西农979抗赤霉病基因Fhb1的分子鉴定及其亲缘关系分析

为明确小麦品种西农979对赤霉病的抗性遗传基础,以西农979和苏麦3号构建的F2代群体以及西农979的主要供体亲本为材料,利用与苏麦3号3BS染色体上Fhb1基因紧密连锁的2对SSR引物及其共分离特异性标记引物UMN10进行检测,并调查西农979大田赤霉病抗性。结果表明,引物Xgwm533、Xgwm493及UMN10对西农979、苏麦3号及西农979/苏麦3号构建的F2群体(510个单株)扩增结果均为单态,表明西农979具有苏麦3号3BS上的Fhb1基因位点。对西农979的主要供体亲本的分子标记及亲缘关系分析,进一步表明西农979具有苏麦3号的抗赤霉病基因,其Fhb1基因位点来源于西农881,这可能是西农979对小麦赤霉病表现为中抗或中感且抗性较为稳定的遗传基础。     

水稻质核互作雄性不育系的微效恢复基因定位和排除方法研究

以232个协青早A/B456///协青早A/B456∥B456植株组成的分离群体为材料,调查花粉可育率和自交结实率,并采用121个在染色体上分布比较均匀的SSR多态性标记进行QTL检测。发现22个SSR标记分别与10个花粉育性位点连锁,分布于第2,5,6,8,10,12染色体上;28个标记分别与16个小穗育性位点连锁,分布于第1,2,4,5,6,8,10和11染色体上;13个标记同时与花粉育性和小穗育性连锁,小穗育性与花粉育性QTL差异是花粉可育度和自交结实率不平行性的遗传基础。各可育位点对花粉育性和小穗育性的提高效应比较小,为微效基因,但每个花粉育性位点的存在都可导致不育系败育不彻底。协青早A中发现1个花粉可育位点Pf5-1,与分子标记Bm55和Rm13紧密连锁,进行分子标记辅助选择,可能排除协青早A的微效恢复基因(可育位点),达到完全不育。协青早A存在8个小穗育性位点,能够提高自交结实率,有助于杂种F1结实率的提高,有利于提高不育系的可恢复性。多数微效恢复基因显示为部分隐性或隐性,是水稻质核互作雄性不育系选育难的重要原因。采用不育系/拟用亲本∥保持系/拟用亲本的方式,观察杂种育性分离,可对拟用亲本的微效恢复基因有所了解,用保持系/部分保持系∥保持系的方式可提高微效恢复基因排除的效率。     

Effects of the number of pollen grains on cold tolerance at the booting stage in rice lines with QTLs for cold tolerance

This study was conducted to clarify the relationship between the number of pollen grains per anther and spikelet fertility under low temperature conditions in the rice cultivars and lines including lines with quantitative trait loci (QTL) for cold tolerance, adapted to the Tohoku region of northern Japan. Cold-water treatment decreased anther length, the number of pollen grains per anther, and spikelet fertility in all cultivars and lines. The number of pollen grains was proportional to anther length in all cultivars and lines and under all temperatures. Spikelet fertility decreased with decreasing the number of pollen grains in cold-water treatments at 18.5 and 19.3?°C. ‘Ouu 415’, with the qLTB3 QTL for cold tolerance, had 28% more pollen grains and 9% higher spikelet fertility than the recurrent parent, ‘Hitomebore’, in the 18.5?°C cold-water treatment, suggesting that qLTB3 increased both parameters. Lines with the qCTB8 QTL for cold tolerance had significantly more pollen grains in two of the three years and significantly higher spikelet fertility in all three years in the 19.3?°C cold-water treatment, suggesting that qCTB8 reinforced cold tolerance. Ctb1, a proposed cold-tolerance QTL, had no effect on the number of pollen grains or spikelet fertility. In conclusion, the cultivars and lines with more pollen grains had a higher cold tolerance. Some of QTLs were inferred to increase the number of pollen grains and reinforce cold tolerance.     

甬优系列杂交水稻SSR标记指纹图谱和籼粳属性

用22个SSR分子标记对13份甬优系列杂交水稻组合及其亲本材料和6份籼粳对照种进行分析,共获得87个等位基因,每对引物扩增的等位基因数为2~8个。所有供试材料间均检测到了差异,从而建立起甬优1号~甬优6号杂交水稻组合及其亲本的DNA指纹图谱。聚类分析结果表明,9份供试材料与3份粳稻对照种聚于一类,属粳稻;4份供试材料与籼稻对照种IR36聚于一类,属籼稻。这与形态分类结果基本一致。从分子水平确认了杂交水稻甬优6号和甬优4号为籼粳亚种间杂交稻。     

Dissection of gene loci underlying pasting temperature in cassava

In this study, a fine genetic map within the quantitative trait loci (QTL) underlying pasting temperature (PT) of cassava (Manihot esculenta Crantz) was constructed using newly developed simple sequence repeat (SSR) markers. The SSRs were designed on the basis of two scaffolds (S11341 and S4043) of the cassava genome, which covered previously identified QTL regions of the PT trait. A total of 55 and 61 SSR markers derived from S11341 and S4043, respectively, representing 0.29% of the cassava genome, were generated; of which 23 and 19 showed informative polymorphic patterns. Consequently, all identified informative polymorphic markers were used to genotype 200 F₁ progeny plants. The genotypic data were then analyzed, and the results showed that 480 markers were distributed across 23 linkage groups (LGs) with total length of 1,334 centimorgans (cM). An analysis of QTL underlying the PT trait revealed that marker EME81 on LG 1 had significant associations (P < 0.0001) in all environments evaluated. Four candidate genes were identified and selected for gene expression analysis in the parents, and among F₁ lines with high and low PT values. Significant differences were observed in relative expression of carbohydrate phosphorylase (CP) and starch synthase II (SSII) between high and low PT in 6-month-old cassava. We found CP and SSII genes to potentially control the PT trait. In addition, the marker EME81 was found to be a promising marker for specific PT trait selection in cassava populations, which should facilitate marker-assisted selection for desired PT traits.     

甘蓝型油菜硫苷含量性状的SSR连锁标记

以硫苷性状差异很大而其它品质性状和农艺性状相近的油菜品系967H和967L及其F1、F2植株为材料,通过建立高硫苷和低硫苷亲本基因池来筛选表现差异的SSR引物,利用差异SSR引物对F2代植株硫苷含量性状进行标记分析,确定其与硫苷性状相连锁的关系。结果表明,SSR引物CB10364与硫苷性状连锁,连锁相关性达极显著水平（F=46.32,p〈0.001）;单因素方差分析表明,标记CB10364对低硫苷性状的贡献率为34.36%。     

一个水稻苗期白条纹叶及抽穗期白穗突变体的鉴定和基因定位

从粳稻中花11组培后代中发现了一个苗期白条纹,抽穗期自穗的突变体.该突变体表现为1叶期叶全白,2叶期从新叶叶尖开始沿叶脉逐渐转绿,至成株期完全变绿,抽穗后内外颖表现为自色,穗轴和小枝梗表现为绿色,成熟后颖壳转黄.根据基因定位结果,将该突变体定名为wslwp(white striped leaf and white pa...     

剑麻斑马纹病菌EST-SSR标记开发与评价

剑麻斑马纹病是剑麻生产中具有破坏性的一种真菌病害。本研究从Gen Bank(http://www.ncbi.nlm.nih.gov/nucest)中随机下载10 525条寄生疫霉EST序列,经过去冗余处理后得到5 519条无冗余EST。利用MISA(MIcro SAtellite identification tool)软件进行SSR发掘,从中共搜索到199个1~6碱基SSR,其中三核苷酸重复基元类型最多,共鉴定到55个,占SSR总数的27.6%;其次是二核苷酸重复基元类型和单核苷酸重复基元类型,分别鉴定到51和47个,各占所鉴定SSR总数的25.6%和23.6%。进一步分析表明,AG/CT二核苷酸重复基元类型为优势重复类型,占二核苷酸SSR总数的76.5%。而AAG/CTT和AGC/CTG 2个基元类型在三核苷酸中出现频率最多,分别为15和11次,分别占三核苷酸SSR总数的27.3%及20.0%。从鉴定的199个SSR中,选取含有SSR的合适区段设计了22对引物,并通过18个剑麻斑马纹病菌菌株的基因组DNA进行了评价,结果只有其中9对引物能从剑麻斑马纹病菌基因组DNA中有效扩增,其扩增移效率为40.9%。由此表明,利用此方法来开发剑麻斑马纹病菌的分子标记具有可行性,只是存在一个转移效率问题。     

籼粳交重组自交系的亚种属性与稻米品质性状的关系

2006—2007年以典型籼稻七山占和典型粳稻秋光构建的重组自交系(RILs)F8的92个株系和F9的142个株系为试材,分别以程氏指数(ChI)和籼粳特异SSR标记(Dj)为群体形态分化与遗传分化的指标,同时测定11项主要稻米品质性状,分析群体稻米品质性状类型间差异及籼粳属性与稻米品质性状的相关性。结果表明,两世代RILs群体形态分化均表现为接近正态的连续分布,籼型和粳型株系数接近,遗传分化则均表现为明显偏粳的连续分布;根据ChI将RILs群体划分为籼型和粳型,除F9粒宽外两世代品质性状类型间差异均未达到显著水平;根据Dj将RILs群体划分为偏粳型和粳型,两世代粳型糙米率、整精米率、粒宽显著或极显著高于偏粳型,长宽比极显著低于偏粳型;两世代RILs群体ChI与所有品质性状的相关性均未达到显著水平;Dj与糙米率、整精米率和粒宽呈显著或极显著正相关,而与长宽比呈显著或极显著负相关。形态分化与北方粳稻品质性状没有直接关系,而遗传分化中粳稻血缘比例增加可能通过粒形间接改善碾磨品质。     

Genetics and mapping of resistance to spore inoculum and culture filtrate of Phaeosphaeria nodorum in spring wheat line ND 735

Stagonospora nodorum blotch (SNB), caused by Phaeosphaeria nodorum, is one of the most devastating foliar diseases on wheat (Triticum aestivum L.) in the northern Great Plains of North America. This study was conducted, under controlled environmental conditions, to elucidate the genetics and map the resistance to SNB caused by spore inoculum and culture filtrate of P. nodorum isolate Sn2000. A hard red spring wheat population was developed from a cross between the susceptible cultivar Steele-ND and the resistant line ND 735 for this study. Two-leaf seedlings of the parents, F₁ and F₂ generations, and F_2:6 recombinant-inbred lines (RILs) were inoculated with spore suspensions while independent two week old seedlings of segregating generations were infiltrated with culture filtrate. Disease reaction was assessed 8 days after inoculation based on a lesion-type scale while plants were evaluated for culture filtrate response four days after infiltration for the presence or absence of necrosis. Genetic analysis revealed that a single recessive gene, Tsn1, in ND 735 confers resistance to both spore suspension and culture filtrate of P. nodorum isolate Sn2000. Mapping analysis using Diversity Arrays Technology (DArT) and simple sequence repeat (SSR) markers indicates the gene, Tsn1, is located on the long arm of chromosome 5B and is flanked by the DArt markers wPt-8285 and wPt-3049 at a distance of 7.0 cM and 2.9 cM, respectively. This gene also controls resistance to tan spot caused by Pyrenophora tritici-repentis race 2. Results of this study reveal that wheat-P. nodorum interaction follows the toxin model of gene-for-gene hypothesis. Additionally, the finding of single gene control in the line ND 735 for both tan spot and SNB enhances the utility of the line ND 735 in wheat breeding program as a source of multiple disease resistance.     

Identification of QTLs and Validation of qCd-2 Associated with Grain Cadmium Concentrations in Rice

Cadmium(Cd) is one of heavy metals harmful to human health. As rice is the main staple food in Asia and Cd is easily contaminated in rice, the molecular regulation of Cd accumulation should be explored. In this study, a recombinant inbred population derived from Xiang 743/Katy was grown in Cd-polluted fields and used to map the quantitative trait loci(QTLs) for Cd accumulation in rice grains. We identified seven QTLs distributed on chromosomes 2, 3, 6, 7, 8 and 10. These QTLs displayed phenotypic variances of 58.50% and 40.59% in 2014 and 2015, respectively. Two QTLs, qCd-2 and qCd-7, were identified in both the two years. qCd-2 was detected on the interval of RM250–RM207 on chromosome 2, with an LOD of 2.51 and a phenotypic contribution of 13.75% in 2014, and an LOD of 3.35 and a phenotypic contribution of 14.16% in 2015. qCd-7 co-localized with the cloned qCdT7 on chromosome 7 and may represent the correct candidate. The other five QTLs were detected only in one year. To further confirm the effects of qCd-2, a residual heterozygous line designated as RHL945, with a heterozygous interval of RM263–RM207 on chromosome 2, was selected from the recombinant inbred population and used to develop an F2 population consisting of 155 individual plants. By incorporating further simple sequence repeat markers into the segmental linkage map of the target region, qCd-2 was delimited in the interval of RM5404–RM3774, with an LOD value of 4.38 and a phenotypic contribution of 15.52%. These results reflected the genetic regulation of grain Cd in rice and paved the way for the future cloning of qCd-2.     

籼粳稻杂交后代群体形态分化与遗传分化的比较

 以典型籼稻七山占和典型粳稻秋光构建的F8、F9重组自交系为试材,研究表型性状所揭示的形态分化与籼粳特异SSR标记所揭示的遗传分化差异,探讨维管束性状与形态分化和遗传分化的关系。形态分化和遗传分化所揭示的群体后代籼粳属性均呈偏粳分布,遗传分化所揭示的更加偏粳。依程氏指数、大小维管束比、大维管束比的分类结果与遗传分化相符度均为50%左右,将维管束性状作为程氏指数补充所得总分值与遗传分化的相符度为80%左右,相符度显著增高。维管束数量比可以作为籼粳分类的补充性状。     

Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.     

Comparison of Morphological and Genetic Differentiations in Filial Generation of Cross Between Indica and Japonica Rice

A recombinant inbred line (RIL) population of F8 and F9 generations derived from a cross between a typical indica rice (Qishanzhan) and a typical japonica rice (Akihikari) was used to study the difference between morphological differentiation based on phenotype characters and genetic differentiation using indica and japonica specific SSR markers, and to evaluate the relationship between vascular bundle characters and morphological and genetic differentiations. The results showed that the frequency distributions of morphological and genetic differentiations were all inclined to japonica type in the filial generation. The population was more inclined to japonica type based on genetic differentiation than on morphological differentiation. The consistent degrees of classification based on the Cheng's index, the ratio of large vascular bundle number to small vascular bundle number in panicle neck (RLSVB) and the ratio of large vascular bundle number in the second internode from the top to that in the panicle neck (RLVB) were all about 50% compared with the genetic differentiation, and the consistent degree of the total scores of the Cheng's index combined with the vascular bundle number ratios was significantly increased to about 80% compared with the genetic differentiation. Therefore, the vascular bundle characters could be used as a helpful supplement for subspecies classification.     

粳稻BT型细胞质雄性不育恢复基因功能标记的开发与应用

 为提高粳稻BT型细胞质雄性不育恢复基因的选择效率,在前人克隆的基础上,根据其恢复系与不育系在Rf1a位点574 bp的碱基插入/缺失,设计出功能标记InDel Rf1a。利用该标记对不同地区来源的72份籼、粳稻材料进行检测,结果表明所有的常规籼稻品种、恢复系及保持系在Rf1a位点并不存在缺失,其基因型为Rf1aRf1a。这些材料对粳稻BT型细胞质雄性不育系具有恢复育性的作用;而绝大多数常规粳稻品种（爱知106和伊粳12号除外）在该位点存在缺失,其基因型为rf1arf1a,它们对BT型细胞质雄性不育系具有保持不育的能力。同时,为进一步验证该标记对不同基因型的检测效果,利用其对粳稻恢复系、不育系、杂交组合以及863A/宁恢 8号 F2分离群体的DNA进行扩增,根据其电泳带型可准确区分出Rf1a位点的3种基因型。     

Construction and Testing of a Primary Microsatellite Database of Major Rice Varieties in China

Sixty-three major inbred varieties and parental lines of major F1 hybrids used in the commercial rice production in China were assayed with rice microsatellites screened in a previous study and additional microsatellites on four chromosomes. A set of 24 markers was selected and proposed for its application in the variety identification of rice, which are distributed on all the 12 rice chromosomes with 2 markers on each chromosome. The 63 major varieties and parental lines, as well as 41 major F1 hybrids, were genotyped with the markers. Alleles detected in each line at each marker locus were verified. By matching marker genotypes of corresponding F1, maternal and paternal lines of hybrid rice, high reliability of the maternal lines was verified, data on the paternal lines were modified, and a false hybrid was removed. A database containing genotype data of 103 major rice varieties and parental lines at the 24 marker loci was constructed and analyzed.     

两系杂交稻亲本SSR指纹图谱的建立及其在种子纯度鉴定中的应用

利用筛选到的20对SSR引物建立了两系杂交稻32个亲本(24个光温敏不育系和8个恢复系)的DNA指纹图谱,针对所涉及的9个杂交稻组合,获得能在父、母本间表现出多态性的特异SSR标记48个.以杂交稻组合两优932为例,在实验室用一个特异SSR标记(RM302)对200粒种子样本进行了纯度鉴定,结果鉴定纯度为90.50%,与田间种植鉴定结果90.60%(海南鉴定)和91.57%(武汉鉴定)基本一致,表明SSR标记技术适用于构建DNA指纹图谱和种子纯度鉴定.     

金粳818抗咪唑啉酮类除草剂基因的功能标记开发与应用

【目的】培育抗除草剂品种是水稻育种中的一个重要方向,开发高效的功能标记可以加快抗除草剂性状的筛选工作。【方法】利用开发的dCAPS和KASP分子标记对试验材料的基因型分型,另结合除草剂喷施试验进行表型鉴定。【结果】测序结果验证了前人发现的金粳818中乙酰乳酸合成酶（acetolactate synthase,ALS）基因第627位氨基酸由丝氨酸（serine,S）突变为天冬酰胺（asparagine,N）是其抗咪唑啉酮类除草剂的主要原因,本文将其命名为ALS^627N。根据此变异位点的DNA序列差异,开发了两个新的功能标记dC-ALS-627和K-ALS-627,可有效区分ALS^627N纯合型、杂合型及非突变纯合型。【结论】两个标记均可以广泛用于ALS^627N基因的标记辅助选择育种,有助于加快培育抗咪唑啉酮类除草剂新品种,具有重要的实际应用价值。     

Development and Utilization of Functional Markers for Imidazolinone Herbicides Resistance Gene in japonica Rice Variety Jinjing 818

【Objective】Cultivating herbicide resistant rice varieties is an important direction in rice breeding. Developing efficient functional markers can accelerate the screening of herbicide resistance trait. 【Method】dCAPS and KASP techniques were used to analyze the genotypes of the experimental materials. In addition, the phenotypes were identified by herbicide spraying test. 【Result】Through sequence alignment analysis, we further confirmed that the mutation of 627 amino acid of acetolactate synthase (ALS) gene from serine (S) into asparagine (N) in Jinjing 818 (here after named ALS627N) produces strong resistance to imidazolinone herbicides. According to the DNA sequence flanking the mutation site, we developed two novel functional markers, dC-ALS-627 and K-ALS-627, for detecting this resistant allele ALS627N. The two markers can effectively distinguish homozygous ALS627N type, heterozygous type and homozygous wild type. Combined with herbicide spraying test, we confirmed that ALS627N is a dominant herbicide resistant gene. 【Conclusion】 We validated that the two markers have great potential in marker-assisted breeding for ALS627N gene. Totally, this study will accelerate the development of new varieties with resistance to imidazolinone herbicides, and have significance in breeding practice.     

Mapping and Comparative Analysis of QTL for Rice Plant Height Based on Different Sample Sizes within a Single Line in a RIL Population

To clarify the most appropriate sample size for obtaining phenotypic data for a single line, we investigated the main-effect QTL (M-QTL) of a quantitative trait plant height (ph) in a recombinant inbred line (RIL) population of rice (derived from the cross between Xieqingzao B and Zhonghui 9308) using five individual plants in 2006 and 2009. Twenty-six ph phenotypic datasets from the completely random combinations of 2, 3, 4, and 5 plants in a single line, and five ph phenotypic datasets from five individual plants were used to detect the QTLs. Fifteen M-QTLs were detected by 1 to 31 datasets. Of these, qph7a was detected repeatedly by all the 31 ph datasets in 2006 and explained 11.67% to 23.93% of phenotypic variation; qph3 was detected repeatedly by all the 31 datasets and explained 5.21% to 7.93% and 11.51% to 24.46% of phenotypic variance in 2006 and 2009, respectively. The results indicate that the M-QTL for a quantitative trait could be detected repeatedly by the phenotypic values from 5 individual plants and 26 sets of completely random combinations of phenotypic data within a single line in an RIL population under different environments. The sample size for a single line of the RIL population did not affect the efficiency for identification of stably expressed M-QTLs.