Recurrent Plant-Specific Duplications of KNL2 and Its Conserved Function as a Kinetochore Assembly Factor.

KINETOCHORE NULL2 (KNL2) plays key role in the recognition of centromeres and new CENH3 deposition. To gain insight into the origin and diversification of the KNL2 gene, we reconstructed its evolutionary history in the plant kingdom. Our results indicate that the KNL2 gene in plants underwent three independent ancient duplications in ferns, grasses and eudicots. Additionally, we demonstrated that previously unclassified KNL2 genes could be divided into two clades αKNL2 and ßKNL2 in eudicots and γKNL2 and δKNL2 in grasses, respectively. KNL2s of all clades encode the conserved SANTA domain, but only the αKNL2 and γKNL2 groups additionally encode the CENPC-k motif. In the more numerous eudicot sequences, signatures of positive selection were found in both αKNL2 and ßKNL2 clades, suggesting recent or ongoing adaptation. The confirmed centromeric localization of ßKNL2 and mutant analysis suggests that it participates in loading of new CENH3, similarly to αKNL2. A high rate of seed abortion was found in heterozygous ßKNL2 plants and the germinated homozygous mutants did not develop beyond the seedling stage. Taken together, our study provides a new understanding of the evolutionary diversification of the plant kinetochore assembly gene KNL2, and suggests that the plant-specific duplicated KNL2 genes are involved in centromere and/or kinetochore assembly for preserving genome stability.

Bimolecular Fluorescence Complementation to Test for Protein-Protein Interactions and to Uncover Regulatory Mechanisms During Gametogenesis.

Bimolecular fluorescence complementation (BiFC) assay is one of the sensitive techniques that allows to investigate direct protein-protein interactions (PPI) in vivo and visualize the subcellular localization of interacting proteins. It is based on splitting of a fluorescent protein into two nonfluorescent parts accordingly fused to two putative interacting partners. If interaction between studied proteins is possible, nonfluorescent parts come to close proximity resulting in reconstitution of the functional fluorescent protein and giving fluorescence under certain wavelength. BiFC analysis implies transient or stable expression of the proteins of interest and can be used as a method to test or validate the direct PPI in various biological pathways, including the regulation of gametogenesis, which is the main focus of this book. In our protocol we give detailed information for beginners about three main steps of BiFC analysis of centromeric protein interactions. These steps include (1) generation of appropriate expression clones with the help of Gateway cloning technology, (2) infiltration of Nicotiana benthamiana plants by Agrobacteria containing generated constructs, and (3) microscopic analysis of plants under fluorescence microscope. Also, we discuss appropriate negative controls that can be used for evaluation as well as recommendable vector systems, possible artifacts and measures to avoid artifactual interactions for BiFC assay.

InDel markers: An extended marker resource for molecular breeding in chickpea.

Chickpea is one of the most important food legumes that holds the key to meet rising global food and nutritional demand. In order to deploy molecular breeding approaches in crop improvement programs, user friendly and cost effective marker resources remain prerequisite. The advent of next generation sequencing (NGS) technology has resulted in the generation of several thousands of markers as part of several large scale genome sequencing and re-sequencing initiatives. Very recently, PCR based Insertion-deletions (InDels) are becoming a popular gel based genotyping solution because of their co-dominant, inexpensive, and highly polymorphic nature. With an objective to expand marker resources for genomics assisted breeding (GAB) in chickpea, whole genome re-sequencing data generated on five parental lines of one interspecific (ICC 4958 × PI 489777) and two intra-specific (ICC 283 × ICC 8261 and ICC 4958 × ICC 1882) mapping populations, were used for identification of InDels. A total of 231,658 InDels were identified using Dindel software with default parameters. Further, a total of 8,307 InDels with ≥20 bp size were selected for development of gel based markers, of which primers could be designed for 7,523 (90.56%) markers. On average, markers appeared at a frequency of 1,038 InDels/LG with a maximum number of markers on CaLG04 (1,952 InDels) and minimum on CaLG08 (360 InDels). In order to validate these InDels, a total of 423 primer pairs were randomly selected and tested on the selected parental lines. A high amplification rate of 80% was observed ranging from 46.06 to 58.01% polymorphism rate across parents on 3% agarose gel. This study clearly reflects the usefulness of available sequence data for the development of genome-wide InDels in chickpea that can further contribute and accelerate a wide range of genetic and molecular breeding activities in chickpea.