Triploid cultivars of Cymbidium act as a bridge in the formation of polyploid plants.

Triploid is considered a reproductive barrier and also a bridge in the formation of polyploids. However, few reports are available in Cymbidium. In this study, diploid 'Xiaofeng', sexual triploid 'Yuchan' and 'Huanghe' of Cymbidium were used to evaluate hybridization compatibility of the triploids. Results showed that the sexual triploids were fertile whether they were used as male or female parents. 'Yuchan' produced male gametes of 1x, 1x~2x, 2x, 2x~3x, and 3x at frequencies of 8.89%, 77.78%, 6.67%, 3.33%, and 3.33%, respectively; while 'Huanghe' produced 3.33% 1x, 80.00% 1x~2x, 8.89% 2x, 5.56% 2x~3x, and 2.22% 3x male gametes. The cross of 'Xiaofeng' with 'Yuchan' produced progenies with a wide range of ploidy levels, including one diploid, 34 2×~3× aneuploids, 12 triploids, and one tetraploid, indicating that male gametes produced by sexual triploid were fertile and could be transmitted and fused with egg cells. On the other hand, 10 progenies obtained from the cross of 'Yuchan' × 'Xiaofeng' were all aneuploids. The cross of 'Yuchan' with 'Huanghe' produced 40 progenies including three 2×~3× aneuploids, nine 3×~4× aneuploids, 21 tetraploids, six 4×~5× aneuploids, and one pentaploid, suggesting that 2x gametes, instead of the unreduced ones played a more important role in the formation of tetraploids. The survival rates of the hybrids were all above 80.00%, with the tetraploids at 96.67%. Cytological analysis revealed that during meiosis of sexual polyploids, two chromosome sets of the 2n gamete were inclined to enter into the same daughter cell, resulting in the production of 2x gametes. Our results indicate that the triploid cymbidiums are not reproductive barrier but serve as a bridge in the formation of polyploid plants.

Unreduced Male Gamete Formation in Cymbidium and Its Use for Developing Sexual Polyploid Cultivars.

Polyploidy plays an important role in crop improvement. Polyploid plants, particularly those produced through unreduced gametes (2n gametes), show increased organ size, improved buffering capacity for deleterious mutations, and enhanced heterozygosity and heterosis. Induced polyploidy has been widely used for improving floriculture crops, however, there are few reported sexual polyploid plants in the floriculture industry. This study evaluated nine cultivars of Cymbidium Swartz and discovered that 2n male gametes occurred in this important orchid. Depending on cultivars, 2n male gamete formation frequencies varied from 0.15 to 4.03%. Interspecific hybrids generally produced more 2n male gametes than traditional cultivars. To generate sexual polyploid plants, seven pairs of crosses were made, which produced five triploid and two tetraploid hybrids. Two triploid hybrids were evaluated for in vitro regeneration and growth characteristics. Compared to the diploid parents, the triploids were more easily regenerated through rhizomes or protocorms, and regenerated plants had improved survival rates after transplanting to the greenhouse. Furthermore, the sexual polyploid plants had more compact growth style, produced fragrant flowers, and demonstrated heterosis in plant growth. Through this study, a reliable protocol for selection of appropriate parents for 2n gamete production, ploidy level evaluation, in vitro culture of polyploid progenies, and development of new polyploid cultivars was established. Our study with Cymbidium suggests that the use of 2n gametes is a viable approach for improving floriculture crops.

Transcriptomic and Hormonal Analyses Reveal that YUC-Mediated Auxin Biogenesis Is Involved in Shoot Regeneration from Rhizome in Cymbidium.

Cymbidium, one of the most important orchid genera in horticulture, can be classified into epiphytic and terrestrial species. Generally, epiphytic Cymbidium seedlings can be easily propagated by tissue culture, but terrestrial seedlings are difficult to propagate. To date, the molecular mechanisms underlying the differences in the ease with which terrestrial and epiphytic cymbidiums can be propagated are largely unknown. Using RNA-sequencing, quantitative reverse transcription PCR and enzyme-linked immunosorbent assay, Cymbidium 'Xiaofeng' (CXF), which can be efficiently micropropagated, and terrestrial Cymbidium sinense 'Qijianbaimo' (CSQ), which has a low regeneration ability, were used to explore the molecular mechanisms underlying the micropropagation ability of Cymbidium species. To this end, 447 million clean short reads were generated, and 31,264 annotated unigenes were obtained from 10 cDNA libraries. A total of 1,290 differentially expressed genes (DEGs) were identified between CXF and CSQ during shoot induction. Gene ontology (GO) enrichment analysis indicated that the DEGs were significantly enriched in auxin pathway-related GO terms. Further analysis demonstrated that YUC and GH3 family genes, which play crucial roles in the regulation of auxin/IAA (indole-3-acetic acid) metabolism, acted quickly in response to shoot induction culture in vitro and were closely correlated with variation in shoot regeneration between CXF and CSQ. In addition, the study showed that IAA accumulated rapidly and significantly during shoot induction in CXF compared to that in CSQ; in contrast, no significant changes in other hormones were observed between CXF and CSQ. Furthermore, shoot regeneration in CXF was inhibited by a yucasin-auxin biosynthesis inhibitor, indicating that increased IAA level is required for high-frequency shoot regeneration in CXF. In conclusion, our study revealed that YUC-mediated auxin biogenesis is involved in shoot regeneration from rhizome in Cymbidium.

[Identification and genetic analysis of pale-green mutant caused by Ds insertion in rice].

Ac/Ds insertion mutation was thought to be one of the powerful tools for identifying gene function in rice. In this study, the rice mutant with pale-green leaves was isolated from the population of Ds-T-DNA and Ac-T-DNA transgenic homozygote in a japonica rice variety Zhonghua 11. The leaves of the mutant turned to be pale-green at three-leaf stage. This mutant was capable of growing slowly and maturing under low-light conditions, but, rapidly died under natural light conditions. The analysis of the photosynthetic activity characteristics by measuring chlorophyll fluorescence in vivo suggested that the mutant was a typical photo-inhibitory mutant. Genetic analysis demonstrated that the mutation was the recessive one resulted from Ds insertion.

[Genetic analysis and gene mapping of an early flowering and multi-ovary mutant in rice (Oryza sativa L.).].

A rice floral mutant was obtained from a japonica variety Zhonghua 11, which was characterized with the phenotype of more stamens, more pistils and early flowering. Genetic analysis showed that the mutant phenotype was controlled by a single recessive gene, which was designed as floral organ number 5 (fon5). To map fon5, an F2 population was derived from the cross between Huajingxian 74 and fon5 mutant. Gene fon5 was roughly mapped between the microsatellite markers RM400 and RM412 on chromosome 6 with genetic distances of 10.5 and 1.6 cM, respectively. Then, six new po-lymorphic InDel markers were developed in the region. According to the physical information of the markers, locus fon5 was finally delimited to a region of 116 kb.

Development of a wide population of chromosome single-segment substitution lines in the genetic background of an elite cultivar of rice (Oryza sativa L.).

Naturally occurring allelic variations underlying complex traits are useful resources for the functional analysis of plant genes. To facilitate the genetic analysis of complex traits and the use of marker-assisted breeding in rice, we developed a wide population consisting of 217 chromosome single-segment substitution lines (SSSLs) using Oryza sativa L. 'Hua-Jing-Xian74' (HJX74), an elite Indica cultivar, as recipient, and 6 other accessions, including 2 Indica and 4 Japonica, as donors. Each SSSL contains a single substituted chromosome segment derived from 1 of the 6 donors in the genetic background of HJX74. The total size of the substituted segments in the SSSL population was 4695.0 cM, which was 3.1 times that of rice genome. To evaluate the potential application of these SSSLs for quantitative trait loci detection, phenotypic variations of the quantitative traits of days to heading and grain length in the population consisting of 210 SSSLs were observed under natural environmental conditions. The results demonstrated that there was a wide range of phenotypic variation in the traits in the SSSL population. These genetic materials will be powerful tools to dissect complex traits into a set of monogenic loci and to assign phenotypic values to different alleles at the locus of interest.

[Developing single segment substitution lines (SSSLs) in rice (Oryza sativa L.) using advanced backcrosses and MAS].

A novel population consisted of 86 single segment substitution lines (SSSLs) were developed from advanced backcrosses between the recipient, Huajingxian 74 and six donors by microsatellite marker-assisted selection (MAS). Fifty-two SSSLs were selected in BC3F2, and 34 others were selected in BC3F3. Every SSSL contains a single chromosome segment introgressed from one donor on the genetic background of Huajingxian 74. The substituted segments in SSSLs were distributed on 12 rice chromosomes. The estimated length of the substituted segments in SSSLs ranged from 1.5 cM to 56.3 cM with an average of 23.0 cM. Total of the substituted segments covered 57.1% of the rice genome.

[Identification of QTLs on substituted segments in single segment substitution lines of rice].

The quantitative trait loci (QTLs) for 21 traits of agronomy importance on ten substituted chromosomal segments were identified using single segment substitution lines (SSSLs) in rice, which were developed by the use of Taichung 65 as a recipient and zhai-ye-qing and dee-geo-woo-gen as donors respectively. Total length of the substituted segments in the SSSLs was 230.00 cM, which was 12.62% of rice genome. T-test was used to detect QTLs in the condition that the difference in phenotypes between a SSSL and the recipient parent (Taichung 65) was significant at the 0.1% level (P< or = 0.001). A total of 57 QTLs for 17 traits had been detected on the ten substituted segments, which were on chromosomes 1, 3, 5, 7, 8, 10 and 12 respectively. The additive effect percentages of the QTLs ranged from 1.10% to 89.73%. Among of 57 QTLs, fifteen QTLs had the additive effect percentage over 10%, thirty QTLs varied from 3% to 10% and other twelve QTLs were below 3%.