Deployment of salt tolerant cultivars is an effective approach to minimize yield loss in a saline soil. In soybean, Glycine max (L.) Merr., substantial genetic variation exists for salt response. However, breeding for salt tolerance is hampered because no economically viable screening method has been developed for practical breeding. To facilitate the development of an effective screening method for salt tolerance in soybean, the present study was conducted to determine the heritability of salt tolerance and to identify associated quantitative trait loci (QTL). F2:5 lines from the cross of 'S-100' (salt tolerant) x 'Tokyo' (salt sensitive) were evaluated in a saline field in Hyde County, N.C., USA, in 1999 and in a greenhouse located in Raleigh, N.C., USA, in 2001. S-100 and Tokyo are ancestors of popular soybean cultivars released for the southern USA. The visual salt tolerance ratings of the F2:5 lines ranged from 0 (complete death) to 5 (normal healthy appearance). The entry-mean heritability for salt tolerance was 0.85, 0.48, and 0.57 in the field (four replications), greenhouse (two replications), and combined environments, respectively. The genotypic correlation between field and greenhouse ratings was 0.55, indicating reasonably good agreement between the two screening environments. To identify QTL associated with salt tolerance, each line was characterized with RFLP markers and an initial QTL single-factor analysis was completed. These results were used to identify genomic regions associated with the trait and to saturate the selected genomic regions with SSR markers to improve mapping precision. Subsequently, a major QTL for salt tolerance was discovered near the Sat_091 SSR marker on linkage group (LG) N, accounting for 41, 60, and 79% of the total genetic variation for salt tolerance in the field, greenhouse, and combined environments, respectively. The QTL allele associated with tolerance was derived from S-100. Pedigree tracking was used to examine the association between the salt tolerance QTL and flanking SSR marker alleles in U.S. cultivars descended from S-100 or Tokyo through 60 years of breeding. The presence of alleles from S-100 at the Sat_091 and Satt237 marker loci was always associated with salt tolerance in descendants. Alleles from Tokyo for these same markers were generally associated with salt sensitivity in descendent cultivars. The strong relationship between the SSR marker alleles and salt tolerance suggests that these markers could be used for marker-assisted selection in commercial breeding.

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

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Drought stress adversely affects soybean at various developmental stages, which collectively results in yield reduction. Unpredictable rainfall has been reported to contribute about 36% to variation of yield difference between the rain-fed and irrigated fields. Among the drought resistance mechanisms, drought avoidance in genotypes with fibrous roots was recognized to be associated with drought resistance in soybean. Plant introduction PI416937 was shown to possess fibrous roots and has been used as a parent in breeding programs to improve soybean productivity. Little information is available on relative contribution and chromosomal location of quantitative trait loci (QTL) conditioning fibrous roots in soybean. To identify the genomic locations and genetic bases of this trait, a recombinant inbred line population was derived from a cross between PI416937 and 'Benning'. To detect associated QTLs, phenotypic data were collected and analyzed for 2 years under rain-fed field conditions. The selective genotyping approach was used to reduce the costs and work associated with conducting the QTL analysis. A total of five QTLs were identified on chromosomes Gm01 (Satt383), Gm03 (Satt339), Gm04 (Sct_191), Gm08 (Satt429), and Gm20 (Sat_299), and together explained 51% of the variation in root score. Detected QTLs were co-localized with QTLs related to root morphology, suggesting that fibrous roots QTL may be associated with other morpho-physiological traits and seed yield in soybean. Genetic dissection of the fibrous roots trait at the individual marker loci will allow for marker-assisted selection to develop soybean genotypes with enhanced levels of fibrous roots.

Evaluation of turfgrass salt tolerance is a basic strategy for selecting grasses that can be grown in areas with salt-affected water or soils. Our objectives were to determine the relative salinity tolerances of 32 grasses and to evaluate potential shoot-based criteria for assessing salinity tolerance. Shoot growth responses to salinity of 28 seashore paspalums (Paspalum vaginatum Swartz) and four bermudagrass [Cynodon dactylon (L.) × C. transvalensis Burtt-Davy] cultivars were investigated under solution/sand culture in a greenhouse. Turfgrasses were grown in a sea-salt amended nutrient solution. Salinity ranges were 1.1 to 41.1 dS·m-1 based on electrical conductivity of the solution (ECw). Selection criteria to assess salt tolerance were absolute growth at 1.1 (ECw0), 24.8 (ECw24), 33.1 (ECw32), and 41.1 dS·m-1 (ECw40); threshold ECw; ECw for 25% and 50% growth reduction based on ECw0 growth; and leaf firing (LF) at ECw0 and ECw40 (LF0 and LF40, respectively). Significant variations among 32 entries were observed for all shoot responses except threshold ECw. Ranges of values for shoot parameters were: inherent growth at ECw0 = 0.10 to 0.98 g dry weight (10-fold difference); growth at 24.8 dS·m-1 = 0.11 to 0.64 g; growth at 33.1 dS·m-1 = 0.09 to 0.54 g; growth at 41.4 dS·m-1 = 0.06 to 0.35 g; threshold ECW = 3.9 to 12.3 dS·m-1; ECw25 % = 14 to 38 dS·m-1; ECw50% = 22 to 43 dS·m-1; and LF40 = 7% to 41%. Results in this study indicated substantial genetic-based variation in salt tolerance within seashore paspalums. When evaluation of salt tolerance based on shoot responses is attempted at wide salinity levels up to 40 dS·m-1, all seven criteria exhibiting a significant F test can be used. Five entries (SI 92, SI 93-1, SI 91, SI 93-2, SI 89) were ranked in the top statistical grouping for all seven-growth parameters, followed by SI 90 ranked in six out of seven, and three paspalums (SI 94-1, `Sea Isle 1', and `Taliaferro') were ranked in five out of seven categories.

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