Cleistogamy (fertilization without flower opening) is a useful trait to suppress outcrossing and to maintain genetic purity of cultivars. In the rice superwoman1-cleistogamy1 (spw1-cls1) mutant, dysfunctional development of lodicules (petal equivalent) causes cleistogamy. In this issue, Ohmori et al. investigated low-temperature responses of the spw1-cls1 cleistogamy caused by alteration of lodicule morphology (This issue, p. 432–441).
(H. Yoshida: Institute of Agrobiological Sciences, NARO)
Analyzing the genetic differences among crop germplasm resources scientifically and accurately is very important for the selection of core accessions, the identification of new cultivars, and the determination of seed purity. However, phenotypic selection per se is not sufficient to identify genetically distinct accessions. In this study, 26 out of 83 simple sequence repeat markers associated/linked with cotton important agronomic traits derived from our previous and other published research, corresponding to the 26 chromosomes of Upland cotton (Gossypium hirsutum L.), were selected as core primers for DNA fingerprinting construction. The 26 markers showed clear band patterns, good repeatability and high polymorphism. The average alleles, gene diversity index and polymorphism information content were 3.12, 0.4312 and 0.3830, respectively. Using TM-1, a genetic standard line for Upland cotton, as the control, DNA fingerprinting pattern and DNA barcodes were obtained based on the core primers. There was a significant positive correlation between genetic distance matrix determined using 26 core primers and that determined using more primers (335) derived from previous research, further suggesting that the core primers were eminently suitable for DNA fingerprinting in Upland cotton. This study provides a molecular basis for assessing identification, authenticity and seed purity of cotton cultivars.
A breeding program was initiated in 2009 to develop temperature stable CMS lines in chilli. ‘CCA 4261’ was used as a CMS donor. From the 11 testcross progeny screened, maintainer plants were identified from ‘SL 461’, ‘SL 462’ and ‘SD 463’. After 6 backcrosses to the maintainer plants, 17 CMS lines in diverse genetic backgrounds were established. The CMS lines were evaluated for stability of sterility over four environments during 2014–15 and 2015–16. The environments E1 and E3 represented the low temperature regime, and E2 and E4 the high temperature regime. The mean square values due to the genotypes and the environments were significant at p = 0.01 for pollen sterility (%), pollen release score, fruit setting (%) and number of seed fruit−1. The G × E interaction effects were significant for pollen sterility (%), fruit setting and number of seed fruit−1 and non-significant for pollen release score. Ten lines namely ‘CMS4611A’, ‘CMS4614A’, ‘CMS4622A’, ‘CMS4624A’, ‘CMS4626A’, ‘CMS46213A’, ‘CMS463D2A’, ‘CMS463D13A’, ‘CMS463D14A’ and ‘CMS463L5A’ were completely male sterile across the environments. Under open pollination conditions, the fruit and the seed setting ability of these lines was normal. The CMS transferred into the diverse genetic backgrounds would broaden the CMS germplasm resources in chilli.
Bacterial grain rot (BGR), caused by the bacterial pathogen Burkholderia glumae, is one of the most destructive rice (Oryza sativa) diseases in Japan; however, there are no BGR-resistant cultivars for use in Japan. We previously developed a cut-panicle inoculation method to assess the levels of BGR resistance in the World Rice Collection (WRC). Here, we evaluated major Japanese cultivars for BGR resistance and found that none showed “strong” or “medium to strong” resistance; most were categorized as “medium to weak”. On the basis of the screening results, standard cultivars for BGR resistance were selected according to resistance level and relative maturity. Our results indicate that it is necessary to introduce quantitative trait loci (QTLs) from indica or tropical japonica resistant cultivars into Japanese temperate japonica to develop BGR-resistant cultivars for Japan. We previously developed a near-isogenic line (RBG2-NIL) by introducing the genomic region containing RBG2 from ‘Kele’ (indica) into ‘Hitomebore’. In this experiment, we confirmed the resistance level of RBG2-NIL. The resistance score of RBG2-NIL was “medium to strong”, indicating its effectiveness against BGR.
Because genomic selection is designed for the population breeding of allogamous species, a successive outcrossing system is required for efficient use of genomic selection in autogamous crops, such as Oryza sativa L. (rice). Transgenic and dominant male-sterility is a suitable tool for efficient outcrossing of autogamous crops. Though there have been some reports of dominant male-sterile rice developed using transgenic technology, the flowering habit was substandard. Here, to isolate promoters that, when linked to a lethal gene, induce dominant male-sterility while retaining a good flowering habit, we identified 38 candidate genes with anther-specific expression by using the ‘RiceXPro’ database. We then evaluated the abilities of the near-upstream regions of these genes to induce male-sterility when linked to the lethal gene barnase and introduced into the rice cultivar ‘Nipponbare’. Seven of the 38 promoters induced clear dominant male-sterility; promoters expressed in the later stage of anther development induced male-sterility while retaining better flowering habits when compared to ones expressed in the early stage. These seven promoters could potentially be used to facilitate development of an efficient outcross-based breeding system in rice.
We reported previously that the rice (Oryza sativa L.) cleistogamous mutation superwoman1-cleistogamy1 (spw1-cls1) was applicable to inhibit outcrossing between genetically modified varieties and their relatives, which causes pollen-mediated gene flow or disturbance of line purity. The cleistogamy of spw1-cls1 is caused by decreased protein-protein interactions between the mutant SPW1 and its partner proteins. Importantly, these interactions are restored under low-temperature conditions, but whether the cleistogamy of spw1-cls1 is affected by this phenomenon was unclear. In this study, we cultivated spw1-cls1 in various regions of Japan and confirmed that its flowers opened at low temperatures. Moreover, we compared the morphology of a series of lodicules generated at various temperatures. The results indicated that the cleistogamy of spw1-cls1 is thermosensitive and is gradually disturbed as the temperature decreases. This was correlated with the protein interaction pattern of the mutant SPW1 as reported previously. Then, we revealed the critical period for the low-temperature-induced instability of the phenotype of spw1-cls1 and examined the effect of daily temperature changes on cleistogamy. The results may facilitate simulation of the phenotype of spw1-cls1 at various temperatures and the prediction of regions where the cleistogamy of spw1-cls1 can be stably used to inhibit outcrossing.
The 100-seed weight (100SW) is one of the most important traits that control soybean yield. To identify the quantitative trait loci (QTL) of 100SW, 120 BC3F5 chromosome segment substitution lines (CSSLs) were cultivated over three years. The CSSLs were developed from a cross between the cultivated soybean variety ‘Jackson’ and the wild soybean accession ‘JWS156-1’, followed by continuous backcrossing using ‘Jackson’ variety as a recurrent parent. A total of nine QTLs (qSW8.1, qSW9.1, qSW12.1, qSW13.1, qSW14.1, qSW16.1, qSW17.1, qSW17.2, and qSW20.1) were detected on eight chromosomes. Of these, qSW12.1 (LOD = 6.78–12.31) was detected over the three successive years on chromosome 12 as a novel, stable, and major QTL. To validate the effect of qSW12.1, a residual heterozygous line (RHL), RHL564, which showed heterozygous at the qSW12.1 region, was selected from the BC3F5 population. Of the two homologous genotypes in the progenies produced by self-pollination of RHL564, a higher seed weight was observed in the ‘Jackson’ genotype plants than that in the ‘JWS156-1’ genotype plants. qSW12.1 was delimited in an interval of approximately 1,348 kb between the BARCSOYSSR_12_1282 and BARCSOYSSR_12_1347 markers on chromosome 12.
The control of insects that consume cereal grains is important for the production and storage of grains. Hull-cracked rice, which has splits in the hull, becomes more susceptible to insects both in the paddy field and during storage. The development of varieties with a low frequency of hull-cracked rice is the most economical and effective strategy to avoid insect damage and the environmental risks from agricultural chemical entering rice grains. In this study, we identified that QTLs for the frequency of hull-cracked rice and for grain width are located on the same chromosome using recombinant inbred lines derived from a cross between the elite rice varieties in Hokkaido, Japan, which are from the same pedigree and are genetically closely related. These QTLs were detected close to different molecular markers, which were separated by 1,101,675 bp, on chromosome 5 in the reference Nipponbare genome. In addition, low coefficient values of the phenotype were found between hull-cracked rice and grain size. These results suggested that the ratio of hull-cracked rice is independent of grain size. Using these QTLs, new varieties with low hull-cracked rice could be developed regardless of grain size.
Spinach is a nutritional leafy green vegetable, and it also serves as a model species for studying sex chromosome evolution. Genetic marker development and genome structure analysis are important in breeding practice and theoretical evolution studies of spinach. In this study, the frequency and distribution of different microsatellites in the recently released draft spinach genome were characterized. A total of 261,002 perfect microsatellites were identified (estimated frequency: ~262.1 loci/Mbp). The most abundant microsatellites were tetranucleotide and trinucleotide, accounting for 33.2% and 27.7% of the total number of microsatellites, respectively. A total of 105 primer pairs were designed and screened, and 34 were polymorphic among the detected spinach cultivars. Combined with seven primer sets developed previously, 41 primer pairs were used to investigate genetic diversity among 43 spinach cultivars in China. The average polymorphism information content value of the 41 markers was 0.43, representing an intermediate level. The spinach cultivars had a low genetic diversity, and no detectable common factors were shared by each group in the UPGMA dendrogram. This study’s findings facilitate further investigations on the organization of the microsatellites in spinach genome and provide clues for future breeding applications of spinach in China.
Transposable elements (TEs), are a rich source for molecular marker development as they constitute a significant fraction of the eukaryotic genome and impact the overall genome structure. Here, we utilize Mutator-based transposon display (Mu-TD), and CACTA-derived sequence-characterized amplified regions (SCAR) anchored by simple sequence repeats and single nucleotide polymorphisms to locate quantitative trait loci (QTLs) linked to agriculturally important traits on a genetic map. Specifically, we studied recombinant inbred line populations derived from a cross between dent corn and waxy corn. The resulting linkage map included 259 Mu-anchored fragments, 34 SCARs, and 614 SSR markers distributed throughout the ten maize chromosomes. Linkage analysis revealed three SNP loci associated with kernel starch synthesis genes (sh2, su1, wx1) linked to either Mu-TD loci or SSR markers, which may be useful for maize breeding programs. In addition, we used QTL analysis to determine the chromosomal location of traits related to grain yield and kernel quality. We identified 24 QTLs associated with nine traits located on nine out of ten maize chromosomes. Among these, 13 QTLs involved Mu loci and two involved SCARs. This study demonstrates the potential use of DNA transposon-based markers to construct linkage maps and identify QTLs linked to agronomic traits.
An intergeneric hybrid was successfully developed between Oryza sativa L. (IRRI 154) and Leersia perrieri (A. Camus) Launert using embryo rescue technique in this study. A low crossability value (0.07%) implied that there was high incompatibility between the two species of the hybrid. The F1 hybrid showed intermediate phenotypic characteristics between the parents but the plant height was very short. The erect plant type resembled the female parent IRRI 154 but the leaves were similar to L. perrieri. Cytological analysis revealed highly non-homology between chromosomes of the two species as the F1 plants showed 24 univalents without any chromosome pairing. The F1 hybrid plant was further confirmed by PCR analysis using the newly designed 11 indel markers showing polymorphism between O. sativa and L. perrieri. This intergeneric hybrid will open up opportunities to transfer novel valuable traits from L. perrieri into cultivated rice.
Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2′-glucoside (Ch2′G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2′G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2′G by chalcone 2′-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2′G simultaneously to give orange color.
DNA markers are useful for identifying genes and developing new genetic materials for breeding and genetic research. High-resolution melting (HRM) analysis can detect a single nucleotide polymorphism (SNP) in two polymerase chain reaction (PCR) fragments as a melting temperature (Tm) difference without additional experimental steps, such as gel electrophoresis. To design a method for developing reliable HRM markers that discriminate between homozygous alleles containing SNPs, we tested new evaluation indexes related to the thermodynamics of double-stranded DNA to find one that maximizes the difference in Tm values between PCR fragments. We found that differences in the change in Gibbs free energy (ΔG°) correlated with actual differences in Tm values. Optimization of the nearest neighboring nucleotide (NNN) of a SNP by nucleotide substitution in the primer and reducing the size of the PCR fragment both enlarged the actual differences in Tm. The genetic DNA markers we developed by NNN substitution, termed NNNs-HRM markers, could be precisely mapped within soybean chromosomes by linkage analysis. We developed a Perl script pipeline to enable the automatic design of a massive number of NNNs-HRM markers; these scripts are freely available and would be useful for practical breeding programs for other plant species.