Analysis of digenic epistatic effects and QE interaction effects QTL controlling grain weight in rice

Immortalized F2 population of rice (Oryza sativa L.) was developed by randomly mating F1 among recombinant inbred (RI) lines derived from (Zhenshan 97B×Minghui 63),which allowed replications within and across environments.QTL (quantitative trait loci) mapping analysis on kilo-grain weight of immortalized F2 population was performed by using newly developed software for QTL mapping,QTL Mapper 2.0. Eleven distinctly digenic epistatic loci included a total of 15 QTL were located on eight chromosomes.QTL main effects of additive,dominance,and additive×additive,additive×dominance,and dominance×dominance interactions were estimated.Interaction effects between QTL main effects and environments (QE) were predicted.Less than 40% of single effects,most of which were additive effects,for identified QTL were significant at 5% level.The directional difference for QTL main effects suggested that these QTL were distributed in parents in the repulsion phase.This should make it feasible to improve kilo-grain weight of both parents by selecting appropriate new recombinants. Only few of the QE interaction effects were significant.Application prospect for QTL mapping achievements in genetic breeding was discussed.

Genetic Diversity of Chinese and Swedish Rapeseed (Brassica napus L.) Analyzed by Inter-Simple Sequence Repeats (ISSRs)

We have compared genetic diversity of 24 Chinese weak-winter, Swedish winter and spring B. napus accessions by inter-simple sequence repeats (ISSRs). By cluster analysis (UPGMA) based on 125 polymorphism bands amplified with 20 primers, the 24 accessions were divided into three groups. Six Swedish winter lines and eight Chinese weak-winter lines were in the group I and the groupⅡwere two Chinese weak-winter lines XiangyoulS and Bao81. The third group contained eight Swedish spring lines. Principal co-ordinates analysis (PCO) showed similar groupings to cluster analysis. Results from cluster analysis and PCO analysis showed very clearly that Chinese weak-winter, Swedish spring and winter accessions were distinguished from each other and Chinese weak-winter accessions in this study were genetically closer to Swedish winter accessions than to Swedish spring accessions. The Chinese weak-winter accessions had larger diversity than Swedish spring or winter accessions did. This study indicated that ISSR is a suitable and effective tool to evaluate genetic diversity among rapeseed germplasm.

Relationship Between Hybrid Performance and Genetic Diversity Based on SSRs and ISSRs in Brassica napus L.

To investigate the relationship between genetic distance (GD) and hybrid performance, two types of molecular markers, microsatellites (simple sequence repeats, SSRs) and intro-simple sequence repeats (ISSRs), were employed to detect the genetic diversity of 3 double low self-incompatible lines and 22 male parental varieties of Brassica napus from different geographical origins. Hybrids were produced in a NCⅡ mating design by hand-pollination. The result indicated that 25 parental varieties (lines) could be divided into six groups by Un-weighted Pair Group Mathematics Average (UPGMA) clustering based on GDs. SI-1300 and SI-1320 could be singly clustered into one group, respectively. Varieties from China could be separated into another group, SI-1310 and varieties from foreign countries could be separated into other three groups. The grouping was generally consistent with parental pedigrees and geographical origins. Significant differences in yield, quality and phenological period traits were observed among these parent groups. Although hybrid yield/plant showed significantly positive correlation with genetic distance based on SSR and ISSR markers, but the determination coefficient was low. It appeared to be unsuitable for using the genetic distance based on SSR and ISSR markers to predict heterosis and hybrid performance in Brassica napus.

Phylogenetic Analysis on SLG and SRK Genes in Brassica and Raphanus

S-locus genes were cloned from three Brassica napus and three B. campestris lines by using PCR walking and homologuesequence methods. A phylogenetic gene tree was constructed based on the six cloned genes and fifty-one previouslyreported SLG/SRK genes of Brassica and Raphanus. The SLGs from R. sativus were dispersed in the phylogenetic treeintermingling with SLG/SRKs from B. oleracea, B. napus and B. campestris. The SLG/SRK genes of classⅡclusteredindependently in one group. The SLG/SRK genes of classⅠshowed to be more divergent than classⅡgenes. Theseresults suggested that the divergence of classⅠand classⅡ should have occurred before the differentiation of thegenera Brassica and Raphanus. In addition, SLG and SRK of the same S haplotypes belonged to the same class. Itsuggested that class-Ⅰ and class-Ⅱ group divergence occurred first, and then SLG and SRK diverged. The three SC SRKgenes from B. napus and B. campestris were grouped into one cluster, displaying difference from the SC SLG of B.oleracea. These three SC SRK genes were close to SI SRK of SI1300, SI271 and guanyou in phylogenetic relationships.These results indicated that SC and SI genes diverged more recently. It is not clear yet whether the differentiation of SCand SI genes was earlier than the differentiation of Brassica and Raphanus. Studies based on more genes are necessaryfor a comprehensive elucidation of the phylogenetic relationships in Brassicaceae.

Analysis of SSR in Citrus Sequences from EMBL Database

Abundance of simple sequence repeat (SSR) in Citrus sequences from EMBL database was investigated by usingcomputer program MISA (MIcroSAtellite), which aimed to provide useful information for the development of SSR markers.Among 32 896 sequences of Citrus, 4 987 SSRs were found in 4 167 sequences and the average distance between SSRs wasapproximately 3.5 kb. Mononucleotide repeats (50.6%) were the most abundant repeats. And di-, tri-, tetra-, penta- andhexa-nucleotide repeats were 22.8, 25.2, 1, 0.08, and 0.36%, respectively. The most abundant motif was A/T followed indescending order by AG/CT, AC/GT, AT/TA. AAT/ATT, AAG/CTT, AGC/CGT, ACG/CTG and C/G. They comprised about90% of all microsatellites. Ten primer pairs were designed, and three of them produced clear visible bands among Citrusand its related genera.

Genetic Transformation of Brassica campestris L.ssp.pekinensis via Agrobacterium-with Bt Gene

By means of Agrobacterium-mediated transformation, 43 kanamycin-resistant buds of Chinese cabbage were got. PCR, PCR-Southern blot and dot blot analysis were used to identify and characterize the putative transgenic plants. 26 plants had the predicted bands of the fragment of npt Ⅱ gene. Insect bioassays of 4 transformants showed that toxic protein had been translated and the translation levels were different among these transformants.

Construction and characterization of a normalized whole-life-cycle cDNA library of rice

A cDNA library with genomic complete cover-age is a powerful tool for functional genomic studies. For studying the functions of rice genes on a large scale, a nor-malized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63, an elite restorer line for a number of rice hybrids that are widely cultivated in China. This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages. For normalization, the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA la-beled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library. This library consists of 62000 clones with an average insert length about 1.4 kb. Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes. Sequencing of 10750 cDNA clones of this library reveals 6399 unique ESTs (expressed sequence tags), indicating that the non-redundancy of the library is about 59.5%. This library has been used to make cDNA mi-croarrays for functional genomic studies.

Transfer of bacterial blight resistance from Oryza meyeriana to O. sativa L. by asymmetric somatic hybridization

Asymmetric somatic hybrid plants were pro-duced between cultivated rice (Oryza sativa L.) and wild spe-cies [O. meyeriana (Zoll. etMor. exSteud.)] with high resis-tance to rice bacterial blight. X-ray-irradiated protoplasts of the wild species were used as donor and chemically fused with iodoacetamide-inactivated protoplasts of rice cv. 02428 to produce hybrids. Seventy-two plants were regenerated from 623 calli based on metabolic complementation. The morphological characters of the plants closely resembled that of the rice. Simple sequence repeats were employed to iden-tify their hybridity. Cytological analysis of root-tips revealed that their chromosome number varied in the range of 27— 38. The somatic hybrids were inoculated with strains of Xanthamonas oryzae pv. oryzae at adult growth stage and demonstrated the resistance to bacterial blight introgression from the O. meyeriana.