Anther Culture, QTL Analyses Of Orange Flesh And Mapping Of Carotenoid Biosynthesis Genes In Cucumber (Cucumis Sativus L.)

Cucumber (Cucumis sativus L.; 2n= 2x= 14) is top ten important vegetable crops grown wildly in the world. Limited by the narrow genetic base of cucumber and the time consuming of conventional breeding ways, more and more emphases have been focused on the haploid breeding and marker-assisted selection (MAS).1 Anther culture in cucumber (Cucumis sativus L.).Haploid and double haploid (DH) plants could be achieved via anther culture technique, but to our knowledge, only three reports have been published on anther culture in cucumber, and low anther culture efficiency (1.6 embryos/anther and 52 haploid/60 anther) has limited its application. To develop an efficient cucumber(Cucumis sativus L.,2n= 2x= 14) anther culture protocol, six experiments (including pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotypic effects) were conducted.The best medium for embryonic callus induction was MS medium supplemented with 1.0 mg/l 6-BA,0.5 mg/l2,4-D,1.0 mg/l KT,3% sucrose and 0.8% agar. For embryo induction was MS medium supplemented with 0.1 mg/1 NAA,3.0 mg/l 6-BA,3%sucrose and 0.8% agar, and for embryo germination was MS medium containing 0.5 mg/16-BA,6% sucrose and 1.2% agar. The results indicated that pretreatment and embryo induction were key factors for successful anther culture.Suitable temperature stress depended on the ecotype, i.e.,cucumbers from cold areas responded well to cold shock whereas those from temperate areas responded well to heat treatment.6-BA/NAA ratio was important for embryo induction in anther culture.Using this protocol, we produced callus from 16 genotypes and regenerated plants from three of 20 evaluated. The origin of regenerated plants were determined by cytological, morphological and AFLP analyses. Three embryos per anther and 42 DH per 45 anthers (93% success) were obtained for cv. Ningjia No.1. 2 QTL analyses of orange flesh and mapping of carotenoid biosynthesis genes in cucumber (Cucumis sativus L.).Carotenoids play an important role in human nutrition. The "Xishuangbanna gourd" (XIS; Cucumis sativus var. xishuangbannanesis Qi et Yuan; 2n= 2x= 14) develops fruit with orange-colored endocarp/mesocarp at full seed maturity, which makes this germplasm attractive to plant improvement programs interested in improving the nutritional quality of cucumber. Therefore a study was designed to identify quantitative trait loci (QTL) associated with orange color fruit mesocarp and endocarp tissue using two F2 populations derived from parental lines with white [EC1 (Deltarstar, Rijk Zwaan Seed Company, European greenhouse type) and'Gy7'(U.S. pickling type)] crossed to orange flesh [SWCC9 (Southwest Chinese type) and USDA#14 (U.S. Pickling type) derived from XIS].1) Constrction of molecular genetic mapsTwo genetic linkage maps were constructed with the molecular markers of RAPD, SCAR, SSR, EST, SNP, AFLP and SSAP. Map 1 contained 74 loci distributed in 16 linkage groups spanning 899.0cM with average distance of 12.0cM. Map 2 contained 23 loci distributed in 7 linkage groups spanning 230.9cM with average distance of 9.6cM.A common collinear region was containing four molecular markers (3 dominant and 1 codominant) on linkage group (LG) LG6 in Map 1 and LG3 in Map 2.These regions contained 3 dominance markers (RAPD_AG13₁200, RAPD_P11₇50 and SNP_N8SNPG2H3₅69) and 1 codominant marker (SNP_AB14SNPG1_H1470/519).2) QTL identification of flesh colorFour QTL associated with mesocarp color (MC, mc6.1, mc6.2, mc9.1 and mc15.1) and five QTL associated with endocarp color (EC, ec6.1, ec6.2, ec6.3, ec9.1 and ec16.1) were detected in Map1. Two QTL associated with mesocarp color (mc2.1 and mc3.1) and three QTL associated with endocarp color (ec2.1, ec3.1, and ec5.1) were identified in Map2.The QTL of flesh color, mc6.1/ec6.1 (Map1) and mc3.1/ec3.1 (Map2), were located in the common collinear region, which indicated that those QTL may be the stable QTL associated with flesh clour under the XIS background.3) QTL identification of carotene contentBiochemical analyses indicated thatβ-carotene (β) and xanthophyll (x) were the two predominant carotenoids in MC and EC tissue. In Map2, three QTL associated with theβ-carotene content of mesocarp and endocarp (mdb2.1, edb2.1 and edb3.1) were detected. Three QTL associated with the xanthophyll content of mesocarp and endocarp (mdx3.1, mdx3.2 and edx3.1) were detected.QTLs controlling the content of P-carotene in endocarp (edb3.1) and xanthophyll in mesocarp (mdx3.1) mapped to the same interval as mc3.1 and ec3.1, respectively.4) Mapping of carotenoid biosynthesis geneOne cucumber carotenoid biosynthesis gene, NCED (9-cis-Epoxycarotenoid dioxygenase), mapped to the same interval as orange flesh color QTLs (mc6.1/ec6.1 and mc3.1/ec3.1) in both maps.5) QTL identification of morphological traitsIdentification of QTLs for cotyledon length (CL), hypocotyl length (HL), node of the first branch (NFB), diameter of the main stem (DM), leaf length (LL), and leaf width (LW) in Map1, and for sugar content (S), fruit length (FL) and fruit width (FW) in Map2 were conducted. The QTL for sugar content (s3.1) mapped to the same interval as fruit color (mc3.1 and ec3.1) and the carotenoid content (edb3.1 and mdx3.1).