Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing.

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M2 rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10-6-9.8×10-6 in the six M2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.

Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice.

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8-71.9% in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8% reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.

Characterization of OsMIK in a rice mutant with reduced phytate content reveals an insertion of a rearranged retrotransposon.

The rice low phytic acid (lpa) mutant Os-lpa-XS110-1(XS-lpa) has ~45 % reduction in seed phytic acid (PA) compared with the wild-type cultivar Xiushui 110. Previously, a single recessive gene mutation was shown to be responsible for the lpa phenotype and was mapped to a region of chromosome 3 near OsMIK (LOC_Os03g52760) and OsIPK1 (LOC_Os03g51610), two genes involved in PA biosynthesis. Here, we report the identification of a large insert in the intron of OsMIK in the XS-lpa mutant. Sequencing of fragments amplified through TAIL-PCRs revealed that the insert was a derivative of the LINE retrotransposon gene LOC_Os03g56910. Further analyses revealed the following characteristics of the insert and its impacts: (1) the inserted sequence of LOC_Os03g56910 was split at its third exon and rejoined inversely, with its 5' and 3' flanking sequences inward and the split third exon segments outward; (2) the LOC_Os03g56910 remained in its original locus in XS-lpa, and the insertion probably resulted from homologous recombination repair of a DNA double strand break; (3) while the OsMIK transcripts of XS-lpa and Xiushui 110 were identical, substantial reductions of the transcript abundance (~87 %) and the protein level (~60 %) were observed in XS-lpa, probably due to increased methylation in its promoter region. The above findings are discussed in the context of plant mutagenesis, epigenetics and lpa breeding.

[Data mining for SSRs in ESTs and development of EST-SSR marker in oilseed rape].

Totally 2803 SSRs distributed in 2443 ESTs were mined out and accounted for 13.58% of 17987 non-redundant ESTs from oilseeed rape, with the average distance of distribution about 4.26 kb. Dinucleotide and trinucleotide repeats are the dominant type, with similar frequency and accounting for 89.05% together in all SSRs. AG/CT and AAG/CTT are the most frequent motifs, accounting for 84.31% and 37.71% in dinucleotide and trinucleotide repeats respectively. Further, 23 primer pairs for EST-SSRs were designed and the suitable annealing temperature for each primer pair was determined by gradient PCR. The amplification and polymorphism displayed by these primers in 10 varieties of oilseed rape were detected by using silver staining of nondenaturing polyacrylamide gel. 21 primer pairs showed the amplification, accounting for 91.30% of total primers, and 12 primer sets showed polymorphisms, accounting for 57.14% of primers available. These results indicate that it is an effective and feasible approach to develop SSR markers based on ESTs in oilseed rape.

[Studies on tea plants (Camellia sinensis) germplasms using EST-SSR marker].

Forty two tea varieties were analyzed by using 16 SSR primer sets derived from tea ESTs in this study, and 13 of the primer sets produced clear bands and 10 of them showed polymorphism, accounting for 76.9%. The PIC (polymorphism information content) for each polymorphic primer set varied from 0.522 to 0.866, with a average about 0.73. Totally 84 Genotypes and 74 alleles were detected in all materials by 10 polymorphic markers, with the range from 4 to 12 and from 3 to 10 for each polymorphic primer set, respectively. The genetic distance among 42 tea varieties varied from 0.074 to 0.667, averagely 0.363, suggesting that the materials used in the experiment possess a broad genetic variation. Based on the similarity coefficient about 0.55, all the tea varieties tested could be classified into 3 groups and most of them were in first group. The results of this study proved that the EST-SSR marker is very effective in evaluation of tea germplasms.

[Development of EST (Expressed Sequence Tags) Marker in Chinese Cabbage and its Transferability to Repeseed.].

28 pairs of primers were designed according to the expressed sequence tags in Chinese cabbage. After testing on the annealing temperature and the concentration of primer, dNTP and MgCl2, a suitable PCR system was established. Under the condition of reaction system developed, primers designed specific to ESTs were screened against genomic DNA of inbreed line A from which the cDNA library was constructed. Among them, 18 pairs of primers showed the amplification. Then all the primers available in line A were subjected to PCR for DNAs from 17 cabbage varieties. Polymorphism was detected by electrophoresis with agarose gel, and 10 of 18 primer sets could reveal polymorphisms among cabbage varieties, which accounted for 55.6% of primers selected. To examine the transferability of EST markers developed in cabbage, all primers were further used for PCR-mediated amplification of genomic DNA from different varieties of rapeseeds. Of 28 pairs of primers, 24 were able to produce amplified product(s) and 18 showed polymorphisms, accounting for 85.7% and 64.3% of total primers respectively. All of 18 primer sets that amplified in cabbage also showed amplified products in rapeseed and 13 of them were polymorphic. Even amongst the 10 primer sets that were unable to amplify in cabbage, 6 pairs produced amplification and 5 could reveal the polymorphism in rapeseeds. Results obtained in the present paper proved that developing polymorphic markers based on EST could be feasible and this kind of marker would be transferable to closed related species.

[Studies on the distorted segregation of foreign genes in transgenic rice progenies].

The segregation mode of transgenes was investigated in the transgenic progenies of three rice varieties (lines) produced by Agrobacterium-mediated transformation. The transgenic lines all contained the Bacillus thuringiensis cry1Ab gene, under the control of a maize ubiquitin promoter, and linked in tandem with gusA and hpt genes. PCR analysis showed the transgenes cry1Ab and gusA co-segregated in all self and crossed progenies tested. Therefore, GUS bio-assay of leaf or endosperm tissues was used to monitor transgene segregation in the experiment. It was found that the ratio of positive to negative plants was significantly smaller than 3:1 in all heterozygous plants derived progenies, which implied the segregation biased from typical Mendelian mode for a single dominant gene. Less GUS positive plants, and consequently less homozygous transgenic lines than expected were recovered from the self progenies. Transgenic heterozygous plants (+/-) were crossed as female or male parent to conventional rice varieties (-/-), and the ratio of gusA positive (+/-) to negative (-/-) plants was investigated in test F1 population. When used as female parent, the segregation fit to 1:1, but significantly smaller than 1:1 when used as male parent. The seed-set of transgenic Nipponbare progeny was investigated individually for GUS positive and negative plants. It was found that the positive plants had an average seed-set of 64.5%, significantly lower than that of negative plants (77.9%). The biological and genetic basis of distorted segregation of transgenes was discussed and deduced on the basis of above results, and the authors are inclined to ascribe these phenomena to the poor competitive ability of pollens carrying transgenes.