矮秆变异株外,其余均缺失了1条50-190 bp的片段,各个不同性状突变株均增加了3-4条44-574 bp的片段;在蛋白质水平,不同性状突变株高分子量麦谷蛋白亚基在Glu-1B和Glu-1D位点呈现出缺失和新增亚基2种情况。这表明突变材料与对照相比,不仅在生物学形态存在明显差异,而且在DNA和蛋白质水平上也产生了突变,为小麦突变产生提供了充分的分子生物学证据。

大豆SSR标记PAGE银染方法的改进

比较改进后的两种最常用的银染方法Bassam和Sanguinetti,建立了一种更加行之有效、简便快捷的应用于大豆SSR标记的PAGE银染检测方法。

小麦基因组DNA快速提取及SSR标记PAGE的银染检测

为了提高SSR-PCR速率,降低试验成本,以8份抗条锈小麦幼苗为材料,比较分析了两种DNA提取方法(CTAB法和SDS法)以及两种SSR标记PAGE银染法(Sanguinetti银染法和Bassam银染法).结果表明:用CTAB法和SDS法均可获得质量较好的DNA,但CTAB法花费的时间较长,提取效率低(在相同条件下SDS法提取速度约为CTAB法的2倍);Sanguinetti银染法染色背景较浅、条带清晰、分辨率高、具有较高的多态性检出率,并且在速度上优于Bassam法,仅需30多min,而Bassam银染法染色步骤烦琐,对试剂质量和水质要求均较高,且未能染色成功.

Bulked Segregant Analysis to Detect QTL Related to Heat Tolerance in Rice(Oryza sativa L.) Using SSR Markers

The study was undertaken to assess the genetic effect of quantitative trait loci （QTLs） conferring heat tolerance at flowering stage in rice. A population consisting of 279 F2 individuals from the cross between 996, a heat tolerant cultivar and 4628, a heat-sensitive cultivar, was analyzed for their segregation pattern of the difference of seed set rate under optimal temperature condition and high temperature condition. The difference of seed set rate under optimal temperature condition and high temperature condition showed normal distribution, indicating the polygenic control over the trait. To identify main effect of QTL for heat tolerance, the parents were surveyed with 200 primer pairs of simple sequence repeats （SSR）. The parental survey revealed 30% polymorphism between parents. In order to detect the main QTL association with heat tolerance, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The association of putative markers identified based on DNA pooling from selected segregants was established by single marker analysis （SMA）. The results of SMA revealed that SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The heat tolerance during flowering stage in rice was controlled by multiple gene. The SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The two genetic loci, especially for RM3735 on chromosome 4, can be used in marker-assistant-selected method in heat tolerance breeding in rice.

棉花品种SSR标记变性与非变性PAGE检测法的比较研究

本研究以12份棉花品种为材料,用三对多态性丰富的SSR引物对变性和非变性PAGE检测法进行比较分析。结果表明,两种检测方法鉴定单株个体基因型的类型一致;变性PAGE检测法检测结果清晰,但操作过程繁琐;非变性PAGE检测法操作相对简单、检测通量高,更适用于材料的高通量检测分析。

Identification and Purity Test of Super Hybrid Rice with SSR Molecular Markers

Five super hybrid rice combinations, i.e. HYS-1/R105, Pei'ai 64S/E32, Liangyoupeijiu (Pei'ai 64S/9311), 88S/0293, and J23A/Q611, and their parental lines were tested by means of SSR analysis. A total of 144 SSR primer pairs distributed on 12 rice chromosomes were used, out of which 47 detected polymorphism among the tested rice lines. Among all these primers, RM337 and RM154 produced polymorphic patterns in four or more of the tested experimental materials respectively, and they could distinguish among most rice genotypes tested. Twenty-four primer pairs, two on each rice chromosome, were selected to make a reference SSR marker-based fingerprinting for the rice lines. For most of the primer pairs, F1 hybrids mainly showed complementary pattern of both parents, which could be very useful to distinguish the F1 from its parental lines. In addition, 5 primer pairs were selected as special primer pairs for five hybrid rice combinations respectively. By combining the rapid, simple method on DNA extraction, it is suggested that SSR technique has wide prospective in variety authentication and purity identification.

Development of a Highly Informative Microsatellite (SSR) Marker Framework for Rice (Oryza sativa L.) Genotyping

To select highly informative microsatellite markers （SSRs） and establish a useful genetic SSR framework for rice genotyping, 15 rice （Oryza sativa L.） cultivars including six indica varieties and nine japonica varieties were used to analyze the polymorphism information content （PIC） value of 489 SSR markers. A total of 1 296 alleles were detected by 405 polymorphic markers with an average of 3.2 per locus. The PIC value of each chromosome was ranged from 0.4039 （chromosome 2） to 0.5840 （chromosome 11）. Among the two rice subspecies, indica （0.3685-0.4952） gave a higher PIC value than japonica （0.1326-0.3164） and displayed a higher genetic diversity. Genetic diversity of indica was high on chromosome 12 （0.4952） and low on chromosome 8 （0.3685）, while that for japonica was high on chromosome 11 （0.3164） and low on chromosome 2 （0.1326）. A SSR framework including 141 highly informative markers for genotyping was selected from 199 SSR markers （PIC〉0.50）. Ninety-three SSR markers distributed on 12 chromosomes were found to be related to indica-japonica differentiation. Of these 93 pairs of SSR primers, 17 pairs were considered as core primers （all the japonica varieties have the same specific alleles, while the indica varieties have another specific alleles）, 48 pairs as the second classic primers （all the japonica or indica varieties have the same specific alleles, while the indica or japanica varieties have two or more other specific alleles ） and 28 pairs as the third classic primers （all the japonica and indica varieties have two or more alleles, but the specific alleles are different between japonica and indica）. Thirty-two SSR markers were selected to be highly informative and useful for genetic diversity analysis of japonica varieties. This work provides a lot of useful information of SSR markers for rice breeding programs, especially for genotyping, diversity analysis and genetic mapping.

Detection of Distorted Segregation in Genotype of Pollen Calli Derived from Hybrid F_1 of Cultivated Rice (Oryza sativa L.) Using SSR Markers

S-a, S-b and S-c are three loci for F1 pollen sterility in cultivated rice （Oryza sativa L.）. Taichung 65 （T65） is all Sj/Sj at these three loci, while its F1 pollen sterile near-isogenic lines, TISL2 （S-b）, TISL4 （S-a） and TISL5 （S-c） is Sj/Sj according to their respective sterility locus. Using SSR molecular marker to detect the segregation of the allele Si and Sj in pollen calli population induced from different hybrid F1, which have different pollen sterility locus, showed that the segregation of allele Si and Sj was distorted. The distorted direction of pollen calli population in vitro was not the same as F2 population in vivo. The quantities of pollen callus carrying Sj were much more than that of carrying Siat S-a and S-c locus, the ratio of Si and Sj were 1：4.81 and 1：1.96 respectively. But the opposite tendency was observed at S-b locus, the ratio of Si and Sj being 1：0.35. At the same time, all these results were undisturbed by either culture medium or culture period.

Construction of DNA Fingerprint Using SSR Marker for Hybrid Rice Cultivars Approved by Hunan Province

[Objective] This study aimed to construct DNA fingerprint for hybrid rice cultivars those have been approved by Hunan Province. [Method] The primers which produced polymorphic and bright DNA bands were selected to construct the DNA molecular fingerprint map for 77 major hybrid rice cultivars approved by Hunan Province. [ Result] A total of 48 SSR primers were selected. Every cultivar had its unique fingerprint map so that the obtained data could identify differ- ent hybrid rice cultivars. [ Conclusion] This study made great contributions to the perfection of hybrid rice germplasm identification.

Analysis on Genetic Differences between Parents of Two-line Medium Indica Hybrid Rice by SSR Markers

The genetic diversity of 50 parents or improved lines of two-line medium Indica hybrid rice were analyzed based on 48 pairs of SSR markers. The results showed that the clustering results were highly accordant with the parental pedigree information of the tested materials. The male sterile lines and the restoring lines were clustered into 2 distinct groups （Ⅰ and Ⅱ）, respectively. There were no significant difference in intra-group average genetic distances （GD） between the 2 groups. Most of the male sterile lines from Hunan Longping High-tech Seed Science Research Institute Co., Ltd. were grouped into a single subgroup （Ⅰ-4） under the male sterile group with the largest average GD compared with the rest male sterile subgroups. Huazhan derived lines and Guangzhou inbred lines were founded to be independent of 9311 and its derivatives or closely-related lines. Meanwhile, they have obviously been differentiated into 2 subgroups （Ⅱ-2 and Ⅱ-3）. The lines derived from Huazhan （or genetic closely related lines） showed a genetic homogenization phenomenon. Therefore, the Huazhan-derived lines should be further improved by the utilization of germplasms with good quality, high resistance and wide adaptability from Southern China to widen the GD between Huazhan derived lines and the male sterile subgroup developed by Hunan Longping High-tech Seed Science Research Institute Co., Ltd., so as to further promote the heterosis level between the groups.

Evaluation of Germplasm Using SSR Markers of Functional Genes in Rice

16 SSR (Simple sequence repeats) primers of functional genes in rice were used to detect genetic diversity among 23 accessions of rice germplasm from 5 countries in the world. The average number of alleles per SSR locus was 5.2 with a range from 2 to 10. Genetic similarities among the 23 rice accessions ranged from 0.13 to 0.64. UPGMA cluster analysis showed that the 23 rice accessions could be classified into two distinct classes at similarities with a coefficient of 0.13. The Japonicas from Brazil, Japan and China were classified into Class I, along with upland rice from Brazil. The Indicas from Pakistan and Korea were classified into Class II. Consequently, the function of genes SSR markers could be used as a useful tool for measuring genetic diversity, assigning rice to geographical distribution, ecotype, and pedigree relationship.

PAGE模型与DICE/RICE模型的比较

采用综合评估模型(IAMs)研究气候变化是当前气候变化研究的趋势,IAMs种类繁多,本文选取DICE/RICE模型以及PAGE模型,比较两种模型的建模理念、温室气体模拟、世界分区、计算方法、关键参数的取值,并简要分析其最新模型的运行结果和气候政策。

Analysis on the Different Results of Seed Purity Identification for the Same Hybrid Rice Variety Using Different SSR Primers

Different results of seed purity identification for Gangyou 158, Ⅱ You 808, Wuyou 308 and Tianfengyou 316 were obtained using different SSR primers in our early work. To find out the reasons, the four hybrid combinations were grown in field to identify their purity according to their phenotypic traits. Then, the results of field identification were compared with that of laboratory tests using different SSR primers. The comparison revealed that only sterile lines （female parent） were distin- guished from true hybrids using the primers RM208, RM264, RM242 and RM164 for the purity identification of Gangyou 158, II You 808, Wuyou 308 and Tianfengyou 316, so the results were higher than that of field identification. In contrast, the primers RM341, RM297, RM21 and RM110 were able to distinguish not only the sterile plants but also the cross-pollinated ones from the true hybrids of Gangyou 158,Ⅱ You 808, Wuyou 308 and Tianfengyou 316, and the results of purity identifi- cation using them were close that of field identification, in summary, several pairs of primers should be used for the purity identification of rice hybrids to distinguish all the off-type plants and thus improve the accuracy.

聚丙烯酰胺凝胶电泳检测水稻SSR标记

以水稻为试验材料,提取水稻基因组DNA,选择多态性SSR标记,并将标记产物进行聚丙烯酰胺凝胶电泳,最后硝酸银染色,试图建立了一种行之有效、简便、快捷的应用于水稻SSR标记的PAGE银染检测方法。

Sensory Phenotypic and Molecular Identification of Aromatic Rice Accessions Cultivated in Benin

Rice is one of the most widely cultivated cereals in the world, and its aroma is increasingly in demand. With the advancement of research, a major rice flavor gene has been identified on rice chromosome 8. It encodes non-functional betaine aldehyde dehydrogenase leading to the accumulation of 2-acetyl-1-pyrroline which is the major olfactory compound that confers the fragrant character to rice. The aroma of rice is considered a special trait of enormous economic importance that determines the prime price in world trade. To satisfy the needs of the population and reduce rice imports into Benin, we conducted this study to identify aromatic rice accessions grown in Benin. Seventy-two rice accessions collected across Benin were PCR amplified with three SSR markers RM 7049, Aro 7, and RM 223, linked to the fgr (fragrance of rice) aroma gene. Molecular analysis revealed that 12 of the 72 accessions, namely Bagou 19, Bagou 22, Tchaka 34, Foun 15, Tchaka 41, Nana 32, Kan 61, Kung 69, Kung 67, Bagou 20, Agbab 101 and Koum 55 possess the fgr gene and can be considered as aromatic rice accessions. A sensory phenotypic test using KOH was carried out on rice accessions carrying fgr gene. Of the twelve positives, only one had the smell of aromatic rice, like the Azucena control. These results show that Benin also has aromatic rice varieties that can be sold on national and international markets.

基于SSR分子标记吉安地区的水稻亲缘关系鉴定分析

稻种亲缘关系分析是生产优质杂交水稻的必要条件。本研究选取均匀分布于水稻基因组12条染色体的SSR分子标记,对8份水稻样品进行SSR分子标记分析,通过聚类分析法将其归类并计算得出遗传相似系数后,对8份水稻样品的亲缘关系进行了鉴定。结果表明:所选取的8份水稻样品中,其相似系数为0.65~1.00,在遗传相似系数0.65处,8份水稻样品聚为两大类群,其中5号‘赣迟A04’单独为一类,其他水稻样品聚为一类。在遗传相似系数0.80处,可划分为三大类群:第一类囊括了6种样品,表明这6个品种亲缘关系较近,其中6号‘赣早A02’与7号‘吉安早A07’疑似同一样品,3号‘井冈山迟A03’与4号‘井冈山早A05’的亲缘关系最近,而6号、7号同时与8号‘吉安迟A09’保持有最近的亲缘关系;第二类只含有2号‘迟-2’样品株;第三类则仅有5号稻株‘赣迟A04’,说明其与其他7份水稻样品的亲缘关系最远。本研究结果为杂交水稻的应用提供重要信息。

Comparison of Genetic Diversity Between Local Cultivated Aromatic and Non-aromatic Rice in Yunnan Province

[ Objective] The genetic diversity of the local cultivated aromatic rice and non-aromatic rice in Yunnan Province were compared to provide further genetic resources for breeding practice. [Method] Genetic diversity of 10 aromatic rice and 45 non-aromatic rice were analyzed by 64 SSR primers covered on 12 rice chromosomes. [ Result] Per locus 5.44 and 7.98 alleles in average were detected, ranging from 2 to 12 and from 2 to 17 in aromatic and non-aromatic rice, respectively. Average genetic multiplicity index（Hs） was 0.46 and 0.67 respectively. The average polymorphism information content （PIC） was 0.43 and 0.58 in aromatic and non-aromatic rice respectively. [ Conclusion] The results indicated that genetic diversity was higher in non-aromatic rice than in aromatic rice.

Physiological Character and Gene Mapping in a New Green-revertible Albino Mutant in Rice

A green-revertible albino mutant-Qiufeng M was found from the japonica rice （Oryza sativa L. ssp. japonica） Qiufeng in the field. The first three leaves of the mutant were albino with some green. The leaf color became pale green since the fourth leaf and the glume had the same phenomenon as the first three leaves. The measuring data of the pigment content confirmed the visually observed results. It truly had a remarkable changing process in the leaf color in Qiufeng M. Comparison of the main agronomic characters between Qiufeng and Qiufeng M indicated that the neck length and grain weight showed significant difference at the 1% level, and other characters were not different. Genetic analysis showed that the green-revertible albino trait was controlled by a single recessive nucleic gene. Using 209 recessive mutant individuals in the F2 population derived from the cross Pei＇ai 64S × Qiufeng M, a gene, tentatively named gra（t）, was located between the SSR markers of RM475 and RM2-22 on the long arm of chromosome 2. The genetic distance were 17.3 cM and 2.9 cM respectively.

应用SSR和ISSR标记分析栽培香稻品种的遗传多样性

本研究利用24对SSR引物和36个ISSR引物,分析33份来源于亚洲10个国家的香稻品种的遗传多样性.分别获得93条和181条多态性片段,每个SSR座位可检测3～8个等位基因,平均为4.23个;每个ISSR引物可检测3～8个多态性位点,平均为5.03个.根据SSR和ISSR标记计算的品种间遗传相似系数分别在0.294～0.884之间和0.595～0.867之间.聚类分析表明,利用两种标记所得的聚类结果基本上一致,与品种所处的3种气候类型变化基本相符.进一步证实SSR和ISSR标记是研究水稻种质资源分类有效的工具.

聚丙烯酰胺凝胶电泳检测南苜蓿SSR标记

以南苜蓿(Medicago polymorpha)为试验材料,用CTAB法提取南苜蓿基因组,选择已开发的蒺藜苜蓿(Medicago truncatula)引物100对,分别在一年生苜蓿SSR-PCR反应体系和苜蓿属变种SSR-PCR反应体系的基础上进行PCR扩增,并将扩增产物进行8%的非变性聚丙烯酰胺凝胶电泳检测,都得到了位点明显、背景清晰的条带。结果表明,南苜蓿均适合于现有的一年生苜蓿SSR-PCR反应体系和苜蓿属变种SSR-PCR反应体系,建立了一套有效、成熟的应用于南苜蓿SSR标记的聚丙烯酰胺凝胶电泳银染检测的方法,该方法可以用于南苜蓿杂交F1代的鉴定以及后期资源的遗传分析。