Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.     

东乡普通野生稻与栽培稻苗期抗旱性的比较

干旱影响水稻生长发育,不论什么时期发生最终都导致产量损失。研究水稻资源抗旱性有助于水稻抗旱改良和稳定干旱胁迫下水稻的产量。东乡普通野生稻被公认为是栽培稻的祖先,对增强水稻抗旱性可能十分重要。对4份来自3个仅存的居群的东乡野生稻与15份栽培稻进行苗期抗旱性比较,考察了3次重复的盆栽土培试验中8个抗旱指标。表明东乡普通野生稻比栽培稻更为抗旱,表现在最大根长、茎长、根干重、根鲜重、根干鲜重比及抗旱指数等6个性状,而不表现在根数及根茎长比;其中茎长、最长根长、根干重、根鲜重及根系相对含水量对水稻苗期抗旱性影响更大。采用抗旱指数和抗旱总级别值法对水稻抗旱性进行评定,结果表明4份东乡野生稻材料间的抗旱性存在很大差异,且来水桃树下居群的抗性最高,东乡野生稻抗旱性可能与其原生境状况有关。结果认为东乡普通野生稻可作为栽培稻抗旱改良的遗传资源。     

普遍野生稻和亚洲栽培稻遗传多样性的研究

用 ４４个 ＲＦＬＰ标记对来自中国、印度、泰国等亚洲 １０个国家的普通野生稻（简称普野,下同）和来自多个国家的７５个栽培稻品种,从多态位点的比率、等位基因数、基因型数、平均杂合度及平均基因多样性等多个方面,比较了不同国家和不同地区的普通野生稻、栽培稻籼粳亚种及栽培稻与普野之间遗传多样性的差异。结果表明：中国普野的遗传多样性最大;其次是印度普野;南亚普野的平均基因多样性大于东南亚普野,而多态位点的比率、等位基因数及基因型数等却低于东南亚普野;栽培稻的遗传多样性明显小于普通野生稻。在所检测的４４个位点中,栽培稻的多态位点数仅为野生稻的３／４,等位基因数约为野生稻的６０％,基因型种类约为野生稻的１／２。栽培稻中籼稻的遗传多样性高于粳稻。在平均每个位点的实际杂合度上,以中国普野杂合度最高,普通野生稻是栽培稻的２倍。说明从野生稻演化成栽培稻的过程中,经过自然选择和人工选择,杂合度降低,等位基因减少,基因多样性下降。     

A Large-Insert (130 kbp) Bacterial Artificial Chromosome Library of the Rice Blast FungusMagnaporthe grisea:Genome Analysis, Contig Assembly, and Gene Cloning

Magnaporthe grisea(Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant–pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitateM. griseagenome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ≈26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis ofM. grisea.Copies of colony hybridization membranes are available upon request.     

An approach to cloning of Pi-b rice blast resistance gene

A contig of clones from BAC rice genomic library encompassing blast resistance gene Pi-b was constructed. On an average eight clones (8 ± 2.6) were picked up by each marker, which was expected basing on the BAC library size (Nakamura et al. 1997). The 2.4 cM distance between flanking RFLP markers G 1234 and RZ 213 (Miyamoto et al. 1996) was spanned with 4 steps of contig including 25 clones. The physical distance of 370 kb between flanking markers corresponds to a small ratio of physical and genetical distances (155 kb/cM) due to a probable structure of the gene locus near the telomeric end of the chromosome. Markers cosegregating with blast resistance against Magnoporthe grisea were localized in a 2 kb restriction fragment. A new border marker was found on the telomeric side of the Pi-b gene, less than 10 kb from cosegregating markers. No clear marker for the centromeric side of the gene was found but the position of Pi-b rice blast resistant gene was narrowed to within at least 50 kb, which is to our knowledge the most precised estimation of the position of this gene.     

Fine mapping of a quantitative trait locus for grain number per panicle from wild rice (Oryza rufipogon Griff.)

SIL040, an introgression line (IL) developed by introgressing chromosomal segments from an accession of Oryza rufipogon into an indica cultivar Guichao 2, showed significantly less grains per panicle than the recurrent parent Guichao 2. Quantitative trait locus (QTL) analysis in F₂ and F₃ generations derived from the cross between SIL040 and Guichao 2 revealed that gpa7, a QTL located on the short arm of chromosome 7, was responsible of this variation. Alleles from O. rufipogon decreased grains per panicle. To fine mapping of gpa7, a high-resolution map with 1,966 F₂ plants derived from the cross between SIL040 and Guichao 2 using markers flanking gpa7 was constructed, and detailed quantitative evaluation of the structure of main panicle of each of F₃ families derived from recombinants screened was performed. By two-step substitution mapping, gpa7 was finally narrowed down to a 35-kb region that contains five predicted genes in cultivated rice. The fact that QTLs for five panicle traits (length of panicle, primary branches per panicle, secondary branches per panicle, grains on primary branches and grains on secondary branches) were all mapped in the same interval as that for gpa7 suggested that this locus was associated with panicle structure, showing pleiotropic effects. The characterizing of panicle structure of IL SIL040 further revealed that, during the domestication from common wild allele to cultivated rice one at gpa7, not only the number of branches and grains per panicle increased significantly, more importantly, but also the ratio of secondary branches per panicle to total branches per panicle and the ratio of grains on secondary branches per panicle to total grains per panicle increased significantly. All these results reinforced the idea that gpa7 might play an important role in the regulation of grain number per panicle and the ratio of secondary branches per panicle during the domestication of rice panicle.Feng Tian and Zuo Feng Zhu contributed equally to this work.     

Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes.     

Construction of a bacterial artificial chromosome (BAC) library of common wild rice (Oryza rufipogon Griff.) for map-based cloning of genes selected during the domestication of rice

As a prerequisite for the map-based cloning of genes from common wild rice (Oryza rufipogon Griff.), which plays an important role in the domestication of cultivated rice (O. sativa L.), we constructed a median-insert size bacterial artificial chromosome (BAC) library of the common wild rice isolate, YJCWR, collected from Yuanjiang, Yunnan Province, China. The library consists of 52,992 clones, with an average insert size of 50 kb, and all clones were pooled into 46 three-dimensional super-pools to facilitate library screening through the PCR method. Seventeen candidate clones were isolated by five markers and some clones containing putative target regions were sequenced. Furthermore, in analyzing the sequences of YJCWR, a retrotransposon, SZ-55, that might contribute to the evolution of Oryza was found.     

Construction and application of a bacterial artificial chromosome (BAC) library of<Emphasis Type="Italic"> Prunus armeniaca</Emphasis> L. for the identification of clones linked to the self-incompatibility locus

To facilitate gene discovery in the Rosaceae, a bacterial artificial chromosome (BAC) library was constructed using high-molecular-weight (HMW) DNA from apricot leaves ( Prunus armeniaca L.). The library contains 101,376 clones (264, 384-well plates) with an average insert size of 64 kb, equivalent to 22-fold genome coverage. In the first application of this library, high-density filters were screened for self-incompatibility genes using apricot DNA probes. Eight positive BAC clones were detected and fingerprinted to determine clone relationships and assemble contigs. These results demonstrate the suitability of this library for gene identification and physical mapping of the apricot genome.Communicated by R. Hagemann     

Identification of an 85-kb DNA fragment containing pms1, a locus for photoperiod-sensitive genic male sterility in rice

Photoperiod-sensitive genic male-sterile rice has a number of desirable characteristics for hybrid rice production. Previous studies identified pms1, located on chromosome 7, as a major locus for photoperiod-sensitive genic male sterility. The objective of this study was to localize the pms1 locus to a specific DNA fragment by genetic and physical mapping. Using 240 highly sterile individuals and a random sample of 599 individuals from an F2 population of over 5000 individuals from a cross between Minghui 63 and 32001S, we localized the pms1 locus by molecular marker analysis to a genetic interval of about 4 cM, 0.25 cM from RG477 on one side and 3.8 cM from R1807 on the other side. A contig map composed of seven BAC clones spanning approximate 500 kb in length was constructed for the pms1 region by screening a BAC library of Minghui 63 DNA using RFLP markers and chromosomal walking. Analysis of recombination events in the pms1 region among the highly sterile individuals reduced the length of the contig map to three BAC clones. Sequencing of one BAC clone, 2109, identified two SSR markers located 85 kb apart in the clone that flanked the pms1 locus on both sides, as indicated by the distribution of recombination events. We thus concluded that the pms1 locus was located on the fragment bounded by the two SSR markers.     

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160-kb DNA segment of the rice genome

The rice lesion mimic mutant spl11 was previously found to confer broad-spectrum disease resistance to both Magnaporthe grisea and Xanthomonas oryzae pv. oryzae. To better understand the molecular basis underlying cell death and disease resistance in rice, a map-based cloning strategy has been employed to isolate Spl11. Five Spl11-linked RAPD markers were developed and four of them were mapped to rice chromosome 12. A high-resolution genetic map was developed using a segregating population consisting of 1138 lesion mimic individuals. Recombination suppression was observed in the vicinity of Spl11. Three molecular markers tightly linked to Spl11 were identified and used to screen a BAC library. A contig spanning the Spl11 locus was constructed and physical mapping delimited Spl11 to a 160-kb DNA segment within a single BAC clone. These results provide the essential information for the final isolation of this important gene in the rice defense pathway.     

High-resolution genetic and physical mapping of the cauliflower high-β-carotene gene<Emphasis Type="Italic"> Or</Emphasis> (<Emphasis Type="Italic">Orange</Emphasis>)

Mutation in the cauliflower gene Or causes high levels of -carotene to accumulate in various tissues of the plant that are normally devoid of carotenoids. To decipher the molecular basis by which Or regulates carotenoid accumulation, we have undertaken the isolation of Or by a map-based cloning strategy. Two previously isolated, locus-specific, sequence-characterized amplified region (SCAR) markers that flank Or were employed for the analysis of a large segregating population consisting of 1632 F₂ individuals, and a high-resolution genetic linkage map of the Or locus region was developed. To facilitate positional cloning, we constructed a cauliflower genomic library in a bacterial artificial chromosome (BAC) vector, using high molecular weight DNA from Or homozygotes. The BAC library comprises 60,288 clones with an average insert size of 110 kb, and represents an estimated 10-fold coverage of the genome. A BAC contig encompassing the Or locus was established by screening the library with a marker that is closely linked to Or and by identifying overlapping BAC clones by chromosome walking. Physical mapping delimited the Or locus to a 50-kb DNA fragment within a single BAC clone, which corresponds to a genetic interval of 0.3 cM.Communicated by R. Hagemann     

Exploitation of Arabidopsis-tomato synteny to construct a high-resolution map of the ovatecontaining region in tomato chromosome 2.

High-resolution genetic and physical maps were constructed for the region of chromosome 2 containing the major fruit-shape locus ovate. A total of 3,000 NIL F2 and F3 NILs derived from Lycopersicon esculentum cv. Yellow Pear (TA503) x L. pennellii (a wild tomato) were used to position ovate adjacent to the marker TG645 and flanked by markers TX700 and BA10R (a 0.03-cM interval). BAC libraries and a BIBAC library were screened with the closest marker, TG645. Genetic mapping with the ends of isolated BAC clones revealed that two BAC clones (100 and 140 kb) both contained the ovate locus. Screening of sequences from these BAC clones revealed synteny between this segment of tomato chromosome 2 and the chromosome-4 region of Arabidopsis containing the BAC clone ATAP22. Microsynteny between the two genomes was exploited to find additional markers near the ovate locus. The placement of ovate on a BAC clone will now allow cloning of this locus and, hence, may open the door to understanding the molecular basis of fruit development and also facilitate the genetic engineering of fruit-shape characteristics. This also represents the first time that microsynteny with Arabidopsis has been exploited for positional cloning purposes in a different plant family.     

Substitution mapping of dth1.1, a flowering-time quantitative trait locus (QTL) associated with transgressive variation in rice, reveals multiple sub-QTL

A quantitative trait locus (QTL), dth1.1, was associated with transgressive variation for days to heading in an advanced backcross population derived from the Oryza sativa variety Jefferson and an accession of the wild rice relative Oryza rufipogon. A series of near-isogenic lines (NILs) containing different O. rufipogon introgressions across the target region were constructed to dissect dth1.1 using substitution mapping. In contrast to the late-flowering O. rufipogon parent, O. rufipogon alleles in the substitution lines caused early flowering under both short- and long-day lengths and provided evidence for at least two distinct sub-QTL: dth1.1a and dth1.1b. Potential candidate genes underlying these sub-QTL include genes with sequence similarity to Arabidopsis GI, FT, SOC1, and EMF1, and Pharbitis nil PNZIP. Evidence from families with nontarget O. rufipogon introgressions in combination with dth1.1 alleles also detected an early flowering QTL on chromosome 4 and a late-flowering QTL on chromosome 6 and provided evidence for additional sub-QTL in the dth1.1 region. The availability of a series of near-isogenic lines with alleles introgressed from a wild relative of rice provides an opportunity to better understand the molecular basis of transgressive variation in a quantitative trait.     

Molecular evolution of the Pi-ta gene resistant to rice blast in wild rice (Oryza rufipogon)

Rice blast disease resistance to the fungal pathogen Magnaporthe grisea is triggered by a physical interaction between the protein products of the host R (resistance) gene, Pi-ta, and the pathogen Avr (avirulence) gene, AVR-pita. The genotype variation and resistant/susceptible phenotype at the Pi-ta locus of wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), was surveyed in 36 locations worldwide to study the molecular evolution and functional adaptation of the Pi-ta gene. The low nucleotide polymorphism of the Pi-ta gene of O. rufipogon was similar to that of O. sativa, but greatly differed from what has been reported for other O. rufipogon genes. The haplotypes can be subdivided into two divergent haplogroups named H1 and H2. H1 is derived from H2, with nearly no variation and at a low frequency. H2 is common and is the ancestral form. The leucine-rich repeat (LRR) domain has a high pi(non)/pi(syn) ratio, and the low polymorphism of the Pi-ta gene might have primarily been caused by recurrent selective sweep and constraint by other putative physiological functions. Meanwhile, we provide data to show that the amino acid Ala-918 of H1 in the LRR domain has a close relationship with the resistant phenotype. H1 might have recently arisen during rice domestication and may be associated with the scenario of a blast pathogen-host shift from Italian millet to rice.     

Evolutionary analysis of the Sub1 gene cluster that confers submergence tolerance to domesticated rice

BACKGROUND AND AIMS: Tolerance of complete submergence is recognized in a small number of accessions of domesticated Asian rice (Oryza sativa) and can be conferred by the Sub1A-1 gene of the polygenic Submergence-1 (Sub1) locus. In all O. sativa varieties, the Sub1 locus encodes the ethylene-responsive factor (ERF) genes Sub1B and Sub1C. A third paralogous ERF gene, Sub1A, is limited to a subset of indica accessions. It is thought that O. sativa was domesticated from the gene pools of the wild perennial species O. rufipogon Griff. and/or the annual species O. nivara Sharma et Shastry. The aim of this study was to evaluate the orthologues of the Sub1 locus in the closest relatives of O. sativa to provide insight into the origin of the gene and allelic variation of the Sub1 locus. METHODS: Orthologues of the Sub1 genes were isolated from O. rufipogon and O. nivara by use of oligonucleotide primers corresponding to the most highly conserved regions of the Sub1 genes of domesticated rice. The phylogenetic relatedness of Sub1 genes of O. sativa and its wild relatives was evaluated. KEY RESULTS AND CONCLUSIONS: Both O. rufipogon and O. nivara possess two Sub1 gene orthologues with strong sequence identity to the Sub1B and Sub1C alleles of cultivated rice. The phylogeny of the Sub1 genes of the domesticated and wild rice suggests that Sub1A arose from duplication of Sub1B. Variation in Sub1B alleles is correlated with the absence or presence of Sub1A. Together, the results indicate that genetic variation at the Sub1 locus is due to gene duplication and divergence that have occurred both prior to and after rice domestication.     

One large-insert plant-transformation-competent BIBAC library and three BAC libraries of Japonica rice for genome research in rice and other grasses

We report one large-insert BIBAC library and three BAC libraries for japonica rice cv Nipponbare. The BIBAC library was constructed in the HindIII site of a plant-transformation-competent binary vector (pCLD04541) and the three BAC libraries were constructed in the BamHI, HindIII and EcoRI sites of a BAC vector (pECBAC1), respectively. Each library contains 23,040 clones, has an average insert size of 130 kb, 170 kb, 150 kb and 156 kb, and covers 6.7x, 8.7x, 7.7x and 8.0 x rice haploid genomes, respectively. The combined libraries contain 92,160 clones in total, covering 31.1 x rice haploid genomes. To demonstrate their utility, we screened the libraries with 55 DNA markers mapped to chromosome 8 of the rice genetic maps and analyzed a number of clones by the restriction fingerprinting and contig assembly method. The results indicate that the libraries completely cover the rice genome and, thus, are well-suited for genome research in rice and other gramineous crops. The BIBAC library represents the first plant-transformation-competent large-insert DNA library for rice, which will streamline map-based cloning, functional analysis of the rice genome sequence and molecular breeding in rice and other grass species. These libraries are being used in the development of a whole-genome, BAC/BIBAC-based, integrated physical, genetic and sequence map of rice and in the research of genome-wide comparative genomics of grass species.     

Polymorphism at the esterase isozyme locus Est10 associated with phylogenetic differentiation in rice

A new esterase isozyme locus, Est10, with 6 alleles including the null form, has been found in rice by using polyacrylamide gel electrophoresis. Thirty F(2) populations of all possible combinations between 5 different band morphs were studied. The segregation pattern indicated that bands 1, 2, 3, 4, and the null form (0) were allelic with each other. The alleles of Est10 were distributed at different frequencies among different varietal groups of rice and also between cultivated rice and its wild relatives (Oryza rufipogon Griff.). Alleles 1 and 2 were frequently found in Japonica and Indica types, respectively. Allele 3 showed a high frequency in Aus and Boro, both Indica types cultivated in South Asia. Allele 4 was frequent in wild rice O. rufipogon. Judging from the linkage between Est10 and RFLP marker RG220 and isozyme marker Est5, Est10 is located on chromosome 1. The importance of this locus in evolutionary studies of rice is discussed.