水稻微卫星标记的发展和应用

微卫星又称简单序列重复。它是由几个核苷酸（一般２～４个）为重复单位组成的串联重复序列。相同座位上的重复序列由于重复次数的不同而造成序列长度的多态性。微卫星标记是一种共显性标记,具有等位基因丰富、检测技术简单等优点。微卫星标记在基因组作图、品种鉴定、种质保存、分子标记辅助选择等方面有着广泛的应用。目前水稻中已发展了３００多个微卫星标记。     

The construction of a genetic linkage map of non-heading Chinese cabbage （Brassica campestris ssp. chinensis Makino）

Non-heading Chinese cabbage （Brassica carnpestris ssp. chinensis Makino） is one of the most important vegetables in eastern China. A genetic linkage map was constructed using 127 doubled haploid （DH） lines, and the DH population was derived from a commercial hybrid ＂Hanxiao＂ （lines SW-13 x L-118）. Out of the 614 polyrnorphic markers, 43.49% were not assigned to any of the linkage groups （LGs）. Chi-square tests showed that 42.67% markers were distorted from expected Mendelian segregation ratios, and the direction of distorted segregation was mainly toward the paternal parent L-118. After sequentially removing the markers that had an interval distance smaller than 1 cM from the upper marker, the overall quality of the linkage map was increased. Two hundred and sixty-eight molecular markers were mapped into 10 LGs, which were anchored to the corresponding chromosome of the B. rapa reference map based on com- mon simple sequence repeat （SSR） markers. The map covers 973.38 cM of the genome and the average interval distance between markers was 3.63 cM. The number of markers on each LG ranged from 18 （R08） to 64 （R07）, with an average interval distance within a single LG from 1.70 cM （R07） to 6.71 cM （R06）. Among these mapped markers, 169 were sequence-related amplified polymorphism （SRAP） molecular markers, 50 were SSR markers and 49 were random amplification polymorphic DNA （RAPD） markers. With further saturation to the LG9 the current map offers a genetic tool for loci analysis for important agronomic traits.     

RiceQTLPro: an integrated database for quantitative trait loci marker mapping in rice plant

The National Agricultural Biotechnology Information Center (NABIC) in South Korea reconstructed a RiceQTLPro database for gene positional analysis and structure prediction of the chromosomes. This database is an integrated web-based system providing information about quantitative trait loci (QTL) markers in rice plant. The RiceQTLPro has the three main features namely, (1) QTL markers list, (2) searching of markers using keyword, and (3) searching of marker position on the rice chromosomes. This updated database provides 112 QTL markers information with 817 polymorphic markers on each of the 12 chromosomes in rice.

Availability

The database is available for free at http://nabic.rda.go.kr/gere/rice/geneticMap/     

Identification of a stable quantitative trait locus for percentage grains with white chalkiness in rice (Oryza sativa)

High chalkiness is a major problem in many rice-producing areas of the world, especially in hybrid rice (Oryza sativa L.) in China. We previously showed a major quantitative trait locus for the percentage of grains with white chalkiness (QTLqPGWC-8) in the interval G1149-R727 on chromosome 8 using a chromosome segment substitution line (CSSL). Here, we selected the line-CSSL50 harboring the QTLqPGWC-8 allele from the CSSLs derived from a cross between Asominori (as a recurrent parent) and IR24 (as a donor parent), which had higher percentage chalkiness, markedly different from that of Asominori. There were also significant differences in starch granules, appearance of amylose content (AAC) and milling qualities between Asominori and CSSL50, but not in grain size or thousand grain weight (TGW). The BC(4) F(2) and BC(4) F(3) populations from a cross between CSSL50 and Asominori were used for fine mapping of qPGWC-8. We narrowed down the location of this QTL to a 142 kb region between Indel markers 8G-7 and 8G-9. QTLqPGWC-8 accounted for 50.9% of the difference in PGWC between the parents. The markers tightly linked to qPGWC-8 should facilitate cloning of the gene underlying this QTL and will be of value for marker-assisted selection in breeding rice varieties with better grain quality.     

Genotyping-by-sequencing map permits identification of clubroot resistance QTLs and revision of the reference genome assembly in cabbage (Brassica oleracea L.)

Clubroot is a devastating disease caused by Plasmodiophora brassicae and results in severe losses of yield and quality in Brassica crops. Many clubroot resistance genes and markers are available in Brassica rapa but less is known in Brassica oleracea. Here, we applied the genotyping-by-sequencing (GBS) technique to construct a high-resolution genetic map and identify clubroot resistance (CR) genes. A total of 43,821 SNPs were identified using GBS data for two parental lines, one resistant and one susceptible lines to clubroot, and 18,187 of them showed >5× coverage in the GBS data. Among those, 4,103 were credibly genotyped for all 78 F₂ individual plants. These markers were clustered into nine linkage groups spanning 879.9 cM with an average interval of 1.15 cM. Quantitative trait loci (QTLs) survey based on three rounds of clubroot resistance tests using F_{2 : 3} progenies revealed two and single major QTLs for Race 2 and Race 9 of P. brassicae, respectively. The QTLs show similar locations to the previously reported CR loci for Race 4 in B. oleracea but are in different positions from any of the CR loci found in B. rapa. We utilized two reference genome sequences in this study. The high-resolution genetic map developed herein allowed us to reposition 37 and 2 misanchored scaffolds in the 02–12 and TO1000DH genome sequences, respectively. Our data also support additional positioning of two unanchored 3.3 Mb scaffolds into the 02–12 genome sequence.     

An integrated database to enhance the identification of SNP markers for rice

The National Academy of Agricultural Science (NAAS) has developed a web-based marker database to provide information about SNP markers in rice. The database consists of three major functional categories: map viewing, marker searching and gene annotation. It provides 12,829 SNP markers information including gene location information on 12 chromosomes in rice. The annotation of SNP marker provides information such as marker name, EST number, gene definition and general marker information. Users are assisted in tracing any new structures of the chromosomes and gene positional functions using specific SNP markers. AVAILABILITY: The database is available for free at http://nabic.niab.go.kr/SNP/     

广西地方稻种资源核心种质构建和遗传多样性分析

以丁颖分类体系分组原则与组内逐层聚类取样方法,对8609份广西地方栽培稻资源表型数据信息进行分析,通过对表型保留比例等评价指标的多重比较确定核心种质总体取样比例,构建出占总体样本5%(414份)的广西地方栽培稻资源初级核心种质。初级核心种质能代表总体遗传变异的89%。用34对SSR分子标记对初级核心种质进行遗传多样性分析,结果表明:广西地方栽培稻资源有较高的遗传多样性(等位基因数A为4.91,Nei’s多样性指数为0.574)。就Nei’s遗传多样性指数而言,粳稻高于籼稻,晚稻高于早稻,水稻高于陆稻,糯稻高于粘稻;来自桂中的稻种资源具有最高的遗传多样性。研究最终利用SSR数据,把414份初级核心种质压缩50%后形成209份核心种质,核心种质基因保留比例达到98%以上,有效代表了广西地方栽培稻资源多样性水平。     

Genetic analysis and gene mapping of a new rolled-leaf mutant in rice (Oryza sativa L.)

To understand the development of rice leaf blades,we identified a new rolled-leaf mutant,w32,from indica cultivar IR64 through EMS mutagenesis. The mutant showed a stable rolled-leaf phenotype throughout the life cycle. Two F2 populations were developed by crossing w32 to cultivar IR24 and PA64. Genetic analysis showed that the rolled-leaf phenotype was controlled by a single recessive gene. To determine the location of the gene,bulked segregant analysis was carried out using mutant and wild-type DNA pools ...     

Linkage Map Construction and Quantitative Trait Loci Analysis for Bolting Based on a Double Haploid Population of Brassica rapa

Early bolting of Chinese cabbage （Brassica rapa L.） during spring cultivation often has detrimental effects on the yield and quality of the harvested products. Breeding late bolting varieties is a major objective of Chinese cabbage breeding programs. In order to analyze the genetic basis of bolting traits, a genetic map of B. rapa was constructed based on amplified fragment-length poiymorphism （AFLP）, sequence-related amplified poiymorphism （SRAP）, simple sequence repeat （SSR）, random amplification of polymorphic DNA （RAPD）, and isozyme markers. Marker analysis was carried out on 81 double haploid （DH） lines obtained by microspore culture from F1 progeny of two homozygous parents： B. rapa L. ssp. pekinensis （BY） （an extra-early bolting Chinese cabbage line） and B. rapa L. ssp. rapifera （MM） （an extra-late bolting European turnip line）. A total of 326 markers including 130 AFLPs, 123 SRAPs, 16 SSRs, 43 RAPDs and 14 isozymes were used to construct a linkage map with 10 linkage groups covering 882 cM with an average distance of 2.71 cM between loci. The bolting trait of each DH line was evaluated by the bolting index under controlled conditions. Quantitative trait loci （QTL） analysis was conducted using multiple QTL model mapping with MapQTL5.0 software. Eight QTLs controlling bolting resistance were identified. These QTLs, accounting for 14.1% to 25.2% of the phenotypic variation with positive additive effects, were distributed into three linkage groups. These results provide useful information for molecular marker-assisted selection of late bolting traits in Chinese cabbage breeding programs.     

Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers.

We searched for SNPs in 417 regions distributed throughout the genome of three Oryza sativa ssp. japonica cultivars, two indica cultivars, and a wild rice (O. rufipogon). We found 2800 SNPs in approximately 250,000 aligned bases for an average of one SNP every 89 bp, or one SNP every 232 bp between two randomly selected strains. Graphic representation of the frequency of SNPs along each chromosome showed uneven distribution of polymorphism-rich and -poor regions, but little obvious association with the centromere or telomere. The 94 SNPs that we found between the closely related cultivars 'Nipponbare' and 'Koshihikari' can be converted into molecular markers. Our establishment of 213 co-dominant SNP markers distributed throughout the genome illustrates the immense potential of SNPs as molecular markers not only for genome research, but also for molecular breeding of rice.     

Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project

As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones.Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa.     

粳稻穗角与稻米品质的相关性及稻米品质遗传分析

测定了粳稻直立穗品种丙8979与弯曲穗品种C堡杂交组合的P1、P2及其重组自交系349个株系的穗角和10个稻米品质性状, 分析了穗角与稻米品质性状之间的相关性, 并运用主基因+多基因混合遗传模型, 对稻米品质10个性状进行了遗传分析。结果表明,穗角与糙米率、整精米率、垩白粒率、垩白度、糊化温度、胶稠度和直链淀粉含量均无显著相关; 与精米率呈显著正相关(r=0.124*); 与粒长和长宽比均呈极显著正相关(相关系数分别为0.470**和0.241**)。糙米率、精米率和直链淀粉含量均受2对主基因+多基因控制, 2对主基因具有累加作用和加性×加性的上位性作用; 整精米率、粒长、长宽比和胶稠度受2对加性-上位性主基因+多基因控制;垩白粒率、垩白度和糊化温度均受3对加性-上位性主基因+多基因控制。糙米率、精米率、整精米率、垩白粒率、垩白度和糊化温度6个品质性状以主基因遗传为主,粒长、长宽比、胶稠度和直链淀粉含量4个性状以多基因遗传为主。     

Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project

As a part of the Multinational Genome Sequencing Project of Brassica rapa,linkage group R9 and R3 were sequenced using a bacterial artificial chromosome(BAC) by BAC strategy.The current physical contigs are expected to cover approximately 90%euchromatins of both chromosomes.As the project progresses,BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific.To support the project,a random sheared fosmid library was constructed.The library consists of 9...     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

BrpSPL9 (Brassica rapa ssp. pekinensis SPL9) controls the earliness of heading time in Chinese cabbage

The leafy heads of cabbage (Brassica oleracea), Chinese cabbage (Brassica rapa ssp. pekinensis), Brussels sprouts (B. oleracea ssp. gemmifera) and lettuce (Lactuca sativa) comprise extremely incurved leaves that are edible vegetable products. The heading time is important for high quality and yield of these crops. Here, we report that BrpSPL9‐2 (B. rapa ssp. pekinensis SQUAMOSA PROMOTER BINDING‐LIKE 9‐2), a target gene of microRNA brp‐miR156, controls the heading time of Chinese cabbage. Quantitative measurements of leaf shapes, sizes, colour and curvature indicated that heading is a late adult phase of vegetative growth. During the vegetative period, miR156 levels gradually decreased from the seedling stage to the heading one, whereas BrpSPL9‐2 and BrpSPL15‐1 mRNAs increased progressively and reached the highest levels at the heading stage. Overexpression of a mutated miR156‐resistant form of BrpSPL9‐2 caused the significant earliness of heading, concurrent with shortening of the seedling and rosette stages. By contrast, overexpression of miR156 delayed the folding time, concomitant with prolongation of the seedling and rosette stages. Morphological analysis reveals that the significant earliness of heading in the transgenic plants overexpressing BrpSPL9‐2 gene was produced because the juvenile phase was absent and the early adult phase shortened, whereas the significant delay of folding in the transgenic plants overexpressing Brp‐MIR156a was due to prolongation of the juvenile and early adult phases. Thus, miR156 and BrpSPL9 genes are potentially important for genetic improvement of earliness of Chinese cabbage and other crops.     

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa. L.)

The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus. Received: 19 October 1999 / Accepted: 16 December 1999