A Cleaved Amplified Polymorphic Sequence Marker to Detect Variation in Wx Locus Conditioning Translucent Endosperm in Rice

The translucent endosperm trait in a japonica rice variety ＇Kantou 194＇ is controlled by a Wx-mq gene which is allelic to Wx locus by genetic analysis and allelic test. The amylose content analysis showed that an intermediate amylose content between those of glutinous and non-glutinous rice existed in endosperm of homozygous Wx-mq genotype. The slight changes of amylose content in different varieties and F1 grains with an identical Wx-mq genotype might be influenced by dissimilar genetic background. To identify the Wx-mq genotype simply and rapidly, a cleaved amplified polymorphic sequence （CAPS） marker was designed. The result from the molecular detection indicated that it could be used for marker-assisted selection for low amylose content varieties in rice breeding.

The Mining of Citrus EST-SNP and Its Application in Cultivar Discrimination

Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The objective of this study was to identify SNP markers useful for discrimination of citrus cultivars, since large numbers of expressed sequence tags （ESTs） of sweet orange are available from the National Center for Biotechnology Information （NCBI）. We now have the opportunity to discover SNP markers suitable for determining the haplotypes with which to distinguish very closely related cultivars and to assess genetic diversity within or between related species of citrus. SNPs and small insertions/deletions （Indels） from ESTs of sweet orange and satsuma were identified by the in silico SNP discovery strategy. 55 296 EST sequences of sweet orange and 2 575 of satsuma retrieved from the NCBI repository were mined for potential SNPs. Cleaved amplified polymorphic sequences （CAPS） and sequencing approaches were used to validate putative SNPs in a sample of 30 citrus accessions. A total of 3 348 putative SNPs were identified based on the abundance of sequences and haplotype cosegregation. Of these 3 348 SNPs, the transitions, transversions and Indels ratios were 47.9, 36.1 and 16.0%, respectively. The SNPs occurred on average at a frequency of 1 per 164 bp in the coding region of citrus. 14 SNPs were randomly selected and genotyped according to 30 citrus accessions including 23 accessions of sweet orange; 11 SNPs displayed polymorphism with an average polymorphism information content （PIC） of 0.20 among 30 citrus accessions. The genetic diversity present in sweet orange was low, so the 14 SNP markers failed to discriminate different cultivars of sweet orange, but they did succeed in distinguishing accessions of inter-species of citrus. In this study, SNPs were mined from EST sequences of sweet orange and satsuma, which displayed potential capability as molecular markers to discriminate inter-species accessions of citrus. It is anticipated that these putative SNPs could be applied in citrus genetics research and breeding.

Conidia of one Fusarium solani isolate from a soybean-production field enable to be virulent to soybean and make soybean seedlings wilted

Fusarium is usually thought to cause soybean root rot, which results in a large quantity of annual yield loss in soybean production, by its secretions including Fusarium toxins and cell wall degrading enzymes, but not by the conidia themselves that do not underlie any virulence so far. Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone. We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis. Of them, except for one isolate that showed diversity of virulence to different soybeans （virulent to one soybean whereas avirulent to another soybean）, the others were all virulent to the two tested soybeans： both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection, and their virulence had dosage effects that only conidia cultures of at least 5×10^6 conidia mL-1 could show virulence to soybean; however, the sole conidia of the F. solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted. Finally, we developed the specific cleaved amplified polymorphic sequences （CAPS） markers to easily differentiate Fusarium isolates. The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.

Genetic Polymorphism of Wx Gene and Its Correlation with Main Grain Quality Characteristics in Rice

The allelic variation of the Wx gene in 50 non-glutinous rice varieties （lines） was analyzed by using the microsatellite marker RM190 [for （CT）n simple sequence repeat （SSR）] and cleaved amplified polymorphic sequence（CAPS） marker 484/W2R-ACCⅠ[for G/T single nucleotide polymorphism （SNP）]. Six homozygous （CT）n types, namely （CT）20, （CT）19, （CT）18, （CT）17, （CT）16, （CT）14, （CT）11 and （CT）10, and a heterozygous genotype （CT）11/（CT）18 were detected for RM190, of which （CT）11 and （CT）18 were predominant. Two homozygous Wx genotypes （G/G and T/T） and one heterozygous （G/T） were detected using 484/W2R-ACC Ⅰ. Most of the materials with a RM190 of （CT）11 were G/G for SNP of 484/W2R-ACC Ⅰ, while T/T for SNP was predominantly appeared in materials with （CT）18. The materials tested could be grouped into 10 categories using the two markers together. Results indicated that 59.3% variance of amylose content was attributed to the polymorphism of Wx gene revealed by RM190, while 56.1% and 24.6% of the variances in amylose content and gel consistency were respectively to the polymorphism of Wx gene revealed by 484/W2R-ACC Ⅰ. Furthermore, with both SSR and CAPS markers, 72.4% of the variance in amylose content could be explained. In addition, the application prospects of the two markers in breeding were also discussed.

Functional Marker Development and Effect Analysis of Grain Size Gene GW2 in Extreme Grain Size Germplasm in Rice

GW2 is an important gene that regulates grain width and weight. We used cDNA clone to obtain the sequences of GW2 from large- and small-grained rice varieties, TD70 and Kasalath, respectively. Then, we developed a dCAPS （derived cleaved amplified polymorphic sequence） marker on the basis of the sequence difference between functional and nonfunctional GW2 genes to analyze the genotypes and phenotypes of recombinant inbred lines. Results showed that the sequence of GW2To7~ had a single nucleotide deletion at site 316 that generates a termination codon. This codon terminated the GW2 protein in advance. By contrast, the sequence of GW2Kasalath encoded an intact protein. A novel dCAPS marker was designed in accordance with a base A deletion at site 316 of the sequence. After the PCR product was digested by Apol, TD70 showed 21 and 30 bp fragments, and Kasalath showed a 51 bp fragment. Up to 82 lines contained GW2TDTO, and 158 lines contained GW2Kasalath. The lines that contained TD70 alleles displayed substantial increases in width and 1000-grain weight. This result suggested that GW2 played a critical role in rice breeding.