A likely paleo-autotetraploidization event shaped the high conservation of Nyssaceae genome

Scientific knowledge about the ancestral genome of core eudicot plant kingdom can potentially have profound impacts on both basic and applied research,including evolution,genetics,genomics,ecology,agriculture,forestry,and global climate.To investigate which plant conserves best the core eudicots common ancestor genome,we compared Arcto-Tertiary relict Nyssaceae and 30 other eudicot plant families.The genomes of Davidia involucrata(a known living fossil),Camptotheca acuminata and Nyssa sinensis,one per existent genus of Nyssaceae,were performed comparative genomic analysis.We found that Nyssaceae originated from a single Nyssaceae common tetraploidization event(NCT)-autotetraploidization 28-31 Mya after the core eudicot common hexaploidization(ECH).We identified Nyssaceae orthologous and paralogous genes,determined its chromosomal evolutionary trajectory,and reconstructed the Nyssaceae most recent ancestor genome.D.involucrata genome contained the entire seven paleochromosomes and 17 ECH-generated eudicot common ancestor chromosomes and was the slowest in mutation among the analyzed 42 species of 31 plant families.Combing both its high retention of paleochromosomes and its low mutation rate,D.involucrata provides the best case in conservation of the core eudicot paleogenome.

Differences in leaf cuticular wax induced by whole-genome duplication in autotetraploid sour jujube

Drought-resistant plants exhibit strong water retention capability.In this regard,the autotetraploid sour jujube leaves exhibit better water retention than diploid leaves.Morphological comparisons and physiological comparisons of diploid and autotetraploid leaves showed that the autotetraploid leaves had thicker leaf cuticles and more leaf wax accumulation than the diploid leaves,which could reduce cuticle permeability and improve the drought tolerance of leaves.In this study,the cuticular wax crystalloids on the adaxial and abaxial sides of young and mature jujube leaves were observed in the two ploidy types,and unique cuticular wax crystalloids covering a large area of the cuticle on autotetraploid sour jujube leaves may provide an advantage in reducing leaf non-stomata transpiration and improving plant drought tolerance.Based on the transcriptome,115 differentially expressed genes between diploids and autotetraploids were further analyzed and found to be involved in the accumulation of cuticular wax components,including terpenoids,fatty acids,and lipids,as well as ABC transporter and wax biosynthetic process.Finally,14 genes differentially expressed between glossy autotetraploid leaves and nonglossy diploid leaves,such as LOC107414787,LOC107411574 and LOC107413721,were screened as candidate genes by qRT-PCR analysis.This findings provided insights into how polyploidization improved drought tolerance.

A likely autotetraploidization event shaped the Chinese mahogany(Toona sinensis)genome

Chinese mahogany(Toona sinensis) is of considerable medical and economic importance, and its genome has been deciphered. However, the process underlying its polyploidy is unclear, and the chromosomal evolutionary trajectory is poorly understood. Here, by reanalysing the T.sinensis genome, we found evidence of a tetraploidization event(T. sinensis special tetraploidization, TST) that occurred approximately 15-17 million years ago(MYA) after the core eudicot-common hexaploidization(ECH or gamma) event. We characterized the synonymous nucleotide substitution rates(Ks values) of collinear genes and found that T. sinensis genes affected by the TST evolve at a slower rate than Acer yangbiense genes. Furthermore, we identified homologous genes related to polyploidization and speciation and constructed multiple alignments with different reference genomes. Notably, the significant balance of gene retention and loss characterized in the two TST-derived subgenomes suggests an autopolyploid nature of the TST. Moreover, we deduced the chromosomal karyotypes of the two subgenomes and identified 7chromosomal fusions that have shaped the T. sinensis genome;more information is available on a newly constructed karyotype platform(http://www.cgrpoee.top/Toona_sinensis/index.html). The T. sinensis genome preserves the ancestral chromosome structure of dicotyledons well and could serve as a good reference for understanding genomic changes in other Meliaceae and Sapindales plants. In addition, we verified that tandem duplication and the ECH have promoted the expansion of terpene synthase(TPS) genes;conversely, the TST seems to have inhibited expansion of these genes. This present effort has clarified the polyploidy events of the T. sinensis genome, filled gaps in the history of karyotype evolution, and laid a solid foundation for further genomic studies in the Meliaceae research community and beyond.

Endosperm Development in Autotetraploid Rice

By using the laser scanning confocal microscope and plastic （Leica 7022 historesin embedding kit） semi-thin sectioning technique, comparative studies on the development of endosperm were carried out between autotetraploid and diploid rices. About one third of the ovaries in the autotetraploid showed normal endosperm development as those in the diploid. In these ovaries, one of the polar nuclei would fuse with the sperm nucleus, and the primary endosperm nucleus formed and underwent the first division in 4 hours after pollination; the anticlinal wall began to grow centripetally between the free nuclei starting from the wall ingrowths of the embryo sac near the micropylar end, and some of the phragmoplasts formed transformed into periclinal walls. In addition, some of the cell wall situated in the middle of the endosperm appeared to originate from phragmoplasts, whereas others seemed to develop randomly without the obvious formation of phragmoplasts. Cellulose began to accumulate in the wall of aleurone cell layer at 6 days after pollination. The cellulose wall of the cells of the aleurone cell layer appeared to have completely formed within 7 to 8 days after pollination. On the other hand, about two thirds of the ovaries in the autotetraploid showed abnormality in endosperm development with various types, such as non-fertilization, abnormal fertilization, endosperm development-delay and non-synchronization in the development of cellulose wall of cells of the aleurone layer. These abnormalities usually resulted in decreased seed setting in autotetraploid rice.

Abnormal Structure of Embryo Sac in Autotetraploid Rice

The structures of mature embryo sacs in 13 genetic stock lines of autotetraploid rice （Oryza sativa L.）, including indica, japonica and javanica, were studied by using the whole-mount stain-clearing laser scanning confocal microscopy （WCLSM）. Among the 13 autotetraploid rice, the majority of ovaries possess normal polygonum-type embryo sacs, while a few ovaries were characterized by abnormal embryo sacs. The abnormalities of embryo sacs could be classified into six categories, i. e. no female germ unit, abnormal polar nuclei, embryo sac degeneration, no egg apparatus, small embryo sac and ＇double set＇ of embryo sacs. The frequency of abnormal embryo sac in japonica （26.6%） was higher than that in indica （19.34%）. In addition, the major abnormalities in each autotetraploid line varied, suggesting that the abnormalities may be related to the genotypes of the varieties.

Observation on Double Fertilization and Early Embryonic Development in Autotetraploid Polyembryonic Rice

The process of double fertilization and the characters of embryo and endosperm development in an autotetraploid polyembryonic mutant rice IR36-Shuang were studied with a laser scanning confocal microscopy. Some abnormalities including degenerated ovary, abortive embryo sac, single fertilization, double-ovule and double-embryo and so on. were found dudng double fertilization and embryo development in IR36-Shuang. The rate of the abnormalities was 46.67% in IR36-Shuang, significantly higher than that in the control, an autotetraploid rice line IR36-4X （33.00%）. Cytological and embryonic evidences were provided for seed setting decline and the initiation of additional embryo in IR36-Shuang.

Effect of Ar^+ Implantation and Maize Genome DNA on Autotetraploid Rice

The effect of Ar^＋ beam implantation and maize genome DNA on autotetraploid rice is studied. Better mutation types and higher mutation rates were discovered in M2 of T3 with ion implantation and immersion in maize genome DNA. In the five agronomic categories investigated, the mutation rate of the seed setting rate was 9.1%, and the total mutation rate was 14.8% in the T3. However, the total mutation rate was 2.1% with the treatment of only ion implantation and 1.3% with the treatment of only immersion in maize genome DNA. Mutant FA36（4） was discovered in M1 with ion beam implantation and immersion in maize genome DNA. Its RuBPCase activity, PEPCase activity and seed setting rate were 32%, 153%, and 36.79%, respectively, higher than its parent IR36（4）. Rapid analysis of polymorphicDNA （RAPD） analysis of three M2 plants of FA36（4） （FMI, FM2, FM3） and two controls （purple maize and IR36（4）） were also conducted with 40 random primers. S5-3 was RAPD fragment amplified with a template of purple maize, FM2 and FM3 genome DNA using primer S5. There was no S5-3 in the RAPD pattern of IR36（4） or FMI.

Biological Effects of Low Energy N^+ Beams Implantation on Calluses of Autotetraploid Rice

Calluses of two autotetraploid rice （DPR （4） and Zijing （4）） were implanted with 30 KeV N＋beams at doses ranging from 1.0×1015 to 9.0×1015 ions cm-2. The differentiation rates of calluses, the concentration of malondialdehyde （MDA）, the activities of superoxide dismutase （SOD）, catalase （CAT） and peroxidase （POD）, and the change of esterase （EST） spectrum were measured and analyzed on the calluses stage. The results showed that differentiation rates of calluses under 3.0×1015 ions cm-2 （（46.12±2.01）%） were higher than the other treatment groups in DPR （4）, and those of Zijing （4） were similar under the doses of 1.0×1015, 3.0×1015 and 5.0×1015 ions cm-2 （（40.55±2.74）, （37.77±3.04） and （34.56±2.65）%） and were higher than that of 7.0×1015 and 9.0×1015 ions cm-2. When the doses were 3.0×1015 and 5.0×1015 ions cm-2, the activities of SOD, POD and CAT were notably enhanced, and the accumulation of MDA content was markedly alleviated in the implanted calluses of two materials, as well as the activity of EST was increased by the additional isforms DPR （4）. Whereas the anti-oxidative systems and enzymes spectrum of EST were destroyed seriously, the accumulation of MDA was signiifcantly aggravated, also the differentiation rate of calluses reduced sharply and almost closed to zero when the implantation doses were 7.0×1015 and 9.0×1015 ions cm-2. It suggested that the proper dose of ion implantation effectively maintained the normal membrane structure and metabolism, and it would be also a feasible approach for autotetraploid rice to improve its genetic characteristics at calluses stage. Additionally, the differences of the radiation resistance between DPR （4） and Zijing （4） could be related to the different genotypes.

Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid

Autotetraploid indic, a/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid dce hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonicaAndica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy （WE-CLSM） technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs （i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac） in autotetraploid japonica/ndica hybdd. Embryo sac fertility was lower in diploid japonica/ndica hybdd than in autotetraploid japonicaAndica hybrid although many abnormal phenomena were observed in autotetraploid hybrid.

Biochemical Composition and Disease Resistance in Newly Synthesized Amphidiploid and Autotetraploid Peanuts

Genetic diversity in peanut (Arachishypogaea L.) is narrow due to its evolution and domestication processes. Amphidiploids and autotetraploids (newly synthesized tetraploids) were created to broaden its genetic base. Molecular analysis has shown that the newly synthesized tetraploids had broader genetic base;and were genetically divergent when compared to cultivated peanut. Nutritional composition relative to oil, fatty acid composition, O/L ratio, protein, iodine value and presence of plant proteinase inhibitors such as trypsin and chymotrypsin inhibitors were studied in the synthesized tetraploids. Some of the newly synthesized tetraploids had higher amounts of proteinase inhibitors. Evaluation of newly synthesized tetraploids revealed several lines resistant to late leaf spot (LLS) and peanut bud necrosis disease (PBND).

Study of Genetics and Embryology of Polyembryonic Mutant of Autotetraploid Rice Induced by N^+ Beam Implantation

In the present study autotetraploid rice IR36-4X was treated by an ion implantation technique with nitrogen ion beams. A polyembryonic mutant （named IR36-Shuang） was identified in the M2 generation. The mutant line and its offspring were systematically investigated in regard to their major agronomic properties and the rate of polyembryonic seedling in the M3-M6 generation. The abnormal phenomena in the embryo sac development and the cytological mechanism of the initiation of additional embryo in IR36-Shuang were observed by Laser Scanning Confocal Microscopy. The results were as follows. 1） The plant height, the panicle length and 1000 grain weight of IRB6-Shuang were lower than that of its control by 35.41%, 5.08% and 15.72% respectively, Moreover, the setting percentage decreased 12.39% compared with that in normal IRB6-4X plants. 2） The polyembryonic trait of IRB6-Shuang was genetically stable and the frequency of the polyembryonic seedlings in the IR36-Shuang line was also relatively stable. 3） The rate of abnormal embryo sacs in IR36-Shuang was significantly higher than that in the control IR36-4X. 4） The additional embryo in IR36-Shuang might arise from the double Jet of embryo sacs in a single owry, antipodal cells or endosperm cells. These results suggest that IRB6-Shuang is a polyembryonic mutant and a new apomixis rice line induced by low energy ion implantation. The prospects for the application in production of the IR36-Shuang line are also discussed. The present study may provide a basis for future investigations of apomixis rice breeding via the ion implantation biotechnology.

Unveiling potential sex-determining genes and sex-specific markers in autotetraploid Carassius auratus

Autotetraploid Carassius auratus is a stable hereditary autotetraploid fish resulting from the hybridization of Carassius auratus red var.(RCC,♀)×Megalobrama amblycephala(BSB,♂),containing four sets of RCC chromosomes.However,the molecular mechanism underlying the determination of sex in this species remains largely unknown.Currently,there lacks a full understanding of the molecular mechanisms governing sex determination and specific molecular markers to differentiate sex in this species.In this study,25,801,677 SNPs(Singlenucleotide polymorphism)and 6,210,306 Indels(insertion-deletion)were obtained from whole-genome resequencing of 100 individuals(including 50 female and 50 male).Further identification confirmed the candidate chromosomes as Chr46B,with the sex-determining region located at Chr46B:22,500,000‒22,800,000 bp.Based on the male-specific insertion(26 bp)within the candidate sex-determining region,a pair of sex-specific molecular markers has been identified.In addition,based on the screening of candidate sex-determining region genes and RT-qPCR validation analysis,ADAM10,AQP9 and tc1a were identified as candidate sex-determining genes.These findings provide a robust foundation for investigating sex determination mechanisms in fish,the evolution of sex chromosomes,and the development of monosex populations.

同源四倍体菜薹新材料的创制与品质分析

[目的]本文旨在创制优质的菜薹四倍体新材料。[方法]用2 g·L^(-1)秋水仙素溶液点滴二倍体菜薹LCX019幼苗的子叶生长点,通过形态学、解剖学、细胞学与流式细胞仪的方式筛选鉴定出同源四倍体植株,并对二、四倍体植株的农艺性状以及品质特性进行分析比较。[结果]形态学与解剖学结果显示,同源四倍体菜薹在株型、叶片、花、种荚、菜薹等方面表现出“巨大性”的特点;气孔孔径变大,气孔密度下降;花粉粒体积相比二倍体增大,形状呈橄榄球形或不规则形状。细胞学结果显示,四倍体菜薹根尖染色体数目为二倍体的2倍;流式细胞仪的分析结果表明,四倍体的DNA荧光强度约为二倍体的2倍。四倍体菜薹的可溶性糖、有机酸及纤维素含量显著增加76.17%、13.71%和80.34%,叶绿素含量、硝态氮含量显著下降25.01%、45.10%,可溶性蛋白含量无显著变化。通过分析二、四倍体的光响应曲线与光合特性,发现四倍体菜薹LCX019对弱光的利用能力强于二倍体植株。[结论]获得了具有优良品质的菜薹同源四倍体新材料,为菜薹育种提供新的种质资源。

黑皮冬瓜同源四倍体与二倍体解剖学分析

以二倍体及同源四倍体黑皮冬瓜为试材,采用光学显微镜和扫描电镜观察,研究了花粉形态及活力、双受精及胚胎发育情况对二倍体及同源四倍体黑皮冬瓜育性和结实率的影响,以期为同源四倍体黑皮冬瓜自交低稔性机理提供参考依据。结果表明:同源四倍体黑皮冬瓜花粉粒大,异形花粉较多,畸形率达42.43%,花粉活力低,花粉管生长缓慢。同源四倍体黑皮冬瓜存在明显的胚囊解体、退化或败育现象,异常胚囊率约占40%;双受精过程延后1 d,且双受精率低;合子休眠期长,胚发育进程比二倍体晚5 d;胚胎发育过程存在较高频率的胚胎发育滞后、非正常发育的合子、大量巨型胚乳游离核及游离核或胚乳细胞在合子期到球胚期大量提前解体、退化等现象。

优质同源四倍体菜心新材料的创制及特性分析

为获得优质的菜心同源四倍体新材料,本研究采用0.2%(W/V)的秋水仙素溶液点滴二倍体菜心LCX017幼苗子叶生长点,通过形态学、解剖学、细胞学以及流式细胞仪等方式鉴定到诱导成功的同源四倍体植株,并对二、四倍体的农艺性状、营养品质、光合特性等方面进行分析比较。结果表明,形态学与农艺性状方面,四倍体植株在叶片、花器官、种荚等方面表现出“巨大性”,叶宽、叶厚增大,菜薹直径变粗,重量增大;解剖学方面,四倍体叶片气孔相对二倍体更大,气孔密度降低;花粉粒相比二倍体变大且呈现棒状或橄榄状;细胞学方面,四倍体植株的根尖染色体数目是二倍体的2倍;流式细胞仪鉴定结果显示,四倍体的DNA荧光强度是二倍体的2倍;营养品质方面,相较于二倍体,四倍体植株中可溶性糖、纤维素、有机酸以及叶绿素含量显著上升,硝态氮含量下降22.92%,可溶性蛋白含量无显著变化;此外,结合光响应曲线分析发现,四倍体在强光和弱光条件下的适应能力均强于二倍体。综上,本研究成功诱导出了优质的同源四倍体菜心新材料,丰富了不结球白菜的种质资源。

CHH methylation of genes associated with fatty acid and jasmonate biosynthesis contributes to cold tolerance in autotetraploids of Poncirus trifoliata

Polyploids have elevated stress tolerance,but the underlying mechanisms remain largely elusive.In this study,we showed that naturally occurring tetraploid plants of trifoliate orange(Poncirus trifoliata(L.) Raf.) exhibited enhanced cold tolerance relative to their diploid progenitors.Transcriptome analysis revealed that whole-genome duplication was associated with higher expression levels of a range of well-characterized cold stress-responsive genes.Global DNA methylation profiling demonstrated that the tetraploids underwent more extensive DNA demethylation in comparison with the diploids under cold stress.CHH methylation in the promoters was associated with up-regulation of related genes,whereas CG,CHG,and CHH methylation in the 3’-regions was relevant to gene down-regulation.Of note,genes involved in unsaturated fatty acids(UFAs) and jasmonate(JA)biosynthesis in the tetraploids displayed different CHH methylation in the gene flanking regions and were prominently up-regulated,consistent with greater accumulation of UFAs and JA when exposed to the cold stress.Collectively,our findings explored the difference in cold stress response between diploids and tetraploids at both transcriptional and epigenetic levels,and gained new insight into the molecular mechanisms underlying enhanced cold tolerance of the tetraploid.These results contribute to uncovering a novel regulatory role of DNA methylation in better cold tolerance of polyploids.

Autotetraploidy induced in Nianmaohuangqin (Radix Scutellariae viscidulae) with colchicine in vitro

OBJECTIVE： To establish and optimize the propaga- tion of Nianmaohuangqin （Radix Scutellariae Viscid- ulae） and induce and characterize polyploidy of Nianmaohuangqin （Radix Scutellariae Viscidulae）. METHODS： Buds from germinating seed-derived explants were induced by tissue culture. With an or- thogonal test, different concentrations of 6-benzyl- aminopurine （BAP）, indole-3-acetic acid （IAA） and kinetin （KT） were used to determine the optimal concentrations for the propagation of Nianmaohuangqin （Radix Scutellariae Viscidulae）. The differ- ent concentrations of IAA and rooting powder （ABT） were used to induce rooting. A 0.3% w/v col- chicine solution was used to induce polyploidy and the induced buds was identified by root-tip chromosome determination and stomatal apparatus ob- servation. RESULTS： A large number of buds could be in- duced directly from epicotyl and hypocotyl ex- plants on Murashige and Skoog （MS） （Murashige and Skoog 1962） medium supplemented with 1.1-1.3 mg/L BAP and 0.2 mg/L IAA. Root induction and development could be observed within 20 days of inoculation on 1/2 MS medium supplemented with 0.2 mg/L IAA and 0.1 mg/L ABTo Furthermore, 27 lines of autotetraploid individuals were ob- tained with a plantlet chromosome number of 2n= 4x=36. CONCLUSION： Autotetraploid lines could be ob- tained through induction with colchicine in vitro, proving that this method might be used for plant selection and breeding.

Resequencing and genome-wide association studies of autotetraploid potato

Potato is the fourth most important food crop in the world.Although with a long history for breeding approaches,genomic information and association between genes and agronomic traits remain largely unknown particularly in autotetraploid potato cultivars,which limit the molecular breeding progression.By resequencing the genome of 108 main cultivar potato accessions with rich genetic diversity and population structure from International Potato Center,with approximate 20-fold coverage,we revealed more than 27 million Single Nucleotide Polymorphisms and~3 million Insertion and Deletions with high quality and accuracy.Domestication analysis and genome-wide association studies(GWAS)identified candidate loci related to photoperiodic flowering time and temperature sensitivity as well as disease resistance,providing informative insights into the selection and domestication of cultivar potato.In addition,GWAS with GWASploy for 25 agronomic traits identified candidate loci by association signals,especially those related to tuber size,small-sized tuber weight and tuber thickness that was also validated by transcriptome analysis.Our study provides a valuable resource that facilitates the elucidation of domestication process as well as the genetic studies and agronomic improvement of autotetraploid potato.

同源四倍体水稻:低育性机理、改良与育种展望

同源四倍体水稻具有籽粒增大、营养成分增加和抗性增强等特点,但其育性普遍偏低,影响产量,无法直接应用。高育性四倍体水稻的成功创制解决了同源四倍体水稻育性偏低的瓶颈问题,然而该类型多倍体水稻能否在生产上推广应用需要进一步探讨。本文总结了同源四倍体水稻及其杂种F1育性偏低的细胞和分子遗传学机理研究的概况,重点介绍了高育性四倍体水稻的主要类型及最新的研究进展,最后提出未来利用新型四倍体水稻开展多代杂种优势等研究的设想,以期为水稻多倍体育种提供参考。

高花青苷同源四倍体紫菜薹新材料的创制及特性分析

为获得优质高花青苷的同源四倍体不结球白菜紫菜薹Lcx025b新材料,本研究利用浓度为0.2%(w/v)的秋水仙素溶液处理二倍体Lcx025b幼苗的子叶生长点,通过形态学、细胞学、流式细胞仪测定等方法筛选鉴定得到同源四倍体植株,通过农艺性状指标、营养品质、光合特性、抗病能力等方面对二、四倍体植株进行比较分析。结果表明,相较于二倍体植株,四倍体植株在形态学方面表现出巨大性,具体表现为叶片的长宽比显著减小,叶片趋于扁圆形,花器官、种荚、种子等明显增大,菜薹颜色加深;叶片气孔明显增大,气孔密度减小;花粉粒表现出明显不规则性;四倍体植株根尖细胞染色体的数目以及通过流式细胞仪分析的四倍体植株DNA的荧光强度都表现为二倍体的两倍;营养品质方面,可溶性糖、菜薹花青苷含量显著增加,纤维素、硝态氮含量显著减少,可溶性蛋白、叶绿素含量无显著变化;结合光响应曲线分析,四倍体光合能力更强,适应性更高;此外,在抗病性鉴定过程中,四倍体植株对灰霉菌的抗性明显优于二倍体植株。综上,本研究获得了高花青苷的同源四倍体不结球白菜紫菜薹Lcx025b新材料,为不结球白菜的种质资源创新提供了重要的材料支撑。