江苏省水稻品质性状遗传和重要基因克隆研究进展

水稻(Oryza sativa L.)是保障我国粮食安全和农业可持续发展的主粮作物,江苏是我国重要的水稻种植区。经过40多年的发展,江苏水稻产量和品质性状研究都取得了重要进展。稻米品质性状组成复杂,涉及生产、加工、销售和消费多方面。本文对江苏省水稻几个主要品质性状的遗传研究历程进行了概括,重点对水稻外观品质、蒸煮食味品质和营养品质的遗传调控研究进行了总结。文章介绍了稻米品质性状的经典遗传规律和分子遗传特点,综述了各个品质性状遗传调控相关的重要功能基因。相关研究成果凸显了江苏水稻品质遗传改良基础研究在国内外的重要地位。特别指出,随着水稻功能基因组学的发展和品质性状遗传调控网络的明晰,未来针对特定或复合品质性状的分子设计育种是江苏水稻品质遗传改良研究中要重点推进的方向。

江苏省植物细胞遗传学研究回顾与展望

20世纪初“遗传的染色体学说”的提出和证明标志着细胞遗传学交叉学科建立,伴随相关学科的发展,20世纪60年代末期细胞遗传学又与分子遗传学相结合,建立发展了分子细胞遗传学交叉学科。分子细胞遗传学以DNA分子原位杂交技术为核心,不断拓展应用领域,为生命科学研究提供了直观、高效的技术手段。原位杂交技术与基因组、细胞生物学等技术结合,被广泛应用于人类、动物、植物的起源、进化、驯化等基础研究和远缘杂交、染色体工程等应用研究。通过形象地展示DNA、RNA、蛋白质在细胞中的实际位置,揭示DNA序列之间的实际位置和顺序、亲缘物种间的进化关系和结构重排、基因组拼接序列的质量、转录水平RNA和翻译水平蛋白质的位置和数量变化等。江苏省遗传学会会员单位南京农业大学、扬州大学、南京林业大学、江苏师范大学、徐淮地区农科院等自20世纪中期开展细胞遗传学理论技术研究,伴随学科发展不断创新,建立了较完善的分子细胞遗传技术体系,并成功应用于开展植物系统进化、远缘杂交、染色体工程、基因组学等研究,取得了一批研究成果。本文将主要综述江苏省在该领域取得的重要进展,并展望未来发展方向。

Mapping QTLs for Heat Tolerance in Rice (Oryza sativa L. ) at Heading Stage Using Chromosome Segment Substitution Lines

In the present study, a japonica rice （ Oryza sativa L. ） variety Nipponbare, an indica variety 9311 and a set of chromosome segment substitution lines （CSSLs） which were generated using Nipponbare as the recipient parent and 9311 as the donor parent were used as the experimental materials. The CSSLs were grown in 2012 （normal temperature condition） and 2013 （high temperature condition） in Yangzhou, Jiangsu, and were used to map the quantitative trait loci （QTLs） for heat tolerance, based on the heat tolerance index [ （The seed setting rate under normal temperature condition -The seed setting rate under high temper- ature condition） / The seed setting rate under normal temperature condition]. As a result, three QTLs related to heat tolerance in rice were mapped on chromo- somes 2, 4 and 12, respectively. They had LOD （logarithm of rntds） scores of 2.56, 4.02 and 2.79, and contributian rates of 4.95%, 7.99% and 5.44%. Among them, qHT12.1 showed positive effect, while qHT2.1 and qHT4. t showed negative effect on heat tolerance. The results lay a foundation for the fine mapping and cloning of the QTLs and genes related to heat tolerance, and for the breeding of heat-tolerant rice varieties.

Wx^lv、the Ancestral Allele of Rice Waxy Gene

In rice grains,the Waxy (Wx) gene is responsible for the synthesis of amylose,the most important determinant for eating and cooking quality.The effects of several Wx alleles on amylose content and the taste of cooked rice have been elucidated.However,the relationship between artificial selection and the evolution of various Wx alleles as well as their distribution remain unclear.Here we report the identification of an ancestral allele,Wx^lv,which dramatically affects the mouthfeel of rice grains by modulating the size of amylose molecules.We demonstrated that WF originated directly from wild rice,and the three major Wx alleles in cultivated rice (Wx^b,Wx^a,and Wx^in) differentiated after the substitution of one base pair at the functional sites.These data indicate that the Wx^lv allele played an important role in artificial selection and domestication.The findings also shed light on the evolution of various Wx alleles,which have greatly contributed to improving the eating and cooking quality of rice.

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.)

Gibberellin （GA） 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs （GA3 or GA4） can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs （GA12 and/or GA53） as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.

A rare Waxy allele coordinately improves rice eating and cooking quality and grain transparency

In rice(Oryza sativa), amylose content(AC) is the major factor that determines eating and cooking quality(ECQ). The diversity in AC is largely attributed to natural allelic variation at the Waxy(Wx)locus. Here we identified a rare Wx allele, Wx^(mw) ,which combines a favorable AC, improved ECQ and grain transparency. Based on a phylogenetic analysis of Wx genomic sequences from 370 rice accessions, we speculated that Wx^(mw) may have derived from recombination between two important natural Wx alleles, Wx^(in) and Wx^(b). We validated the effects of Wx^(mw) on rice grain quality using both transgenic lines and near-isogenic lines(NILs). When introgressed into the japonica Nipponbare(NIP) background, Wx^(mw) resulted in a moderate AC that was intermediate between that of NILs carrying the Wx^(b)allele and NILs with the Wx^(mp) allele. Notably, mature grains of NILs fixed for Wx^(mw) had an improved transparent endosperm relative to soft rice. Further, we introduced Wx^(mw) into a high-yielding japonica cultivar via molecular marker-assisted selection: the introgressed lines exhibited clear improvements in ECQ and endosperm transparency. Our results suggest that Wx^(mw) is a promising allele to improve grain quality, especially ECQ and grain transparency of high-yielding japonica cultivars, in rice breeding programs.

A Pyramid Breeding of Eight Grain-yield Related Quantitative Trait Loci Based on Marker-assistant and Phenotype Selection in Rice(Oryza sativa L.)

1000-Grain weight and spikelet number per panicle are two important components for rice grain yield. In our previous study, eight quantitative trait loci （QTLs） conferring spikelet number per panicle and 1000-grain weight were mapped through sequencing-based genotyping of 150 rice recombinant inbred lines （RILs）. In this study, we validated the effects of four QTLs from Nipponbare using chromosome segment substitution lines （CSSLs）, and pyramided eight grain yield related QTLs. The new lines containing the eight QTLs with positive effects showed increased panicle and spikelet size as compared with the parent variety 93-11. We further proposed a novel pyramid breeding scheme based on marker-assistant and phenotype selection （MAPS）. This scheme allowed pyramiding of as many as 24 QTLs at a single hybridization without massive cross work. This study provided insights into the molecular basis of rice grain yield for direct wealth for high-yielding rice breeding.

ELE restrains empty glumes from developing into lemmas

Although there is evident homology among reproductive organs when comparing Poaceae （grass） and eudicots, the identity of grass specific organs, such as lodicules, palea, lemma, and glumes has been the subject of a vast and largely inconclusive discussion. Here we provide some direct evidence to support the idea that the empty glumes of rice （Oryza sativa） are counterparts of lemmas. We show that the development of empty glumes is regulated by ELE （elongated empty glume）, which belongs to a plant specific novel gene family. Mutations at the ELE locus cause elongated empty glumes, which mimic the lemmas and have the epidermal morphology of lemmas with four or five vascular bundles. As a nuclear-localized gene, ELE is specifically expressed at the empty glumes of immature spikelets, and its ectopic expression causes many floral development defects, including lemma-like palea, extra palea-like structures, elongated lodicules, extra stamens and stigmas. Our result suggests that empty glumes are lemmas of the sterile florets located at the lateral side of the rice spikelet, and ELE acts as a regulator restraining its growth to maintain its small size in wild-type plants.

Development and characterization of synthetic amphiploids of Oryza sativa and Oryza latifolia

Oryza sativa and Oryza latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, amphiploids were obtained from the tube seedlings of O. sativa 9 O. latifolia F1 hybrids by treatment with colchicine, an agent for chromosome doubling. Subsequently, amphiploids were investigated using the methods of morphology, genomic in situ hybridization, and molecular markers. Amphiploids were characterized by a shorter plant height, larger diameter of stem, longer and wider leaves, darker leaf color, decreased spikelets per panicle and panicle length, and larger spikelets and anthers than the original F1 hybrid. Based on the mitotic metaphase chromosome number of the investigated root tips, the somatic chromosome number of the amphiploid is 2n = 72.Additionally, the amphiploid is an allohexaploid, and its genomic constitution is AACCDD by genomic in situ hybridization analysis. Finally, the amphiploids were identified to be true using 37 polymorphic markers at the DNA level.

Two sequence alterations,a 136 bp InDel and an A/C polymorphic site,in the S5 locus are associated with spikelet fertility of indica-japonica hybrid in rice

The rice indica/japonica hybrid shows strong heterosis.However,such inter-subspecific hybrid can＇t be directly used in rice production due to its low spikelet fertility.The S5 locus was proved to be associated with fertility of indica/japonica hybrid and its S5n allele from wide-compatibility variety （WCV） is capable to overcome fertility barrier.In the present study,we reported the causal sites in the S5 locus responsible for compatibility of indica/japonica hybrid.Fine-mapping of the S5 locus using the 11 test-cross families pinpoints a candidate S5 locus encoding aspartic protease （Asp）.Intragenic recombination within the Asp gene happened in a number of recombinants,resulting in chimeric S5j-S5n alleles.Just like S5n,the chimeric S5j-S5n allele displayed higher spikelet fertility when combined with the S5i allele.In the complementary test,however,the S5n allele from WCVs failed to enhance fertilities of the indica/japonica hybrids.Compared to both indica and japonica varieties,all nine WCVs from different resources are characterized with a 136 bp deletion in the Asp N-terminus,which probably renders the S5n allele non-functional.Furthermore,an A/C polymorphic site is detected 1,233 bp downstream of the Asp start codon.The heterozygous A/C site of the Asp gene in indica/japonica hybrid is believed to be the casual factor to cause partial sterility.The functional makers based on the two polymorphic sites will be broadly used in developing wide-compatibility rice varieties.

DsMTOPVIB Promotes Meiotic DNA Double-Strand Break Formation in Rice

Dear Editor,Meiotic recombination is initiated by the generation of DNA double-strand breaks （DSBs）, which are catalyzed by the Spo11 protein （de Massy, 2013）. Two key findings point to the role of Spo11 as the initiator of DSB formation in budding yeast： （1） Spo11 is linked to the 5＇ termini of the broken DNA molecules in rad50s mutants, whose meiosis has been blocked at the stage of the cleavaqe reaction （＇Keenev et al., 1997）.

Isolation and Identification of a Functional Centromere Element in the Wild Rice Species Oryza granulata with the GG Genome

The centromere of eukaryotic chromosomes is the crucial locus responsible for sister chromatid cohesion and for correct segregation of chromosomes to daughter cells during cell division. In the structural genomics era, centromeres represent the last frontiers of higher eukaryotic genomes because of their densely methylated, highly repetitive and, heterochromatic DNA （Hall et al., 2004）. Although these functions are conserved among all eukaryotes, centromeric DNA sequences are evolving rapidly （Jiang et al., 2003）.

Microdissection and amplification of the chromosome arm 5S in a rice telo-tetrasomic

The rice aneuploids with telochromosomes are ideal genetic stocks for chromosome arm identification and microdissection. A rice telo-tetrasomic line with two extra short arms of chromosome 5 (5S) was used in the present study. The arms of 5S were microdissected from the prometaphase cells in mitosis and amplified with linker adaptor PCR (LA-PCR). The amplified fragments, ranging from 200-3000 bp, were confirmed to be from the rice chromosome arm 5S by the rice STS and microsatellite markers.