Research progress of plant cytogenetics in Jiangsu province.

Cytogenetics was established based on the "Chromosome theory of inheritance", proposed by Boveri and Sutton and evidenced by Morgan's lab in early stage of the 20 th centrary. With rapid development of related research areas, especially molecular genetics, cytogenetics developed from traditional into a new era, molecular cytogenetics in late 1960s. Featured by an established technique named DNA in situ hybridization (ISH), molecular cytogenetics has been applied in various research areas. ISH provids vivid and straightforward figures showing the virtual presence of DNA, RNA or proteins. In combination with genomics and cell biology tools, ISH and derived techniques have been widely used in studies of the origin, evolution, domestication of human, animal and plant, as well as wide hybridization and chromosome engineering. The physical location and order of DNA sequences revealed by ISH enables the detection of chromosomal re-arrangments among related species and gaps of assembled genome sequences. In addition, ISH using RNA or protein probes can reveal the location and quantification of transcripted RNA or translated protein. Since the 1970s, scientists from universities or institutes belonging to the Jiangsu Society of Genetics have initiated cytogenetics researches using various plant species. In recent years, research platforms for molecular cytogenetics have also been well established in Nanjing Agricultural University, Yangzhou University, Nanjing Forestry University, Jiangsu Xuhuai Academy of Agricultural Sciences, and Jiangsu Normal University. The application of molecular cytogenetics in plant evolution, wide hybridization, chromosome engineering, chromosome biology, genomics has been successful. Significant progresses have been achieved, both in basic and applied researches. In this paper, we will review main research progresses of plant cytogenetics in Jiangsu province, and discuss the potential development of this research area.

Progress on inheritance and gene cloning for rice grain quality in Jiangsu province.

In China, rice (Oryza sativa L.) is a major cereal crop of great importance maintaining the food security and sustainable agricultural development. Jiangsu is one of the main provinces for rice production. After more than 40 years of development, the yield and quality of rice grain have made great progress. Rice grain quality is a complex trait involving production, processing, marketing and consumption of the grain. In this review, we summarize the progress on the genetic basis of main grain quality traits in the rice variety breeding in Jiangsu province and point out the achievement of each milestone. With a focus on the genetic regulation of grain appearance, eating and cooking quality and nutritional quality, we describe the classic genetic rules and molecular basis of rice grain quality traits and review the function of major genes that regulate corresponding traits. The genetics and improvement of grain quality achieved in Jiangsu province was highlighted on the domestic and international rice breeding programs. In particular, with the advance of breeding conception in terms of functional genomics and genetic regulatory networks, the specific molecular design for grain quality improvement will be the future direction of rice genetic breeding program of Jiangsu Province.

Clinical characteristics and phenotype distribution in 10 Chinese patients with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is the most frequent type of inherited demyelinating peroxisomal disease caused by mutations in the ATP binding cassette subfamily D member 1 (ABCD1) gene. The rate of early recognition and genetic diagnosis of X-ALD remains low due to its variable clinical manifestations. The present study summarized the clinical features Chinese X-ALD patients and performed a follow-up study to further precisely characterize this disease. A total of 10 patients diagnosed with X-ALD between 1994 and 2016 at Shandong Provincial Hospital Affiliated to Shandong University (Jinan, China) were included in the present study. Through reviewing their medical records and performing telephone follow-ups, the clinical features, biochemical laboratory data, brain images, treatments and long-term outcomes were retrospectively summarized. Mutation analysis of the ABCD1 gene was performed in certain patients. Most of the patients (8/10) had the childhood cerebral form of X-ALD. One patient presented with the olivo-ponto-cerebellar form, the rarest form of X-ALD. In all patients, brain magnetic resonance images revealed abnormalities with typical T2-weighted hyperintensity. Analysis of very long chain fatty acid revealed high plasma levels of hexacosanoic acid in all patients. Increased adrenocorticotropic hormone, decreased cortisol and neurophysiological manifestations were also observed. Three different mutations of the ABCD1 gene were identified in the 3 patients subjected to genotyping. During the follow-ups, most patients took neurotrophic drugs and received hydrocortisone replacement when required. One patient received a hematopoietic stem cell transplantation, but died 1 year following the transplantation. Chronic myelopathy and peripheral neuropathy progressed with time, gradually leading to a vegetative state or paralysis within several years of clinical symptom onset. In conclusion, male patients with adrenocortical insufficiency should be further investigated for X-ALD. Early detection is critical to prevent the progression of X-ALD with mutation analysis of ABCD1 the most accurate method to confirm diagnosis.

Down-Regulation of SSSII-2 Gene Expression Results in Novel Low-Amylose Rice with Soft, Transparent Grains.

Although soft rice, with low amylose content (AC), has high eating and cooking quality (ECQ), its appearance is poor due to the opaque endosperm. Here, a novel soft rice with low AC but a transparent appearance was generated by knocking-down the expression of SSSII-2, a gene encoding one isoform of soluble starch synthase (SSS). The physicochemical properties of the SSSII-2 RNAi rice are quite different from the control but more like the popular soft rice "Nanjing 46". The taste value assay further demonstrated that the ECQ of SSSII-2 RNAi rice was as high as "Nanjing 46", but only SSSII-2 RNAi rice retained the transparent endosperm under low moisture conditions. Further examination showed that the different morphologies and fine structures of the starch granules may contribute to the specific properties of SSSII-2 RNAi rice. Therefore, SSSII-2 has potential application in future high quality rice breeding programs.

GS9 acts as a transcriptional activator to regulate rice grain shape and appearance quality.

Identification of grain shape determining genes can facilitate breeding of rice cultivars with optimal grain shape and appearance quality. Here, we identify GS9 (Grain Shape Gene on Chromosome 9) gene by map-based cloning. The gs9 null mutant has slender grains, while overexpression GS9 results in round grains. GS9 encodes a protein without known conserved functional domain. It regulates grain shape by altering cell division. The interaction of GS9 and ovate family proteins OsOFP14 and OsOFP8 is modulated by OsGSK2 kinase, a key regulator of the brassinosteroids signaling pathway. Genetic interaction analysis reveals that GS9 functions independently from other previously identified grain size genes. Introducing the gs9 allele into elite rice cultivars significantly improves grain shape and appearance quality. It suggests potential application of gs9, alone or in combination with other grain size determining genes, in breeding of rice varieties with optimized grain shape.

A Connection between Lysine and Serotonin Metabolism in Rice Endosperm.

Cereal endosperms produce a vast array of metabolites, including the essential amino acid lysine (Lys). Enhanced accumulation of Lys has been achieved via metabolic engineering in cereals, but the potential connection between metabolic engineering and Lys fortification is unclear. In mature seeds of engineered High Free Lysine (HFL) rice (Oryza sativa), the endosperm takes on a characteristic dark-brown appearance. In this study, we use an integrated metabolomic and transcriptomic approach combined with functional validation to elucidate the key metabolites responsible for the dark-brown phenotype. Importantly, we found that serotonin biosynthesis was elevated dramatically and closely linked with dark-brown endosperm color in HFL rice. A functional connection between serotonin and endosperm color was confirmed via overexpression of TDC3, a key enzyme of serotonin biosynthesis. Furthermore, we show that both the jasmonate signaling pathway and TDC expression were strongly induced in the late stage of endosperm development of HFL rice, coinciding with serotonin accumulation and dark-brown pigmentation. We propose a model for the metabolic connection between Lys and serotonin metabolism in which elevated 2-aminoadipate from Lys catabolism may play a key role in the connection between the jasmonate signaling pathway, serotonin accumulation, and the brown phenotype in rice endosperm. Our data provide a deeper understanding of amino acid metabolism in rice. In addition, the finding that both Lys and serotonin accumulate in HFL rice grains should promote efforts to create a nutritionally favorable crop.

Improved growth performance, food efficiency, and lysine availability in growing rats fed with lysine-biofortified rice.

Rice is an excellent source of protein, and has an adequate balance of amino acids with the exception of the essential amino acid lysine. By using a combined enhancement of lysine synthesis and suppression of its catabolism, we had produced two transgenic rice lines HFL1 and HFL2 (High Free Lysine) containing high concentration of free lysine. In this study, a 70-day rat feeding study was conducted to assess the nutritional value of two transgenic lines as compared with either their wild type (WT) or the WT rice supplemented with different concentrations of L-lysine. The results revealed that animal performance, including body weight, food intake, and food efficiency, was greater in the HFL groups than in the WT group. Moreover, the HFL diets had increased protein apparent digestibility, protein efficiency ratio, and lysine availability than the WT diet. Based on the linear relationship between dietary L-lysine concentrations and animal performance, it indicated that the biological indexes of the HFL groups were similar or better than that of the WT20 group, which was supplemented with L-lysine concentrations similar to those present in the HFL diets. Therefore, lysine-biofortified rice contributed to improved growth performance, food efficiency, and lysine availability in growing rats.

Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice.

A residue substitution in the plastid ribosomal protein L12/AL1 produces defective plastid ribosome and causes early seedling lethality in rice.

The plastid ribosome is essential for chloroplast biogenesis as well as seedling formation. As the plastid ribosome closely resembles the prokaryotic 70S ribosome, many plastid ribosomal proteins (PRPs) have been identified in higher plants. However, their assembly in the chloroplast ribosome in rice remains unclear. In the present study, we identified a novel rice mutant, albino lethal 1 (al1), from a chromosome segment substitution line population. The al1 mutant displayed an albino phenotype at the seedling stage and did not survive past the three-leaf stage. No other apparent differences in plant morphology were observed in the al1 mutant. The albino phenotype of the al1 mutant was associated with decreased chlorophyll content and abnormal chloroplast morphology. Using fine mapping, AL1 was shown to encode the PRPL12, a protein localized in the chloroplasts of rice, and a spontaneous single-nucleotide mutation (C/T), resulting in a residue substitution from leucine in AL1 to phenylalanine in al1, was found to be responsible for the early seedling lethality. This point mutation is located at the L10 interface feature of the L12/AL1 protein. Yeast two-hybrid analysis showed that there was no physical interaction between al1 and PRPL10. In addition, the mutation had little effect on the transcript abundance of al1, but had a remarkable effect on the protein abundance of al1 and transcript abundance of chloroplast biogenesis-related and photosynthesis-related genes. These results provide a first glimpse into the molecular details of L12's function in rice.

Major QTLs reduce the deleterious effects of high temperature on rice amylose content by increasing splicing efficiency of Wx pre-mRNA.

KEY MESSAGE: We discovered four QTLs that maintain proper rice amylose content at high temperature by increasing the splicing efficiency of Wx gene. Amylose content mainly controlled by Wx gene is a key physicochemical property for eating and cooking quality in rice. During the grain filling stage, high temperature can harm rice grain quality by significantly reducing the amylose content in many rice varieties. Here, we provide genetic evidences between Wx gene expression and rice amylose content at high temperature, and identified several quantitative trait loci (QTLs) in this pathway. We performed a genome-wide survey on a set of chromosome segment substitution lines (CSSLs) which carried chromosomal segments from the heat resistant indica 9311 in the heat-sensitive japonica Nipponbare background. Four QTLs, qHAC4, qHAC8a, qHAC8b and qHAC10, which can reduce the deleterious effects of amylose content at high temperature, were identified and mapped to chromosome 4, 8, 8 and 10, respectively. The major QTL qHAC8a, with the highest LOD score of 6.196, was physically mapped to a small chromosome segment (~300 kb). The CSSLs carrying the qHAC8a, qHAC8b and/or qHAC4 from 9311 have the high pre-mRNA splicing efficiency of Wx gene and likely lead to stable amylose content at high temperature. Thus, increasing pre-mRNA processing efficiency of Wx gene could be an important regulation mechanism for maintaining stable amylose content in rice seeds at high temperature. In addition, our results provide a theoretical basis for breeding heat-stable grain in rice.

Development and high-throughput genotyping of substitution lines carring the chromosome segments of indica 9311 in the background of japonica Nipponbare.

Chromosome segment substitution lines (CSSLs) are useful for the precise mapping of quantitative trait loci (QTLs) and dissection of the genetic basis of complex traits. In this study, two whole-genome sequenced rice cultivars, the japonica Nipponbare and indica 9311 were used as recipient and donor, respectively. A population with 57 CSSLs was developed after crossing and back-crossing assisted by molecular markers, and genotypes were identified using a high-throughput resequencing strategy. Detailed graphical genotypes of 38 lines were constructed based on resequencing data. These CSSLs had a total of 95 substituted segments derived from indica 9311, with an average of about 2.5 segments per CSSL and eight segments per chromosome, and covered about 87.4% of the rice whole genome. A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight. The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice. These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome discovery and functional study of essential genes/QTLs in rice, and offer ideal materials and foundations for japonica breeding.

A major locus qS12, located in a duplicated segment of chromosome 12, causes spikelet sterility in an indica-japonica rice hybrid.

Chromosome segment duplications are integral in genome evolution by providing a source for the origin of new genes. In the rice genome, besides an ancient polyploidy event known in the rice common ancestor, it had been identified that there was a special segmental duplication involving chromosomes 11 and 12, but the biological role of this duplication remains unknown. In this study, by using a set of chromosome segment substitution lines (CSSLs) and near isogenic lines (NILs) derived from the indica cultivar 9311 and japonica cultivar Nipponbare, a major QTL (qS12) resulting in hybrid male sterility was mapped within ~400 kb region adjacent to the special duplicated segment on the short arm of chromosome 12. Compared to the japonica cultivar Nipponbare, the two sides of the qS12 candidate region were inverted in the indica cultivar 9311. Among 47 of the 111 rice genotypes evaluated by molecular markers, the inverted sides were detected, and found completely homologous to indica cultivar 9311. These results suggested that the two inverted sides protect the sequence in the qS12 regions from recombination. On the short-arm of chromosome 12, two QTLs S-e and S25, in addition to qS12, were previously detected as a distinct segregation distortion and pollen semi-sterility loci. We propose these three hybrid sterility loci are the same locus, and the duplicated segment on chromosome 12 may play a prominent role in diversification, i.e., sub-speciation of cultivated rice.

The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice.

WRKY proteins are a large super family of transcriptional regulators primarily involved in various plant physiological programs. In present study, the expression profile and putative function of the WRKY transcriptional factor, WRKY78, in rice were identified. Real-time RT-PCR analysis showed that OsWRKY78 transcript was most abundant in elongating stems though its expression was detected in all the tested organs. The expression profiles were further confirmed by using promoter-GUS analysis in transgenic rice. OsWRKY78::GFP fusion gene transient expression analysis demonstrated that OsWRKY78 targeted to the nuclei of onion epidermal cell. Furthermore, OsWRKY78 RNAi and overexpression transgenic rice lines were generated. Transgenic plants with OsWRKY78 overexpression exhibited a phenotype identical to the wild type, whereas inhibition of OsWRKY78 expression resulted in a semi-dwarf and small kernel phenotype due to reduced cell length in transgenic plants. In addition, a T-DNA insertion mutant line oswrky78 was identified and a phenotype similar to that of RNAi plants was also observed. Grain quality analysis data showed no significant differences, with the exception of minor changes in endosperm starch crystal structure in RNAi plants. Taken together, these results suggest that OsWRKY78 may acts as a stem elongation and seed development regulator in rice.

Rice OsGL1-1 is involved in leaf cuticular wax and cuticle membrane.

Cuticular wax forms a hydrophobic barrier on aerial plant organs; it plays an important role in protecting a plant from damage caused by many forms of environmental stress. In the present study, we characterized a rice leaf wax-deficient mutant osgl1-1 derived from a spontaneous mutation, which exhibited a wax-deficient and highly hydrophilic leaf phenotype. We cloned the OsGL1-1 gene by the map-based cloning method and performed a complementation test to confirm the function of the candidate gene. Molecular studies revealed that OsGL1-1 was a member of the OsGL1 family, and contained regions that were homologous to some regions in sterol desaturases and short-chain dehydrogenases/reductases. Compared to the wild-type, the osgl1-1 mutant showed decreased cuticular wax deposition, thinner cuticular membrane, decreased chlorophyll leaching, increased rate of water loss, and enhanced sensitivity to drought. OsGL1-1 is expressed ubiquitously in rice. The transient expression of OsGL1-1-green fluorescent protein fusion protein indicated that OsGL1-1 is localized in the cytoplasm, plasma membrane, and nucleus.

Genetic analysis of starch paste viscosity parameters in glutinous rice (Oryza sativa L.).

Starch paste viscosity plays an important role in estimating the cooking, eating, and processing quality of rice. The inheritance of starch paste viscosity in glutinous rice remains undefined. In the present study, 118 glutinous rice accessions were collected, and the genotypes of 17 starch synthesis-related genes (SSRG) were analyzed by using 43 gene-specific molecular markers. Association analysis indicated that 10 of 17 SSRGs were involved in controlling the rapid visco analyzer (RVA) profile parameters. Among these, the PUL gene was identified to play an important role in control of peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), peak time (PeT), and paste temperature (PaT) in glutinous rice. Other SSRGs involved only a few RVA profile parameters. Furthermore, interactions between SSRGs were found being responsible for PeT, PaT, and BDV. Some of the RVA parameters, including PKV, HPV, CPV, CSV, and PaT, were mainly governed by single SSRG, whereas other parameters, such as BDV, SBV, and PeT, were controlled by a few SSRGs, functioning cooperatively. Further, three near-isogenic lines (NIL) of a japonica glutinous cv. Suyunuo as genetic background, with PUL, SSIII-1, and SSIII-2 alleles replaced with those of indica cv. Guichao 2, were employed to verify the genetic effects of the various genes, and the results were consistent with those obtained from the association analysis. These findings indicated that starch paste viscosity in glutinous rice had a complex genetic system, and the PUL gene played an important role in determining the RVA profile parameters in glutinous rice. These results provide important information for potentially improving the quality of glutinous rice.

Characterization of expression of the OsPUL gene encoding a pullulanase-type debranching enzyme during seed development and germination in rice.

Starch-debranching enzymes (DBEs) are key enzymes involved in starch metabolism in cereals, having a dual function, in both starch synthesis and degradation. However, their precise roles in this pathway, particularly their expression profiles, remain unclear. In the present study, we performed a quantitative real-time PCR (Q-PCR) analysis of the expression pattern of the OsPUL gene encoding a pullulanase-type DBE in different tissues as well as in seeds at different developmental stages. The results showed that this gene was expressed only in seeds. In addition, the 1177-bp OsPUL promoter sequence was cloned, and some endosperm-specific motifs such as the GCN4 and AACA motifs were observed to exist in this region. The promoter was then fused with the GUS reporter gene and its expression was carefully investigated in transgenic rice. The data from both histochemical and fluorometric analyses showed that the OsPUL promoter was capable of driving the target gene to have a high level of endosperm-specific expression. The OsPUL gene maintained a relatively high expression level during the entire period of seed development, and peaked in the middle and late stages. This observation was very consistent with that of the endogenous transcription analysis by Q-PCR. Furthermore, the seed germination experiment showed that the OsPUL promoter actively functions in the late stage of seed germination. The expression of the OsPUL gene was maintained at a significant level during the entire grain filling period and in the late stage of seed germination, which coincided with its involvement in starch anabolism and catabolism.

[Molecular mechanism of leaf development].

Leaf plays important roles during plant development for their function of photosynthesis and transpiration. Leaf development includes initiation of leaf primordium and establishment of leaf polarity. Various studies indicate that leaf development is controlled through the interaction of transcription factors, small RNAs and auxin. This review focuses on re-cent advances in studying on leaf development and morphogenesis, and provides information on the regulation network in the process.

ROLLED LEAF 9, encoding a GARP protein, regulates the leaf abaxial cell fate in rice.

Leaves, the collective organ produced by the shoot apical meristem (SAM), are polarized along their adaxial-abaxial axis. In this study, we characterized two rice (Oryza sativa) allelic rolled-leaf mutants, rolled leaf 9-1 (rl9-1) and rl9-2, which display very similar phenotypes with completely adaxialized leaves and malformed spikelets. We cloned the RL9 gene by way of a map-based cloning strategy. Molecular studies have revealed that RL9 encodes a GARP protein, an orthologue of Arabidopsis KANADIs. RL9 is mainly expressed in roots, leaves, and flowers. The transient expression of a RL9-GFP (green fluorescent protein) fusion protein has indicated that RL9 protein is localized in the nucleus, suggesting that RL9 acts as a putative transcription factor.

Identification and characterization of a major QTL responsible for erect panicle trait in japonica rice (Oryza sativa L.).

Panicle erectness (PE) is one of the most important traits for high-yielding japonica cultivars. Although several cultivars with PE trait have been developed and released for commercial production in China, there is little information on the inheritance of PE traits in rice. In the present study, 69 widely cultivated japonica cultivars and a double haploid (DH) population derived from a cross between a PE cultivar (Wuyunjing 8) and a drooping panicle cultivar (Nongken 57) were utilized to elucidate the mechanisms of PE formation and to map PE associated genes. Our data suggested that panicle length (PL) and plant height (PH) significantly affected panicle curvature (PC), with shorter PL and PH resulting in smaller PC and consequently more erect. A putative major gene was identified on chromosome 9 by molecular markers and bulk segregant analysis in DH population. In order to finely map the major gene, all simple sequence repeats (SSR) markers on chromosome 9 as well as 100 newly developed sequence-tagged site (STS) markers were used to construct a linkage group for quantitative trait locus (QTL) mapping. A major QTL, qPE9-1, between STS marker H90 and SSR marker RM5652, was detected, and accounted for 41.72% of PC variation with pleiotropic effect on PH and PL. another QTL, qPE9-2, was also found to be adjacent to qPE9-1. In addition, we found that H90, the nearest marker to qPE9-1, used for genotyping 38 cultivars with extremely erect and drooping panicles, segregated in agreement with PC, suggesting the H90 product was possibly part of the qPE9-1 gene or closely related to it. These data demonstrated that H90 could be used for marker-aided selection for the PE trait in breeding and in the cloning of qPE9-1.

[Isolation and characterization of the centromeric BAC clones from different genomes in genus Oryza].

Centromeres play an important role in ensuring the correct segregation and transmission of chromosome during mitosis and meiosis in eukaryotes. In this research, we constructed five BAC libraries for diploid wild rice with different genomes. Together with the technique of colony blot hybridization and fluorescence in situ hybridization (FISH), centromere-related BAC clones were screened and characterized from different genomes. Meanwhile, co-hybridization was detected between these clones and the five genomes. The results from this study demonstrated that: (1) there were centromere-specific satellite repeat in Oryza officinalis (CC genome) and O. brachyantha (FF genome), respectively, and centromere-specific CRR-related sequence was found in O. brachyantha; (2) homology sequences of CentO and CRR of O. sativa (AA genome) were detected on all centromeres of O. glaberrima (AA genome), O. punctata (BB genome) and O. australiensis (EE genome); And (3) the two somatic chromosomes of O. officinalis comprised of homology sequences of CentO satellites as revealed FISH analysis probed with RCS2. Homology sequences of CRR of O. sativa were also detected on all centromeres of O. officinalis. The results provided a foundation toward cloning the centromeric sequences from different genomes of genus Oryza, studying centromere organization and evolution of different genome, analyzing the relationship between centromeric structure and function among different genome.