Читать книгу Cucurbits - James R. Myers - Страница 41

Speed breeding has advanced as new technologies have emerged. Doubled haploid production has made it faster to produce inbred lines, although it is sometimes difficult to produce enough dihaploids for each cross that plant breeders are interested in evaluating. Often, hybrids are produced before the parental lines have been thoroughly evaluated. In that way, the best hybrids are quickly available when the data from field and greenhouse trials have identified those that should be advanced to the next stage of breeding. In the same way, lines are used to start new crosses before they have been thoroughly evaluated. Seedling evaluations using molecular markers to select individuals with traits of interest are also decreasing generation times.

Numerous systems to detect variability in plant DNA have been developed and used to facilitate genetic mapping. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) are two of the most common molecular marker systems used for generating genetic maps currently. One use of genetic mapping is for the identification of molecular markers associated with traits of interest (high yield, fruit quality, disease resistance). Linkage between a molecular marker and a trait of interest allows one to identify the presence of the trait by checking the genotype at the marker locus. This testing can occur as early as the seed stage, allowing for rapid selection of plants that have the trait of interest. In cucumber, watermelon, squash and melon, markers are available to select for improved fruit quality and disease resistance.

In cases where simply inherited sources of disease resistance have not been found (for example, resistance to CMV in melon and squash), genetic engineering and transfer of the coat protein or replicase genes of the virus to cucurbits has given these cucurbits disease resistance. For example, Chee and Slightom (1991) developed the coat protein-mediated mechanism for CMV protection in cucumber. They used a disarmed strain of Agrobacterium tumefaciens to transfer the engineered viral coat protein to cucumber. Small pieces of young, growing cotyledon tissue were soaked in a solution containing the recombinant DNA-carrying bacterial plasmids. The transformed embryogenic calli were regenerated and the resulting plants were inoculated and shown to have resistance to CMV. Gonsalves et al. (1992) went on to prove that the transgenic plants had a high level of CMV resistance under natural field conditions, where viral exposure is usually via insect vectors.

Similar field performance experiments have been conducted for transgenic squash lines (C. pepo). The virus-susceptible hybrid ‘Pavo’ was compared with two transgenic selections from this cultivar that possessed CMV and WMV resistance (Arce-Ochoa et al., 1995). Under field conditions in Texas, the two transgenic selections had only 3% and 14% symptomatic plants, compared with 53% for ‘Pavo’.

In the USA, the first commercially available transgenic cucurbit was ‘Freedom II’ (previously called ‘ZW-20’), a yellow summer squash of the crookneck type (C. pepo). This cultivar was engineered with resistance to WMV and ZYMV. Again, A. tumefaciens was used, this time to transfer the coat protein genes of WMV and ZYMV to crookneck squash. Since the introduction of ‘Freedom II’ additional cultivars have been released, including ‘Liberator III’ and ‘Independence II’.

More recently, gene editing techniques have gained popularity. The clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein system is universally recognized as a powerful genome editing tool. In brief, CRISPR is an RNA-guided editing tool that uses a short (21–72 nucleotides) seed sequence flanked by palindromic repeats to recognize and cleave a specific sequence of DNA. Unlike genetic engineering, where genes are inserted at random, genome editing allowed for specific changes with no to few off-target effects or foreign genes remaining in the new cultivar. CRISPR represents a highly efficient, low-cost genome editing tool and is now being developed for cucurbits. While the CAS9 system is the most common, multiple CRISPR systems are being developed to fully utilize this technology. In cucumber, CRISPR has been used to incorporate resistance to zucchini yellow mosaic virus, papaya ringspot mosaic virus-W and cucumber vein yellowing virus into a non-transgenic plant (Chandrasekaran et al., 2016). In watermelon, a CRISPR system has been tested using phytoene desaturase, but new quality traits have not yet been successfully incorporated (Tian et al., 2017).