PLANT DEVELOPMENTAL MUTANTS AS MODELS FOR PLANT DEVELOPMENTAL MUTANTS AS MODELS FOR CROP SYSTEMS BIOLOGY STUDIESCROP SYSTEMS BIOLOGY STUDIES

FACCIOLI P., FACCIOLI P., BALDASSARRE V., PAGANI D., STANCA A.M.,BALDASSARRE V., PAGANI D., STANCA A.M., TERZI V.TERZI V.CRA-CPG Genomic Research Centre, via S. Protaso 302, I-29107 Fiorenzuola d’Arda (Pc), Italy.

Crop Systems Biology is a promising approach to fulfil challenges in improving complex traits. It combines modern functional genomics and traditional approaches, such as Crop Systems Biology is a promising approach to fulfil challenges in improving complex traits. It combines modern functional genomics and traditional approaches, such as crop physiology and biochemistry, to understand phenotype at the crop level (e.g. grain yield). Data integration thus plays a fundamental role in systems-based approaches crop physiology and biochemistry, to understand phenotype at the crop level (e.g. grain yield). Data integration thus plays a fundamental role in systems-based approaches and numerous studies are underway to deal with this issue.and numerous studies are underway to deal with this issue.Our study shows an example of such a working strategy for the analysis of awn development in barley. The role of awn photosynthetic activity, particularly during stress Our study shows an example of such a working strategy for the analysis of awn development in barley. The role of awn photosynthetic activity, particularly during stress periods, on grain yield has been largely proven: as a consequence awn morphology and physiology have received quite a lot of attention from the breeders. periods, on grain yield has been largely proven: as a consequence awn morphology and physiology have received quite a lot of attention from the breeders. Here, a barley double recessive mutant named “Here, a barley double recessive mutant named “leafy lemmaleafy lemma” (” (lellel) and characterized by a modified awn shape and increased awn area has been utilized as a black box in ) and characterized by a modified awn shape and increased awn area has been utilized as a black box in comparison with its wild type counterpart, through the development of pairs of near-isogenic lines obtained by initially crossing comparison with its wild type counterpart, through the development of pairs of near-isogenic lines obtained by initially crossing lellel ( (lel1lel1lel1lel1//lel2lel2lel2lel2) to the cultivar Kaskade ) to the cultivar Kaskade ((Lel1Lel1Lel1Lel1//Lel2Lel2Lel2Lel2). Pairs of NILs were developed using a program of single seed descent. During the first generations, heterozygosity was maintained at the ). Pairs of NILs were developed using a program of single seed descent. During the first generations, heterozygosity was maintained at the Lel/lelLel/lel loci and loci and identified with progeny testing to verify segregation for the awn morphology. The program was terminated at the end of F7 generation at which 27 pairs of NILs were identified with progeny testing to verify segregation for the awn morphology. The program was terminated at the end of F7 generation at which 27 pairs of NILs were obtained. Each pair of lines thus includes a mutant and a wild type genotypes that are expected to be strongly homogeneous in most part of the genome excluding the obtained. Each pair of lines thus includes a mutant and a wild type genotypes that are expected to be strongly homogeneous in most part of the genome excluding the mutated chromosomic regions (which have been previously mapped on barley chromosomes 5 and 7, Pozzi et al., Genetics 2000, 154: 1335-1346). Each pair is also expected mutated chromosomic regions (which have been previously mapped on barley chromosomes 5 and 7, Pozzi et al., Genetics 2000, 154: 1335-1346). Each pair is also expected to be characterized by a specific combination of chromosomic regions coming randomly from one of the two parental genotypes.to be characterized by a specific combination of chromosomic regions coming randomly from one of the two parental genotypes.Currently, each pair of NILs is being studied from the genetic, morphological, physiological and agronomical points of view and comparisons are in progress both at Currently, each pair of NILs is being studied from the genetic, morphological, physiological and agronomical points of view and comparisons are in progress both at intra-pair/inter-pairs level. intra-pair/inter-pairs level. As it is evident from the photos reported below, the expressivity of the mutant alleles is strongly influenced by the underlying genome structure, i.e. which linkage blocks from As it is evident from the photos reported below, the expressivity of the mutant alleles is strongly influenced by the underlying genome structure, i.e. which linkage blocks from the two parental genotypes are present. In some pairs the awn morphology of the mutant genotype is very different from that of the wt one, while in some other pairs the the two parental genotypes are present. In some pairs the awn morphology of the mutant genotype is very different from that of the wt one, while in some other pairs the structure of the two lemmas, though distinguishable, are just slightly different.structure of the two lemmas, though distinguishable, are just slightly different.

wtwt mutantmutant

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Phenotype at crop level is the result of the interaction among many Phenotype at crop level is the result of the interaction among many genes whose expression is often dependent on environmental genes whose expression is often dependent on environmental conditions and developmental stage. Multilevel data integration thus conditions and developmental stage. Multilevel data integration thus plays a fundamental role in the understanding of many important plays a fundamental role in the understanding of many important agronomic traits. Here data integration for the study of a plant agronomic traits. Here data integration for the study of a plant developmental mutant has been used as a working strategy for the developmental mutant has been used as a working strategy for the future design of an effective virtual crop modelling. In particular, future design of an effective virtual crop modelling. In particular, the the 27 pairs of NILs may offer unique germplasm for the study of awn 27 pairs of NILs may offer unique germplasm for the study of awn development.development.

AFLP analysis of the whole set of genotypes involved in the present study was carried AFLP analysis of the whole set of genotypes involved in the present study was carried out: “mixlel” sample was obtained by bulking the out: “mixlel” sample was obtained by bulking the lellel genotypes from the 27 pairs of genotypes from the 27 pairs of lines (leaf bulking previous to DNA extraction), “mixwt” sample was obtained by bulking lines (leaf bulking previous to DNA extraction), “mixwt” sample was obtained by bulking the 27 the 27 wtwt genotypes, genotypes, lk2lk2 is the short awn mutant from which is the short awn mutant from which lellel has been obtained. Two has been obtained. Two hundred fourteen fragments from 5 AFLP primer combinations have been used for hundred fourteen fragments from 5 AFLP primer combinations have been used for obtaining the binary matrix. As expected, Kaskade is closer to the mixlel/wt cluster obtaining the binary matrix. As expected, Kaskade is closer to the mixlel/wt cluster being one of the parent from which the being one of the parent from which the lellel//wtwt pairs come from. On the basis of what it is pairs come from. On the basis of what it is reported above, it was obviously also expected to find a tight relationship between reported above, it was obviously also expected to find a tight relationship between lellel and and lk2lk2 genotypes. genotypes.

AFLP-based testing of the genome homogeneity inside each pair of lines is here AFLP-based testing of the genome homogeneity inside each pair of lines is here reported, as an example, for pairs named W97 and W612. AFLP fragments were scored reported, as an example, for pairs named W97 and W612. AFLP fragments were scored as 1 (presence) or 0 (absence) to obtain a binary matrix. The cluster analysis was as 1 (presence) or 0 (absence) to obtain a binary matrix. The cluster analysis was performed with TREECON package (Van de Peer et al., Comput. Applic. Biosci 1994, 10: performed with TREECON package (Van de Peer et al., Comput. Applic. Biosci 1994, 10: 569-570).569-570).AFLP markers (256 fragments from 6 primer combinations) were able to group each AFLP markers (256 fragments from 6 primer combinations) were able to group each mutant component of a pair with its wild type counterpart (two technical replications mutant component of a pair with its wild type counterpart (two technical replications were made for each genotype).were made for each genotype).

Rachilla hair type; anthocyanin coloration of nerves of lemma; spiculation of inner lateral nerves of lemma of dorsal side; hairiness of ventral furrow; disposition of lodicules; color of aleurone layer; lenght (cm); width (cm); weight of 100 grains; lenght of rachilla (cm).

GRAINGRAIN

Growth habit; frequency of plants with recurved flag leaves; lenght(cm); number of internode; lenght of the last internode (cm); colour of the base of the culm; seasonal type.

WHOLE WHOLE PLANTPLANT

Anthocyanin coloration of tips; intensity of anthocyanin coloration of tips, lenght(cm); lenght relative to ear; ruggedness, area.

AWNSAWNS

Flag leaf (anthocyanin coloration of auricles, intensity of anthocyanin coloration of auricles, glaucosity of sheath, leaf posture); lenght (cm); width (cm); lowest leaves (hairiness of leaf sheaths).

LEAVESLEAVES

Time of ear emergence; attitude; glaucosity; attitude at milk-waxy stage; number of rows; shape; density; compactness; lenght (cm); lenght of anther; rachis (lenght of first segment; curvature of first segment); sterile spikelet (attitude, shape);median spikelet (lenght of glume and its awn relative to grain).

EAREAR

MORPHO-PHYSIOLOGICAL MORPHO-PHYSIOLOGICAL CHARACTERSCHARACTERS

PART OF PART OF PLANTPLANT

Morpho-physiological characters which are in use for the study of 27 pairs of NILs.