Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution     

Fingerprinting temperate <Emphasis Type="Italic">japonica</Emphasis> and tropical <Emphasis Type="Italic">indica</Emphasis> rice genotypes by comparative analysis of DNA markers

This paper describes the relative efficiency of three marker systems, RAPD, ISSR, and AFLP, in terms of fingerprinting 14 rice genotypes consisting of seven temperatejaponica rice cultivars, three indica near-isogenic lines, three indica introgression lines, and one breeding line of japonica type adapted to high-altitude areas of the tropics with cold tolerance genes. Fourteen RAPD, 21 ISSR, and 8 AFLP primers could produce 970 loci, with the highest average number of loci (92.5) generated by AFLP. Although polymorphic bands in the genotypes were detected by all marker assays, the AFLP assay discriminated the genotypes effectively with a robust discriminating power (0.99), followed by ISSR (0.76) and RAPD (0.61). While significant polymorphism was detected among the genotypes of japonica and indica through analysis of molecular variance (AMOVA), relatively low polymorphism was detected within the genotypes of japonica rice cultivars. The correlation coefficients of similarity were significant for the three marker systems used, but only the AFLP assay effectively differentiated all tested rice lines. Fingerprinting of backcross-derived resistant progenies using ISSR and AFLP markers easily detected progenies having a maximum rate of recovery for the recurrent parent genome and suggested that our fingerprinting approach adopting the ‘undefined-element-amplifying’ DNA marker system is suitable for incorporating useful alleles from the indica donor genome into the genome of temperate japonica rice cultivars with the least impact of deleterious linkage drag.     

Analysis on the spectra and level of wide-compatibility conferred by three neutral alleles in inter-subspecific hybrids of rice (Oryza sativa L.) using near isogenic lines

Wide‐compatibility varieties are a special class of rice germplasm that is able to produce fertile hybrids when crossed to either indica or japonica subspecies. Previous studies determined the f5 allele from ‘Dular’ (f5‐Du), f6 allele from ‘Dular’ (f6‐Du) and S5 allele from ‘02428’ (S5‐08) as neutral alleles conferring wide‐compatibility. However, the possible extent of the effect of the three neutral alleles has not been fully characterized because of the narrow range of the tester varieties used and the highly complex differentiation in Asian cultivated rice. In this study, we further developed the five near‐isogenic lines with higher recovery rates of the recurrent parent genome, and testcrossed to 14 japonica varieties, which have been widely used in rice breeding programmes in China. The results clearly revealed that all three neutral alleles exhibited substantial effects on spikelet fertility in most of the indica–japonica testcrosses, which indicated that these hybrid sterility loci have been extensively differentiated between indica and japonica varieties. The magnitudes of effects on spikelet fertility averaged over various crosses seem to be similar among the three neutral alleles, with f5‐Du, f6‐Du and S5‐08 alleles increasing spikelet fertility by 15.09%, 13.99% and 14.25%, respectively. The testcrosses involving f5‐Du allele generally showed much smaller variation in pollen fertility than others. The pyramiding lines with two neutral alleles showed a wider spectrum and a higher level of wide compatibility than others, whereas most of the increases in hybrid fertility couldn’t be simply explained by additive effects, suggesting the very complexity of wide compatibility and hybrid sterility. The indica–japonica hybrids involving restorer lines as one of their parents showed much higher pollen fertility (almost normal) and also higher spikelet fertility. The implications of the findings in rice breeding programmes are also discussed.     

Variation of <Emphasis Type="Italic">Wx</Emphasis> microsatellite allele,waxy allele distribution and differentiation of chloroplast DNA in a collection of Thai rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Eighty-two varieties of rice from different regions in Thailand were selected to explore the Waxy (Wx)gene diversity and indica-japonica differentiation of chloroplast DNA. A comparison of the 5 splice site in the first intron was made between glutinous and nonglutinous rice. It revealed that non-glutinous with low-amylose content and glutinous rice were characterized as the Wx^b allele based on the G-to-T base substitution, whereas non-glutinous rice with intermediate and high amylose carried the Wx^a allele. Four Wx microsatellite alleles, (CT)_n repeat, (n = 16,17,18 and 19) were found in glutinous rice. In contrast, non-glutinous rice showed five Wx microsatellite alleles (n = 11, 16, 17, 18 and 19). The (CT)₁₇ allele was prominent allele in Thai population, while the (CT)₁₁ allele was found only in intermediate and high amylose rice varieties from southern Thailand. Almost all of upland rice grown by various ethnic groups in northern Thailand were characterized as japonica type based on their having the PstI-12 fragment in their cpDNA, whereas most of rainfed lowland varieties from other regions of Thailand were indica. This exploration of DNA-based genetic markers is important, as it enhances our ability to describe and manipulate sources of genetic variation for rice breeding programs.     

Development of gene-tagged markers for quantitative trait loci underlying rice yield components

Higher yields of rice have always been a predominant goal in rice breeding techniques. However, the inheritances of rice yield and its components are still unknown, and no information regarding suitable alleles can be directly provided for improving the rice yield level until three major quantitative trait loci (QTLs) have been cloned and functionally characterized. These QTLs contain Gn1a for grain number per panicle and GS3 and GW2 for grain weight. It has been proven that these three genes show a potential in improving the rice yield level. However, the distribution of suitable alleles on these three loci in rice cultivars and germ plasm are yet to be elucidated, this retards the progress of the utilization of suitable alleles in rice breeding techniques to produce higher yields. In the present study, we developed a set of gene-tagged markers based on the gene mutation sites Gn1a-M1 and Gn1a-M2 for Gn1a, GW2-HapI for GW2, and GS3-PstI for GS3. The results demonstrated that these STS markers could clearly differentiate between the different alleles at each gene locus. A survey of the allele distributions of the abovementioned three genes was performed with 156 cultivars. It was observed that the 5150-Gn1a allele was absent on the Gn1a locus and only two type alleles (Ha-Gn1a and Ko-Gn1a) were present, of which 54.3% indica and 21.5% japonica cultivars contained the Ha-Gn1a allele. Two alleles (MH-GS3 and ZS-GS3) were detected on the GS3 locus, and 48.6% indica and 9.9% japonica cultivars harbored the suitable allele MH-GS3. Further, all the cultivars contained the FA-GW2 allele on GW2, whereas the WY-GW2 allele was not found. These results further suggested that some of the alleles residing in the indica subspecies have introgressed into the japonica group with a very low frequency. The gene-tagged markers developed in the present study can be directly used as a tool for marker-aided selection (MAS) in rice breeding techniques to produce higher yields. Chang-Jie Yan and Song Yan contributed equally to this work.     

Suitability of mapped sequence tagged microsatellite site markers for establishing distinctness, uniformity and stability in aromatic rice

At present, testing for distinctness, uniformity and stability (DUS) of crop varieties relies on a set of morphological characters. These characters suffer fromthe limitations of number, interaction with the environment in which the variety grows and subjectivity in decision-making. The potential of DNA-based markers such as sequence tagged microsatellite site (STMS), for establishing DUS merits investigation. In the present study, a set of 55 mapped STMS markers, selected from 12 linkage groups of rice genome, was used to examine distinctness of 23 aromatic rice genotypes including the commercially important Basmati varieties. Forty-one of these markers (74.5%) showed polymorphism between the varieties. The number of alleles per locus ranged from 2–4 with an average of 2.3. The polymorphism information content (PIC) of the markers varied from 0.083 to 0.665 with an average of 0.338. All the varieties could be differentiated from each other at a low probability (0.07×10^-13) of identical match by chance. The marker-based clustering of the varieties corresponded with the known phenotypic classification, thereby providing confidence in the distinctness established by the mapped STMS markers. The utility of these markers to study uniformity and stability was analysed using a commercially important crossbred Basmati rice variety Pusa Basmati 1(IET-10364) that contributes about 40–50% of Basmati rice export from India. Genotyping of twenty individual plants, grown from the nucleus, breeder, foundation, certified and farmer's saved seed samples using all the 55 markers revealed no variation among the plants. These observations suggested that the set of mapped markers employed in this study could be further used for establishing distinctness of aromatic rice varieties and for studying DUS of the important commercial variety Pusa Basmati 1. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pyramiding of two bacterial blight resistance and a semidwarfing gene in Type 3 Basmati using marker-assisted selection

A traditional Type 3 Basmati rice cultivar grown in India is tall and lodges even under low nitrogen fertilizer dose. In addition to lodging, it is highly susceptible to several diseases and pests including bacterial blight (BB). BB resistance genes (Xa21 and xa13) and a semidwarfing gene (sd-1) were pyramided in Type 3 Basmati from a rice cultivar PR106-P2 using marker-assisted selection (MAS). Foreground selection for BB resistance genes, Xa21 and xa13 and reduced plant height gene, sd-1 was carried on the basis of linked molecular markers pTA248, RG136 and ‘h’, respectively. The BC₂F₃ progenies with both the BB resistance genes were highly resistant with lower lesion length than either of the genes individually. Background profiling of the selected 16 BC₂F₃ progenies was done using 95 anchored SSR and 12 ISSR markers. Among the selected 16 BC₂F₃ progenies, 38-5-2 and 38-5-36 closely clustered along with the recipient parent Type 3 Basmati showing above 85% genetic similarity with the same. Further selection was continued till F₅ generation for higher recovery for Type 3 Basmati characteristics. The desirable alleles of intermediate amylose content (wx) and aroma (fgr) loci of Type 3 Basmati were also tracked using the linked SSR markers. The BC₂F₅ pyramid lines T3-4, T3-5, T3-6 and T3-7 homozygous for the three target genes Xa21, xa13 and sd-1 from the donor parent with wx and fgr alleles of Type 3 Basmati had excellent cooking quality and strong aroma.     

Indica-japonica differentiation in Chinese rice landraces

Summary Ninety Chinese rice landraces were examined with special reference to the indica-japonica differentiation in terms of traditional criteria, isozyme analysis and PCR analysis of the chloroplast DNA (cpDNA). Cultivars were separated into indica and japonica defined by a discriminant function (Z) based on key characters, as well as by isozyme genotypes. Most indica landraces had chloroplast DNAs with a deletion at the Pst-12 fragment, while most japonica landraces had cpDNAs without the deletion. Two traditionally recognized varietal groups in China, keng and hsien, corresponded largely to the respective japonica and indica revealed in our study. The results obtained in this study showed good agreement for classification of indica and japonica types by the three methods: discriminant analysis by Z value, isozyme analysis, and PCR analysis for cpDNA.     

The indica-japonica classification of Asian rice ecotypes and Japanese lowland and upland rice (Oryza sativa L.)

Summary Rice cultivars (Oryza sativa L.) belonging to five ecotypes (aus, aman, boro, bulu and tjereh) and to two groups of Japanese rice (lowland and upland) are examined with respect to KClO₃ resistance, phenol reaction and apiculus hair length. These characters have been used as available criteria to classify rice into two types indica and japonica, for the last thirty years.The findings of this study are that the aman, boro and tjereh ecotypes should be classified as typical indica; and that the Japanese lowland rice cultivars are mainly typical japonica. Some of the aus, bulu and Japanese upland rice cultivars differ from typical indica and typical japonica, so the respective terms aus type, bulu type and J.u.r. type, are proposed. Aman, boro tjereh and Japanese lowland rice are cultivated in lowland. Some of the aus, bulu and Japanese upland rice cultivars have the characteristics of upland rice. In general, lowland rice cultivars can be clearly classified into indica or japonica, while upland cultivars cannot.Abbreviations Aph dominant gene for apiculus hair length > 0.7 mm - aph recessive gene for apiculus hair length < 0.7 mm - J.u.r. type Japanese upland rice type     

Detection of quantitative trait locus for leaffolder (Cnaphalocrocis medinalis (Guenée)) resistance in rice on linkage group 1 based on damage score and flag leaf width

Rice leaffolder (RLF) (Cnaphalocrocis medinalis (Guenée) is a destructive and widespread insect pest throughout the rice growing regions in Asia. The genetics of resistance to RLF in rice is very complex and not thoroughly explored. The present study was conducted to detect the quantitative trait loci (QTL) associated with RLF resistance involving 176 recombinant inbred lines (RILs) of F₈ generation derived from a cross between IR36, a leaffolder susceptible variety and TNAULFR831311, a moderately resistant indica rice culture. Simple sequence repeat (SSR) markers were used to construct specific linkage groups of rice. All the RILs were screened to assess their level of resistance to RLF by measuring the leaf area damaged. Besides this, the length and width of the flag leaf of each RIL were measured since these two parameters were considered as correlated traits to the RLF resistance in rice. All the above parameters observed across the RILs showed quantitative variation. Correlation analysis revealed that damage score based on greenhouse screening was positively correlated with length and width of the flag leaf. Out of 364 SSR markers analysed, 90 were polymorphic between the parents. Multi-point analysis carried out on segregating 69 SSR marker loci linkage group wise resulted in construction of linkage map with eleven groups of 42 SSR markers. Through single marker analysis, 19 SSR markers were found to have putative association with the three phenotypic traits studied. Of these markers, RM472 was identified as a locus having major effect on RLF resistance trait based on length of the flag leaf. Interval mapping detected two QTLs on linkage group 1. Among these QTLs, the QTL flanked by RM576–RM3412 were found to be associated with width of the flag leaf and RLF resistance. The putative SSR markers associated with leaffolder resistance identified in the present study may be one of the loci contributing resistance to RLF in rice.     

Molecular characterization of the recombinant inbred line population derived from a japonica-indica rice cross

Recombinant inbred line (RIL) populations of rice are useful genetic sources for map-based cloning of agronomically important genes. Zhe733 is a high-yielding indica cultivar from China conferring resistance to rice blast (RB), rice water weevil (RWW) and straighthead; whereas Kaybonnet low-phytic acid 1-1 (KBNTlpa) is a mutant of a tropical japonica cultivar from the US containing low-phytic acid with average yield, and is susceptible to some RB races, RWW, and straighthead. A 355 RIL F₁₀₋₁₁ population derived from the cross of KBNTlpa × Zhe733 was recently released. Simple sequence repeat (SSR) markers were used to evaluate 269 RILs of this population. A total of 107 polymorphic markers were mapped on all rice chromosomes representing a total of 1,016.3 cM of genetic distance. Two hundred and thirty-five KBNTlpa × Zhe733 RILs (KZRILs) were clustered into seven groups based on allele frequencies of SSR markers. Twenty-three markers (21.1%) on chromosomes 3, 6, 7, 9, and 11 were found to favor Zhe733 (χ ² = 16.8−189.7 and P < 0.01) and five markers (4.6%) on chromosome 1 and 6 were found to favor KBNTlpa (χ ² = 18.5−46.6 and P < 0.01). Marker segregations were observed to be normal for both parents except 26 (10.2%) KZRILs were found to skew toward Zhe733 (χ ² > 15.7 and P < 0.01). Furthermore, the average frequencies of heterozygosity and non-parental alleles per KZRIL were 1.3% (0.0−38.9%) and 0.4% (0.0−15.0%), respectively. Thirteen heterozygous KZRILs were found at more than five markers loci and nine KZRILs were found with more than five non-parental alleles representing 5.1 and 3.5% of 255 KZRILs. Overall, this KZRIL population is a good population with relatively low frequencies of heterozygosity and non-parental alleles, and with relatively low percentages of skewed markers and skewed KZRILs. The profiles of these SSR markers should facilitate molecular tagging critical genes controlling yield, RB, RWW, and straighthead resistance.     

Development and validation of microsatellite markers linked to the rice blast resistance gene <Emphasis Type="Italic">Pi-z</Emphasis> of Fukunishiki and Zenith

Rice blast resistance gene ‘Pi-z’ present in rice genotypes, Zenith and Fukunishiki, represents a potential source of blast resistance for the north-western Himalayan region of India. We tested the reliability of microsatellite markers linked to Pi-z for assessing blast resistance phenotype in crosses of commercial importance. A new set of microsatellite markers linked to Pi-z was also developed by exploiting the publicly available marker and genomic resources of rice. Of the three previously reported markers for Pi-z, only MRG5836 was suitable for the marker assisted selection of Pi-z. Among the 17 microsatellites selected from the putative region of Pi-z locus, two, RM8225 and RM8226 cosegregated with MRG5836 and were located at distance of 1.2–4.5 cM from the gene. A new microsatellite marker ‘SSR236’ was developed from the (CT)₁₆ repeat of PAC clone P0502B12, which exhibited closer linkage (0.6–1.2 cM) to Pi-z. Survey of the allelic diversity at the loci of the Pi-z linked microsatellite markers revealed that the Fukunishiki and Zenith type alleles were not present in majority of the local indica rice genotypes. As these markers are polymorphic between the Pi-z donors and a great majority of local indica rices tested, they can be used as a selection tool in rice breeding programs aimed at improving the blast resistance of local rices.     

Genetic analysis and fine mapping of a semi-dwarf gene in a centromeric region in rice (Oryza sativa L.)

Superior plant architecture is a key means of enhancing yield potential in high yielding varieties. A newly identified recessive gene, named sd-c, controls plant height and tiller number. Genetic analysis of an F₂ population from a cross between the semi-dwarf mutant and japonica cv. Houshengheng showed that the sd-c locus was flanked by SSR markers RM27877 and RM277 on chromosome 12. Thirty nine InDel markers were developed in the region and the sd-c gene was further mapped to a 1 cM centromeric region between InDel markers C11 and C12. These sequenced markers can be used to distinguish wild type and mutants and thus can be used in marker-assisted selection. The sd-c mutant decreases culm length by about 26% and doubles the tiller number without changing seed weight. Until now only sd-1 has been used in indica rice breeding programs. The sd-c mutant seems to have no undesirable pleiotropic effects and is therefore a potential genetic resource for breeding semi-dwarf indica rice cultivars.     

Molecular diversity and genome-wide linkage disequilibrium patterns in a worldwide collection of <Emphasis Type="Italic">Oryza sativa</Emphasis> and its wild relatives

Genetic diversity analysis within a species is vital for understanding evolutionary processes at the population and genomic levels. We report a detailed study of molecular diversity, polymorphism and linkage disequilibrium in three groups of rice (Oryza) germplasm accessions based on 176 SSR markers. The first group included 65 rice (O. sativa L.) accessions introduced from seven countries, including five regions of China. The second group included 58 US rice varieties released in the past 25 years. The third group consisted of 54 accessions of rice wild relatives represented by ten different species. The number of alleles per SSR marker ranged from 4 to 32 with a mean of 16 alleles and the polymorphism information content values ranged from 0.43 to 0.91 with a mean of 0.70. The variation in SSR alleles was a significant contribution to the genetic discrimination of the 177 accessions within the three Oryza groups. Analysis of molecular variance identified deviation from Hardy–Weinberg equilibrium. Principal coordinates analysis clearly separated the accessions into their respective three groups. Neighbor-joining phylogenetic cluster reflects the ordination of each accession. Linkage disequilibrium (D′) averaged 0.75 in wild Oryza spp., and about 0.5 in both US and international O. sativa accessions. Our results showed that LD among adjacent loci in both O. sativa and Oryza spp. accessions is strong enough to be detecting marker-trait association via genome-wide scans.     

Anther culture of recalcitrant indica × Basmati rice hybrids

Fertile, green, di-haploid plants were obtained at high frequencies from several indica × Basmati rice F₁ hybrids and/or F₂ plant populations using an improved anther culture procedure. Anthers from cold-pretreated (10 ^°C for 10 d) panicles of six indica (HKR120, HKR86-3, HKR86-217, PR106, Gobind andCH2 double dwarf) and two Basmati rice (Basmati 370,Taraori Basmati) varieties and 14 heterotic indica ×Basmati F₁/F₂ hybrids were cultured in modified agarose-solidified N6M, Heh5M and RZM media. Best callus induction frequencies (2.6–78%) were obtained in RZM medium containing 4% (w/v) maltose,2,4-D, NAA and kinetin. F₂ plants compared to F₁ hybrids and parental rice varieties, were more responsive to anther culture. Androgenesis frequencies of 31–78% were obtained for indica × Basmati F₂ plants in RZM medium in just 30 d which are comparable to or higher than that reported for japonica rice varieties and hybrids involving japonica rice parent(s). Agarose (1.0% w/v)-solidified MS medium containing 3.0% maltose, kinetin, BAP, and NAA, induced green shoot regeneration in 0–51% of the anther-derived callide pending upon the genotype. High plant regeneration frequencies (67–337 green plants per 1000 anthers)were obtained from anther calli of several F₁hybrids (Gobind × Basmati 370 and HKR120 ×Taraori Basmati) and F₂ plants (Gobind × Basmati370, Gobind × Taraori Basmati, HKR86-3 × TaraoriBasmati). A sample of 498 plants obtained from the above hybrids, were transferred to pots with>90% survival; 8–78% of these plants had >5%spikelet fertility and were diploid. In addition,18% of the haploid plants could be diploidized by submerging in 0.1% colchicine solution for 16–18 h. The improved anther culture procedure reported here, resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice hybrids compared to previous published procedures. The study may accelerate the introgression of desirable genes from indica into Basmati rice using anther culture as a breeding tool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cold tolerance at the early growth stage in wild and cultivated rice

The present study was conducted to understand the pattern of variation and the genetic bases for cold tolerance at the early growth stage in Asian rice. The genetic variation was investigated at the germination, plumule and seedling stages among 57 strains including cultivated rice (Oryza sativa ssp. indica and ssp. japonica) and its wild progenitor (Oryza rufipogon). The significant differentiation of cold tolerance was observed among the taxonomically divided groups. At the germination stage, both indica and japonica subspecies tended to be more tolerant than O. rufipogon, whereas at the plumule and seedling stages, ssp. japonica tended to be more tolerant than ssp. indica and O. rufipogon. Furthermore, in cold tolerance at the plumule stage, the clinal variation across the latitude of origins was observed within O. rufipogon and ssp. japonica, suggesting that the current pattern of variation seems to have been shaped by both their phylogenetic histories and on-going adaptation to the local environments. QTL analysis between O. sativa ssp. japonica (tolerant) and O. rufipogon (susceptible) revealed five putative QTLs for cold tolerance at the plumule and seedling stages but not at the germination stage. Substitution mapping was also carried out to precisely locate the two major QTLs for cold tolerance at the plumule stage, which could be used for improvement of tolerance to cold stress in ssp. indica.     

Simple sequence repeats for genetic analysis in pear

The development of highly informative DNA markers, such as simple sequence repeats (SSRs), is essential for breeding to select agronomically important traits and for genetic studies in pear. We developed SSR markers by using two approaches, RAHM (random amplified hybridization microsatellites) and 5' anchored PCR methods. Segregation analysis of the SSRs revealed that amplified fragments were derived from the same loci, using 3 sets of progenies from crosses between pear varieties. Genetic diversity was characterized using 32 varieties, including 10 from Japanese pear (Pyrus pyrifolia), 9 from Chinese pear (P. bretschneideri, P. ussuriensis), 10 from European pear (P. communis) as well as 3 wild relatives (P. calleryana). Diversity of SSR genotypes was observed among species as well as within species and 65 putative alleles were detected. The use of seven SSR markers was sufficient to differentiate between all of the 32 varieties. This revised version was published online in August 2006 with corrections to the Cover Date.     

Multiplex PCR genotyping for five bacterial blight resistance genes applied to marker‐assisted selection in rice (Oryza sativa)

Bacterial blight (BB) is the most economically damaging disease of rice in Asia and other parts of the world. In this study, a multiplex PCR genotyping method was developed to simultaneously identify genotypes of five BB resistance genes, Xa4, xa5, Xa7, xa13 and Xa21. The resistance R alleles were amplified using five functional markers (FMs) to generate amplicons of 217, 103, 179, 381 and 595 bp in IRBB66. Amplicons of 198, 107, 87, 391 and 467 bp corresponded to susceptible alleles in Taiwanese japonica rice cultivars. In backcross breeding programmes, the multiplex PCR assay was integrated into selection from a population using BB resistance donor IRBB66 crossed to rice cultivar ‘Tainung82’. Two plants with homozygosity for Xa4, xa5, Xa7, xa13 and Xa21 were selected from 1100 BC₂F₂ plants. In addition, the five BB resistance genes were also accurately identified in F₂ populations. This multiplex PCR method provides a rapid and efficient method for detecting various BB resistance genes, which will assist in pyramiding genes to improve durability of BB resistance in Taiwanese elite rice cultivars.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.