As a first step toward cloning the genic multiple allele inherited male-sterile gene Ms in Chinese cabbage (Brassica rapa L. ssp pekinensis (Lour.) Olsson), we attempted the molecular tagging of this male-sterility locus using the amplified fragment length polymorphism (AFLP) technique. A BC1 mapping population (130 plants) segregating for male sterility/fertility was constructed for the tagging using the bulked segregant analysis (BSA) method. Screening 144 primer combinations, four AFLP markers were identified to be tightly linked to the Ms gene, the nearest one of which, AFLP01, was converted to a SCAR marker for the marker-assisted selection (MAS). Finally, a genetic map of the Ms gene was constructed with four AFLP markers and one SCAR marker, covering a total interval of 13.9 cM. Two markers AFLP01(SCAR01) and AFLP04 flanked the Ms locus at distances of 2.3 and 7.8 cM, respectively.
There are prior reports of Pyricularia grisea—the causal agent of blast of rice—causing disease in barley. In order to determine the specificity of this resistance in barley, we extended our previous mapping efforts to include blast isolates from barley and rice grown in Thailand and we assessed two resistance phenotypes: leaf blast (LB) and neck blast (NB). The largest-effect resistance QTL, on chromosome 1H, was associated with NB and LB and is located in a region rich in resistance genes, including QTL conferring resistance to stripe rust (incited by Puccinia striiformis f. sp. hordei) and the mildew (Blumeria graminis f. sp. hordei) resistance gene Mla. The LB, NB and mildew resistance alleles trace to one parent (Baronesse) whereas the stripe rust resistance allele traces to the other parent (BCD47) of the mapping population. Baronesse is the susceptible recurrent parent of a set of near-isogenic lines (NILs) for three stripe rust resistance QTL, including one on 1H. Unigene (EST) derived single nucleotide polymorphism haplotypes of these NILs were aligned with the blast mapping population QTL using Mla as an anchor. Baronesse and all NILs without the 1H introgression were resistant to LB and NB. However, two NILs with the 1H introgression were resistant to LB and NB. Both are resistant to stripe rust. Therefore, the QTL conferring resistance to stripe rust is separable by recombination from the blast resistance QTL.
A full-length cDNA clone encoding granule-bound starch synthase I (GBSSI = Waxy gene) from grain amaranth (Amaranthus cruentus L.) perisperm was isolated and characterized. Segregation of amylose content in F2 population suggested that the amylose content of A cruentus is controlled by a single gene, Waxy (GBSSI). cDNA clone of this gene is 2076 bp in length and contains an open reading frame of 1821 bp corresponding to a polypeptide of 606 amino acids residues, including a transit peptide of 77 amino acids. Comparison of the cDNA and genomic sequences (3492 bp) suggested that the amaranth GBSSI gene has 12 introns, of which exons 1–13 contributed to the coding sequence. The mature protein shares 70.2–75.3% sequence identity with GBSSI of dicots and about 64.0–67.8% identity with those of monocots. This protein contains the conserved motif KTGGL found in other GBSSI proteins, which has been implicated as the active site in glycogen synthase. Sequence analysis predicted that GBSSI of amaranth has a transit peptide of 77 amino acids including FIR↓S, which is different cleavage site that of the other dicot species. These results will provide more useful information for understanding the structure/function relationship of this protein from amaranths perisperm.
To reveal the genetic properties of an Afghan wheat collection maintained in Kyoto University, variations in morphological character and HMW glutenin subunit composition were investigated in 410 Afghan wheat accessions together with 65 accessions of Iranian and Pakistani wheat. The majority of the samples had primitive morphological characteristics, namely, tall plant height and lax spikes with a spelt head. The most frequent combination of HMW glutenin subunit was Glu-A1c (encoding subunit null), Glu-B1b (7 + 8), and Glu-D1a (2 + 12). Alleles preferable for modern breeding such as Glu-B1c and Glu-D1d were rare in our samples. Consequently, we concluded that the Afghan wheat accessions investigated in this study have genetic characteristics typical of landraces. As for their genetic diversity, the level of variation in the HMW glutenin subunit in Afghanistan was found to be equal to or lower than that in the neighboring countries. The largest variation was observed in Western Iran and Pakistan. AFLP analysis using 91 representative accessions also showed a comparatively low level of diversity in Afghanistan. These results suggest that a decrease in genetic diversity has occurred in Afghanistan. A novel HMW glutenin subunit (2.8) was found in this study, indicating the potential usefulness of Afghan landraces for wheat breeding.
Spelt wheat [Triticum aestivum ssp. spelta (L.) Thell.] is becoming a valuable crop due to its reputation as a healthy food. In Australia, this crop has not been targeted for systematic breeding. Identification of spelt genotypes having low PPO activity and resistance to acid soils (Al3+) are desirable attributes for future cultivar development. We evaluated 51 genebank accessions of spelts from the Australian Winter Cereals Collection for polyphenol oxidase (PPO) activity and for resistance to aluminium (Al). PPO activity was measured both visually and spectrophotometrically, using L-DOPA substrate. PPO activity for genotypes ranged from 0.15 to 1.3 and could be grouped into ‘low’, ‘medium’ and ‘high’ categories. At least eight accessions exhibited low PPO activity (not different to the durum check cultivar Arrivato). After measuring PPO activity, the same kernels were further evaluated for Al resistance using a nutrient solution culture method with haematoxylin staining test of root tips. Thirty-three accessions were resistant to Al. Functional gene markers associated with loci conditioning Al resistance gene (TaALMT1) and PPO activity (XPPO-2A) in common wheat confirmed their association with target phenotypes within spelt accessions. Genetic variation within spelt wheat for important agronomic and quality traits such as Al resistance and PPO suggested that progress in spelt improvement can be made by molecular plant breeding. Diversity Array Technology (DArT) based allelic data revealed that these spelt accessions are genetically diverse.
The lateral spikelets of two-rowed barley are reduced in size and sterile, but in six-rowed barley all three spikelets are fully fertile. The trait is largely controlled by alleles at the vrs1 locus on chromosome arm 2HL, as modified by the allele present at the I locus on chromosome arm 4HS. Molecular markers were developed to saturate the 4HS region by exploiting expressed sequence-tags, either previously mapped in barley to this region, or present in the syntenic region of rice chromosome 3. Collinearity between rice and barley was strong in the 4.8 cM interval BJ468164–AV933435 and the 10 cM interval AV942364–BJ455560. A major QTL for lateral spikelet fertility (the I locus) explained 44% of phenotypic variance, and was located in the interval CB873567–BJ473916. The genotyping of near-isogenic lines for I placed the locus in a region between CB873567 and EBmac635, and therefore the most likely position of the I locus was proximal to CB873567 in a 5.3 cM interval between CB873567–BJ473916.
We developed 73 novel expressed sequence tag (EST)—simple sequence repeat (SSR) markers based on ESTs derived from 11 different cDNA libraries of the Japanese pear (Pyrus pyrifolia Nakai) cultivar ‘Housui’, comprising 30, 36, two, one, and four SSRs with repeat motifs of di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively. Forty-eight EST sequences showed significant homology to Arabidopsis thaliana genes. The linkage groups and positions of 57 loci generated from 53 SSR markers covering all 17 linkage groups, were identified. These EST-SSRs will be useful for high-throughput genotyping of marker-assisted selection. The numbers of alleles and the observed and expected heterozygosities at single-locus EST-SSR markers in ten Japanese and ten European pears suggest high polymorphism and heterozygosity of the EST-SSRs. Di-nucleotide EST-SSRs showed two to three times the polymorphism and heterozygosity of tri-nucleotide EST-SSRs. Some of the EST-SSRs proved transferable to other species in the Rosaceae, paving the way for the development of markers for the same phenotypic traits among species of the Rosaceae.
In order to clarify the origin of weedy rice (Oryza sativa L.) in Okayama Prefecture, Japan, we used a set of 15 sequence-tagged site primers to analyze polymorphisms among 27 weedy rice accessions and 88 cultivated rice varieties accessions including common rice varieties from Okayama. The band patterns indicated that 13 of the japonica weedy accessions corresponded exactly with one of the japonica cultivated rice varieties. Furthermore, with the exception of a strong shattering habit, these japonica weedy accessions closely resembled each corresponding rice variety in morphophysiological characteristics. Consequently, in view of the close genetic homology and high morphophysiological resemblance between weedy rice and cultivated rice varieties in Okayama, we conclude that weedy rice originated from cultivated rice varieties as an “off-type” caused by genetic mutation accompanied by the development of a strong shattering habit.
F1 sterility, one of the most common post-zygotic reproductive barriers, is frequently observed in both interspecific and intraspecific crosses of rice. Elucidating the genetic and cytological mechanisms of F1 pollen sterility is important to exploit genetic resources and to understand the evolutionary dynamics of post-zygotic reproductive isolation in rice. Here, we report two F1 pollen sterility loci, designated S36 and S25, found in an interspecific cross between O. sativa ssp. japonica (Taichung 65) and O. nivara (IRGC105444), and an intraspecific cross between O. sativa ssp. japonica (Asominori) and ssp. indica (IR24). Genetic analyses revealed that both loci are located on distal end of the short arm of chromosome 12 and that allelic interaction at the heterozygous locus caused the sterility of male gametes carrying the japonica alleles in both cases. Comparison of map positions of S36 and S25 suggested that these two loci might be the same locus. Cytological investigation revealed that abnormality of sterile pollen grains caused by S36 occurred mainly at the late bicellular stage after initiation of starch accumulation. This study would provide the better understanding on the genetic nature and the cytological mechanism of F1 pollen sterility in rice.
Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The inhibition of seed coat pigmentation is due to naturally occurring posttranscriptional gene silencing (PTGS) of the chalcone synthase (CHS) genes. In the present study, RNA gel blot analysis in different tissues indicated that endogenous CHS siRNAs are accumulated only in the seed coat, supporting the suggestion that CHS PTGS is seed coat-specific. The probe region of a CHS gene was divided into several parts, each of which was used for RNA gel blot analysis in the seed coat. Interestingly, endogenous CHS siRNAs were only detected with probes corresponding to the exon 2 region. The results were confirmed by deep sequencing analysis of CHS siRNAs in the seed coat, i.e., CHS siRNAs were predominantly derived from the 3′-half of exon 2 of the CHS gene. Mapping of CHS siRNA sequences on CHS genes and an inverted repeat region of the CHS pseudogene (pseudoCHS3) suggested that primary CHS siRNAs may be generated from double-stranded RNA of pseudoCHS3, and subsequently secondary CHS siRNAs may be produced according to the two-hit model, which has been proposed to explain siRNA amplification by the RNA-dependent RNA polymerase.
The teosinte Zea nicaraguensis forms constitutive aerenchyma in the root cortex as observed in flooding tolerant wetland plants. We have previously identified a quantitative trait locus (QTL) controlling aerenchyma formation under non-flooding conditions on chromosome 1 (Qaer1.06) using 214 individuals of a maize Mi29 × Z. nicaraguensis BC2F1 population. The present studies objective was to increase the marker density around the Qaer1.06 locus, which is essential for the development of near-isogenic lines possessing the aerenchyma-forming gene with only a small region of the Z. nicaraguensis chromosome segment. A survey of 62 SSR and 38 insertion/deletion (INDEL) markers identified a total of 36 useful markers in the region of interest. These were selected for the construction of a linkage map in a 214 individual BC2F1 and a 123 individual BC4F1 population from a cross between Mi29 × Z. nicaraguensis. Using the BC4F1 population, we performed QTL mapping and the results indicated that a QTL for aerenchyma formation under non-flooding condition was located on chromosome 1 (bin 1.05) at a position that is near to Qaer1.06. The markers obtained here should be useful for the development of high quality near-isogenic lines possessing the Qaer1.05-6.
A major quantitative trait locus (QTL) controlling pod dehiscence (shattering) in soybean, designated qPDH1, has previously been identified using progeny of shattering-resistant cultivars derived from a Thai cultivar, SJ2. The QTL was located near a simple sequence repeat marker, Sat_366, on linkage group J. To determine whether shattering-resistance genes originating from different resources are located at qPDH1 in general, we conducted genetic analysis using DNA markers for several populations. In an F2 population derived from a cross between a shattering-susceptible cultivar, Toyomusume, and a shattering-resistant cultivar, Harosoy, a major QTL for pod dehiscence was identified in the region near qPDH1, which was confirmed in the progeny of F4:5 populations. A major QTL was identified near qPDH1 also in F2 populations derived from crosses including Wasekogane and Kariyutaka as shattering-resistant parents. The heterozygous genotypes at the QTL showed high degrees of pod dehiscence, suggesting that shattering resistance behaves as a nearly recessive trait. In F2 populations derived from crosses between shattering-resistant cultivars, heterozygous genotypes at the Sat_366 locus were shattering-resistant. These results suggest that shattering-resistant cultivars harbor recessive shattering-resistance allele(s) at qPDH1 regardless of their origin and that molecular markers near qPDH1 could be used for marker-assisted selection for shattering resistance in soybean.
Hybrid sterility is a serious barrier in the utilization of wild rice for breeding, but little is known regarding hybrid sterility between the cultivated rice, Oryza sativa, and its wild relative, O. longistaminata. In order to understand further the nature of interspecific hybrid sterility, pollen and spikelet fertility were investigated in two BC7F2 populations derived from a semisterile individual of BC5F1 between Oryza sativa L. and O. longistaminata. One main-effect QTL for pollen and spikelet fertility qpsf6 was detected on the short arm of chromosome 6 close to RM587, around it favoring O. longistaminata allele was found. Comparing the position and effect with other studies indicated that this QTL coincides with the gamete eliminator, S1. It suggests that there exists an orthologous hybrid sterility locus that controls the reproduction barrier between O. sativa and its AA genome relatives. QTL mapping for plant height was also conducted in one of the BC7F2 populations. One QTL, qph1, was detected on the long arm of chromosomes 1 close to RM6333 and coincides with the semi-dwarf gene, sd-1. These new QTL information will increase the efficiency of cultivar development via interspecific hybridization involving O. longistaminata, and offset the stage for fine mapping of these QTL.
To find out an efficient and accurate way to estimate the genetic relationship among cultivars having within-variety genetic variation by using different types of approaches, we compared among-varieties genetic similarity estimated based on individual samples and also on bulked population samples of six Japanese turnip (Brassica rapa ssp. rapa) cultivars using RAPD and ISSR markers. In both individual- and bulked-samples-based approaches, 37 individuals were sampled from each cultivar, amplified individually and also as bulked DNA, respectively. Genetic similarity matrices among six cultivars were estimated based on four similarity measures estimated with 125 or 124 polymorphic bands scored in the individual- or bulked-samples-based approach, respectively, and the correlations between the similarity matrices were calculated to evaluate the degree of agreement between matrices. Correlations between similarity matrices calculated based on individuals on the one hand and those calculated based on bulked samples on the other were also high and significant in all combinations of the four similarity measures. Use of computational analysis indicated that 6 to 8 individuals per cultivar were enough to accurately estimate similarity with the individual-based approach.