Agro-morphological and genetic diversity studies in Rice (Oryza sativa L.) germplasm using microsatellite markers

Molecular Biology Reports - Knowledge of the genetic diversity and population structure of germplasm collections is an important foundation for crop improvement. Rice production across a broad...     

Genetic diversity in basmati rice (Oryza sativa L.) germplasm as revealed by microsatellite (SSR) markers

Genetic diversity among rice genotypes, including 15 indica basmati advance lines and 5 basmati improved varieties were investigated by 28 SSR markets including one indel marker. The SSRs covered all the 12 chromosomes that distributed across the rice genomes. The mean number of alleles per locus was 3.60, showing average number of polymorphism information content was 0.48. A total of 101 alleles were also identified from the microsatellite marker loci. A number of SSR markers were also identified that could be utilized to differentiate between rice genotypes. Pair wise Nei,s genetic distance between rice genotypes ranged from 0.07 to 0.95. The dendrogram based on cluster analysis by using SSR polymorphism that grouped the 20 genotypes of rice in to five clusters based on their genetic similarity. The result could be useful for the identification and selection of the diverse genotypes for the future cross breeding program and development of new rice varieties.     

Genetic diversity analysis of traditional and improved Indonesian rice (Oryza sativa L.) germplasm using microsatellite markers

The archipelago of Indonesia has a long history of rice production across a broad range of rice-growing environments resulting in a diverse array of local Indonesian rice varieties. Although some have been incorporated into modern breeding programs, the vast majority of these landraces remain untapped. To better understand this rich source of genetic diversity we have characterized 330 rice accessions, including 246 Indonesian landraces and 63 Indonesian improved cultivars, using 30 fluorescently-labeled microsatellite markers. The landraces were selected across 21 provinces and include representatives of the classical subpopulations of cere, bulu, and gundil rices. A total of 394 alleles were detected at the 30 simple sequence repeat loci, with an average number of 13 alleles per locus across all accessions, and an average polymorphism information content value of 0.66. Genetic diversity analysis characterized the Indonesian landraces as 68% indica and 32% tropical japonica, with an indica gene diversity of 0.53 and a tropical japonica gene diversity of 0.56, and a Fst of 0.38 between the two groups. All of the improved varieties sampled were indica, and had an average gene diversity of 0.46. A set of high quality Indonesian varieties, including Rojolele, formed a separate cluster within the tropical japonicas. This germplasm presents a valuable source of diversity for future breeding and association mapping efforts.     

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。     

Population structure and genetic diversity analysis of Indian and exotic rice (Oryza sativa L.) accessions using SSR markers

In order to understand the population structure and genetic diversity among a set of 82 rice genotypes collected from different parts of the Asian countries including India were characterized using 39 microsatellite loci. The Population structure analysis suggested that the optimum number of subpopulations was four (K = 4) among the rice genotypes, whereas phylogenetic analysis grouped them into three populations. The results obtained from phylogenetic and STRUCTURE analysis proved to be very powerful for the differentiation of rice genotypes based on their place of origin. The genetic diversity analysis using 39 SSR loci yielded 183 scorable alleles, out of which 182 alleles were observed to be polymorphic with an average of 4.8 alleles per locus. The Polymorphism Information Content (PIC) values for all the polymorphic primers across 82 rice genotypes varied from 0.02 to 0.77, with an average of 0.50. Gene diversity (He) was found to be in the range of 0.02 (RM484) to 0.80 (OSR13) with an average value of 0.55, while heterozygosity (Ho) was observed with an average of 0.07, ranging from 0.01 (RM334) to 0.31 (RM316). The present study resulted in identification of seven highly polymorphic SSR loci viz., OSR13, RM152, RM144, RM536, RM489, RM259 and RM271 based on the parameters like PIC value (≥0.70), gene diversity (≥0.71), and polymorphic alleles (≥6). These seven polymorphic primers can effectively be used in further molecular breeding programs and QTL mapping studies of rice since they exhibited very high polymorphism over other loci. SSR analysis resulted in a more definitive separation of clustering of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions.     

Fluorescent-labeled microsatellite panels useful for detecting allelic diversity in cultivated rice ( Oryza sativa L.)

Four multiplex panels of fluorescent-labeled rice microsatellite markers were designed to survey allelic diversity at a total of 27 simple sequence repeat loci. Automated fragment detection, size calling and binning were used to identify the microsatellite alleles. The panels were tested on 72 accessions representing the diversity of ecotypes and isozyme groups in cultivated rice ( Oryza sativa L.). Genetic diversity was evaluated by estimating the distribution and range of allele sizes, as well as the polymorphism information content (PIC), for each microsatellite locus. The multiplex panels were shown to be useful for fingerprinting and for clustering rice varieties, and genetic associations determined in this analysis agreed well with previous isozyme- and RFLP-based studies of these genotypes. Because of the wide cross-section of germplasm represented, we expect that the allele size-ranges reported here are close to the maximum for cultivated rice. In the future, panels of microsatellites from each rice chromosome will be made to facilitate the mapping of segregating populations and the identification of genes and QTLs underlying traits of interest.     

Population structure and genetic diversity in a rice core collection (Oryza sativa L.) investigated with SSR markers

The assessment of genetic diversity and population structure of a core collection would benefit to make use of these germplasm as well as applying them in association mapping. The objective of this study were to (1) examine the population structure of a rice core collection; (2) investigate the genetic diversity within and among subgroups of the rice core collection; (3) identify the extent of linkage disequilibrium (LD) of the rice core collection. A rice core collection consisting of 150 varieties which was established from 2260 varieties of Ting's collection of rice germplasm were genotyped with 274 SSR markers and used in this study. Two distinct subgroups (i.e. SG 1 and SG 2) were detected within the entire population by different statistical methods, which is in accordance with the differentiation of indica and japonica rice. MCLUST analysis might be an alternative method to STRUCTURE for population structure analysis. A percentage of 26% of the total markers could detect the population structure as the whole SSR marker set did with similar precision. Gene diversity and MRD between the two subspecies varied considerably across the genome, which might be used to identify candidate genes for the traits under domestication and artificial selection of indica and japonica rice. The percentage of SSR loci pairs in significant (P<0.05) LD is 46.8% in the entire population and the ratio of linked to unlinked loci pairs in LD is 1.06. Across the entire population as well as the subgroups and sub-subgroups, LD decays with genetic distance, indicating that linkage is one main cause of LD. The results of this study would provide valuable information for association mapping using the rice core collection in future.     

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice. Received: 19 May 1999 / Accepted: 26 April 2000     

Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.)

In order to enhance the resolution of an existing genetic map of rice, and to obtain a comprehensive picture of marker utility and genomic distribution of microsatellites in this important grain species, rice DNA sequences containing simple sequence repeats (SSRs) were extracted from several small-insert genomic libraries and from the database. One hundred and eighty eight new microsatellite markers were developed and evaluated for allelic diversity. The new simple sequence length polymorphisms (SSLPs) were incorporated into the existing map previously containing 124 SSR loci. The 312 microsatellite markers reported here provide whole-genome coverage with an average density of one SSLP per 6 cM. In this study, 26 SSLP markers were identified in published sequences of known genes, 65 were developed based on partial cDNA sequences available in GenBank, and 97 were isolated from genomic libraries. Microsatellite markers with different SSR motifs are relatively uniformly distributed along rice chromosomes regardless of whether they were derived from genomic clones or cDNA sequences. However, the distribution of polymorphism detected by these markers varies between different regions of the genome. Received: 5 May 1999 / Accepted: 16 August 1999     

Studies on some genetic parameters of rice (Oryza sativa L.)

Summary In order to utilize the available useful variation in breeding rice to improve yield and quality of grain, the performance of a wide range of rice germ plasm comprising 30 varieties was evaluated in northern India, a major rice growing belt. Plant performance revealed high genetic divergence and phenotypic variability in the crop, with the maximum range of variation being for grain number per panicle and the minimum for grain dimensions. There were also significant differences among varietal means for ten phenotypic traits. Genotypic and phenotypic variance contributed profoundly to the variance of the phenotypic traits studied, but, since genetic variability in the traits related to yield was considerable, there is scope for further improvement in yielding ability. Grain number per panicle, number of effective tillers per plant and culm length exhibit high heritability, and genotypic coefficient of variation and therefore a high genetic advance. Thus, selection for these traits would be effective in crop improvement. Moreover, grain number per panicle shows a significant positive correlation with yield, and this trait could profitably form a reliable index for the yielding capacity of this crop.     

Contrasting genetic diversity relationships are revealed in rice (Oryza sativa L.) using different marker types

Genetic variation between samples of Oryza sativa from 19 localities in Bangladesh and Bhutan was assessed using two PCR-based molecular marker systems: RAPD (random amplification of polymorphic DNA) and ISSR-PCR (inter-simple sequence repeat polymerase chain reaction). Employing RAPD, a set of 14 decanucleotides of arbitrary sequence directed the amplification of 94 reproducible marker bands, 47 (50%) of which were polymorphic. In addition, a set of 9 ISSR primers were used to direct amplification of 71 PCR products, 40 (56%) of which were polymorphic. Multivariate analyses of the two PCR-based molecular marker data sets provided evidence that the patterns of variation correspond with the classification described by Glaszmann [9] using isozyme analysis. Subtle differences in the relationships revealed between rice groups using the two types of PCR-based marker led to investigations of their map positions using an intraspecific doubled haploid mapping population. The observation that the chromosomal locations of markers can influence diversity assessments is presented and the significance of this is discussed.     

Generation and characterization of low phytic acid germplasm in rice (Oryza sativa L.)

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate), or its salt form, phytate, is commonly regarded as the major anti-nutritional component in cereal and legume grains. Breeding of low phytic acid (lpa) crops has recently been considered as a potential way to increase nutritional quality of crop products. In this study, eight independent lpa rice mutant lines from both indica and japonica subspecies were developed through physical and chemical mutagenesis. Among them, five are non-lethal while the other three are homozygous lethal. None of the lethal lines could produce homozygous lpa plants through seed germination and growth under field conditions, but two of them could be rescued through in vitro culture of mature embryos. The non-lethal lpa mutants had lower PA content ranging from 34 to 64% that of their corresponding parent and four of them had an unchanged total P level. All the lpa mutations were inherited in a single recessive gene model and at least four lpa mutations were identified mutually non-allelic, while the other two remain to be verified. One mutation was mapped on chromosome 2 between microsatellite locus RM3542 and RM482, falling in the same region as the previously mapped lpa1-1 locus did; another lpa mutation was mapped on chromosome 3, tightly linked to RM3199 with a genetic distance of 1.198 cM. The latter mutation was very likely to have happened to the LOC_Os03g52760, a homolog of the maize myo-inositol kinase (EC 2.7.1.64) gene. The present work greatly expands the number of loci that could influence the biosynthesis of PA in rice, making rice an excellent model system for research in this area.     

Phylogenetic distribution and genetic mapping of a (GGC)n microsatellite from rice (Oryza sativa L.)

DNA microsatellites are ubiquitously present in eukaryotic genomes [30] and represent a vast source of highly informative markers [30, 33, 34, 2]. We describe in this article a (GGC)_n microsatellite which is widely distributed in eukaryotic genomes. Using polymerase chain reaction (PCR) techniques and DNA sequencing, we demonstrated for the first time in plant species that a (GGC)_n microsatellite locus is moderately polymorphic. Six alleles are present at this locus in rice and length polymorphisms are caused by variation in the number of tandem GGC repeats. By scoring a backcross mapping population, we were able to demonstrate that this locus is stably inherited and does not link to any known RFLP markers on the rice RFLP map. Our results suggest that DNA microsatellites should be useful in plants for construction of genetic linkage maps, extension of the existing genetic linkage maps, linkage analysis of disease and pest resistance genes, and the study of population genetics.     

The physical location of fourteen RFLP markers in rice (Oryza sativa L.)

A biotin-labeled in situ hybridization technique was used in order to physically map RFLP markers to the chromosomes of rice (Oryza sativa L.). Fourteen RFLP markers, associated with the ends of the linkage groups on rice chromosomes 7, 8, 11, 12, were physically mapped onto specific regions of the chromosomes. The average detection rate of in situ hybridization was 5.91%. The markers were located on seven different chromosome arms. Ten of the fourteen markers were distributed near the chromosome ends. This demonstrated that the RFLP linkage groups involved covered a wide physical distance and that the centromeric region was bisected by all but one linkage group. Two markers covered a short genetic distance but were physically distant, while two covering a longer genetic distance were physically closer together. This indicates that considerable variation can, and does, exist between genetic and physical maps.This paper is a contribution of the U.S. Department of Agriculture, Agricultural Research Service, and Missouri Agricultural Experiment Station, Journal Series No. 11 882All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicapThis paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the U.S. Department of Agriculture or the University of Missouri     

Heterosis breeding in rice (Oryza sativa L.)

Summary Studies conducted at the International Rice Research Institute (IRRI) during 1980 and 1981 have shown up to 73% heterosis, 59% heterobeltiosis and 34% standard heterosis for yield in rice. The latter was estimated in comparison to commercial varieties: IR36 and IR42 (yield 4–5 t/ha in wet season trials and 7–8 t/ha in dry season trials). Generally speaking, absolute yield was lower and extent of standard heterosis was higher in wet season than in dry season with some exception. Yields up to 5.9 t/ha (22% standard heterosis) in the wet season and 10.4 t/ha (34% standard heterosis) in the dry season were obtained. Most of the hybrids performed better in some season while some performed better in both seasons. Hybrids showed better lodging resistance although they were 5–10 cm taller. F₁ hybrids had significant positive correlations with the parental traits viz., yield (r = 0.446), tillering (r = 0.746), height (r = 0.810) and flowering (r = 0.843). Selection of parents among elite breeding lines on the basis of their per se yield performance, diverse origin and resistance to insects and diseases should give heterotic combination. Yield advantage of hybrids was due primarily to increase in number of spikelets per unit area even though tiller number was reduced. Grain weight was either the same or slightly higher. High yielding hybrids also showed significant heterosis and heterobeltiosis for total dry matter and harvest index. For commercial utilization of heterosis in rice, effective male sterility and fertility restoration systems are available and up to 45% natural outcrossing on male sterile lines has been observed. Consequently, F₁ rice hybrid have been successfully developed and used in China. Prospects of developing hybrid rice varieties elsewhere appear bright especially in countries that have organized seed production, certification and distribution programs and where hybrid seed can be produced at a reasonable cost.     

Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)

Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives ofOryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three relatedindica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.