Aberrant endosperm formation caused by reduced production of major allergen proteins in a rice flo2 mutant that confers low-protein accumulation in grains.

Rice flo2 mutation produces grains showing a reduced amount of storage proteins. Using Nipponbare and the flo2 mutant, we created rice transformants that showed defective production of major allergen proteins RA14 and RA33 (14-16 kDa and 33 kDa allergen proteins, respectively) by RNAi introduction. The knock-down transformant generated using Nipponbare showed greatly reduced accumulation of both allergen proteins, normal growth, and production of a sufficient amount of normal-shaped seeds. F1 seeds were obtained by crossing between the transformants containing RNAi genes to RA14 and RA33, and showed decreased accumulation of both proteins. However, a peculiar phenotype was observed in the flo2 transformants that lacked accumulation of RA14 or RA33. They showed significantly reduced fertility. A wrinkled grain feature was found on the transformant lacking accumulation of RA14. F1 seeds obtained by crossing these transformants showed significantly lower fertility. F2 seeds showed decreases in both allergen proteins but morphological abnormality with small and severely wrinkled features. These results indicated that it is hard to obtain any transformant lacking accumulation of these allergen proteins using the flo2 mutant, whereas a knock-down transformant of both allergen protein genes was obtained when a wild-type Nipponbare was used as a host. These facts strongly suggest that RA14 and RA33 have some roles in rice seeds.

Effects of correcting missing daily feed intake values on the genetic parameters and estimated breeding values for feeding traits in pigs.

Daily feed intake (DFI) is an important consideration for improving feed efficiency, but measurements using electronic feeder systems contain many missing and incorrect values. Therefore, we evaluated three methods for correcting missing DFI data (quadratic, orthogonal polynomial, and locally weighted (Loess) regression equations) and assessed the effects of these missing values on the genetic parameters and the estimated breeding values (EBV) for feeding traits. DFI records were obtained from 1622 Duroc pigs, comprising 902 individuals without missing DFI and 720 individuals with missing DFI. The Loess equation was the most suitable method for correcting the missing DFI values in 5-50% randomly deleted datasets among the three equations. Both variance components and heritability for the average DFI (ADFI) did not change because of the missing DFI proportion and Loess correction. In terms of rank correlation and information criteria, Loess correction improved the accuracy of EBV for ADFI compared to randomly deleted cases. These findings indicate that the Loess equation is useful for correcting missing DFI values for individual pigs and that the correction of missing DFI values could be effective for the estimation of breeding values and genetic improvement using EBV for feeding traits.

Selection for testis size and correlated responses of female reproductive traits in golden hamsters.

Golden hamsters were selected for large (high selection direction: H) and small (low selection direction: L) testis size, as measured in live males at age 13 weeks over six generations. The selection response and correlated responses in female reproductive traits were evaluated in terms of the divergence between H and L lines for testis size. In males, the differences in testis size at 13 weeks, testis weight at 19 weeks, and body weight at 13 and 19 weeks were significant at the 1% level from generation 1 onward. The realized heritability of testis size, as estimated from regression of the selection responses on effective selection differentials, was 0.29. This was similar to the heritability estimated by using the multiple-trait animal model restricted maximum likelihood (0.30). In females, significant differences in ovulation rate at the 5% level were detected in generations 5 and 6. Litter size 1 day after birth also diverged between the H and L lines, but the difference was not consistently significant. The realized genetic correlation between testis size and ovulation rate was 0.67.

Comparison of production systems for efficient use of indigenous pig breeds in developing countries.

Conserving pig genetic resources and improving their productivity is important to increase returns over investment in developing countries. The purebred, first-cross, rotational cross and backcross matings representing production systems based on pig breeds indigenous to the country and exotic pig breeds were investigated. The number of pigs in the nucleus and commercial herds necessary to produce a defined quantity of pork was considered. The amount of heterosis between the indigenous and exotic breeds, superiority in meat production, and degree of inferiority in reproductive performance of the exotic breed compared with that of the indigenous breed were investigated. The number of breeding pigs in the whole system was in the following order: pure breeding (PB) > first-cross (F1) > rotational cross (RC) > backcross (BC) systems. The number of breeding pigs in the nucleus herds of the RC and BC systems was smaller than that in the nucleus herds of the PB and F1 systems. The degree of inferiority in reproductive performance of the exotic breed compared with that of the indigenous breed affected the efficiency of the production system.

Estimation of the time of divergence between Japanese Mishima Island cattle and other cattle populations using microsatellite DNA markers.

We applied the theory of random genetic drift to determine the divergence history of a closed cattle population over the relatively short timescale of several hundred years. The divergence history of the closed population of Mishima Island cattle, a national natural treasure of Japan, was examined, and the results were compared with historical documents. Inbreeding depression in the isolated population was investigated for body size and fertility. Twenty-one DNA microsatellite markers in Mishima Island cattle and 3 major breeds from the mainland were genotyped. For the mainland breeds, all 21 or 20 markers were segregating. However, nearly half the number of loci (9 of 21) was fixed in the Island cattle. The average number of alleles per locus of Island cattle was markedly lower than that in the mainland breeds. These results support the theory that Island cattle have been isolated for a considerable period of time. The number of generations of isolation was estimated as 14.1-22.6, and the year of divergence was calculated as 1778-1846. In view of these findings, we propose that Island cattle diverged from the mainland population at around 1800 and were isolated for about 200 years. These estimates are in agreement with historical documents showing that divergence occurred between 1672 and 1880. The total inbreeding coefficient of the present population was predicted to be in the range of 0.51-0.60. However, historical reports dated over 100 years do not support changes in fertility, so that there is no evidence for inbreeding depression.

The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp.

Different colors, such as purple, brown, red and white, occur in the pericarp of rice. Here, two genes affecting proanthocyanidin synthesis in red- and brown-colored rice were elucidated. Genetic segregation analysis suggested that the Rd and A loci are identical, and both encode dihydroflavonol-4-reductase (DFR). The introduction of the DFR gene into an Rcrd mutant resulted in red-colored rice, which was brown in the original mutant, demonstrating that the Rd locus encodes the DFR protein. Accumulation of proanthocyanidins was observed in the transformants by the introduction of the Rd gene into the rice Rcrd line. Protein blot analysis showed that the DFR gene was translated in seeds with alternative translation initiation. A search for the Rc gene, which encodes a transacting regulatory factor, was conducted using available DNA markers and the Rice Genome Automated Annotation System program. Three candidate genes were identified and cloned from a rice RcRd line and subsequently introduced into a rice rcrd line. Brown-colored seeds were obtained from transgenic plants by the introduction of a gene containing the basic helix-loop-helix (bHLH) motif, demonstrating that the Rc gene encodes a bHLH protein. Comparison of the Rc locus among rice accessions showed that a 14-bp deletion occurred only in the rc locus.

Quantitative analysis of gene expression in preimplantation mouse embryos using green fluorescent protein reporter.

We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluorescent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluorescence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available.