The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes.

The Mi locus of tomato confers resistance to root knot nematodes. Tomato DNA spanning the locus was isolated as bacterial artificial chromosome clones, and 52 kb of contiguous DNA was sequenced. Three open reading frames were identified with similarity to cloned plant disease resistance genes. Two of them, Mi-1.1 and Mi-1.2, appear to be intact genes; the third is a pseudogene. A 4-kb mRNA hybridizing with these genes is present in tomato roots. Complementation studies using cloned copies of Mi-1.1 and Mi-1.2 indicated that Mi-1.2, but not Mi-1.1, is sufficient to confer resistance to a susceptible tomato line with the progeny of transformants segregating for resistance. The cloned gene most similar to Mi-1.2 is Prf, a tomato gene required for resistance to Pseudomonas syringae. Prf and Mi-1.2 share several structural motifs, including a nucleotide binding site and a leucine-rich repeat region, that are characteristic of a family of plant proteins, including several that are required for resistance against viruses, bacteria, fungi, and now, nematodes.

The nematode resistance gene Mi of tomato confers resistance against the potato aphid.

Resistance against the aphid Macrosiphum euphorbiae previously was observed in tomato and attributed to a novel gene, designated Meu-1, tightly linked to the nematode resistance gene, Mi. Recent cloning of Mi allowed us to determine whether Meu-1 and Mi are the same gene. We show that Mi is expressed in leaves, that aphid resistance is isolate-specific, and that susceptible tomato transformed with Mi is resistant to the same aphid isolates as the original resistant lines. We conclude that Mi and Meu-1 are the same gene and that Mi mediates resistance against both aphids and nematodes, organisms belonging to different phyla. Mi is the first example of a plant resistance gene active against two such distantly related organisms. Furthermore, it is the first isolate-specific insect resistance gene to be cloned and belongs to the nucleotide-binding, leucine-rich repeat family of resistance genes.

An endochitinase gene expressed at high levels in the stylar transmitting tissue of tomatoes.

A gene (pMON9617; Chi2;1) identified by screening a tomato pistil cDNA library has been found to encode a protein similar in sequence to class II chitinases. Using a baculovirus expression system we show that the Chi2;1 protein is an active endochitinase. The Chi2;1 protein is most similar in sequence to a previously described stylar chitinase (SK2) isolated from potato. Both proteins lack the diagnostic N-terminal cysteine-rich regions and the C-terminal vacuolar targeting signals of class I chitinases and appear to define a novel type of class II endochitinases associated with flowers. Chi2;1 is expressed predominantly in floral organs and its expression within these organs is temporally regulated. The highest level of expression is found in the transmitting tissue of the style where Chi2;1 mRNA begins to accumulate just prior to anthesis. In vegetative tissue, low levels of Chi2;1 mRNA were detected, and these levels did not increase in response to wounding or treatment with ethephon. mRNA from Chi2;1 orthologs was not detected in most other angiosperms tested, even including some members of the Solanaceae, and it is therefore unlikely that Chi2;1 is essential for stylar function. A fragment containing 1.4 kilobase pairs of 5'-flanking DNA from the Chi2;1 gene was shown to drive high-level expression of an attached reporter gene in the styles of transgenic tomatoes. This fragment has utility for engineering expression of other coding regions in styles.

Nature and regulation of pistil-expressed genes in tomato.

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.

Arabidopsis floral homeotic gene BELL (BEL1) controls ovule development through negative regulation of AGAMOUS gene (AG).

Ovules are the developmental precursors of seeds. In angiosperms the ovules are enclosed within the central floral organs, the carpels. We have identified a homeotic mutation in Arabidopsis, "bell" (bel1), which causes transformation of ovule integuments into carpels. In situ hybridization analysis shows that this mutation leads to increased expression of the carpel-determining homeotic gene AGAMOUS (AG) in the mutant ovules. Introduction of a constitutively expressed AG transgene into wild-type plants causes the ovules to resemble those of bel1 mutants. We propose that the BEL1 gene product directs normal integument development, in part by suppressing AG expression in this structure. Our results allow expansion of the current model of floral organ identity to include regulation of ovule integument identity.

Estimating the potential of sugarcane families to produce elite genotypes using bivariate prediction methods.

Three bivariate statistical methods to predict the family potential to produce elite progeny were studied to improve the efficiency of a sugarcane (Saccharum spp.) breeding program. Progeny from 15 piparental crosses were evaluated in plant cane and first ratoon seedlings, and in clonal plant cane plots during 1989 and 1990. The bivariate predictions of Brix combined with cane yield components (stalk number, stalk weight, stalk diameter, stalk length, and stool weight) were investigated. The best linear unbiased predictors (BLUPs) and the sum of ranks based on family mean values of two traits (RANK) were repeatable among tests in the estimation of family potential. Bivariate normal probabilities (PROB) estimated with family means, phenotypic standard deviations, and genetic correlations generally demonstrated poor repeatability among tests. The three statistical predictions were compared with the progeny selection rate within the crosses through three selection stages. Predictions were not correlated to the selection rates of eight crosses with smaller initial progeny populations (< 500 progeny). However, when the predictions were compared with the 7 of 15 families over which 1,000 progeny for each cross had been evaluated, the rankings based on BLUP and RANK bivariate predictions of Brix and stool weight identified the better crosses. PROB was inconsistent in this regard. Early selection work is highly subjective. We speculate that near-random selection occurs for stalk number at the initial selection stage and that the high selection rate at this stage (≈5%) generates a first clonal population (10 to 25) that is too small to accurately base selection rates for stalk number. Larger initial progeny populations produce sufficiently large clonal populations (>50) to appraise crosses using selection rates. The study suggested that family evaluations for breeding programs can use bivariate predictions. The comparative ease of calculating the RANK estimate versus the BLUP along with the absence of any apparent loss of predictive value suggests that the RANK method would be the most suitable statistic to use for bivariate predictions.

Estimating the potential of sugarcane families to produce elite genotypes using univariate cross prediction methods.

A quick, accurate method to determine the potential of a sugarcane (Saccharum spp.) cross to produce elite progeny is needed for maximizing genetic gain. Development of a practical cross appraisal method was initiated by evaluating 1,800 progeny from 15 crosses among 23 parents at two intrarow plant spacings (41 cm and 82 cm). Plant spacing was examined for its affect on stool weight variability. The goals were to identify the most reliable and/or easily obtained cross appraisal statistic and to determine the earliest breeding program stage and crop to collect these statistics. Three tests, on plant cane (PC) and first ratoon (FR) single stool seedlings and clonal plant cane plots, were conducted. Four statistics, the family mean, the estimated elite proportion (PROB), the observed elite proportion, and the best linear unbiased predictor (BLUP) were estimated and examined for each cross. These statistics were strongly correlated within each test (0.69≤r≤1.00). Family worth estimates based on single stool data were moderately correlated (ca. range 0.5≤r≤0.7) to the family worth estimates based on clonal plots.The research suggested that the potential of a cross to produce elite progeny for a trait could be accurately predicted by the cross mean of that trait. Data for the mean were the most easily obtained and, hence, would be the most practical family appraisal statistic to use in a breeding program. Correlations of statistics among the PC and FR seedlings and the clonal plots showed that the PC estimates of Brix, stalk weight, and its components, stalk length and stalk diameter, could be used for cross appraisal. Genotypic selection by the Louisiana Sugarcane Variety Development Program (LSVDP) occurs among the FR seedlings. FR stalk number and PC Brix and stalk weight data could be used to perform family selection prior to the currently practiced individual plant selection. The benefits of family selection to the LSVDP were demonstrated by the expected genetic gains for two selection scenarios. The gains were consistently larger for an initial 50% family selection and subsequent 20% individual selection than they were for simple individual selection at a 10% selection intensity. Our research also suggests that the use of a wider intrarow spacing may improve the ability to discern among seedlings due to its enhancement of stool weight variability.

Sequence analysis and comparison of avocado fruit and bean abscission cellulases.

A 1700 nucleotide cDNA clone for a bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase (endo-(1,4)-beta-d-glucanase) has been identified and sequenced. This cDNA clone contains a 1485 nucleotide open reading frame which includes coding sequences for a putative signal peptide and mature protein. The nucleotide and deduced amino acid sequences for the bean abscission cellulase are compared to the previously reported sequences of an avocado fruit ripening cellulase. Optimal alignment of these sequences shows 64% and 50% identically matched nucleotides and amino acids, respectively. Analysis of the deduced amino acid sequences for the mature bean and avocado cellulases indicates that these two proteins share similar molecular weights, position of cysteine residues, and hydropathic character, but have very different isoelectric points and glycosylation. Genomic blot data suggest that the avocado fruit cellulase belongs to a small gene family, whereas the bean abscission cellulase appears to be encoded by a single gene or a few very closely related genes.

The role of pith, tube, and stalk density in determining sugarcane sucrose content and stalk weight.

Selection against pith and tube is one of the major criteria used to eliminate inferior sugarcane (Saccharum spp.) cultivars in early stages of new cultivar evaluation. Understanding the genetic relationships for these traits would facilitate crossing and selection decisions. This study was conducted to determine heritability, genetic coefficient of variation, and the potential for genetic advance by selection for pith, tube, and stalk density. Correlation and path-coefficient analysis studies were conducted to determine the effects of Brix, pith, and stalk density on sucrose content and the effects of stalk volume, tube, and pith on stalk weight. Eighty randomly selected cultivars (four progeny from each of 20 crosses), representing a first clonal stage of a Louisiana sugarcane breeding population, and their parents were planted at St. Gabriel/LA, and yield data were collected in 1986, 1987, and 1988. Pith and tube exhibited large genotype and genotype-by-year variation, whereas variation in stalk density was nonsignificant. Broad-sense and narrow-sense heritabilities were high, moderately high, and low for pith, tube, and stalk density, respectively. Path-coefficient analysis revealed that stalk volume was the major factor determining stalk weight. Tube and pith were factors that decreased stalk weight. As expected, Brix was the single most important factor determining sucrose content, however, high stalk density and low pith were also associated with high sucrose content. Sugarcane breeders should practice stringent selection for low pith across years to increase stalk weight and sucrose content. Since stalk density was effectively increased by high sucrose content and low pith, the use of stalk density in breeding and selection should be avoided. The minimal effect of tube on stalk weight suggests that its use as selection criteria be minimized or dropped.