Genome-wide association mapping focusing on a rice population derived from rice breeding programs in a region

Plant breeding programs in local regions may generate genetic variations that are desirable to local populations and shape adaptability during the establishment of local populations. To elucidate genetic bases for this process, we proposed a new approach for identifying the genetic bases for the traits improved during rice breeding programs; association mapping focusing on a local population. In the present study, we performed association mapping focusing on a local rice population, consisting of 63 varieties, in Hokkaido, the northernmost region of Japan and one of the northern limits of rice cultivation worldwide. Six and seventeen QTLs were identified for heading date and low temperature germinability, respectively. Of these, 13 were novel QTLs in this population and 10 corresponded to the QTLs previously reported based on QTL mapping. The identification of QTLs for traits in local populations including elite varieties may lead to a better understanding of the genetic bases of elite traits. This is of direct relevance for plant breeding programs in local regions.     

Quantitative trait loci for rice blast resistance detected in a local rice breeding population by genome-wide association mapping

Plant breeding programs aim to develop cultivars with high adaptability to the specific conditions in a local region. As a result, unique genes and gene combinations have been accumulated in local elite breeding populations during the long history of plant breeding. Genetic analyses on such genes and combinations may be useful for developing new cultivars with more-desirable agronomic traits. Here, we attempted to detect quantitative trait loci (QTL) for rice blast resistance (BR) using a local breeding rice population from Hokkaido, Japan. Using genotyping data on single nucleotide polymorphisms and simple sequence repeat markers distributed throughout the whole genomic region, we detected genetic regions associated with phenotypic variation in BR by a genome-wide association mapping study (GWAS). An additional association analysis using other breeding cultivars verified the effect and inheritance of the associated region. Furthermore, the existence of a gene for BR in the associated region was confirmed by QTL mapping. The results from these studies enabled us to estimate potential of the Hokkaido rice population as a gene pool for improving BR. The results of this study could be useful for developing novel cultivars with vigorous BR in rice breeding programs.     

Quantitative trait loci for whiteness of cooked rice detected in improved rice cultivars in Hokkaido

Improving the eating quality of cooked rice has been one of the most important objectives in rice breeding programs. Eating quality of cooked rice is a complex trait including several components, such as external appearance, taste, aroma, and texture. Therefore, dissection of these components followed by marker-assisted selection of detected QTL(s) may be a useful approach for achieving desirable eating quality in rice breeding. Whiteness of cooked rice (WCR) is an important factor related to the external appearance of cooked rice. WCR is known to be associated with the amylose and protein contents of the endosperm. However, the genetic basis of WCR remains unclear. In this study, we evaluated phenotypic variation in WCR among recently developed rice cultivars from Hokkaido, Japan. Then, we developed doubled haploid lines (DHLs) derived from a cross between two cultivars from Hokkaido, Joiku No. 462 (high WCR) and Jokei06214 (low WCR). Using the DHLs, we detected two QTLs for WCR, qWCR3 and qWCR11, on chromosomes 3 and 11, respectively. We also examined the dosage effect of the two QTLs based on both the categorized segregants in the DHLs and the relationship between the WCR phenotype and inheritance around the QTL regions in cultivars from Hokkaido.     

QTL pyramiding for improving of cold tolerance at fertilization stage in rice

Vigorous cold tolerance at the fertilization stage (CTF) is a very important characteristic for stable rice production in cold temperature conditions. Because CTF is a quantitatively inherited trait, pyramiding quantitative trait loci (QTLs) using marker-assisted selection (MAS) is effective for improving CTF levels in rice breeding programs. We previously identified three QTLs controlling CTF, qCTF7, qCTF8 and qCTF12, using backcrossed inbred lines derived from a cross between rice cultivar Eikei88223 (vigorous CTF) and Suisei (very weak CTF). However, pyramiding of these QTLs for the application of MAS in practical rice breeding programs have not yet been elucidated. In this study, we examined the effect of pyramiding QTLs for improvement of CTF level using eight possible genotype classes from the 152 F₃ population derived from a cross between Eikei88223 and Suisei. Increasing of CTF levels in combinations between qCTF7 and qCTF12 and between qCTF8 and qCTF12 were detected. Furthermore, we compared the haplotype pattern around the QTLs for CTF among the rice cultivars from Hokkaido. These results are useful for improvement of new cultivars with high CTF levels using MAS and identification of genetic resources with the novel QTL(s) for CTF.     

水稻InDel和SSR标记多态性的比较分析

为评价插入缺失（InDel）标记在水稻分子育种中的应用价值,本研究用日本晴和9311序列筛选得到遍布每条染色体的20对InDel标记和53对SSR标记,分析46份粳稻和47份籼稻的遗传多样性。研究表明这些InDel标记在籼粳亚种间具有很高的多态性,亚种内多态性较低,平均多态性水平显著小于SSR标记,InDel标记具有数量多,扩增产物稳定和易于检测等优点,将InDel标记应用到遗传图谱构建、基因定位分析和标记辅助选择中可以加速研究进程。     

Characterization of a mini core collection of Japanese wheat varieties using single-nucleotide polymorphisms generated by genotyping-by-sequencing

A core collection of Japanese wheat varieties (JWC) consisting of 96 accessions was established based on their passport data and breeding pedigrees. To clarify the molecular basis of the JWC collection, genome-wide single-nucleotide polymorphism (SNP) genotyping was performed using the genotyping-by-sequencing (GBS) approach. Phylogenetic tree and population structure analyses using these SNP data revealed the genetic diversity and relationships among the JWC accessions, classifying them into four groups; “varieties in the Hokkaido area”, “modern varieties in the northeast part of Japan”, “modern varieties in the southwest part of Japan” and “classical varieties including landraces”. This clustering closely reflected the history of wheat breeding in Japan. Furthermore, to demonstrate the utility of the JWC collection, we performed a genome-wide association study (GWAS) for three traits, namely, “days to heading in autumn sowing”, “days to heading in spring sowing” and “culm length”. We found significantly associated SNP markers with each trait, and some of these were closely linked to known major genes for heading date or culm length on the genetic map. Our study indicates that this JWC collection is a useful set of germplasm for basic and applied research aimed at understanding and utilizing the genetic diversity among Japanese wheat varieties.     

水稻核心种质及成恢448回交后代的稻米延伸性研究

对列入全球水稻分子育种计划的73个水稻供体品种的稻米延伸性进行测试分析,并对其中的部分品种与成恢448（CH448）不同回交世代的自交种进行整精米长（HRL）、饭粒长（CRL）和稻米延伸性（CRE）的相关性分析,结果表明：这73个品种之间的CRE存在极显著的差异性,可聚为6个类型;在10个亲本与CH448的BC2F2群体中,HRL与CRL具有正相关性并存在差异,9个群体的HRL与CRE的负相关性达显著或极显著水平。在成恢448／Basmati370的BC2F3、BC2F4及BC3F3群体中,随着育种世代增加,HRL与CRE从负相关性趋向于0,增加一个回交世代只在一定程度上对HRL和CRL的相关性产生一定的影响,在BC2F4群体可以对CRE开始单株选择。在不同供体品种与CH448的BC3F2群体中,不同回交组合内CRE的变异有所不同。     

Adaptation of tropical maize germplasm to temperate environments

Maize (Zea mays L.) is one of few crops that can offer significant genetic gains with the utilization of genetic diversity. Genetically broad-based germplasm has the potential to contribute useful and unique alleles to U.S. Corn Belt breeding programs not present in current U.S. genome sequences (e.g. B73, NAM, etc.). Our objectives were to determine if unique tropical genetic materials have been effectively adapted to temperate environments and how their agronomic performance was relative to adapted populations. An important long-term objective of the Iowa and North Dakota maize breeding programs has been, in addition to the typical elite by elite line pedigree selection cultivar development process, to adapt exotic and unique germplasm, maximize their genetic improvement, and develop unique products for breeding and commercial uses. Stratified mass selection methodology for earliness has been utilized for the adaptation of tropical and temperate populations to Iowa and North Dakota environments. This method has allowed screening of up to 25,000 genotypes per population cycle at a rate of one cycle per year. In addition, the estimated cost per year our programs had for the adaptation of each population was less than $2,000 which could successfully be applied in any breeding program across the globe. This cost has been less than 1 % of the total cost for finding minor genes on the same trait. Our results showed the successful adaptation of exotic populations was independent from genetic background. We can speculate there are a few major genes responsible for most of flowering date expression. We encourage the use of technology to target traits according to their genetic complexity. Stratified mass selection at the phenotypic level has been successful. Each of the populations with either 25 of 100 % tropical germplasm are available for anyone who may desire to expand the germplasm base of their breeding programs with tropical germplasm adapted to temperate mid- and short-season U.S. Corn Belt environments.     

Genome-wide InDel marker system for application in rice breeding and mapping studies

In order to widen the genetic basis for local rice breeding programs, a set of 506 PCR-based novel insertion/deletion (InDel) markers were designed. The markers have size differences larger than 30 base-pairs and were generated by published genome sequences of rice (Oryza sativa L.) indica and japonica subspecies. Among the 506 InDel markers, 133 markers are polymorphic between the selected parents Taiken 2 and Taichung Sen 10, which are elite Taiwanese japonica and indica varieties, respectively. The InDel to amplicon size ratios, based on the japonica template, were in the range of 8.8–45.6 %, with an average of 21.6 %. As a result, the PCR product can be efficiently separated and easily scored by running 30 min of non-submerging electrophoresis on 2–3 % agarose gels. Commonly observed non-parental bands originated from heteroduplex are considered beneficial in distinguishing homo- and hetero-zygotes. A linkage map constructed solely on these markers arranged in 12 linkage groups and extended over 1,413.9 cM with an average distance of 9.8 cM between markers, based on a segregating population of 286 F₂ individuals derived from a cross between Taiken 2 and Taichung Sen 10. The result indicated that InDel markers with moderate size differences were abundant and uniformly distributed across the whole genome of rice. Due to their simplicity in design and robustness in genotyping, these InDel markers have been routinely used in our quantitative trait loci mapping studies and marker-assisted selection programs for rice. We suggest that InDel markers with moderate size differences could be a valuable alternative in addition to the widely used SSR and SNP markers for rice breeding, and particularly suitable for field laboratories at breeding stations and/or laboratories with limited resources.     

Characterization of near-isogenic lines carrying QTL for high spikelet number with the genetic background of an indica rice variety IR64 (Oryza sativa L.)

Total spikelet number per panicle (TSN) is one of the most important traits associated with rice yield potential. This trait was assessed in a set of 334 chromosomal segment introgression lines (ILs: BC₃-derived lines), developed from new plant type (NPT) varieties as donor parents and having the genetic background of an indica-type rice variety IR64. Among the 334 ILs, five lines which had different donor parents and showed significantly higher TSN than IR64 were used for genetic analysis. Quantitative trait locus (QTL) analysis was conducted using F₂ populations derived from crosses between IR64 and these ILs. As a result, a QTL for high TSN (one from each NPT donor variety) was detected on common region of the long arm of chromosome 4. The effect of the QTL was confirmed by an increase in TSN of five near-isogenic lines (NILs) developed in the present study. The variation in TSN was found among these NILs, attributing to the panicle architecture in the numbers of primary, secondary and tertiary branches. The NILs for TSN and the SSR markers linked to the TSN QTLs are expected to be useful materials for research and breeding to enhance the yield potential of rice varieties.     

单核苷酸多态性在水稻遗传育种中的应用

单核苷酸多态性(SNP)是最新发展起来的继限制性片段多态性(RFLP)、微卫星标记(SSR)后的新一代分子标记,具有分布不均匀、丰度高、检测易实现自动化的特点。SNPs日益成为功能基因组学和分子标记辅助选择等领域研究的主要手段。本文主要介绍SNPs在水稻基因组中的分布、特点、检测及其在遗传育种中的应用。     

Collection,preservation and distribution of Oryza genetic resources by the National Bioresource Project RICE (NBRP-RICE)

Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.     

Genetic region responsible for the differences of starch properties in two glutinous rice cultivars in Hokkaido,Japan

Starch properties are major determinants of grain quality and food characteristics in rice (Oryza sativa L.). Control of starch properties will lead to the development of rice cultivars with desirable characteristics. We performed quantitative trait locus analysis and detected a putative region on chromosome 2 associated with phenotypic variation of starch properties in two glutinous rice varieties developed in the Hokkaido region of Japan: ‘Kitayukimochi’, which has a low pasting temperature and creates soft rice cakes, and ‘Shirokumamochi’, which has a high pasting temperature and creates hard rice cakes. Starch branching enzyme IIb (SbeIIb) was identified as a candidate gene within the region. Sequence analysis of SbeIIb in parental lines identified two single-nucleotide polymorphisms (SNPs) with non-synonymous mutations in the coding region of the ‘Shirokumamochi’ genotype (SbeIIb^sr). We genotyped over 100 rice cultivars, including 28 rice varieties in the Honshu region of Japan, using the CAPS marker, which was designed using one of the SNPs. However, SbeIIb^sr was not found in rice cultivars in Honshu. Distribution analysis indicated that SbeIIb^sr was introduced to the rice breeding population in Hokkaido from the American variety ‘Cody’ via the Hokkaido cultivar ‘Kitaake’. As a result, SbeIIb^sr was distributed only in progenies of ‘Kitaake’.     

黑龙江省水稻品种的遗传多样性

为了研究黑龙江省当前水稻品种的遗传基础及其亲缘关系,利用农业行业标准《水稻品种鉴定技术规程SSR标记法(NY/T 1433—2014)》中公布的47对SSR标记,对来自黑龙江省不同积温区的231份水稻品种进行遗传多样性分析。结果显示：47对SSR标记在231份水稻中共检测到136个等位基因,每个位点等位基因变幅为2~5个,平均2.92个;基因多样性变幅为0.11~0.79,平均0.56;多态性信息量(PIC)变化范围为0.11~0.76,平均0.49;标记指数(MI)的变化范围在3.18~18.39,平均6.52。聚类分析将231份水稻分为3类7群,聚类结果与主成分分析结果一致。综上所述,黑龙江省水稻品种遗传多样性不够丰富（平均PIC值为0.49）,同一积温区品种间亲缘关系较近。育种中注意南北部品种的杂交,以便拓宽遗传背景,培育出绿色优质高产新品种。     

Identification of microsatellite markers linked to the blast resistance gene <Emphasis Type="Italic">Pi-1(t)</Emphasis> in rice

The present work was conducted to identify microsatellite markers linked to the rice blast resistance gene Pi-1(t) for a marker-assisted selection program. Twenty-four primer pairs corresponding to 19 microsatellite loci were selected from the Gramene database (www. gramene.org) considering their relative proximity to Pi-1(t) gene in the current rice genetic map. Progenitors and DNA bulks of resistant and susceptible families from F₃ segregating populations of a cross between the near-isogenic lines C101LAC (resistant) and C101A51 (susceptible) were used to identify polymorphic microsatellite markers associated to this gene through bulked segregant analysis. Putative molecular markers linked to the blast resistance gene Pi-1(t) were then used on the whole progeny for linkage analysis. Additionally, the diagnostic potential of the microsatellite markers associated to the resistance gene was also evaluated on 17 rice varieties planted in Latin America by amplification of the specific resistant alleles for the gene in each genotype. Comparing with greenhouse phenotypic evaluations for blast resistance, the usefulness of the highly linked microsatellite markers to identify resistant rice genotypes was evaluated. As expected, the phenotypic segregation in the F₃ generation agreed to the expected segregation ratio for a single gene model. Of the 24 microsatellite sequences tested, six resulted polymorphic and linked to the gene. Two markers (RM1233*I and RM224) mapped in the same position (0.0 cM) with the Pi-1(t) gene. Other three markers corresponding to the same genetic locus were located at 18.5 cM above the resistance gene, while another marker was positioned at 23.8 cM below the gene. Microsatellite analysis on elite rice varieties with different genetic background showed that all known sources of blast resistance included in this study carry the specific Pi-1(t) allele. Results are discussed considering the potential utility of the microsatellite markers found, for MAS in rice breeding programs aiming at developing rice varieties with durable blast resistance based on a combination of resistance genes. Centro Internactional de Agricultura Tropical (CIAT) institute where the research was carried out     

Trisomic genetic analysis of aroma in three Japanese native rice varieties (Oryza sativa L.)

Information on the genetics of aroma in rice facilitates breeding and selection of new aromatic varieties with high yield and good quality. Objective of the present study was to make clear the number of genes controlling aroma, and the allelism of aroma genes and the location of aroma gene(s) on the chromosome in three Japanese native aromatic rice varieties (Kabashiko, Shiroikichi and Henroyori). Lack of leaf aroma in all F₁ plants of non-aromatic/aromatic crosses indicated the recessive nature of aroma, and the segregation ratios (3:1) of non-aromatic to aromatic plants in its F₂ populations from Nipponbare/aromatic varieties crosses revealed that each of the three aromatic varieties contains a single recessive gene for aroma. Through trisomic analysis, the segregation of non-aromatic and aromatic plants in all F₂ populations from the crosses between trisomics lines NT8, with an extra chromosome 8, and aromatic varieties deviated significantly from disomic segregation of 3:1 ratios, and fitted to trisomic segregation, however, in other F₂ populations derived from other 7 types of trisomic F₁ plants, the segregation ratios of non-aromatic to aromatic were 3:1, indicating that the single recessive aroma gene was located on chromosome 8 in three aromatic varieties. This revised version was published online in July 2006 with corrections to the Cover Date.     

中国不同地理来源籼稻地方品种的亲缘关系研究

利用61对SSR引物对原产于中国14个省的440份籼稻地方品种进行了SSR分析,并对各省、经度和纬度间的籼稻地方品种进行了遗传距离和聚类分析。结果表明,籼稻地方品种遗传距离变异范围在各省份间为0.0707~0.4076;经度间为0.0680~0.3032;纬度间为0.0385~0.2696。相邻的省份、经度、纬度的籼稻地方品种基本归为同一类群,遗传距离和亲缘关系较近,而地理位置相隔较远的省份、经度、纬度的籼稻地方品种基本归为不同类群,遗传距离和亲缘关系较远,说明籼稻地方品种的亲缘关系与地理位置有着密切的相关性。但按经度和纬度划分较按省份划分的品种间遗传距离更具有规律性。     

作物驯化和品种改良所选择的关键基因及其特点

近15~20年作物基因组学迅速发展,特别是第2代测序技术的普及,显著降低了测序成本,使单核苷酸多态性(SNP)分析和单元型区段(也称单倍型区段)分析渗透到生命科学的各个领域,对系统生物学、遗传学、种质资源学和育种学影响最为深刻,使其进入基因组学的全新时代。一批驯化选择基因的克隆,特别是对一些控制复杂性状形成的遗传基础及其调控机制的解析,更清晰地揭示了作物驯化和品种改良的历史,提升了人们对育种的认知,推动育种方法的改进。驯化和育种既有相似之处,也存在明显的差异。驯化选择常常发生在少数关键基因或位点,对基因的选择几乎是一步到位;而现代作物育种虽然只有100年左右的历史,但其对基因组影响更为强烈,是一些重要代谢途径不断优化的过程。随着生态环境或栽培条件的变化,育种选择目标基因(等位变异)会发生相应的变化或调整,因此对基因(等位变异)的选择是逐步的。此外,强烈的定向选择重塑了多倍体物种的基因组,使其亚基因组与供体种基因组明显不同。在群体水平上,系统分析驯化和育种在作物基因组和基因中留下的踪迹,凝炼其中的规律,将为品种改良和育种提供科学理论和指导,本文也简要介绍了"十三五"国家重点研发计划专项"主要农作物优异种质资源形成与演化规律"的基本研究思路。     

水稻稻瘟病抗性基因Bsr-d1功能标记的开发和利用

稻瘟病是危害水稻最严重的病害之一, 选育抗病品种是防治该病害最有效的方法。Bsr-d1是对稻瘟病菌具有广谱抗性的一个重要基因。为提高Bsr-d1基因在育种中的选择效率, 根据Bsr-d1与其感病等位基因bsr-d1在功能区域存在的单核苷酸差异, 设计和筛选出Bsr-d1基因不同类型的基因功能标记CAPs5-1和3Bsr-d1/3bsr-d1, 结合测序分析验证, 可准确鉴定出Bsr-d1的不同基因型。利用3Bsr-d1/3bsr-d1对34份籼稻品种、江苏历年来主要推广的110份粳稻品种、其他省份的13份粳稻品种、148份太湖流域地方粳稻资源和19份太湖流域地方籼稻资源进行了Bsr-d1基因型检测, 筛选到携带Bsr-d1基因的籼型资源11份, 271份粳型资源中均不携带Bsr-d1基因, 这也说明Bsr-d1主要分布在籼型水稻资源中, 在粳型资源中几乎不存在。本研究为Bsr-d1基因的育种利用和分子标记辅助选择奠定了基础。     

亚洲不同生态区水稻群体遗传多样性与特异性分析

研究不同生态区水稻群体的遗传多样性及其遗传结构分化、群体特异性及其相互关系,为水稻起源及亲本遴选提供遗传基础。本研究利用119对SSR标记对太湖、黑龙江省和越南的440种水稻品种进行分析。结果在三个不同生态区中的太湖流域水稻中检测到783个等位基因,平均多态信息含量（PIC）为0.49;在黑龙江水稻中检测到296个等位基因,平均多态信息含量（PIC）为0.21;在越南水稻中共检测到374个等位基因,平均多态信息含量（PIC）为0.28,这表明越南水稻和黑龙江水稻的多样性低于太湖水稻。特缺等位变异数最多的是黑龙江亚群（132）,最少的是太湖亚群（4）;特有等位变异数最多的是太湖亚群（284）,最少的是黑龙江亚群（25）。从亚群间补充等位变异数来看：其补充等位变异数最多的是太湖亚群对黑龙江亚群的补充（447）,最少的是黑龙江亚群对太湖亚群的补充（29）。利用Structure2.2软件将三个地区的440个品种分为7类血缘,发现每个血缘都不是独立的而是相互渗透的。